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Timaeus 12

Summary of guidance for PrEP providers {#jve11-sec-0001} ====================================== ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Men who have sex with men Trans and gender diverse people Heterosexuals People who inject drugs ----------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ---------------------------------------------------------------------------------------- ---------------------------------------------------------------------------------- -------------------------------------------------------------- **When to *offer* PrEP** If the risk of acquiring HIV infection is rated as *high* according to the eligibility criteria discussed (see: Behavioural eligibility for PrEP below) **When to *consider* PrEP** If the risk of acquiring HIV infection is rated as *medium* according to the eligibility criteria discussed (see: Behavioural eligibility for PrEP below) **PrEP for those not meeting eligibility criteria** In all four scenarios, the individual may not necessarily meet the high- or medium-risk criteria. However, the clinician may deem, after taking a detailed history, that the individual is at high or medium risk, and may recommend or consider PrEP accordingly\ Note that individuals who inject drugs may have high or medium risk of HIV acquisition through sexual exposure **Clinical eligibility** Documented negative HIV test result using 4th-generation Ag/Ab test within 7 days of starting PrEP\ No signs or symptoms of acute HIV infection\ Normal renal function (eGFR \>60 mL/min/1.73 m^2^)\ No contraindicated medications (those that reduce renal function or compete for active tubular secretion may increase serum concentrations of tenofovir and emtricitabine: see Patients with chronic renal failure below) **Prescribe** Daily, continuing, oral dose of coformulated tenofovir and emtricitabine ≤90-day supply\ Patients need to take a daily dose of PrEP for 7 days before high levels of protection are achieved for both vaginal and rectal exposure to HIV **Other services** At baseline, document hepatitis B and C infection and vaccinate for hepatitis B for those not immune (see Monitoring HBV and HCV infections below)\ Follow-up visits at month 1 after PrEP initiation (optional) and at least every 3 months after initiation to provide: •HIV testing using 4th-generation Ag/Ab test (at every follow-up visit), assessment for primary HIV infection if suspected (see Testing for HIV below)•Medication-adherence assessment and support (at every follow-up visit)•Discussion about the reduction of risk of HIV and sexually transmitted infections (STIs) (at every follow-up visit)•Side effects (at every follow-up visit)•STI symptom assessment at every visit and management as required•Complete HBV vaccination if commenced or chronic hepatitis B monitoring and management, as required•Assessment of renal function at 3 months and every 6 months thereafter, or more frequently as indicated•Assessment of hepatitis C status (at least every 12 months or more frequently if necessary) **Additional testing** **Men who have sex with men**\ **Trans and gender diverse people**\ **Heterosexuals**\ **People who inject drugs**\ Every 3 months, STI testing as per Australian STI testing guidelines [@jve11-bib-0001] Every 3 months, STI testing as per Australian STI testing guidelines [@jve11-bib-0001] Assess pregnancy intent and conduct pregnancy test every 3 months if appropriate Test for STI if indicated and hepatitis C\ Access to clean needles/syringes and drug treatment services **Optional testing** HIV testing at 1 month or sooner at clinician\'s discretion based upon clinician\'s concerns around adherence, or that a high-risk HIV exposure occurred 3 or more days prior to PrEP initiation\ Bone mineral density in patients at risk on initial or subsequent assessment\ Vitamin D levels\ Proviral DNA testing if early (primary) HIV infection is suspected ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Introduction {#jve11-sec-0002} ============ Co-formulated tenofovir and emtricitabine for use as HIV pre-exposure prophylaxis (PrEP) by populations at high risk of HIV infection is now recommended in guidelines in the United States, Europe and Australia [@jve11-bib-0002], as well as globally through the WHO guidelines [@jve11-bib-0005]. These clinical PrEP guidelines update the 2015 Australasian Society for HIV, Viral Hepatitis and Sexual Health Medicine\'s (ASHM) PrEP guidelines and they represent an adaptation and update of the 2014 United States Centers for Disease Control\'s PrEP guidelines [@jve11-bib-0003]. Between May 2016 and January 2017, the Australian Therapeutic Goods Administration (TGA) approved tenofovir disoproxil fumarate and emtricitabine (TDF/FTC, Truvada), tenofovir disoproxil maleate and emtricitabine (TDM/FTC, Trucitavir) and tenofovir disoproxil phosphate and emtricitabine (TDP/FTC, Tenofovir EMT Lupin) for entry onto the Australian Register of Therapeutic Goods (ARTG). These medications are registered for daily use in individuals aged 18 years and over. However, these medications are not yet subsidised by the Pharmaceutical Benefits Scheme for use as HIV PrEP [@jve11-bib-0006]. Other drugs used for PrEP but not registered in Australia can be legally imported into Australia using the TGA Personal Importation Scheme (PIS) [@jve11-bib-0007]. For simplicity, the form TD\* is used to denote the tenofovir disoproxil component present in the three registered drugs. The recommendations included here are designed to: •support the prescription of PrEP using the ARTG-listed drugs and other bioequivalent generic drugs that are available in Australia, or are being used in Australian PrEP trials•assist clinicians in their evaluation of patients who are seeking PrEP•to assist clinicians in commencing and monitoring their patients on PrEP by providing information on PrEP dosing schedules, management of side-effects and toxicity, use of PrEP in pregnancy and in chronic hepatitis B infection and how to cease PrEP.Daily PrEP with TD\*/FTC is recommended in CDC, WHO and earlier Australian guidelines as a key HIV-prevention option for men who have sex with men (MSM), transgender men and women, heterosexual men and women, and people who inject drugs (PWID) at substantial risk of HIV acquisition. Based on available evidence, this guideline does not recommend the coitally timed use of PrEP. Such use can be considered only for patients at risk of HIV infection who are unable to take daily PrEP, and it is contraindicated for people with chronic hepatitis B infection. The intended users of this guideline include: •general practitioners who provide care to persons at risk of acquiring HIV infection•sexual health physicians who provide care to persons at risk of acquiring HIV infection•infectious disease and HIV treatment specialists who may provide PrEP, or serve as consultants to primary-care physicians about the use of antiretroviral medications•trainees and registrars in each of the above categories•nurse practitioners•nurses working in nurse-led clinics in consultation with doctors•peer workers•counsellors and individuals performing HIV testing, including point-of-care testing•health program policymakers•health consumers and others with an interest in HIV PrEP HIV prevention in Australia {#jve11-sec-0003} =========================== Australia endorsed the 2011 UN Declaration of commitment on HIV/AIDS, and Australian Health Ministers have endorsed the UN Declaration\'s prevention and treatment targets adapted to the Australian context. These targets informed the development of the Seventh National HIV Strategy 2014--2017 and a number of jurisdictional HIV strategies, with the ambitious goal of virtually eliminating HIV transmission in Australia by 2020 [@jve11-bib-0008]. Primary HIV-prevention strategies in Australia focus on the use of safer sex practices, including condoms, clean needles and biomedical strategies. Other prevention strategies promoted in Australia include HIV testing, immediate HIV treatment to reduce HIV viral load, and HIV post-exposure prophylaxis. Australian testing guidelines recommend that all sexually active MSM test for HIV and retest every 12 months, and that those who are at high risk of HIV infection should retest up to four times a year [@jve11-bib-0009]. Among priority populations considered to be at high risk of HIV infection due to sexual or injecting behaviour, the proportion tested annually has generally been high [@jve11-bib-0010]. Among MSM, both the proportion tested in the last year and the average number of HIV tests per year increased during the period 2011--2015. Indications for PrEP in Australia {#jve11-sec-0004} ================================= HIV epidemiology {#jve11-sec-0005} ---------------- Australia has a concentrated HIV epidemic, with sexual contact between men (with or without injection drug use) accounting for 73% of new HIV diagnoses in 2015 [@jve11-bib-0011]. A further 20% of cases were attributed to heterosexual sex. In these cases, 40% occurred in people from a country with high HIV prevalence, or who had a partner from such a country. Only 3% of new HIV diagnoses were attributable to injecting drug use alone. Other or unknown exposures contributed only 3.8% of infections in 2015 [@jve11-bib-0011]. Table [1](#jve11-tbl-0002){ref-type="table"} summarises the key factors associated with an increased risk of HIV acquisition among gay and bisexually identified men in the Sydney-based Health in Men (HIM) study. Four factors were associated with HIV incidence of above 1.8 per 100 person years; these factors formed a group of criteria for identifying people at high risk of HIV. Two more factors with an HIV incidence above 1.0 and below 1.8 per 100 person years formed this guideline\'s criteria for medium HIV risk. The HIM study collected data from 2001 to 2007. While treatment rates are now higher, the annual number of HIV diagnoses has increased by 8% since 2007, and the same risk factors for increased HIV infection risk are likely to remain relevant in Australia today. Therefore, the HIV risk factors reported by the HIM study provided a basis for formulating criteria to identify MSM at increased HIV risk and eligible for PrEP. Due to the specifics of data collection for the HIM study, not all indicators were available to support each individual eligibility criterion. Some indicators were collected in different forms, or have a different denominator or reference period. Most importantly, the HIV treatment and viral load of HIV-positive regular partners is now known to have a significant impact on HIV transmission; however, these data were not collected in the HIM study. Similarly, infectious syphilis was uncommon and not associated with HIV transmission in the HIM cohort, but its incidence has increased greatly since 2007. Syphilis is associated with an increased risk of HIV among MSM globally, and is therefore included in the risk criteria. Finally, the reference period for PrEP eligibility assessment is set up in these guidelines to reflect behaviour over the previous 3 months. ###### Factors associated with elevated risk of HIV acquisition among MSM in the Health in Men (HIM) study, Australia, 2001--2007, and their translation into eligibility criteria for PrEP Findings of the HIM study Criteria to identify increased risk of HIV ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------- -------------------------------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- All gay and bisexual men regardless of behavioural practices 0.78 (0.59--1.02) n/a **HIV risk factors: high risk** A regular sexual partner of an HIV-positive man with whom condoms were not consistently used in the last 6 months 5.36 (2.78--10.25) A regular sexual partner of an HIV-infected men (***not on treatment and/or detectable viral load***) with whom condoms were not consistently used in the last 3 months At least one episode of receptive, unprotected anal intercourse with any casual HIV-infected male partner or a male partner of unknown HIV status during the last 6 months 2.31 (1.48--3.63) At least one episode of receptive condomless anal intercourse (CLAI) with any casual HIV-infected male partner or a male partner of unknown HIV status in the last 3 months Rectal gonorrhoea diagnosis in last 6 months 7.01 (2.26--21.74) Rectal gonorrhoea, rectal chlamydia or ***infectious syphilis*** diagnosis in the last 3 months Rectal chlamydia diagnosis in last 6 months 3.57 (1.34--9.52) Methamphetamine use in last 6 months 1.89 (1.25--2.84) Methamphetamine use in last 3 months **HIV risk factors: medium HIV risk to be used in individual clinical assessment** More than one episode of anal intercourse during the last 3 months when proper condom use was not achieved (e.g. condoms slipped off or broke) 1.30 (0.95--1.77) More than one episode of anal intercourse during the last 3 months when proper condom use was not achieved (e.g. condoms slipped off or broke) A regular sexual partner of CLAI or having at least one episode of insertive CLAI where the serostatus of partner is not known or is HIV-positive 0.94 (0.35--2.52) n/a In uncircumcised men having at least one episode of insertive CLAI where the serostatus of partner is not known or is HIV-positive 1.73 (0.43--6.90) (If patient is uncircumcised) having in the last three months more than one episode of insertive CLAI where the serostatus of partner was not known or was HIV-positive and not on treatment In circumcised men (comparison group, low risk, PrEP not recommended) 0.65 (0.16--2.61) n/a Note that while the HIM study uses the terminology of 'gay and bisexual men', this guideline uses 'men who have sex with men' (MSM) to focus on behaviour, rather than identity. Behavioural eligibility for PrEP {#jve11-sec-0006} ================================ Providers need to obtain a thorough sexual and drug-use history at baseline to determine PrEP eligibility. Thereafter, they must regularly discuss HIV-risk practices with their patients to assess their ongoing eligibility for PrEP. Medium-risk behaviours have been included to assist clinicians in the case-by-case approach to assessing eligibility. These guidelines acknowledge that as an HIV-prevention option, PrEP may be considered by anyone who is at risk of acquiring HIV. Some patients will not fall within the recommended eligibility criteria outlined below, but may still benefit from accessing PrEP. Examples of why a case-by-case approach is important when evaluating a person for PrEP include: •those patients without recent evidence of sexually transmitted infections (STIs) or high-risk sexual practices, but who report that they have recently left a monogamous relationship and will be having condomless sex with casual partners in the future•a person whose HIV risk does not meet the high- or medium-risk criteria, but is so anxious about HIV infection that it impedes their quality of life and may prevent them from having regular HIV testingClinicians with limited experience in sexual health or management of patients on PrEP are encouraged to discuss cases where PrEP eligibility is not straightforward with a clinician who is experienced in HIV PrEP. Behavioural eligibility criteria for PrEP for men who have sex with men (Box [1](#jve11-fea-0001){ref-type="boxed-text"}) {#jve11-sec-0007} ------------------------------------------------------------------------------------------------------------------------- ###### Risk criteria for MSM to identify their eligibility for PrEP ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ----- --------------------------------------------------------------------------------------------------------------------------------------------------------- **A. High risk -- recommend prescribing daily PrEP if the patient acknowledges** **Having had any of the following in the last 3 months** •At least one episode of condomless anal intercourse (CLAI) with a regular HIV+ partner (not on treatment and/or detectable viral load)•At least **one** episode of receptive CLAI with any casual HIV+ male partner or a male partner of unknown status•Rectal gonorrhoea, rectal chlamydia or infectious syphilis diagnosis (during the last 3 months or at screening for PrEP)•Methamphetamine use, which may increase the risk of HIV acquisition AND **Being likely to have in the next 3 months** (indicating sustained risk) •Multiple episodes of CLAI with or without sharing intravenous drug equipment **B. Medium risk -- consider prescribing daily PrEP, based on a case-by-case approach if discussion reveals** **Having had any of the following in the last 3 months** •More than one episode of anal intercourse when proper condom use was not achieved (e.g. condom slipped off or broke) where the serostatus of partner was not known, or was HIV+ and not on treatment or with a detectable viral load•(If patient uncircumcised) more than one episode of insertive CLAI where the serostatus of partner was not known, or was HIV+ and not on treatment or with a detectable viral load AND **Being likely to have in the next 3 months** (indicating sustained risk) •Multiple episodes of CLAI with or without sharing intravenous drug equipment **Case-by-case approach**\ Based on a complete sexual and alcohol and other drug-using history and the personal circumstances of the patient, if the clinician is of the opinion that the patient is likely to be at high risk of HIV, then PrEP prescription may be considered despite the absence of reported high- or medium-risk factors above. ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ----- --------------------------------------------------------------------------------------------------------------------------------------------------------- PrEP for trans and gender diverse people (Box [2](#jve11-fea-0002){ref-type="boxed-text"}) {#jve11-sec-0008} ------------------------------------------------------------------------------------------ Trans and gender diverse individuals have rarely been included in PrEP studies. As a result, limited data are available for these individuals. Incorrect assumptions can be made about trans people and their sexual practices, although they may practice vaginal/neovaginal and anal intercourse, or insertive and receptive sex. Trans and gender diverse people who are at high risk of acquiring HIV on the basis of their sexual history are eligible to access PrEP. ###### Risk criteria for trans and gender diverse people to identify their eligibility for PrEP --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ----- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------ **A. High risk -- recommend prescribing daily PrEP if the patient acknowledges** **Having had any of the following in the last 3 months** •Being a regular sexual partner of an HIV+ person (not on treatment and/or detectable viral load) with whom condoms have not been consistently used•At least **one** episode of receptive condomless intercourse (CLI) with any casual HIV+ partner or a male partner of unknown status•Rectal or vaginal gonorrhoea, rectal or vaginal chlamydia or infectious syphilis diagnosis (during the last 3 months or at screening for PrEP)•Methamphetamine use, which may increase the risk of HIV acquisition AND **Being likely to have in the next 3 months** (indicating sustained risk) •Multiple episodes of anal or vaginal CLI with or without sharing intravenous drug equipment **B. Medium risk -- consider prescribing daily PrEP, based on a case-by-case approach if discussion reveals** **Having had any of the following in the last 3 months** •More than one episode of anal or vaginal intercourse when proper condom use was not achieved (e.g. condom slipped off or broke) and where the serostatus of partner was not known, or was HIV+ and not on treatment or with a detectable viral load•(If patient uncircumcised) more than one episode of insertive CLAI where the serostatus of partner was not known, or was HIV+ and not on treatment or with a detectable viral load AND **Being likely to have in the next 3 months** (indicating sustained risk) •Multiple episodes of anal or vaginal CLI with or without sharing intravenous drug equipment **Case-by-case approach**\ Based on a complete sexual and alcohol and other drug-using history and the personal circumstances of the patient, if the clinician is of the opinion that the patient is likely to be at high risk of HIV, then PrEP prescription may be considered despite the absence of reported high- or medium-risk factors above. --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ----- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------ PrEP for heterosexual people (Box [3](#jve11-fea-0003){ref-type="boxed-text"}) {#jve11-sec-0009} ------------------------------------------------------------------------------ ###### Risk criteria for heterosexual people to identify their eligibility for PrEP ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ----- ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- **A. High risk -- recommend prescribing daily PrEP if the patient acknowledges** **Having had any of the following in the last 3 months** •Being a regular sexual partner of an HIV+ person (not on treatment and/or with detectable viral load) with whom condoms have not been consistently used•At least **one** episode of receptive anal or vaginal condomless intercourse (CLI) with any casual HIV+ partner or a male homosexual or bisexual partner of unknown status•A female patient in a serodiscordant heterosexual relationship, who is planning natural conception in the next 3 months AND **Being likely to have in the next 3 months** (indicating sustained risk) •Multiple episodes of CLI with or without sharing intravenous drug equipment **B. Medium risk -- consider prescribing daily PrEP, based on a case-by-case approach if discussion reveals** **The patient acknowledges having had any of the following in the last 3 months** •At least one episode of CLI with a heterosexual partner, not known to be HIV--, from a country with high HIV prevalence AND **Being likely to have in the next 3 months** (indicating sustained risk) •Multiple episodes of CLI with a heterosexual partner, not known to be HIV+, but at high risk of being HIV+ with or without sharing injecting equipment **Case-by-case approach**\ Based on a complete sexual and alcohol and other drug-using history and the personal circumstances of the patient, if the clinician is of the opinion that the patient is likely to be at high risk of HIV, then PrEP prescription may be considered despite the absence of reported high- or medium-risk factors above. Specific consideration should be given to recommending PrEP to a patient who is travelling to one or more countries with high HIV prevalence and is likely to be sexually active while travelling. ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ----- ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- PrEP for people who inject drugs (Box [4](#jve11-fea-0004){ref-type="boxed-text"}) {#jve11-sec-0010} ---------------------------------------------------------------------------------- In the first instance, PWID should be advised of and provided with options for using sterile needles, syringes and other injecting equipment, or offered opioid substitution therapy. PWID can be referred to local needle and syringe programs (NSPs) or the Australian Injecting and Illicit Drug Users League affiliates in their state or territory. Because PWID may face many health threats, PrEP and other HIV-prevention interventions should be integrated with prevention and clinical care services for hepatitis B and C infection and other infectious diseases services. This includes screening for both viruses and vaccination for hepatitis B where indicated, and injection-related injuries and disease, including abscesses, septicaemia, endocarditis and overdose [@jve11-bib-0012]. The ASHM PrEP guidelines panel is cognisant of the concerns of the International Network of People who Use Drugs. The Network cautions against prioritising PrEP at the expense of other proven interventions as the prime HIV-prevention strategy for PWID, and emphasises that access to harm-reduction services remains a critical component of HIV prevention in PWID [@jve11-bib-0013]. The ASHM PrEP guidelines panel will monitor the outcomes of the few ongoing studies of PrEP in PWID. ###### Risk criteria for PWID to identify their eligibility for PrEP -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ----- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ **A. High risk -- recommend prescribing daily PrEP if the patient acknowledges** **Having in the last 3 months** •Shared injecting equipment with an HIV+ individual or with a gay or bisexual man of unknown HIV status AND **Being likely to have in the next 3 months** (indicating sustained risk) •Multiple events of sharing injecting equipment with an HIV+ individual or a gay or bisexual man of unknown HIV status•Inadequate access to safe injecting equipment **Case-by-case approach**\ Based on a complete sexual and alcohol and other drug-using history and the personal circumstances of the patient, if the clinician is of the opinion that the patient is likely to be at high risk of HIV, then PrEP prescription may be considered despite the absence of reported high- or medium-risk factors above. -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ----- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ PWID may also be at elevated risk of HIV acquisition through sexual exposure. Therefore, they should be evaluated for PrEP using risk criteria outlined in Boxes [1](#jve11-fea-0001){ref-type="boxed-text"}, [2](#jve11-fea-0002){ref-type="boxed-text"} and [3](#jve11-fea-0003){ref-type="boxed-text"}. Clinical assessment before starting PrEP {#jve11-sec-0011} ======================================== All patients whose sexual or drug injection history indicates the recommendation or consideration of PrEP, and who are interested in taking PrEP, must undergo laboratory testing. The tests identify those for whom this intervention would be harmful, or in whom it could present specific health risks that would require close monitoring. HIV testing {#jve11-sec-0012} ----------- For patients' safety, ***a negative HIV test result must be performed and documented at the time that the patient is evaluated for PrEP****.* This is because daily TD\*/FTC combination alone is insufficient for treatment of undiagnosed acute or chronic HIV infection. ***HIV testing should be repeated every 3 months*** (before prescriptions are refilled or reissued). This requirement should be explained to patients during the discussion about whether PrEP is appropriate for them. ***A fourth-generation HIV antibody/antigen test should be used,*** and performed within 7 days of the patient being evaluated for PrEP. Clinicians should tell patients to start PrEP within 7 days of the day that their HIV-negative test was performed. Rapid, point-of-care tests (PoCT) should not be used alone to screen for HIV infection when considering PrEP, because they are less sensitive than blood tests [@jve11-bib-0014]. A PoCT can exclude potential PrEP users who are found to be HIV-positive, and any reactive PoCT should be confirmed by conventional laboratory testing in line with the Australian HIV Testing Policy. Seroconversions to HIV have been described in the published literature that appeared to be related to stage 0 or pre-seroconversion HIV, and were acquired very soon before testing for the trial entry, but could not be detected by PoCT [@jve11-bib-0015]. Clinicians should not accept patient-reported test results, or documented anonymous test results. ***Any positive HIV antibody test*** ***result*** must be managed according to the Australian HIV Testing Policy and local management guidelines ( [www.testingportal.ashm.org.au/](www.testingportal.ashm.org.au/)). ***A course of nPEP may be required before transitioning to PrEP***, in accordance with the PEP and nPEP Guidelines [@jve11-bib-0017] if a patient had a recent high-risk exposure (within 72 hours). See nPEP and PrEP for more information. ***Patients who have had a recent high-risk exposure outside the window for the commencement of nPEP*** should be started on PrEP and closely monitored for seroconversion using a fourth-generation HIV test for the next 2--8 weeks before reverting to standard PrEP monitoring. ***Acute HIV infection should be suspectedin individuals at high risk of HIV who have been exposed recently to HIV*** (e.g. no condom or a condom broke during sex with an HIV-positive partner not on treatment, or casual partner of MSM; recent injection drug use with shared injection equipment with MSM, or person known to be HIV-positive). In a prospective study of 2226 people at high risk of HIV infection who underwent twice-weekly HIV nucleic acid testing, 50 people were evaluated for their clinical signs and symptoms during acute HIV infection. Symptoms occurred in 94% of participants with acute HIV infection, just before and around the time of peak HIV viraemia [@jve11-bib-0018]. Importantly, when individuals were examined during study visits during the period of acute infection, 71% reported no symptoms and 50% reported no symptoms or signs. Hence, clinical detection of acute HIV infection may not be possible in all patients who present for PrEP. The most common symptoms were fever, headache and malaise, while the most common signs were related to the head, eyes, ears, nose, throat, tachycardia and lymphadenopathy (see Table [2](#jve11-tbl-0007){ref-type="table"}). ###### Symptoms and abnormalities associated with primary or acute HIV infection, overall and by region [@jve11-bib-0018] Africa (*n*=31) Thailand (*n*=17) Overall (*n*=48) ----------------------------------------------------------- ----------------- ------------------- ------------------ ----- ---- ---- **Symptom** Fever 18 55 7 41 25 50 Headache 17 52 6 35 23 46 Feeling of illness 14 42 5 29 19 38 Coughing 10 30 9 535 19 38 **Abnormality** HEENT[^a^](#jve11-fn-0004){ref-type="table-fn"} 6 18 16 94 22 44 Lymphadenopathy[^b^](#jve11-fn-0005){ref-type="table-fn"} 9 9 16 94 19 38 Tachycardia 11 33 5 29 16 32 Head, ears, eyes, nose and throat A condition or disease affecting the lymph glands of the body resulting in lymph nodes that are abnormal in size, consistency or number Initiation of TD\*/FTC PrEP in individuals with undiagnosed primary or acute (symptomatic) HIV infection has been associated with the development of resistance to TD\*/FTC, mostly commonly to the FTC component [@jve11-bib-0019]. Individuals who present with signs or symptoms consistent with acute HIV infection should not be commenced on PrEP until HIV infection has been excluded. ***Patients with indeterminate HIV test results at baseline*** should not be started on PrEP. They should be assessed for early HIV infection and treated according to local antiretroviral treatment guidelines [@jve11-bib-0023]. Such patients can only be started on PrEP if and when HIV infection is excluded. Concerns about TD\* or FTC resistance {#jve11-sec-0013} ------------------------------------- Overall, the risk of developing TD\* or FTC resistance among participants on PrEP is low [@jve11-bib-0024]. According to a World Health Organization (WHO) meta-analysis of HIV resistance data from randomised clinical trials of PrEP, participants on PrEP versus placebo who started PrEP at the time of acute HIV infection had a higher risk of developing resistance, with more cases of resistance developing to FTC than to TD\*. Only a few TD\* or FTC mutations were recorded among participants who seroconverted after randomisation [@jve11-bib-0024]. Assessment of renal function at baseline {#jve11-sec-0014} ---------------------------------------- In a meta-analysis, tenofovir use in HIV-positive patients was associated with a statistically significant loss of renal function, with the effect being judged as clinically modest [@jve11-bib-0025]. Tenofovir use was not associated with increased risk of fractures, hypophosphataemia or severe proteinuria [@jve11-bib-0025]. Rarely, proximal renal tubular dysfunction (including Fanconi\'s syndrome) may occur with TD\* use [@jve11-bib-0025]. Overall, tenofovir use in PrEP studies has not been associated with significant renal problems, although some patient populations may be at a higher risk for renal decline while taking TD\*/FTC for PrEP. In the US PrEP Demo Project, 23 occurrences of elevated creatinine levels were seen in 13 of 557 individuals (2.3%), including 22 grade 1 and one grade 2 events [@jve11-bib-0028]. On repeat testing, only three elevations among three participants were confirmed, and all episodes of creatinine elevation resolved within 2--20 weeks without stopping PrEP [@jve11-bib-0029]. In a recent conference report that updated the US PrEP Demo Project findings, the median decline in creatinine clearance was 6 mL/min (5%) from baseline to week 12. It then remained stable through to week 48 (*P*=0.96), with no differences by race/ethnicity, weight or use of non-steroidal agents. However, new-onset estimated glomerular filtration rate (eGFR) \<70 mL/min/1.73 m^2^ was more common in those with baseline eGFR \<90 mL/min/1.73 m^2^, and was seen more obviously in people over the age of 45 [@jve11-bib-0028]. However, in a multivariable analysis, the following factors were independently associated with greater creatinine clearance loss: age younger than 25 years, use of medications for hypertension or diabetes, and red blood cell tenofovir diphosphate (TFV-DP) levels consistent with taking two or more doses/week of TD\*/FTC. The authors reported that younger PrEP users may warrant increased monitoring of renal function [@jve11-bib-0028]. In another study, factors associated with greater odds of the eGFR falling below 60 mL/min/1.73 m^2^ were: age greater than 40 years, a baseline creatinine clearance of less than 90 mL/min and evidence of good adherence [@jve11-bib-0030]. Finally, older age was associated with greater decline in renal function in the Bangkok Tenofovir study [@jve11-bib-0031]. For all patients considered for PrEP, their risk factors for renal disease should be assessed. These include diabetes, hypertension, smoking, concurrent medications and a known history of renal impairment. Measurements of baseline serum creatinine, eGFR, urine protein: creatinine ratio (PCR) and blood pressure should also be taken. The Cockcroft--Gault formula (see Appendix 1) is regarded as the ideal way to measure the eGFR. However, for most practitioners, this is not practical. Instead, it is reasonable to measure the patient\'s renal function using the eGFR. A patient with an eGFR of less than 60 mL/min/1.73 m^2^ should not be prescribed PrEP. These guidelines recommend that individuals' creatinine, eGFR and urinary PCR are evaluated at 3 months after commencing PrEP then 6 monthly thereafter, while they receive PrEP. However, based on currently available evidence, more intensive monitoring may be warranted in individuals under the age of 25 years or over the age of 40 years, and in those with a baseline eGFR of less than 90 mL/min/1.73 m^2^. Assessment for and management of sexually transmissible infections at baseline {#jve11-sec-0015} ------------------------------------------------------------------------------ Individuals at high risk for HIV infection are also at high risk for STIs. Clinicians should screen for STIs (specifically gonorrhoea, chlamydia and infectious syphilis) using the standard of care tests and procedures, and manage any detected STI as recommended by the Australian STI Management Guidelines [@jve11-bib-0009]. The presence of an STI at baseline should not delay the commencement of PrEP. Patients starting on PrEP should be informed about: •prevention of STI acquisition and transmission•frequency of STI testing•signs and symptoms of STIs•the need to present for testing and treatment whenever signs or symptoms of STIs appear Assessment for hepatitis B and C status {#jve11-sec-0016} --------------------------------------- Patients assessed as high or medium risk for HIV can also be at risk of acquiring hepatitis B virus (HBV) [@jve11-bib-0032] and hepatitis C virus (HCV) [@jve11-bib-0033] infection. HBV and HCV infection status should be documented by screening serology when PrEP is initiated. Vaccination against HBV is recommended for all susceptible priority populations, which include MSM; sex workers; people from countries with a high HIV, HBV or HCV prevalence, and their sexual partners; and PWID [@jve11-bib-0034]. Individuals identified at baseline as having undiagnosed chronic hepatitis B should be referred to a clinician experienced in the management of hepatitis B for treatment assessment. Individuals on TD\*/FTC for HIV PrEP should be counselled on the importance of strict adherence to PrEP to prevent both a flare in their hepatitis B infection and the development of hepatitis B resistance to TD\*/FTC. Individuals identified at baseline with undiagnosed hepatitis C infection should be referred to a clinician experienced in hepatitis C management for consideration of hepatitis C treatment. A diagnosis of hepatitis B or hepatitis C is not an impediment to HIV PrEP initiation. Other considerations and testing before commencement of PrEP {#jve11-sec-0017} ------------------------------------------------------------ Additional testing may be warranted in some special subpopulations. Assessment of bone health Low bone mineral density (BMD) has been observed at baseline in approximately 10% of individuals receiving TD\*/FTC for PrEP in two studies [@jve11-bib-0028]. Hence, individuals should be counselled about the effects of TD\* on BMD and counselled to decrease alcohol and cigarette use, undertake weight-bearing exercise and ensure their diet provides adequate amounts of calcium and vitamin D [@jve11-bib-0036]. A clinician may suspect that an individual is vitamin D deficient and may wish to test their vitamin D levels. There is no evidence that over-the-counter vitamin D supplements reduce tenofovir-related BMD changes. A small but significant decline in BMD has been observed in three studies using TD\* or TD\*/FTC for PrEP. The decline in BMD correlates directly with levels of intracellular TD\*-DP and is reversible following PrEP cessation [@jve11-bib-0036]. A person with a history of osteoporosis will require careful monitoring while on PrEP. If the clinician suspects that a person may have osteoporosis, they may recommend BMD testing. BMD testing is rebated in Australia under specific clinical circumstances (more information about BMD rebates can be found at: [www.health.gov.au/internet/main/publishing.nsf/content/diagnosticimaging-bd.htm](www.health.gov.au/internet/main/publishing.nsf/content/diagnosticimaging-bd.htm)). Assessment for pregnancy (in women of child-bearing age, where applicable or suspected) PrEP can be used in pregnancy. In observational studies of HIV-positive women treated throughout pregnancy with TD\*-containing regimens, TD\* during pregnancy has not been associated with adverse outcomes, but lowered BMD was observed in their newborns [@jve11-bib-0037]. In HIV-negative women in PrEP trials, PrEP was promptly discontinued for those who became pregnant. Therefore, the safety for exposed fetuses could not be adequately assessed. Some women with an HIV-positive partner may prefer to continue PrEP while pregnant, due to increased risk of acquisition of HIV during pregnancy. Providing PrEP {#jve11-sec-0018} ============== Goals of PrEP {#jve11-sec-0019} ------------- The ultimate goal of PrEP is to reduce the acquisition of HIV infection and its resulting morbidity, mortality, and cost to individuals and society. Therefore, clinicians initiating the provision of PrEP should: •prescribe medication regimens that are proven safe and effective for HIV-negative persons who meet recommended criteria to reduce their risk of HIV acquisition•educate patients about the medications and the regimen to maximise their safe use•provide medication-adherence support and counselling to help patients achieve and maintain protective levels of medication in their bodies•provide HIV risk-reduction support and prevention services or service referrals to help patients minimise their risk of acquiring HIV•provide effective contraception to women who are taking PrEP and do not wish to become pregnant•monitor patients to detect HIV infection, medication toxicities, STIs and levels of risk behaviour, make indicated changes in strategies to support patients' long-term health, and initiate timely treatment of infections. The choice of PrEP schedule: daily *vs* on-demand {#jve11-sec-0020} ------------------------------------------------- PrEP can be used daily for long periods of time, and this is the most commonly prescribed PrEP regimen (also known as daily PrEP). PrEP can also be effective when used on demand, e.g. daily for short periods of time, or around single events of HIV exposure. The choice as to how to take PrEP should be made by the individual with advice from their PrEP provider. ### Daily PrEP {#JVEv4-sec-1003} Daily PrEP should be recommended to people who have ongoing high or medium risk of acquiring HIV. In Australia, TD\*/FTC has been registered for use as a daily medication. ### On-demand PrEP {#JVEv4-sec-2003} If exposure happens only for relatively short periods of time (e.g. during travel), or irregularly, on-demand PrEP can be recommended to cover the events of exposure and this strategy can be used on an ongoing basis. It may also suit those who have adverse events, such as nausea and diarrhoea, or changes in their kidney function, due to previous use of daily PrEP. #### Evidence in support of on-demand PrEP dosing {#JVEv4-sec-1004} In France, where gay men are routinely offered the choice of daily PrEP or on-demand PrEP, approximately two-thirds opt for the latter regimen. In the AMPrEP (Netherlands) and Be PrEPared (Belgium) implementation studies, about one-third of men opted to take PrEP on demand ( [www.prepineurope.org/en/faqs/does-prep-work/intermittent-prep/](www.prepineurope.org/en/faqs/does-prep-work/intermittent-prep/)). In a recent report from the PrELUDE study from New South Wales, one third of individuals enrolling in the study expressed a preference for non-daily PrEP [@jve11-bib-0038]. Data on the efficacy of non-daily PrEP dosing are available only for MSM and transgender women, and only from a single randomised, placebo-controlled trial, IPERGAY and its open-label extension IPERGAY OLE. IPERGAY evaluated the efficacy of an on-demand regimen comprising two pills of TDF/FTC (versus placebo) taken 2--24 hours before potential sexual exposure to HIV, followed by one pill daily until 48 hours had passed after the last act of sexual intercourse. If sexual activity is resumed within a week, a single, rather than a double, dose before sex was recommended. If sexual activity resumed more than a week later, the loading dose schedule (two tablets) was recommenced, followed by one pill daily until 48 hours had passed after the last act of sexual intercourse. The incidence of HIV was high in the placebo group, and a risk reduction of 86% and 97% was reported in the randomised and open-label phases, respectively [@jve11-bib-0039]. The IPERGAY participants reported using a median of 15 PrEP pills per month (interquartile range 9--21), which is approximately four pills per week. Previously, the results from a pharmacological TDF/FTC dosing study in 21 HIV-negative volunteers were extrapolated to the Pre-exposure Prophylaxis Initiative (iPrEx) study participants who had taken TD\*/FTC daily. The results suggested that four daily TDF/FTC doses per week were highly protective for MSM and transgender women [@jve11-bib-0041]. The efficacy of an on-demand regimen in MSM who have infrequent HIV exposure (less than one episode per week) was examined in a subsequent analysis of the IPERGAY data aiming to establish whether men having sex less often were still protected by an on-demand PrEP strategy. The sub-study looked at time periods during which participants used 15 or fewer pills per month and said they used PrEP 'from time to time or never' and not 'systematically or often'. Patterns of PrEP use varied over time for individuals and between participants. Overall, 269 participants had this pattern of PrEP use at some point during the study. In total, these 269 participants accumulated 134 person years of follow-up. During these periods of lower PrEP use, participants used a median of 9.5 pills per month, had sex a median of five times per month, and no HIV infections occurred. All participants who acquired HIV during these follow-up periods were in the placebo group and were not taking PrEP. The study investigators concluded that an on-demand PrEP strategy remains highly effective in MSM even when they have infrequent sex [@JVEv4-bib-0042]. Additionally, the feasibility of different TD\*/FTC dosing methods was investigated in a single Phase II, randomised, open-label trial of pharmacokinetics and behaviour -- the HIV Prevention Trials Network HPTN 067 (also known as the ADAPT study and available at: [www.hptn.org/research/studies/hptn067](www.hptn.org/research/studies/hptn067)). It is important to emphasise that on-demand PrEP should not be used in cis-gender women or heterosexual men owing to a lack of data about its efficacy in these populations. #### PEP {#JVEv4-sec-1005} If an individual is not taking PrEP but presents within 72 hours of a potential HIV exposure they should be assessed for nPEP as a matter of urgency and should be offered nPEP immediately according to current nPEP guidelines ( [www.ashm.org.au/HIV/PEP/](www.ashm.org.au/HIV/PEP/)) if appropriate. If risks are likely to continue into the future, PrEP should be offered as it will generally be superior to attempting to access nPEP on an ongoing basis. ### Recommendation {#JVEv4-sec-1006} Based on evidence described above, the ASHM PrEP guidelines panel continues to recommend daily TD\*/FTC dosing for all population groups eligible for PrEP, but on-demand PrEP should be considered an alternative regimen for MSM and transgender women and offered where appropriate. On-demand PrEP should not be used in cis-gender women and heterosexual men as there is currently no evidence of effectiveness in these populations. The panel will monitor developments concerning PrEP and provide updated guidance as additional evidence becomes available. Duration of PrEP use and follow-up schedule {#jve11-sec-0021} ------------------------------------------- Along with encouraging safer sex practices and safer injection techniques (if applicable), clinicians should help their patients decide when to use PrEP and when to discontinue its use. ***The duration of PrEP use*** will depend on whether the individual\'s risk of HIV continues over time. PrEP should only be prescribed to those patients who are able to adhere to a regimen that has been shown to be efficacious and express a willingness to do so. ***Initial prescription*** should offer a 90-day medication supply. ***Extensions of PrEP prescriptions*** should cover no more than 90 days of TD\*/FTC supply at a time, renewable only after HIV testing confirms that a patient remains HIV-uninfected and still eligible for PrEP. The recommended schedule of testing and follow-up is outlined in Table [3](#jve11-tbl-0008){ref-type="table"}. ***Adherence to PrEP*** ***should be assessed*** at each follow-up visit. PrEP users who otherwise declare non-adherence, but are willing and eligible to continue on PrEP, should be offered additional adherence education (see Improving medication adherence, including offering referral to peer-based support services). If a patient repeatedly reports adherence that is sufficiently suboptimal to compromise both PrEP\'s efficacy (i.e. fewer than four pills per week) and the patient\'s safety, the clinician should discontinue prescribing PrEP. See also nPEP and PrEP for the course of action if a patient was not adherent to PrEP and had a risk of exposure in the last 72 hours. Indicated medication {#jve11-sec-0022} -------------------- The medications proven safe and effective, and currently approved by the TGA for PrEP in healthy adults at risk of acquiring HIV infection, are the fixed-dose combination of TD\* and FTC in a single daily dose. Therefore, TD\*/FTC or other generic versions of TD\*/FTC are the recommended medications that should be prescribed for PrEP for MSM, trans and gender diverse individuals heterosexuals, and PWID who meet recommended criteria. TDF alone has been proven effective in trials with PWID and heterosexuals (with efficacy comparable to TDF/FTC [@jve11-bib-0024]). As a result, WHO recommends that TDF alone can be considered as an alternative regimen in these specific populations. TDF alone is not recommended as PrEP for MSM, because no trials have been performed to assess the efficacy of this regimen in MSM. There have been some recent overseas reports of HIV seroconversion in MSM taking unprescribed antiretroviral medication for PrEP [@jve11-bib-0045]. What not to use for PrEP {#jve11-sec-0023} ------------------------ DO NOT use: •other antiretroviral medications, either in place of, or in addition to TD\* or FTC•other than daily dosing, such as intermittent, coitally timed, or episodic (pre/post sex only)DO NOT provide PrEP as expedited partner therapy (i.e. do not prescribe for an uninfected person not in your care). PrEP medication side effects {#jve11-sec-0024} ---------------------------- Patients taking PrEP should be informed of TD\*/FTC side effects experienced by participants in PrEP trials. These include headache, nausea, flatulence, and the potential for renal injury or hepatotoxicity. In these trials, side effects were uncommon and usually resolved within the first month of taking PrEP ('start-up syndrome'). Clinicians should discuss the use of over-the-counter medications for headache, nausea and flatulence should they occur. Patients should also be counselled about signs or symptoms that indicate a need for urgent evaluation (e.g. those suggesting possible acute renal injury or acute HIV infection). PrEP medication drug interactions {#jve11-sec-0025} --------------------------------- In addition to the safety data obtained in PrEP clinical trials, data on drug interactions and longer-term toxicities have been obtained by studying the component drugs individually for their use in treatment of HIV-infected persons. Studies have also been performed in small numbers of HIV-uninfected, healthy adults. No significant effect was seen and no dosage adjustment was necessary for TD\*, but there are no data on FTC [@jve11-bib-0046]. FTC and TD\* are primarily excreted by the kidneys by a combination of glomerular filtration and active tubular secretion. Since both drugs are primarily eliminated by the kidneys, co-administration of TD\*/FTC with drugs that reduce renal function or compete for active tubular secretion may increase serum concentrations of TD\* and FTC, and those of other renally eliminated drugs. Examples include (but are not limited to) cidofovir, aciclovir, valaciclovir, ganciclovir, valganciclovir, aminoglycosides and high-dose or multiple non-steroidal anti-inflammatory drugs [@jve11-bib-0046]. Cocaine, methamphetamine and alcohol use was not seen to influence the concentrations of PrEP drugs [@jve11-bib-0048]. Time to achieving and maintaining protection {#jve11-sec-0026} -------------------------------------------- The pharmacokinetics of TD\* and FTC vary by tissue [@jve11-bib-0049]. Data from exploratory pharmacokinetic studies conducted with HIV-uninfected men and women [@jve11-bib-0050] suggest that maximum intracellular concentrations of TFV-DP are reached in blood after approximately 20 days of daily oral dosing. Current evidence suggests that for both rectal and vaginal exposures, high protection is achieved after 7 days of daily dosing [@jve11-bib-0052]. Women need to have high adherence to daily dosing of TD\*/FTC to maintain adequate drug levels in vaginal/cervical tissues [@jve11-bib-0052]. No data are yet available about intracellular drug concentrations in penile tissues susceptible to HIV infection to inform considerations of protection for male insertive sex partners. Limited data exist for trans and gender diverse people; therefore (similarly to women), extra attention to daily dosing is recommended. Intermittent (coitally timed or on-demand) PrEP was assessed among MSM in the IPERGAY study, and has been recommended as an acceptable regimen for this population group in France. The high frequency with which this dosing was performed afforded blood levels similar to daily dosing levels. Patients who would only like to use PrEP on rare occasions may not reach the therapeutic (protective) drug level after a long period without PrEP, and may not maintain these levels long enough to prevent HIV infection. In the absence of strong evidence of efficacy of intermittent PrEP dosing in such users, the ASHM guidance panel has taken the more cautious approach of recommending only daily dosing as described in the section on Indicated medication. Clinicians should familiarise themselves with the reasons for this recommendation, because it is likely that patients will want to experiment with less rigorous dosing and reduce their pill burden. Therefore, it may be necessary to explore the frequency with which patients will begin to use PrEP for HIV prevention. For example, if an MSM patient wants to take PrEP while on an overseas trip, he can be advised to start daily PrEP a week before departure and to cease PrEP once it is no longer needed (i.e. 28 days after the last high-risk episode). Taking a break and discontinuing PrEP {#jve11-sec-0027} ------------------------------------- ### Starting and stopping PrEP {#jve11-sec-1027} For some patients, HIV exposure may be episodic, and people who start PrEP may stop and restart it periodically. Learning when and how to start and stop PrEP is important for effective PrEP use [@jve11-bib-0053]. The need for PrEP may end when a partner with HIV achieves viral suppression, or when a patient enters a mutually monogamous relationship with a seroconcordant partner. Patients may want to restart PrEP when: •entering a period of engaging in condomless sex•leaving a long-term relationship•starting a new relationship with an HIV-positive partner who is not on antiretroviral treatment, or a partner whose HIV status is unknown•moving to a new region or country with high or unknown prevalence of HIV and unknown sex partners•entering the field of sex work ### Discontinuing PrEP {#jve11-sec-2027} It is important to note that protective levels of the PrEP drugs are thought to persist only for up to 7 days after ceasing PrEP. One US study recommends that people continue to take daily PrEP for 28 days after the last sexual exposure that put them at high risk of HIV infection [@jve11-bib-0054] and we recommend that clinicians should offer this advice until more information is available. Upon discontinuation for any reason, the following should be documented in the health record: •HIV status at the time of discontinuation•reasons for PrEP discontinuation•recent medication adherence and reported sexual risk behaviourAny person who wishes to restart taking PrEP medications should repeat the baseline evaluations (see sections on Eligibility for PrEP based on risk exposure and Assessment before starting PrEP). Previous discontinuation of PrEP for any reason (except seroconversion to HIV) should not exclude the person from restarting PrEP. Clinical follow-up and monitoring of patients on PrEP {#jve11-sec-0028} ===================================================== Recommended schedule of testing and follow-up for individuals on PrEP (Box [5](#jve11-fea-0005){ref-type="boxed-text"}) {#jve11-sec-0029} ----------------------------------------------------------------------------------------------------------------------- Once PrEP is initiated, patients should return for follow-up every 3 months. Clinicians may wish to see patients more frequently at the beginning of PrEP (e.g. 1 month after initiation) to assess and confirm HIV-negative test status, assess for early side effects, discuss any difficulties with medication adherence, and answer questions. Table [3](#jve11-tbl-0008){ref-type="table"} and Box [5](#jve11-fea-0005){ref-type="boxed-text"} set out the recommended schedule of testing and follow-up for individuals who are prescribed PrEP. Patients who are accessing PrEP through the PIS should allow a lead time of up to 4--6 weeks if ordering online to allow for the drug to arrive in Australia and pass customs clearance. ###### Laboratory evaluation and clinical follow-up of individuals who are prescribed PrEP Test Baseline (Week 0) About day 30 after initiating PrEP (optional) 90 days after initiating PrEP Every subsequent 90 days on PrEP Other frequency ---------------------------------------------------------------------------------------------------------- ------------------- ----------------------------------------------- ------------------------------- ---------------------------------- -------------------------- HIV testing and assessment for signs or symptoms of acute infection Y Y Y Y N Assess side effects N Y Y Y N Hepatitis B serology Y N N N N Hepatitis C serology Y N N N At least every 12 months STI (i.e. syphilis, gonorrhoea, chlamydia) as per Australian STI Management Guidelines [@jve11-bib-0009] Y N Y Y N eGFR and urine protein: creatinine ratio (PCR) at 3 months and then every 6 months Y N Y N At least every 6 months Pregnancy test (for women of child-bearing potential) Y Y Y Y N ###### PrEP follow-up procedures **At least every 3 months:** •Repeat HIV testing and assess for signs or symptoms of acute infection to document that patients are still HIV-negative•Repeat pregnancy testing for women who may become pregnant•Provide a prescription or refill authorisation of daily TD\*/FTC for no more than 90 days (until the next HIV test)•Assess side effects, adherence and HIV risk behaviours•Provide support for medication adherence and risk-reduction behaviours•Respond to new questions and provide any new information about PrEP use•Test for STIs•Evaluate the need to continue PrEP as a component of HIV prevention•Test for HCV in PWID who report continued sharing and MSM with elevated risk of HCV acquisition**At least every 6 months:** •Monitor eGFR, creatinine and urinary PCR•If other conditions are present that may impair renal function (e.g., hypertension, diabetes), renal function may require more frequent monitoring or may need to include additional tests•A rise in serum creatinine is not a reason to withhold treatment if the eGFR remains ≥60 mL/min/1.73 m^2^•If eGFR is declining steadily (but still ≥60 mL/min/1.73 m^2^), consultation with a nephrologist or other evaluations of possible causes for declining renal function may be indicated**At least every 12 months:** •Evaluate the need to continue PrEP as a component of HIV prevention•Test for hepatitis C, in those not tested more frequently Testing for HIV {#jve11-sec-0030} =============== ***HIV testing should be repeated every 3 months*** (before prescriptions are refilled or reissued). Risk practices and adherence to PrEP should also be ascertained when requesting an HIV test (see section on Improving medication adherence). Patients should be familiar with this requirement for subsequent PrEP prescriptions from the discussion conducted when starting PrEP. Clinicians should consider writing on the prescription a date past which dispensing the script should not occur. This may help ensure that patients who have poor medication adherence, or who have only used PrEP intermittently and have unknowingly become infected with HIV, do not receive three months of dual antiretroviral therapy. See Appendix 2 for up-to-date HIV tests approved in Australia (K Wilson, National Serum Reference Library, personal communication) and time to detection of HIV infection [@jve11-bib-0055]. ***A positive HIV test*** ***result*** must be managed as per HIV Testing Policy and local management guidelines. ***A course of nPEP may be required*** if a patient had a recent high-risk exposure (within 72 hours) but only if they did not take PrEP during those days. See section on nPEP and PrEP below for management of such cases. ***Acute HIV infection should be suspectedin individuals at high risk for HIV who have been exposed recently*** (e.g. no condom or a condom broke during sex with an HIV-positive partner who is not on antiretroviral treatment, or has a detectable HIV viral load; recent injection drug use with shared injection equipment with an HIV-positive partner) ***and were not taking PrEP during that time***. Also, infection with TD\* and/or FTC resistant HIV is extremely rare but possible while on PrEP, with two cases reported internationally [@jve11-bib-0056]. Therefore, in addition to sexual behaviour and injecting drug use, clinicians should solicit a history of non-specific signs or symptoms of viral infection during the preceding month, or on the day of evaluation. See Table [2](#jve11-tbl-0007){ref-type="table"} for clinical symptoms and abnormalities of acute (primary) HIV infection. If a patient is diagnosed with HIV infection while taking PrEP, their health and wellbeing rather than the emphasis on what their medication adherence was like and how they acquired HIV, should be the chief priority. HIV drug resistance testing should be performed in all cases and if the individual reports high PrEP adherence they may agree to have their blood, or hair tested for tenofovir and emtricitabine drug levels. In this setting referral to an HIV specialist is recommended. Indeterminate HIV test results in the first 3 months on PrEP {#jve11-sec-1030} ------------------------------------------------------------ There is a potential for PrEP to delay or attenuate seroconversion in people who may have been exposed just before starting PrEP [@jve11-bib-0058]. As yet, there has been little consideration, and no common recommendations of how to manage these patients. Patients with an indeterminate HIV test result while on PrEP (particularly, with repeated indeterminate test results) should be closely monitored in conjunction with an HIV specialist. Proviral DNA testing should be considered. The ASHM PrEP guidelines panel will continue to monitor this issue with a view to providing further guidance. Monitoring of renal function {#jve11-sec-0031} ---------------------------- Renal function should be monitored at 3 months and 6 monthly thereafter, or more frequently in certain populations (see Assessment of renal function at baseline). The management of individuals at high and ongoing risk of HIV infection, but whose eGFR has declined below 60 mL/min/1.73 m^2^ since commencing TD\*/FTC, is challenging. This situation typically requires consultation with a physician who is expert in PrEP. Cessation of TD\*/FTC for 1 month may restore eGFR to above 60 mL/min/1.73 m^2^, following which TD\*/FTC may be recommenced with cautious monitoring. In these circumstances, consideration should be given to using coitally based TD\*/FTC, or possibly second-daily TD\*/FTC. However, there are no data to show that either of these options will stabilise the eGFR above 60 mL/min/1.73 m^2^. Testing for and management of STI {#jve11-sec-0032} --------------------------------- Because PrEP users are at high risk for STIs, clinicians should screen for STIs (specifically gonorrhoea, chlamydia and infectious syphilis) every 3 months using the standard of care tests and procedures, and manage any detected STI as recommended by the Australian STI Management Guidelines [@jve11-bib-0009]. At each follow-up visit, patients taking PrEP should be reminded about: •prevention of STI acquisition and transmission•the need for quarterly STI testing•the need to present for testing and treatment whenever signs or symptoms of STI appearThe presence of an STI at follow-up testing should not prevent the ongoing prescription of PrEP. Monitoring HBV and HCV infections {#jve11-sec-0033} --------------------------------- ### HBV monitoring {#jve11-sec-1033} Both TD\* and FTC are active against HBV [@jve11-bib-0060]. If people living with chronic HBV infection stop taking these medications, hepatic flares can occur, which can be severe [@jve11-bib-0060]. A person taking PrEP who has chronic HBV infection should be assessed by a clinician experienced in the management of hepatitis B before ceasing PrEP. If PrEP is discontinued, close monitoring is strongly advised. For additional guidance about the management of PrEP in persons with chronic active HBV infection, see the section on Special clinical considerations. ### HCV monitoring {#jve11-sec-2033} MSM and trans and gender diverse people should be monitored for HCV if they engage in sexually adventurous sex. The incidence of HCV has currently been low at \~1% per annum in PrEP studies of MSM [@jve11-bib-0038], and higher in HIV-positive MSM [@jve11-bib-0062]. However, there is concern that HCV incidence may increase following changes in sexual and sero-sorting behaviour in the era of PrEP. Therefore, in this context, HCV should be viewed as an STI. It should be tested at least annually, and more frequently if necessary, following sexual history taking. Managing side effects {#jve11-sec-0034} --------------------- Patients taking PrEP should be assessed for common side effects associated with TD\*/FTC use, most importantly those suggesting possible acute renal injury or hepatotoxicity. A recent review of symptoms experienced in the iPrEx study showed that potential PrEP-associated symptoms peaked at 1 month, when 39% of participants reported symptoms, compared with 22% at baseline. Gastrointestinal (GIT) symptoms occurred in a median of 28% of individuals across study sites (range 11--70%) and non-GIT symptoms occurred in a median of 24% of individuals (range 3--59%). The odds of GIT symptoms were higher in those with evidence of high adherence to PrEP. By 3 months, symptoms had returned to pre-PrEP levels. Bodybuilding increases muscle mass, which may result in increased creatinine levels in blood. When evaluating and managing PrEP users with creatinine clearance changes, clinicians should solicit and take into consideration the history of steroid use and bodybuilding. The ASHM PrEP guidance panel will monitor evidence in this area and update the guidelines as appropriate. Optional assessments {#jve11-sec-0035} -------------------- ### Therapeutic drug monitoring {#jve11-sec-1035} As for HIV treatment, the limited availability of clinical laboratories that can quantify TD\*/FTC concentrations under rigorous quality assurance and quality control standards prevents the routine use of therapeutic drug monitoring in PrEP. Initial demonstration projects in Australia conducted therapeutic drug monitoring as part of research protocols to understand why seroconversions occur among study participants. Their results revealed a high correlation between self-reports of pill taking and blood concentrations of TD\* and FTC, and high adherence to PrEP at \>90% [@jve11-bib-0064]. Therapeutic drug monitoring is unlikely to become available in routine clinical practice. Special clinical considerations {#jve11-sec-0036} =============================== Women taking PrEP during conception, pregnancy and breastfeeding {#jve11-sec-0037} ---------------------------------------------------------------- ### Conception in serodiscordant couples {#jve11-sec-1036} Serodiscordant couples may desire pregnancy. Women without HIV infection who have sex partners with documented HIV infection are at substantial risk of HIV acquisition during natural attempts to conceive (i.e. without a condom) if their HIV-positive partner has a detectable or variably detectable plasma viral load. Providers should discuss the available information about the potential risks and benefits of PrEP in these circumstances. ### Pregnancy {#jve11-sec-2036} Among HIV-uninfected women, pregnancy is a period of elevated risk of HIV infection. In addition, infection acquired during pregnancy may be more likely to be transmitted to the infant than a pre-existing infection. PrEP exposure among couples desiring pregnancy was not associated with adverse pregnancy outcomes in the Partners PrEP study [@jve11-bib-0019]. TD\* use for HIV treatment has not been associated with adverse pregnancy outcomes, although lower BMD in newborns is observed if the mother received antiretroviral therapy during pregnancy, especially if regimens containing TD\* are used. Providers should discuss available information about potential risks and benefits of beginning or continuing PrEP during pregnancy so that patients can make an informed decision. After weighing the risks and benefits, PrEP may be continued during pregnancy in women at substantial risk for HIV acquisition. ### Breastfeeding {#jve11-sec-3036} Although experience with PrEP during breastfeeding is still lacking, there is substantial experience with the use of TD\*/FTC during the breastfeeding period by women with HIV taking TD\*/FTC based ART. TD\* and FTC are secreted in breast milk, although at very low concentrations (0.3 and 2%, respectively, of the doses recommended for treatment of HIV infection in infants [@jve11-bib-0066]). If a woman becomes infected with HIV during breastfeeding, the risk of transmission to her infant is higher than in an established infection, because of high viral load soon after seroconversion. Therefore, PrEP may be continued during breastfeeding in women at substantial risk for HIV acquisition. Patients with chronic active HBV infection {#jve11-sec-0038} ------------------------------------------ Both TD\* and FTC are active against HIV and HBV infections. They may prevent the development of significant liver disease by suppressing HBV replication. Only TD\*, however, is currently approved for this use in Australia. Therefore, ongoing treatment with TD\*/FTC may be especially indicated in persons with substantial risk of both HIV acquisition and active HBV infection. Of note are two case reports of patients who were receiving TD\* for treatment of hepatitis B and acquired HIV infection [@jve11-bib-0067]. Plasma levels of tenofovir and prescription refills suggested that the patients' medication adherence was good. These guidelines recommend that individuals with established hepatitis B infection who require treatment for hepatitis B infection receive combined TD\*/FTC and have ongoing monitoring for HIV PrEP and hepatitis B infection. ***All persons who test positive for hepatitis B surface antigen (HBsAg)*** should be evaluated by a clinician experienced in the treatment of HBV infection. For clinicians without this experience, co-management with an infectious disease or a liver specialist should be considered. ***People living with chronic HBV infection*** should be tested for HBV DNA by the use of a quantitative assay to determine the level of HBV replication [@jve11-bib-0068] before PrEP is prescribed, and at regular intervals (e.g. every 3--6 months) while taking PrEP. TD\* presents a very high barrier to the development of HBV resistance. However, it is important to reinforce the need for consistent adherence to the daily doses of TD\*/FTC to prevent reactivation of HBV infection with the attendant risk of hepatic injury, and to minimise the possible risk of developing TD\*-resistant HBV infection [@jve11-bib-0069]. Patients with chronic hepatitis B should be strongly advised against intermittent PrEP for these reasons. ***If PrEP is no longer needed to prevent HIV infection***, a separate determination should be made about whether the patient requires ongoing treatment for HBV infection. Acute flares resulting from the reactivation of HBV infection have been seen in HIV-infected and HIV-uninfected persons after stopping TD\* and other medications used to treat HBV infection. When people living with chronic hepatitis B elect to discontinue PrEP, they should first be evaluated by a clinician experienced in the management of HBV infection to ascertain their need for ongoing HBV treatment, and to monitor for any hepatic flares that occur if PrEP is ceased. Patients with chronic renal failure {#jve11-sec-0039} ----------------------------------- HIV-uninfected patients with an eGFR of less than 60 mL/min/1.73 m^2^ should not be prescribed PrEP. The only PrEP regimen proven effective to date is TD\*/FTC and approved by the TGA for PrEP is not indicated for persons with chronic renal failure [@jve11-bib-0046]. Adolescent minors {#jve11-sec-0040} ----------------- As a part of primary health care, HIV screening should be discussed with all adolescents who are sexually active or have a history of injection drug use. Parental or guardian involvement in an adolescent\'s health care is often desirable, but is sometimes contraindicated for the safety of the adolescent, and can compromise full disclosure. Clinicians should carefully consider the lack of data on safety and effectiveness of PrEP taken by persons under 18 years of age, including the possibility of BMD loss or other toxicities among youth who are still growing, and the safety evidence available when TD\*/FTC is used in treatment regimens for HIV-infected youth [@jve11-bib-0070]. Adherence to PrEP in adolescents may be suboptimal; a PrEP demonstration program for 18--22-year-old HIV-negative MSM reported that TFV-DP intracellular levels consistent with good adherence peaked at 56% at month 1, and declined thereafter [@jve11-bib-0071]. A recent study of 15--22-year-old HIV-negative MSM demonstrated that over 48 weeks, TD\*/FTC was not associated with a decline in eGFR, but was associated with a significant decline in BMD [@jve11-bib-0072]. However, another study has suggested that age younger than 25 years is associated with a more rapid decline in eGFR in individuals receiving TD\*/FTC for PrEP [@jve11-bib-0029]. The above factors should be weighed against the potential benefit of providing PrEP for an individual adolescent at substantial risk of HIV acquisition. Clinicians are encouraged to seek expert advice in complex cases. Of note PrEP is approved for use by the TGA for individuals aged 18 years and older. nPEP and PrEP {#jve11-sec-0041} ============= Persons not receiving PrEP who seek care within 72 hours after an isolated sexual or injection-related HIV exposure should be evaluated for the potential need for nPEP [@jve11-bib-0017]. If the exposure is isolated (e.g. sexual assault, infrequent condom failure), nPEP should be prescribed, but continued antiretroviral medication is not indicated after completion of the 28-day nPEP course. If such exposures are not isolated, and the person is determined not to have an HIV infection, clinicians should consider beginning PrEP immediately (or prescribe nPEP immediately followed by PrEP) if the individual needs a three-drug nPEP regimen. The decision to commence nPEP should be made in line with local nPEP Guidelines [@jve11-bib-0017]. The decision to transition to PrEP should rely on eligibility for PrEP (including confirmatory HIV test result) and willingness to continue taking daily TD\*/FTC. A course of nPEP may be required if a PrEP user had a recent high-risk exposure (within 72 hours), but only if they did not take PrEP during those days. The decision to return to PrEP should rely on eligibility for PrEP (including confirmatory HIV test result) and willingness to continue taking daily TD\*/FTC. Improving medication adherence {#jve11-sec-0042} ============================== Medication adherence is critical to achieving the maximum prevention benefit of PrEP and reducing the risk of selecting for a drug-resistant virus in the event of HIV acquisition [@jve11-bib-0073]. Medication education and adherence counselling (also called medication self-management) will need to be provided to support daily PrEP use (Box [6](#jve11-fea-0006){ref-type="boxed-text"}). Various approaches can be used to effectively support medication adherence [@jve11-bib-1073]. These include: •educating patients about the medications•helping patients anticipate and manage side effects•helping patients establish dosing routines that mesh with their work and social schedules•providing reminder systems and tools•addressing financial, substance abuse or mental-health needs that may impede adherence•facilitating social supportWhen initiating a PrEP regimen, clinicians must educate patients about how to take their medications (i.e. when to take them, how many pills to take) and what to do if they experience problems (e.g. what constitutes a missed dose \[i.e. number of hours after the failure to take the daily dose\], what to do if they miss a dose). Patients should be told to take a single missed dose as soon as they remember it, unless it is almost time for the next dose. If it is almost time for the next dose (\<12 hours), patients should skip the missed dose and continue with the regular dosing schedule. Side effects can lead to non-adherence. Clinicians should inform patients about the most common side effects and should work with patients to develop a specific plan for handling them, including the use of specific over-the-counter medications that can mitigate symptoms. The importance of using condoms during sex, especially for patients who decide to stop taking their medications, should be emphasised. ###### Key components of medication-adherence counselling **Establish trust and bidirectional communication** **Provide simple explanations and education** ■Medication dosage and schedule■Management of common side effects■Relationship of adherence to the efficacy of PrEP■Signs and symptoms of acute HIV infection and recommended actions**Support adherence** ■Tailor daily dose taking to patient\'s daily routine (e.g. with tooth brushing, before bed)■Identify reminders and devices to minimise forgotten doses■Identify and address barriers to adherence**Monitor medication adherence in a non-judgemental manner** ■Normalise occasional missed doses, while ensuring patient understands importance of daily dosing for optimal protection■Reinforce success■Identify factors interfering with adherence and plan with patient to address these**Assess side effects and plan how to manage them** Behavioural strategies to risk reduction {#jve11-sec-0043} ======================================== In the era of PrEP, behavioural methods of risk reduction -- including condom use -- retain their importance in preventing HIV infection and remain the pillar of STI prevention (Box [7](#jve11-fea-0007){ref-type="boxed-text"}). ###### Discussion points on behavioural reduction of HIV and STI risk **Establish trust and two-way communication** **Provide feedback on HIV risk factors identified during sexual and substance use history taking** ■Elicit barriers to, and facilitators of, consistent condom use■Elicit barriers to, and facilitators of, reducing substance abuse**Support risk-reduction efforts** ■Help patient identify 1 or 2 feasible, acceptable, incremental steps toward risk reduction■Identify and address anticipated barriers to accomplishing planned actions to reduce risk**Monitor medication adherence in a non-judgemental manner** ■Acknowledge the effort required for behaviour change■Reinforce success**If not fully successful, assess factors interfering with completion of planned actions and help** **patient identify the next steps (including PrEP prescriptions)** How to access PrEP in Australia {#jve11-sec-0044} =============================== At the time of writing, PrEP has not been approved for subsidy by the Pharmaceutical Benefits Advisory Committee. Availability in Australia is limited to personal importation and the state government medical trials listed below: NSWEpic Trial[www.endinghiv.org.au/nsw/stay-safe/epic/](www.endinghiv.org.au/nsw/stay-safe/epic/)QLDQPrEP Study[www.comeprepd.info/accessing-prep/](www.comeprepd.info/accessing-prep/)SAPrEPX-SA[www.samesh.org.au/prep.html](www.samesh.org.au/prep.html)VICPrEPX[www.alfredhealth.org.au/research/research-areas/infectious-diseases-research/prepx-study](www.alfredhealth.org.au/research/research-areas/infectious-diseases-research/prepx-study)WATrial coming soon[www.waaids.com/hiv/prep-easier-than-you-think.html](www.waaids.com/hiv/prep-easier-than-you-think.html) Other jurisdictions are in the process of negotiating access to demonstration projects and clinical trials. This section will be updated as changes come into place. The WA Department of Health provides advice to clinicians supporting patients to import PrEP: [www.waaids.com/hiv/prep-easier-than-you-think.html](www.waaids.com/hiv/prep-easier-than-you-think.html) Information about personal importation is provided at the following links: [www.tga.gov.au/personal-importation-scheme](www.tga.gov.au/personal-importation-scheme) [www.ashm.org.au/hiv/PrEP](www.ashm.org.au/hiv/PrEP) [www.endinghiv.org.au/nsw/wp-content/uploads/2015/02/PrEP_Access_Options_Paper1.pdf\#page=2](www.endinghiv.org.au/nsw/wp-content/uploads/2015/02/PrEP_Access_Options_Paper1.pdf#page=2) Acknowledgements {#jve11-sec-0045} ================ The Australian PrEP guidelines panel wishes to acknowledge and thank the CDC PrEP guidelines project manager, Dawn Smith, for allowing us to use and adapt the structure and content of the 2014 US Public Health Service Pre-exposure prophylaxis for the prevention of HIV infection in the United States -- 2014 clinical practice guidelines. Contributors {#jve11-sec-0046} ------------ Alfred Health, Monash University (VIC): Christina Chang; Michelle Giles Australian Federation of AIDS Organisations: Heath Paynter Australian Injecting and Illicit Drug Users League: Angella Duvnjak Centers for Disease Control and Prevention (USA): Silvina Masciotra Centre Clinic -- St Kilda: Pauline Cundall Peter Doherty Institute for Infection and Immunity WHO Collaborating Centre for Viral Hepatitis: Ben Cowie Gender Centre: Phinn Borg Harm Reduction Victoria: Jenny Kelso Holdsworth House Medical Practice (Sydney): Mark Bloch Kirby Institute: Lisa Maher National Association of People with HIV Australia: Aaron Cogle National HIV Reference Laboratory (NRL): Kim Wilson Peer Advocacy Network for the Sexual Health of Trans Masculinities: Ted Cook Positive Women Victoria: Alison Broughy Prahan Market Clinic: Vincent Cornelisse Queensland AIDS Council: Michael Scott Royal Prince Alfred Hospital: David Gracey St Vincent\'s Hospital: Phillip Cunningham Sydney Sexual Health Centre: Anna McNulty Taylor Square Private Clinic: Robert Finlayson Western Sydney Sexual Health Centre: Cationa Ooi Abbreviations {#jve11-sec-0047} ============= AIDS: acquired immunodeficiency syndrome ART: antiretroviral therapy ARTG: Australian Register of Therapeutic Drugs ASHM: Australasian Society for HIV, Viral Hepatitis and Sexual Health Medicine BMD: bone mineral density eCrCl: estimated creatinine clearance rate eGFR: estimated glomerular filtration rate FTC: emtricitabine (trade name Emtriva) HBV: hepatitis B virus HCV: hepatitis C virus HIV: human immunodeficiency virus iPrEx: Pre-exposure Prophylaxis Initiative MSM: men who have sex with men nPEP: non-occupational post-exposure prophylaxis NSP: needle and syringe program PEP: post-exposure prophylaxis PIS: Personal Importation Scheme PoCT: point-of-care tests PWID: people who inject drugs PrEP: pre-exposure prophylaxis PCR: urine protein: creatinine clearance STI: sexually transmitted infection TD\*: tenofovir disoproxil maleate or fumarate or phosphate TDF: tenofovir disoproxil fumarate (trade name Viread) TDM: tenofovir disoproxil maleate (trade name Trucitavir) TDP: tenofovir disoproxil phosphate (trade name Tenofovir EMT Lupin) TDF/FTC: tenofovir disoproxil fumarate coformulated with emtricitabine (trade name Truvada, or in generic form Tenvir). In Australia, the TGA has also approved the generic Trucitavir, which is coformulated tenofovir disoproxil maleate and emtricitabine and Tenofovir EMT Lupin which is co-formulated tenofovir disoproxil phosphate and emtricitabine TFV-DP: tenofovir diphosphate TGA: Therapeutic Goods Administration WHO: World Health Organization Appendix 1 {#jve11-sec-0048} ========== Cockcroft--Gault formula {#jve11-sec-0049} ------------------------ Basic formula [@jve11-bib-0075] eCrCl~CG~=\[\[(140−age)×IBW×0.85 for females\]÷(serum creatinine×72)\] IBW=ideal body weight Males: IBW=50 kg+2.3 kg for each inch over 5 feet Females: IBW=45.5 kg+2.3 kg for each inch over 5 feet, age in years, weight in kg, and serum creatinine in mg/100 mL Optional adjustment for low actual body weight [@jve11-bib-0076] If the actual body weight is less than the IBW use the actual body weight for calculating the eCrCl. Optional adjustment of high actual body weight [@jve11-bib-0076] Used only if the actual body weight is 30% greater than the IBW. Otherwise, the IBW is used. eCrCl=\[\[(140−age)×AjBW\]÷(serum creatinine×72)\] (×0.85 for females) AjBW=IBW+0.3( ABW−IBW) AjBW=adjusted body weight; ABW=actual body weight Optional adjustment for body surface area (BSA) [@jve11-bib-0077] Can be used if actual body weight is greater or less than IBW eCrClBSAadj=1.73 m^2^×eCrCl~CG~ (mL/min)÷BSA of the patient (m^2^) BSA (DuBois and DuBois formula [@jve11-bib-0078])=(height (m) 0.725×weight (kg) 0.425)÷139.2 Appendix 2 {#jve11-sec-0050} ========== This appendix contains the TGA-approved products for HIV tests. TGA-approved HIV rapid tests typically used for point-of-care testing or in clinicians' offices {#jve11-sec-0051} ----------------------------------------------------------------------------------------------- ### Inverness Medical Innovations Australia Pty Ltd T/A Alere {#jve11-sec-0052} 1. **ARTG ID:** 276049 2. **Product name:** Alere HIV Combo -- HIV1/HIV2 antigen/antibody IVD, kit, immunochromatographic test (ICT), rapid 3. **Sponsor:** Inverness Medical Innovations Australia Pty Ltd T/A Alere ### Immuno Pty Ltd {#jve11-sec-0053} 1. **ARTG ID:** 240814 2. **Product name:** Uni-Gold HIV -- HIV1/HIV2 antibody IVD, kit, immunochromatographic test (ICT), rapid 3. **Sponsor:** Immuno Pty Ltd ### Integrated Sciences Pty Ltd {#jve11-sec-0054} 1. **ARTG ID:** 240813 2. **Product name:** OraQuick ADVANCE Rapid HIV-1/2 Antibody Test and Kit Controls -- HIV1/HIV2 antibody IVD, kit, immunochromatographic test (ICT), rapid 3. **Sponsor:** Integrated Sciences Pty Ltd ### [Inverness Medical Innovations Australia Pty Ltd T/A Alere](Inverness Medical Innovations Australia Pty Ltd T/A Alere) {#jve11-sec-0055} 1. **ARTG ID:** 232594 2. **Product name:** Determine HIV-1/2 -- HIV1/HIV2 antibody IVD, kit, immunochromatographic test (ICT), rapid 3. **Sponsor:** Inverness Medical Innovations Australia Pty Ltd T/A Alere TGA-approved diagnostic laboratory-based hiv antibody tests {#jve11-sec-0056} ----------------------------------------------------------- ### [DiaSorin Australia Pty Ltd](DiaSorin Australia Pty Ltd) {#jve11-sec-0057} 1. **ARTG ID:** 279803 2. **Product name:** LIAISON XL MUREX HIV Ab/Ag HT -- HIV1/HIV2 antigen/antibody IVD, kit, chemiluminescent immunoassay 3. **Sponsor:** DiaSorin Australia Pty Ltd ### [Ortho-Clinical Diagnostics Australia Pty Ltd](Ortho-Clinical Diagnostics Australia Pty Ltd) {#jve11-sec-0058} 1. **ARTG ID:** 251957 2. **Product name:** VITROS Anti-HIV 1+2 -- HIV1/HIV2 antibody IVD, kit, chemiluminescent immunoassay 3. **Sponsor:** Ortho-Clinical Diagnostics Australia Pty Ltd ### [Siemens Healthcare Pty Ltd](Siemens Healthcare Pty Ltd) {#jve11-sec-0059} 1. **ARTG ID:** 239117 2. **Product name:** ADVIA Centaur HIV 1/O/2 Enhanced (EHIV) -- HIV1/HIV2 antibody IVD, kit, chemiluminescent immunoassay 3. **Sponsor:** Siemens Healthcare Pty Ltd ### [Biomerieux Australia Pty Ltd](Biomerieux Australia Pty Ltd) {#jve11-sec-0060} 1. **ARTG ID:** 233218 2. **Product name:** bioMerieux SA VIDAS HIV DUO Ultra -- HIV1/HIV2 antigen/antibody IVD, kit, enzyme immunoassay (EIA) 3. **Sponsor:** Biomerieux Australia Pty Ltd ### [Bio-Rad Laboratories Pty Ltd](Bio-Rad Laboratories Pty Ltd) {#jve11-sec-0061} 1. **ARTG ID:** 229064 2. **Product name:** Geenius™ HIV 1/2 Confirmatory Assay -- HIV1/HIV2 antibody IVD, kit, immunochromatographic test (ICT), rapid 3. **Sponsor:** Bio-Rad Laboratories Pty Ltd ### [Roche Diagnostics Australia Pty Limited](Roche Diagnostics Australia Pty Limited) {#jve11-sec-0062} 1. **ARTG ID:** 226069 2. **Product name:** Elecsys HIV Combi PT -- HIV1 antigen/antibody IVD, kit, chemiluminescent immunoassay 3. **Sponsor:** Roche Diagnostics Australia Pty Limited ### [Bio-Rad Laboratories Pty Ltd](Bio-Rad Laboratories Pty Ltd) {#jve11-sec-0063} 1. **ARTG ID:** 220632 2. **Product name:** Genscreen ULTRA HIV Ag-Ab -- HIV1/HIV2 antigen/antibody IVD, kit, enzyme immunoassay (EIA) 3. **Sponsor:** Bio-Rad Laboratories Pty Ltd ### [Bio-Rad Laboratories Pty Ltd](Bio-Rad Laboratories Pty Ltd) {#jve11-sec-0064} 1. **ARTG ID:** 220068 2. **Product name:** Genscreen HIV-1 Ag Confirmatory Assay -- HIV1 antigen neutralization IVD, kit, enzyme immunoassay (EIA) 3. **Sponsor:** Bio-Rad Laboratories Pty Ltd ### [Bio-Rad Laboratories Pty Ltd](Bio-Rad Laboratories Pty Ltd) {#jve11-sec-0065} 1. **ARTG ID:** 220067 2. **Product name:** Genscreen HIV-1 Antigen Assay -- HIV1 antigen IVD, kit, enzyme immunoassay (EIA) 3. **Sponsor:** Bio-Rad Laboratories Pty Ltd ### [Bio-Rad Laboratories Pty Ltd](Bio-Rad Laboratories Pty Ltd) {#jve11-sec-0066} 1. **ARTG ID:** 220061 2. **Product name:** Genscreen™ HIV-1/2 Version 2 -- HIV1/HIV2 antibody IVD, kit, enzyme immunoassay (EIA) 3. **Sponsor:** Bio-Rad Laboratories Pty Ltd ### [Abbott Australasia Pty Ltd Diagnostic Division](Abbott Australasia Pty Ltd Diagnostic Division) {#jve11-sec-0067} 1. **ARTG ID:** 213306 2. **Product name:** ARCHITECT HIV Ag/Ab Combo assay -- HIV1/HIV2 antigen/antibody IVD, kit, chemiluminescent immunoassay 3. **Sponsor:** Abbott Australasia Pty Ltd Diagnostic Division ### [Abbott Australasia Pty Ltd Diagnostic Division](Abbott Australasia Pty Ltd Diagnostic Division) {#jve11-sec-0068} 1. **ARTG ID:** 212528 2. **Product name:** PRISM HIV Ag/Ab combo assay -- HIV1/HIV2 antigen/antibody IVD, kit, chemiluminescent immunoassay 3. **Sponsor:** Abbott Australasia Pty Ltd Diagnostic Division ### [MP Biomedicals Australasia Pty Ltd](MP Biomedicals Australasia Pty Ltd) {#jve11-sec-0069} 1. **ARTG ID:** 212462 2. **Product name:** MP Diagnostics HIV Blot 2.2 assay -- HIV1/HIV2 antibody IVD, kit, immunoblot 3. **Sponsor:** MP Biomedicals Australasia Pty Ltd ### [DiaSorin Australia Pty Ltd](DiaSorin Australia Pty Ltd) {#jve11-sec-0070} 1. **ARTG ID:** 212434 2. **Product name:** LIAISON XL MUREX HIV Ab / Ag -- HIV1/HIV2 antigen/antibody IVD, kit, chemiluminescent immunoassay 3. **Sponsor:** DiaSorin Australia Pty Ltd ### [Siemens Healthcare Pty Ltd](Siemens Healthcare Pty Ltd) {#jve11-sec-0071} 1. **ARTG ID:** 205090 2. **Product name:** ADVIA Centaur HIV Ag/Ab Combo (CHIV) -- HIV1/HIV2 antigen/antibody IVD, kit, chemiluminescent immunoassay 3. **Sponsor:** Siemens Healthcare Pty Ltd ### [Bio-Rad Laboratories Pty Ltd](Bio-Rad Laboratories Pty Ltd) {#jve11-sec-0072} 1. **ARTG ID:** 207994 2. **Product name:** Access HIV Combo -- HIV1/HIV2 antigen/antibody IVD, kit, chemiluminescent immunoassay 3. **Sponsor:** Bio-Rad Laboratories Pty Ltd TGA-approved diagnostic laboratory-based HIV NAT tests {#jve11-sec-0073} ------------------------------------------------------ ### [Gen-Probe Australia Pty Ltd](Gen-Probe Australia Pty Ltd) {#jve11-sec-0074} 1. **ARTG ID:** 269680 2. **Product name:** Aptima HIV-1 Quant DX Assay Kit, PRD-03000 -- HIV1 nucleic acid IVD, kit, nucleic acid technique (NAT) 3. **Sponsor:** Gen-Probe Australia Pty Ltd [^1]: Senior co-authors

Related literature   {#sec1} ==================== For the synthesis and biological activity of the title compound, see; Doğruer *et al.* (2007[@bb5]). For related structures, see: Aydın *et al.* (2008[@bb3]); Girisha *et al.* (2008[@bb8]). For puckering parameters, see: Cremer & Pople (1975[@bb4]). For standard bond lengths, see: Allen *et al.* (1987[@bb1]). Experimental   {#sec2} ============== {#sec2.1} ### Crystal data   {#sec2.1.1} C~29~H~27~ClN~4~O~2~*M* *~r~* = 499.00Triclinic,*a* = 10.7929 (10) Å*b* = 10.8527 (10) Å*c* = 12.7815 (13) Åα = 97.745 (8)°β = 104.041 (7)°γ = 115.635 (7)°*V* = 1259.3 (2) Å^3^*Z* = 2Mo *K*α radiationμ = 0.19 mm^−1^*T* = 296 K0.66 × 0.53 × 0.35 mm ### Data collection   {#sec2.1.2} Stoe IPDS 2 diffractometerAbsorption correction: integration (*X-RED32*; Stoe & Cie, 2002[@bb10]) *T* ~min~ = 0.784, *T* ~max~ = 0.94815922 measured reflections4945 independent reflections3837 reflections with *I* \> 2σ(*I*)*R* ~int~ = 0.059 ### Refinement   {#sec2.1.3} *R*\[*F* ^2^ \> 2σ(*F* ^2^)\] = 0.041*wR*(*F* ^2^) = 0.111*S* = 1.054945 reflections326 parametersH-atom parameters constrainedΔρ~max~ = 0.29 e Å^−3^Δρ~min~ = −0.37 e Å^−3^ {#d5e500} Data collection: *X-AREA* (Stoe & Cie, 2002[@bb10]); cell refinement: *X-AREA*; data reduction: *X-RED32* (Stoe & Cie, 2002[@bb10]); program(s) used to solve structure: *SIR97* (Altomare *et al.*, 1999[@bb2]); program(s) used to refine structure: *SHELXL97* (Sheldrick, 2008[@bb9]); molecular graphics: *ORTEP-3 for Windows* (Farrugia, 1997[@bb6]); software used to prepare material for publication: *WinGX* (Farrugia, 1999[@bb7]). Supplementary Material ====================== Crystal structure: contains datablock(s) global, I. DOI: [10.1107/S160053681203543X/su2489sup1.cif](http://dx.doi.org/10.1107/S160053681203543X/su2489sup1.cif) Structure factors: contains datablock(s) I. DOI: [10.1107/S160053681203543X/su2489Isup2.hkl](http://dx.doi.org/10.1107/S160053681203543X/su2489Isup2.hkl) Supplementary material file. DOI: [10.1107/S160053681203543X/su2489Isup3.cml](http://dx.doi.org/10.1107/S160053681203543X/su2489Isup3.cml) Additional supplementary materials: [crystallographic information](http://scripts.iucr.org/cgi-bin/sendsupfiles?su2489&file=su2489sup0.html&mime=text/html); [3D view](http://scripts.iucr.org/cgi-bin/sendcif?su2489sup1&Qmime=cif); [checkCIF report](http://scripts.iucr.org/cgi-bin/paper?su2489&checkcif=yes) Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: [SU2489](http://scripts.iucr.org/cgi-bin/sendsup?su2489)). The authors acknowledge the Faculty of Arts and Sciences, Ondokuz Mayıs University, Turkey, for the use of the Stoe IPDS 2 diffractometer (purchased under grant F.279 of the University Research Fund). Comment ======= 1-\[4-(4-Chlorophenyl)piperazin-1-yl\]-3-(6-oxo-3,4-diphenyl-1,6-dihydropyridazin-1-yl)propan-1-one has analgesic and anti-inflammatory effect. Its *in vivo* analgesic and anti-inflammatory activities were tested in mice. This compound showed higher analgesic activity than aspirin at 100 mg/kg. Analgesic activity results of the compound also shows good correlation with its anti-inflammatory activity and produced anti-inflammatory activity in both phases of carrageenan-induced edema (Doğruer *et al.*, 2007). In the present study, the title compound has been synthesized for first time by (Doğruer *et al.*, 2007) and characterized by spectroscopic techniques. Herein we report on the synthesis and its crystal structure. In the title compound, Fig. 1, the six-membered ring of the pyridazin-3(2*H*)-one system is nearly planar with maximum deviations of -0.062 (2) Å for N1, 0.052 (2) Å for C1 and -0.045 (2) Å for C3 from the mean plane. The dihedral angles between the mean plane of the six-membered ring (N1/N2/C1--C4) and the phenyl rings C11---C16 and C5---C10 are 44.71 (10)° and 43.05 (9)°, respectively. As seen in Fig. 1, the Cl1---C27---C28---C29 and N3---C20---C21---N4 torsion angles are 178.69 (14) and 57.22 (18)°, respectively. The double-bond length for C19---O2 is 1.2192 (19) Å and the C27---Cl1 bond length is 1.7450 (19) Å. All bond lengths (Allen *et al.*, 1987) and angles are within normal ranges and are comparable to those reported for similar structures (Aydın *et al.*, 2008; Girisha *et al.*, 2008). The piperazine ring (N3/N4/C20--C23) has a chair conformation with puckering parameters: Q~T~ = 0.566 (2) Å, θ = 0.5 (2) ° and φ = 67 (10) ° (Cremer & Pople, 1975). The mean plane of the six-membered ring forms a dihedral angle of 83.20 (16)° with the benzene ring (C24--C29). The phenyl rings (C11--C16) and (C5---C10) make dihedral angles of 67.84 (10)° and 32.76 (9)° with the benzene ring (C24--C29), respectively, whilst the dihedral angle between them is 60.42 (10)°. In the crystal, molecules are linked via two pairs of C---H···O interactions which result in the formation of chains propagating along \[1 0 -1\], (Table 1 and Fig. 2). Neighbouring chains are linked via C---H···π interactions (Table 1). Experimental {#experimental} ============ The title compound was synthesized according to the literature procedure (Doğruer *et al.*, 2007). 0.01 Mol of compound 3-\[5,6-Diphenyl-3(2*H*)-pyridazinone-2-yl\] propanoic acid in 40 ml dichloromethane at 273 K (ice-bath) was treated with triethylamine (1 ml) and 0.01 mol of ethyl chloroformate. After stirring the reaction mixture at 273 K for 15 min, 0.011 mole of 4-chlorophenylpiperazine was added to this solution. This mixture was stirred at 273--298 K for 24 h and evaporated to dryness. The product was then solidified with ice-cold water and recrystallized from ethanol (yield 52%, M.p. 432 K). IR *v* cm^-1^ (KBr): 1652 (CO ring, amide). Refinement {#refinement} ========== All the H atoms were positioned geometrically and refined using a riding model: C---H = 0.93 and 0.97 Å for CH and CH~2~ H atoms, respectively, with *U*~iso~(H) = 1.2*U*~eq~(C). Figures ======= {#Fap1} ![A view along the a axis of the crystal packing of the title compound. The C-H···O interactions are shown as dashed lines \[see Table 1 for details; H atoms not participating in these interactions have been omitted for clarity\].](e-68-o2720-fig2){#Fap2} Crystal data {#tablewrapcrystaldatalong} ============ ----------------------- ---------------------------------------- C~29~H~27~ClN~4~O~2~ *Z* = 2 *M~r~* = 499.00 *F*(000) = 524 Triclinic, *P*1 *D*~x~ = 1.316 Mg m^−3^ Hall symbol: -P 1 Mo *K*α radiation, λ = 0.71073 Å *a* = 10.7929 (10) Å Cell parameters from 24474 reflections *b* = 10.8527 (10) Å θ = 2.2--27.9° *c* = 12.7815 (13) Å µ = 0.19 mm^−1^ α = 97.745 (8)° *T* = 296 K β = 104.041 (7)° Prism, colourless γ = 115.635 (7)° 0.66 × 0.53 × 0.35 mm *V* = 1259.3 (2) Å^3^ ----------------------- ---------------------------------------- Data collection {#tablewrapdatacollectionlong} =============== ------------------------------------------------------------------ -------------------------------------- Stoe IPDS 2 diffractometer 4945 independent reflections Radiation source: sealed X-ray tube, 12 x 0.4 mm long-fine focus 3837 reflections with *I* \> 2σ(*I*) Plane graphite monochromator *R*~int~ = 0.059 Detector resolution: 6.67 pixels mm^-1^ θ~max~ = 26.0°, θ~min~ = 2.2° ω scans *h* = −13→13 Absorption correction: integration (*X-RED32*; Stoe & Cie, 2002) *k* = −13→13 *T*~min~ = 0.784, *T*~max~ = 0.948 *l* = −15→15 15922 measured reflections ------------------------------------------------------------------ -------------------------------------- Refinement {#tablewraprefinementdatalong} ========== ---------------------------------------------------------------- ---------------------------------------------------------------------------------------------------- Refinement on *F*^2^ Secondary atom site location: difference Fourier map Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites *R*\[*F*^2^ \> 2σ(*F*^2^)\] = 0.041 H-atom parameters constrained *wR*(*F*^2^) = 0.111 *w* = 1/\[σ^2^(*F*~o~^2^) + (0.0478*P*)^2^ + 0.165*P*\] where *P* = (*F*~o~^2^ + 2*F*~c~^2^)/3 *S* = 1.05 (Δ/σ)~max~ = 0.001 4945 reflections Δρ~max~ = 0.29 e Å^−3^ 326 parameters Δρ~min~ = −0.37 e Å^−3^ 0 restraints Extinction correction: *SHELXL97* (Sheldrick, 2008), FC^\*^=KFC\[1+0.001XFC^2^Λ^3^/SIN(2Θ)\]^-1/4^ Primary atom site location: structure-invariant direct methods Extinction coefficient: 0.021 (2) ---------------------------------------------------------------- ---------------------------------------------------------------------------------------------------- Special details {#specialdetails} =============== ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Geometry. Bond distances, angles *etc*. have been calculated using the rounded fractional coordinates. All su\'s are estimated from the variances of the (full) variance-covariance matrix. The cell e.s.d.\'s are taken into account in the estimation of distances, angles and torsion angles Refinement. Refinement on *F*^2^ for ALL reflections except those flagged by the user for potential systematic errors. Weighted *R*-factors *wR* and all goodnesses of fit *S* are based on *F*^2^, conventional *R*-factors *R* are based on *F*, with *F* set to zero for negative *F*^2^. The observed criterion of *F*^2^ \> σ(*F*^2^) is used only for calculating -*R*-factor-obs *etc*. and is not relevant to the choice of reflections for refinement. *R*-factors based on *F*^2^ are statistically about twice as large as those based on *F*, and *R*-factors based on ALL data will be even larger. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å^2^) {#tablewrapcoords} ================================================================================================== ------ -------------- -------------- -------------- -------------------- -- *x* *y* *z* *U*~iso~\*/*U*~eq~ Cl1 1.08842 (7) 1.37656 (7) 0.23195 (6) 0.0985 (3) O1 0.77526 (16) 0.94002 (13) 0.89191 (12) 0.0766 (5) O2 0.37402 (15) 0.69982 (19) 0.55504 (11) 0.0806 (6) N1 0.63418 (14) 0.70263 (13) 0.85637 (11) 0.0502 (4) N2 0.61006 (14) 0.57387 (13) 0.87067 (10) 0.0470 (4) N3 0.52244 (15) 0.69229 (17) 0.46036 (11) 0.0588 (5) N4 0.68833 (14) 0.88867 (16) 0.35873 (11) 0.0550 (5) C1 0.75365 (19) 0.83035 (17) 0.91999 (14) 0.0557 (5) C2 0.84157 (19) 0.82159 (17) 1.01957 (14) 0.0560 (5) C3 0.81424 (16) 0.69621 (16) 1.04284 (12) 0.0475 (5) C4 0.69752 (16) 0.56941 (15) 0.95936 (12) 0.0446 (4) C5 0.66902 (16) 0.42329 (15) 0.96261 (12) 0.0445 (4) C6 0.7836 (2) 0.39377 (19) 0.99172 (15) 0.0587 (6) C7 0.7563 (2) 0.2564 (2) 0.98782 (17) 0.0680 (7) C8 0.6157 (2) 0.1486 (2) 0.95736 (16) 0.0695 (7) C9 0.5011 (2) 0.17680 (18) 0.93023 (15) 0.0612 (6) C10 0.52791 (18) 0.31370 (16) 0.93212 (13) 0.0505 (5) C11 0.89780 (17) 0.69523 (16) 1.15342 (13) 0.0493 (5) C12 1.04711 (19) 0.78512 (19) 1.20005 (16) 0.0609 (6) C13 1.1225 (2) 0.7904 (2) 1.30592 (18) 0.0758 (7) C14 1.0503 (3) 0.7066 (2) 1.36637 (17) 0.0793 (8) C15 0.9027 (2) 0.6189 (2) 1.32177 (15) 0.0679 (7) C16 0.8258 (2) 0.61227 (18) 1.21591 (14) 0.0556 (5) C17 0.53089 (18) 0.69840 (18) 0.75579 (13) 0.0533 (5) C18 0.58384 (18) 0.69042 (19) 0.65707 (13) 0.0546 (5) C19 0.48415 (18) 0.69447 (18) 0.55362 (14) 0.0545 (5) C20 0.45077 (19) 0.7272 (2) 0.36582 (15) 0.0644 (6) C21 0.54978 (19) 0.8760 (2) 0.36406 (15) 0.0622 (6) C22 0.76026 (19) 0.8518 (2) 0.45262 (14) 0.0579 (6) C23 0.66045 (19) 0.7045 (2) 0.45535 (15) 0.0601 (6) C24 0.78162 (17) 1.00788 (18) 0.33070 (13) 0.0526 (5) C25 0.7588 (2) 1.1234 (2) 0.32311 (16) 0.0648 (6) C26 0.8519 (2) 1.2354 (2) 0.29196 (18) 0.0739 (7) C27 0.9681 (2) 1.2340 (2) 0.26832 (16) 0.0665 (6) C28 0.9917 (2) 1.1201 (2) 0.27347 (16) 0.0643 (6) C29 0.89932 (19) 1.00883 (19) 0.30419 (15) 0.0592 (6) H2 0.92010 0.90490 1.06990 0.0670\* H6 0.87930 0.46690 1.01400 0.0700\* H7 0.83350 0.23700 1.00590 0.0820\* H8 0.59780 0.05620 0.95500 0.0830\* H9 0.40580 0.10380 0.91070 0.0740\* H10 0.45020 0.33220 0.91270 0.0610\* H12 1.09660 0.84210 1.15980 0.0730\* H13 1.22270 0.85080 1.33670 0.0910\* H14 1.10180 0.70970 1.43740 0.0950\* H15 0.85390 0.56340 1.36310 0.0810\* H16 0.72550 0.55220 1.18610 0.0670\* H17A 0.52040 0.78290 0.76920 0.0640\* H17B 0.43600 0.61620 0.73890 0.0640\* H18A 0.68130 0.76950 0.67610 0.0660\* H18B 0.58920 0.60320 0.64150 0.0660\* H20A 0.36040 0.72030 0.37220 0.0770\* H20B 0.42740 0.66010 0.29630 0.0770\* H21A 0.50310 0.89750 0.29950 0.0750\* H21B 0.56740 0.94370 0.43110 0.0750\* H22A 0.78630 0.91950 0.52250 0.0700\* H22B 0.84920 0.85650 0.44460 0.0700\* H23A 0.64250 0.63590 0.38880 0.0720\* H23B 0.70680 0.68350 0.52030 0.0720\* H25 0.68000 1.12540 0.33910 0.0780\* H26 0.83530 1.31200 0.28710 0.0890\* H28 1.06990 1.11840 0.25620 0.0770\* H29 0.91600 0.93200 0.30730 0.0710\* ------ -------------- -------------- -------------- -------------------- -- Atomic displacement parameters (Å^2^) {#tablewrapadps} ===================================== ----- ------------- ------------- ------------- ------------- ------------- ------------- *U*^11^ *U*^22^ *U*^33^ *U*^12^ *U*^13^ *U*^23^ Cl1 0.0811 (4) 0.0793 (4) 0.1288 (5) 0.0272 (3) 0.0379 (4) 0.0461 (4) O1 0.0907 (10) 0.0489 (7) 0.0827 (9) 0.0260 (7) 0.0238 (7) 0.0331 (6) O2 0.0747 (9) 0.1370 (13) 0.0629 (8) 0.0719 (10) 0.0320 (7) 0.0370 (8) N1 0.0563 (8) 0.0463 (7) 0.0512 (7) 0.0236 (6) 0.0212 (6) 0.0214 (6) N2 0.0524 (7) 0.0427 (6) 0.0488 (7) 0.0214 (6) 0.0215 (6) 0.0184 (5) N3 0.0588 (8) 0.0806 (10) 0.0523 (8) 0.0420 (8) 0.0234 (6) 0.0261 (7) N4 0.0508 (8) 0.0698 (9) 0.0524 (8) 0.0349 (7) 0.0176 (6) 0.0207 (7) C1 0.0640 (10) 0.0456 (8) 0.0587 (9) 0.0226 (8) 0.0263 (8) 0.0215 (7) C2 0.0590 (10) 0.0425 (8) 0.0540 (9) 0.0145 (7) 0.0175 (7) 0.0141 (7) C3 0.0484 (8) 0.0461 (8) 0.0487 (8) 0.0202 (7) 0.0213 (7) 0.0159 (6) C4 0.0464 (8) 0.0442 (7) 0.0463 (8) 0.0202 (7) 0.0215 (6) 0.0167 (6) C5 0.0521 (8) 0.0431 (7) 0.0421 (7) 0.0234 (7) 0.0198 (6) 0.0147 (6) C6 0.0562 (10) 0.0582 (10) 0.0687 (10) 0.0310 (8) 0.0251 (8) 0.0194 (8) C7 0.0813 (13) 0.0719 (12) 0.0746 (12) 0.0530 (11) 0.0305 (10) 0.0278 (10) C8 0.1002 (15) 0.0526 (10) 0.0695 (11) 0.0433 (11) 0.0336 (11) 0.0268 (9) C9 0.0687 (11) 0.0464 (9) 0.0605 (10) 0.0202 (8) 0.0213 (8) 0.0184 (7) C10 0.0539 (9) 0.0462 (8) 0.0501 (8) 0.0224 (7) 0.0174 (7) 0.0160 (7) C11 0.0519 (9) 0.0457 (8) 0.0483 (8) 0.0233 (7) 0.0159 (7) 0.0099 (6) C12 0.0536 (10) 0.0559 (10) 0.0696 (11) 0.0258 (8) 0.0190 (8) 0.0125 (8) C13 0.0593 (11) 0.0720 (12) 0.0794 (13) 0.0338 (10) 0.0009 (10) 0.0048 (10) C14 0.0937 (16) 0.0884 (14) 0.0572 (11) 0.0577 (13) 0.0049 (10) 0.0136 (10) C15 0.0880 (14) 0.0730 (12) 0.0547 (10) 0.0479 (11) 0.0235 (9) 0.0231 (9) C16 0.0602 (10) 0.0568 (9) 0.0512 (9) 0.0284 (8) 0.0190 (7) 0.0180 (7) C17 0.0561 (9) 0.0576 (9) 0.0544 (9) 0.0297 (8) 0.0225 (7) 0.0250 (7) C18 0.0559 (9) 0.0624 (10) 0.0555 (9) 0.0327 (8) 0.0225 (7) 0.0248 (8) C19 0.0556 (9) 0.0622 (10) 0.0532 (9) 0.0328 (8) 0.0211 (7) 0.0179 (7) C20 0.0545 (10) 0.0927 (13) 0.0500 (9) 0.0378 (10) 0.0173 (7) 0.0246 (9) C21 0.0595 (10) 0.0881 (13) 0.0544 (9) 0.0461 (10) 0.0196 (8) 0.0281 (9) C22 0.0567 (10) 0.0717 (11) 0.0546 (9) 0.0396 (9) 0.0170 (7) 0.0182 (8) C23 0.0649 (10) 0.0738 (11) 0.0610 (10) 0.0439 (10) 0.0297 (8) 0.0249 (9) C24 0.0527 (9) 0.0586 (9) 0.0440 (8) 0.0291 (8) 0.0111 (7) 0.0084 (7) C25 0.0676 (11) 0.0726 (11) 0.0692 (11) 0.0439 (10) 0.0275 (9) 0.0215 (9) C26 0.0813 (13) 0.0645 (11) 0.0876 (14) 0.0436 (11) 0.0283 (11) 0.0267 (10) C27 0.0609 (11) 0.0609 (10) 0.0667 (11) 0.0238 (9) 0.0152 (9) 0.0165 (8) C28 0.0529 (10) 0.0674 (11) 0.0674 (11) 0.0273 (9) 0.0181 (8) 0.0129 (9) C29 0.0568 (10) 0.0604 (10) 0.0636 (10) 0.0326 (9) 0.0191 (8) 0.0136 (8) ----- ------------- ------------- ------------- ------------- ------------- ------------- Geometric parameters (Å, º) {#tablewrapgeomlong} =========================== ---------------------- -------------- ----------------------- -------------- Cl1---C27 1.745 (2) C24---C25 1.389 (3) O1---C1 1.231 (2) C24---C29 1.388 (3) O2---C19 1.219 (3) C25---C26 1.383 (3) N1---N2 1.3532 (19) C26---C27 1.365 (3) N1---C1 1.373 (2) C27---C28 1.372 (3) N1---C17 1.464 (2) C28---C29 1.372 (3) N2---C4 1.309 (2) C2---H2 0.9300 N3---C19 1.354 (2) C6---H6 0.9300 N3---C20 1.458 (2) C7---H7 0.9300 N3---C23 1.455 (3) C8---H8 0.9300 N4---C21 1.461 (3) C9---H9 0.9300 N4---C22 1.466 (2) C10---H10 0.9300 N4---C24 1.410 (2) C12---H12 0.9300 C1---C2 1.433 (3) C13---H13 0.9300 C2---C3 1.353 (2) C14---H14 0.9300 C3---C4 1.439 (2) C15---H15 0.9300 C3---C11 1.485 (2) C16---H16 0.9300 C4---C5 1.488 (2) C17---H17A 0.9700 C5---C6 1.386 (3) C17---H17B 0.9700 C5---C10 1.381 (3) C18---H18A 0.9700 C6---C7 1.380 (3) C18---H18B 0.9700 C7---C8 1.370 (3) C20---H20A 0.9700 C8---C9 1.375 (3) C20---H20B 0.9700 C9---C10 1.381 (3) C21---H21A 0.9700 C11---C12 1.386 (3) C21---H21B 0.9700 C11---C16 1.394 (3) C22---H22A 0.9700 C12---C13 1.379 (3) C22---H22B 0.9700 C13---C14 1.378 (3) C23---H23A 0.9700 C14---C15 1.367 (4) C23---H23B 0.9700 C15---C16 1.379 (3) C25---H25 0.9300 C17---C18 1.513 (3) C26---H26 0.9300 C18---C19 1.508 (3) C28---H28 0.9300 C20---C21 1.510 (3) C29---H29 0.9300 C22---C23 1.504 (3) Cl1···H7^i^ 3.0900 H6···C11 2.7500 O1···C18 3.252 (2) H6···H6^vii^ 2.5200 O1···C12^ii^ 3.323 (2) H7···Cl1^xi^ 3.0900 O2···C25^iii^ 3.289 (3) H7···H12^vii^ 2.5800 O1···H17A 2.4600 H8···O1^xiv^ 2.9100 O1···H8^iv^ 2.9100 H8···C8^viii^ 3.0000 O1···H2^ii^ 2.8400 H8···C9^viii^ 3.0200 O1···H12^ii^ 2.4400 H8···H8^viii^ 2.5700 O1···H18A 2.7800 H8···H9^viii^ 2.6000 O2···H17B 2.6700 H9···C24^v^ 2.9900 O2···H20A 2.3500 H9···H8^viii^ 2.6000 O2···H17A 2.6100 H10···N2 2.6600 O2···H25^iii^ 2.5300 H10···C3^vi^ 2.9400 N3···N4 2.830 (2) H10···C4^vi^ 2.9400 N4···N3 2.830 (2) H12···C2 2.7900 N2···H10 2.6600 H12···H2 2.3800 N2···H20B^v^ 2.9000 H12···O1^ii^ 2.4400 N3···H18B^v^ 2.8600 H12···C7^vii^ 3.1000 C5···C16 3.178 (2) H12···H7^vii^ 2.5800 C6···C11 3.154 (3) H13···H20A^x^ 2.4700 C6···C16 3.262 (3) H14···C25^ii^ 2.9700 C9···C21^v^ 3.595 (3) H16···C4 2.8800 C9···C24^v^ 3.524 (3) H16···C5 2.8000 C11···C6 3.154 (3) H16···H17B^vi^ 2.4100 C12···O1^ii^ 3.323 (2) H17A···O1 2.4600 C16···C5 3.178 (2) H17A···O2 2.6100 C16···C6 3.262 (3) H17B···O2 2.6700 C18···O1 3.252 (2) H17B···C16^vi^ 3.1000 C21···C9^v^ 3.595 (3) H17B···H16^vi^ 2.4100 C24···C9^v^ 3.524 (3) H18A···O1 2.7800 C25···O2^iii^ 3.289 (3) H18A···C1 2.9300 C1···H18A 2.9300 H18A···C23 2.7400 C2···H12 2.7900 H18A···H23B 2.2100 C3···H6 2.8700 H18A···C28^xiii^ 3.0600 C3···H10^vi^ 2.9400 H18A···H28^xiii^ 2.2800 C4···H16 2.8800 H18B···C23 2.8800 C4···H10^vi^ 2.9400 H18B···H23B 2.2700 C5···H20B^v^ 3.0900 H18B···N3^v^ 2.8600 C5···H16 2.8000 H18B···C23^v^ 3.0700 C7···H12^vii^ 3.1000 H20A···O2 2.3500 C8···H8^viii^ 3.0000 H20A···C13^xv^ 2.9500 C9···H21A^v^ 2.9200 H20A···H13^xv^ 2.4700 C9···H8^viii^ 3.0200 H20B···H23A 2.4800 C11···H29^ix^ 2.9200 H20B···N2^v^ 2.9000 C11···H6 2.7500 H20B···C5^v^ 3.0900 C12···H2 2.7100 H21A···C25 2.6900 C12···H29^ix^ 2.9500 H21A···H25 2.2600 C13···H20A^x^ 2.9500 H21A···C9^v^ 2.9200 C15···H26^xi^ 3.0300 H21B···C25 2.9300 C16···H17B^vi^ 3.1000 H21B···H22A 2.5100 C18···H23B 2.4500 H21B···H25 2.4500 C21···H25 2.5600 H22A···H21B 2.5100 C22···H29 2.7800 H22B···C29 2.6000 C23···H18B 2.8800 H22B···H29 2.1800 C23···H18B^v^ 3.0700 H23A···H20B 2.4800 C23···H18A 2.7400 H23B···C18 2.4500 C24···H9^v^ 2.9900 H23B···H18A 2.2100 C25···H21A 2.6900 H23B···H18B 2.2700 C25···H21B 2.9300 H25···C21 2.5600 C25···H14^ii^ 2.9700 H25···H21A 2.2600 C28···H2^xii^ 2.9400 H25···H21B 2.4500 C28···H18A^xiii^ 3.0600 H25···O2^iii^ 2.5300 C29···H22B 2.6000 H26···C15^i^ 3.0300 H2···C12 2.7100 H28···H18A^xiii^ 2.2800 H2···C28^ix^ 2.9400 H29···C11^xii^ 2.9200 H2···H12 2.3800 H29···C12^xii^ 2.9500 H2···O1^ii^ 2.8400 H29···C22 2.7800 H6···C3 2.8700 H29···H22B 2.1800 N2---N1---C1 125.35 (15) C7---C6---H6 120.00 N2---N1---C17 114.86 (13) C6---C7---H7 120.00 C1---N1---C17 119.35 (14) C8---C7---H7 120.00 N1---N2---C4 118.26 (13) C7---C8---H8 120.00 C19---N3---C20 120.13 (18) C9---C8---H8 120.00 C19---N3---C23 125.31 (16) C8---C9---H9 120.00 C20---N3---C23 111.42 (15) C10---C9---H9 120.00 C21---N4---C22 111.60 (15) C5---C10---H10 120.00 C21---N4---C24 118.29 (16) C9---C10---H10 120.00 C22---N4---C24 115.21 (16) C11---C12---H12 120.00 O1---C1---N1 120.49 (17) C13---C12---H12 120.00 O1---C1---C2 125.55 (17) C12---C13---H13 120.00 N1---C1---C2 113.92 (15) C14---C13---H13 120.00 C1---C2---C3 122.23 (16) C13---C14---H14 120.00 C2---C3---C4 116.98 (15) C15---C14---H14 120.00 C2---C3---C11 119.65 (15) C14---C15---H15 120.00 C4---C3---C11 123.27 (14) C16---C15---H15 120.00 N2---C4---C3 122.15 (14) C11---C16---H16 120.00 N2---C4---C5 114.01 (13) C15---C16---H16 120.00 C3---C4---C5 123.79 (14) N1---C17---H17A 110.00 C4---C5---C6 120.46 (16) N1---C17---H17B 110.00 C4---C5---C10 120.58 (17) C18---C17---H17A 110.00 C6---C5---C10 118.90 (16) C18---C17---H17B 110.00 C5---C6---C7 120.31 (19) H17A---C17---H17B 108.00 C6---C7---C8 120.2 (2) C17---C18---H18A 109.00 C7---C8---C9 120.12 (19) C17---C18---H18B 109.00 C8---C9---C10 119.85 (19) C19---C18---H18A 109.00 C5---C10---C9 120.63 (19) C19---C18---H18B 109.00 C3---C11---C12 120.49 (16) H18A---C18---H18B 108.00 C3---C11---C16 120.48 (17) N3---C20---H20A 110.00 C12---C11---C16 118.84 (16) N3---C20---H20B 110.00 C11---C12---C13 120.20 (18) C21---C20---H20A 110.00 C12---C13---C14 120.4 (2) C21---C20---H20B 110.00 C13---C14---C15 119.9 (2) H20A---C20---H20B 108.00 C14---C15---C16 120.38 (19) N4---C21---H21A 110.00 C11---C16---C15 120.28 (19) N4---C21---H21B 110.00 N1---C17---C18 110.42 (17) C20---C21---H21A 110.00 C17---C18---C19 111.72 (17) C20---C21---H21B 110.00 O2---C19---N3 121.94 (17) H21A---C21---H21B 108.00 O2---C19---C18 120.78 (17) N4---C22---H22A 110.00 N3---C19---C18 117.29 (18) N4---C22---H22B 110.00 N3---C20---C21 109.95 (16) C23---C22---H22A 110.00 N4---C21---C20 110.00 (17) C23---C22---H22B 110.00 N4---C22---C23 110.26 (16) H22A---C22---H22B 108.00 N3---C23---C22 110.62 (18) N3---C23---H23A 110.00 N4---C24---C25 123.46 (19) N3---C23---H23B 110.00 N4---C24---C29 119.06 (17) C22---C23---H23A 110.00 C25---C24---C29 117.41 (18) C22---C23---H23B 110.00 C24---C25---C26 120.8 (2) H23A---C23---H23B 108.00 C25---C26---C27 120.3 (2) C24---C25---H25 120.00 Cl1---C27---C26 120.62 (17) C26---C25---H25 120.00 Cl1---C27---C28 119.29 (18) C25---C26---H26 120.00 C26---C27---C28 120.1 (2) C27---C26---H26 120.00 C27---C28---C29 119.7 (2) C27---C28---H28 120.00 C24---C29---C28 121.76 (19) C29---C28---H28 120.00 C1---C2---H2 119.00 C24---C29---H29 119.00 C3---C2---H2 119.00 C28---C29---H29 119.00 C5---C6---H6 120.00 C1---N1---N2---C4 8.3 (3) C2---C3---C4---N2 −7.7 (3) C17---N1---N2---C4 −179.47 (16) N2---C4---C5---C10 −42.1 (2) N2---N1---C1---O1 170.70 (18) C3---C4---C5---C6 −42.2 (2) C17---N1---C1---O1 −1.2 (3) C3---C4---C5---C10 140.53 (18) N2---N1---C1---C2 −11.6 (3) N2---C4---C5---C6 135.15 (17) C17---N1---C1---C2 176.54 (17) C6---C5---C10---C9 −0.2 (2) N2---N1---C17---C18 −88.78 (18) C10---C5---C6---C7 1.4 (3) C1---N1---C17---C18 84.0 (2) C4---C5---C10---C9 177.05 (15) N1---N2---C4---C3 2.0 (3) C4---C5---C6---C7 −175.90 (16) N1---N2---C4---C5 −175.41 (15) C5---C6---C7---C8 −1.4 (3) C20---N3---C19---O2 13.4 (3) C6---C7---C8---C9 0.2 (3) C23---N3---C19---O2 171.58 (19) C7---C8---C9---C10 1.0 (3) C23---N3---C20---C21 −58.0 (2) C8---C9---C10---C5 −1.0 (3) C19---N3---C20---C21 103.0 (2) C12---C11---C16---C15 −0.7 (3) C23---N3---C19---C18 −8.3 (3) C3---C11---C16---C15 −175.65 (18) C19---N3---C23---C22 −102.3 (2) C16---C11---C12---C13 0.8 (3) C20---N3---C19---C18 −166.45 (16) C3---C11---C12---C13 175.75 (18) C20---N3---C23---C22 57.50 (19) C11---C12---C13---C14 0.0 (3) C24---N4---C21---C20 165.70 (14) C12---C13---C14---C15 −0.8 (4) C21---N4---C22---C23 56.4 (2) C13---C14---C15---C16 0.9 (4) C22---N4---C21---C20 −57.14 (19) C14---C15---C16---C11 −0.2 (3) C24---N4---C22---C23 −165.05 (16) N1---C17---C18---C19 −176.70 (14) C22---N4---C24---C25 −126.42 (19) C17---C18---C19---N3 178.08 (16) C21---N4---C24---C29 −167.77 (15) C17---C18---C19---O2 −1.8 (3) C21---N4---C24---C25 9.3 (2) N3---C20---C21---N4 57.2 (2) C22---N4---C24---C29 56.6 (2) N4---C22---C23---N3 −55.78 (19) N1---C1---C2---C3 5.0 (3) N4---C24---C25---C26 −178.06 (17) O1---C1---C2---C3 −177.4 (2) C29---C24---C25---C26 −1.0 (3) C1---C2---C3---C11 −172.67 (19) N4---C24---C29---C28 178.30 (16) C1---C2---C3---C4 3.8 (3) C25---C24---C29---C28 1.1 (3) C2---C3---C4---C5 169.46 (18) C24---C25---C26---C27 −0.1 (3) C11---C3---C4---C5 −14.2 (3) C25---C26---C27---Cl1 −178.59 (16) C4---C3---C11---C12 140.70 (19) C25---C26---C27---C28 1.1 (3) C4---C3---C11---C16 −44.4 (3) Cl1---C27---C28---C29 178.70 (15) C11---C3---C4---N2 168.63 (18) C26---C27---C28---C29 −1.0 (3) C2---C3---C11---C12 −43.0 (3) C27---C28---C29---C24 −0.1 (3) C2---C3---C11---C16 131.8 (2) ---------------------- -------------- ----------------------- -------------- Symmetry codes: (i) *x*, *y*+1, *z*−1; (ii) −*x*+2, −*y*+2, −*z*+2; (iii) −*x*+1, −*y*+2, −*z*+1; (iv) *x*, *y*+1, *z*; (v) −*x*+1, −*y*+1, −*z*+1; (vi) −*x*+1, −*y*+1, −*z*+2; (vii) −*x*+2, −*y*+1, −*z*+2; (viii) −*x*+1, −*y*, −*z*+2; (ix) *x*, *y*, *z*+1; (x) *x*+1, *y*, *z*+1; (xi) *x*, *y*−1, *z*+1; (xii) *x*, *y*, *z*−1; (xiii) −*x*+2, −*y*+2, −*z*+1; (xiv) *x*, *y*−1, *z*; (xv) *x*−1, *y*, *z*−1. Hydrogen-bond geometry (Å, º) {#tablewraphbondslong} ============================= Cg1 and Cg4 are the centroids of the N1/N2/C1--C4 and C11--C16 rings, respectively. ------------------------ --------- --------- ----------- --------------- *D*---H···*A* *D*---H H···*A* *D*···*A* *D*---H···*A* C12---H12···O1^ii^ 0.93 2.44 3.323 (2) 158 C25---H25···O2^iii^ 0.93 2.53 3.289 (3) 139 C10---H10···*Cg*1^vi^ 0.93 2.88 3.431 (2) 119 C29---H29···*Cg*4^xii^ 0.93 2.86 3.762 (2) 165 ------------------------ --------- --------- ----------- --------------- Symmetry codes: (ii) −*x*+2, −*y*+2, −*z*+2; (iii) −*x*+1, −*y*+2, −*z*+1; (vi) −*x*+1, −*y*+1, −*z*+2; (xii) *x*, *y*, *z*−1. ###### Hydrogen-bond geometry (Å, °) *Cg*1 and *Cg*4 are the centroids of the N1/N2/C1--C4 and C11--C16 rings, respectively. *D*---H⋯*A* *D*---H H⋯*A* *D*⋯*A* *D*---H⋯*A* ---------------------- --------- ------- ----------- ------------- C12---H12⋯O1^i^ 0.93 2.44 3.323 (2) 158 C25---H25⋯O2^ii^ 0.93 2.53 3.289 (3) 139 C10---H10⋯*Cg*1^iii^ 0.93 2.88 3.431 (2) 119 C29---H29⋯*Cg*4^iv^ 0.93 2.86 3.762 (2) 165 Symmetry codes: (i) ; (ii) ; (iii) ; (iv) .

Background {#Sec1} ========== Desmopressin (1-deamino-8-[d]{.smallcaps}-arginine vasopressin or dDAVP) is a peptide analog of the naturally occurring human antidiuretic hormone, vasopressin. It was first synthesized by Zaoral et al. ([@CR32]), being a selective agonist for the vasopressin V2 cell membrane receptor (V2R) present in kidney tubules and endothelia of blood vessels. Activation of endothelial V2R by dDAVP causes cAMP-mediated signaling followed by the release of von Willebrand factor (vWF), coagulation factor VIII and tissue-type plasminogen activator into the blood (Juul et al. [@CR12]). The hemostatic effects of dDAVP at doses as low as 0.2--0.3 µg per kg of body weight make it an often-used treatment for the management of bleeding disorders, and is also being evaluated as a blood-saving agent in surgery or trauma (Mannucci [@CR15]; Svensson et al. [@CR29]). The evidence for elevated vWF levels as a risk factor for venous thromboembolism is weak and dDAVP appears safe for perioperative use. The compound has few side effects but it is recommended caution in small children and elderly, due to the risk of fluid retention and hyponatremia after repeated administration (Svensson et al. [@CR29]). Beyond its role in hemostasis, vWF has emerged as a pivotal regulator of tumor cell metastasis. Using a vWF-deficient mouse model, it was demonstrated that vWF plays a protective role against tumor cell dissemination in vivo by inducing apoptosis of metastatic cells, presumably early after their arrest in the microvasculature of the target organ (Terraube et al. [@CR30], [@CR31]). Interestingly, aggressive human breast cancer cells expressing high levels of ADAM28 (a disintegrin and metalloproteinase 28) are capable of avoiding vWF-induced apoptosis in the circulatory system at micrometastatic sites. ADAM28 specifically binds to vWF and renders it inactive by cleaving, thus favoring the survival of metastatic cells (Mochizuki et al. [@CR18]). Since physiological levels of vWF can induce cancer cell apoptosis, an attractive strategy could be to stimulate endothelial secretion of vWF by a pharmacological intervention, such as dDAVP infusion, aimed at increasing host resistance to metastasis (Ripoll and Alonso [@CR25]). Vasopressin receptors have been detected in many human cancer cell lines (Petit et al. [@CR22]), including breast cancer (North et al. [@CR19], [@CR20]), and it is known that dDAVP exerts some direct antiproliferative effect against V2R-expressing human breast carcinoma cells (Keegan et al. [@CR13]). Such action is mediated through agonist V2R signaling, involving activation of adenylate cyclase with consequent intracellular cAMP elevation and protein kinase A activation. The cytostatic effect could be blocked by the selective nonpeptide V2R antagonists satavaptan (Keegan et al. [@CR13]) and tolvaptan (Iannucci et al. [@CR11]). In mouse mammary tumor models, intravenous (IV) administration of dDAVP prevented the development of blood-borne metastases (Alonso et al. [@CR1]), and also decreased axillary lymph node involvement when administered at high doses during manipulation and surgical removal of the primary tumor (Giron et al. [@CR7]). In addition, more recent studies in human and mouse mammary cancer cells have found that dDAVP can induce anti-angiogenic effects associated with the proteolytic conversion of plasminogen to angiostatin (Ripoll et al. [@CR26]). A pilot veterinary clinical trial in dogs with locally-advanced mammary cancer showed that a perioperative infusion of dDAVP at high doses of 1 μg/kg significantly prolonged disease-free and overall survival (Hermo et al. [@CR9]). It seems that dDAVP infusion during the surgical phase not only inhibits perioperative metastatic events but also combats micrometastases that occurred before surgery. An extended veterinary trial confirmed these observations, demonstrating a reduced incidence of local relapses and lung metastasis in perioperatively treated animals, and a particular survival benefit in cases with high-grade carcinoma (Hermo et al. [@CR10]). Considering the well-known hemostatic effect and tolerability of dDAVP as well as its potential antimetastatic properties, we conducted a phase II dose-escalation trial in patients with breast cancer, administering a lyophilized formulation of dDAVP by IV infusion in saline, before and after surgical resection of primary tumor. Patients and methods {#Sec2} ==================== Patients {#Sec3} -------- Patients were enrolled from the "Eva Peron" Hospital, San Martin and the Italian Hospital, La Plata (Argentina). Eligible patients were otherwise healthy women between 18 and 65 years of age, with histological and/or cytological diagnosis of breast carcinoma (Stage 0, I, II) and managed by mastectomy or lumpectomy as primary treatment, including sentinel lymph node biopsy. Exclusion criteria included pregnancy or breast-feeding, hormonal treatment, known hypersensitivity to dDAVP or vasopressin, severe von Willebrand's disease or hemophilia, syndrome of inadequate secretion of antidiuretic hormone, renal impairment or hyponatremia, congestive heart failure, blood hypertension, heart arrhythmia, thromboembolic disease, diabetes type I or II, any underlying coronary disease detected in pre-surgical evaluations, symptoms or evidence of metastasis on images and other malignant diseases. All patients provided written informed consent. The study was approved by the ethics committee at each site and by the National Administration of Drugs, Food and Medical Technology (ANMAT) in Argentina (No. NCT01606072). Study design {#Sec4} ------------ This was an open-label, dose-escalation phase II trial. Primary endpoints were safety and tolerability in breast cancer patients undergoing surgery as first treatment, as well as selection of the best dose of dDAVP for perioperative use in oncology. Secondary endpoints included surgical bleeding, plasma levels of vWF, and circulating tumor cells (CTCs). Perioperative administration of study treatment and anesthesia {#Sec5} -------------------------------------------------------------- Eligible patients were administered with dDAVP divided into 2 IV infusions, the first started preoperatively 30--60 min before surgery and the second postoperatively 24 h later. A lyophilized formulation of dDAVP (Surprex TM, Elea Laboratories, Buenos Aires, Argentina) was diluted in 100 mL of saline solution and slowly infused over the course of approximately 20--30 min. Five groups of at least four patients each received increasing total dDAVP doses of 0.5, 1.0, 1.25, 1.5 and 2.0 μg/kg, according to the scheme in Table [1](#Tab1){ref-type="table"}. If no dose-limiting toxicity occurred, dosages were escalated to the next cohort of patients.Table 1Treatment groups, dosage and schedule of administration of perioperative dDAVPGroupFirst dose (µg/kg) 30--60 min before surgerySecond dose (µg/kg) 24 h after surgeryTotal dose (µg/kg)10.250.250.520.50.51.030.750.51.2541.00.51.551.01.02.0 Anesthesia was induced and maintained by target-controlled infusion of remifentanil (1--4 μg/kg) and propofol (1.5 mg/kg). Vecuronium bromide (0.1 mg/kg) was administered for muscle relaxation and endotracheal intubation. All patients were pre-oxygenated for 3 min with 100 % oxygen using face mask ventilation. Safety assessments {#Sec6} ------------------ Safety and tolerability were assessed for all enrolled patients from the time the patient signs the informed consent through post-treatment follow-up. Adverse events were graded according to the NCI Common Toxicity Criteria for Adverse Events (CTCAE, Version 4.0). Serious adverse events were reported to the sponsor and the ethics committees and were followed up until resolution. Biochemical analysis {#Sec7} -------------------- Blood was drawn within 7 days prior to surgery to obtain a baseline, and postoperatively 90--120 min after the first and the second dose of dDAVP. All laboratory assays were performed by investigators blinded to the clinical data, as described elsewhere (Sanchez-Luceros et al. [@CR27]). The vWF antigen (vWF:Ag) was measured by ELISA. The functional activity of vWF was analyzed by the von Willebrand ristocetin cofactor (vWF:RCo) assay using formalin-fixed platelets. The factor VIII levels (FVIII:C) were assayed applying the one-stage method. The standard pool was periodically calibrated against the WHO International Standard for FVIII and vWF in plasma (07/316). Quantitative real-time reverse transcription-PCR (qPCR) detection of CTCs {#Sec8} ------------------------------------------------------------------------- CTCs were measured by qPCR assay for expression of cytokeratin-19 (CK-19) mRNA in whole blood (Ring et al. [@CR24]). Total RNA was purified from peripheral blood stored in guanidine thiocyanate (Promega, Madison, WI) using QuickZol reagent (Kalium Technologies, Buenos Aires, Argentina). DNase treatment was carried out using a DNase I amplification grade kit (Life Technologies, Breda, the Netherlands) according to the manufacturer's instructions. The RNA pellets were dissolved in nuclease-free water and stored at −70 °C prior to use. RNA was reverse transcribed with SuperScript III first-Strand kit (Life Technologies) according to the manufacturer's protocol. Real-time PCR was performed with SYBR Green PCR Master Mix (Life Technologies) and StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The following specific primers were used as described elsewhere (Ring et al. [@CR24]): for CK-19, forward: 5′-TGC GGG ACA AGA TTC TTG GT-3′ and reverse: 5′-TCT CAA ACT TGG TTC GGA AGT CA-3′; for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), forward: 5′-CAT GGG TGT GAA CCA TGA GA-3′ and reverse: 5′-CAG TGA TGG CAT GGA CTG TG-3′. All sample plates were run with positive controls (RNA from MCF-7 human breast cancer cell line) and no template negative controls. The following thermal cycling conditions were used: 48 °C for 30 min, 95 °C for 10 min, 40 cycles of 95 °C for 15 s followed by 60 °C for 60 s. Each sample was analyzed in triplicate and mean cycle threshold (Ct) values were used for further analysis. Ct values for CK-19 were normalized for GAPDH expression levels and expressed in relation to positive control samples. Relative quantification (RQ) values were calculated as 2^−ΔΔCt^. Immunohistochemical detection of V2R {#Sec9} ------------------------------------ Breast tumor samples were fixed in 10 % formalin, embedded in paraffin, and tissue sections of 4 µm were cut and placed on silane coated slides. Immunohistochemistry was performed on a Bond automated system (Leica Biosystems, Newcastle, UK). Sections were dewaxed and pretreated with the epitope retrieval solution 2 (EDTA buffer, pH 8.8) at 100 °C for 20 min. Immunostaining was carried out using polyclonal rabbit antibodies against the human V2R (V5514; 1:100 dilution, Sigma-Aldrich) at room temperature for 20 min, and a biotin-free, polymeric horseradish peroxidase (HRP)-linked antibody conjugate as a secondary antibody. Sections were counterstained with hematoxylin. Kidney tubules, as well as V2R-expressing MCF-7 human breast cancer xenografts generated in nude mice (Garona et al. [@CR6]), were used as positive controls for V2R expression. Statistical analysis {#Sec10} -------------------- PRISM 6, Version 6.01 (GraphPad Software Inc, La Jolla, CA, USA) was used to conduct all statistical analyses. P values less than 0.05 were considered statistically significant. For multiple group comparisons one-way or two-way ANOVA, followed by Tukey post hoc test were applied after normal distribution of data was confirmed using the Shapiro--Wilk normality test. In addition, the homoscedasticity was determined with Bartlett's test. For non-normally distributed data or when homoscedasticity was not supported, Kruskal--Wallis test was performed. The cut-off value for CK-19 mRNA was determined with receiver operating characteristics (ROC) curve analysis (minimal false-negative and false-positive results). Results {#Sec11} ======= The trial accrued a total of 21 patients from April 2012 to February 2014. One patient who developed a hypertensive episode during the night before surgery was ineligible and excluded from the study. Characteristics of the enrolled patients are summarized in Table [2](#Tab2){ref-type="table"}. Among the 20 patients evaluable for toxicity, adverse events attributable to dDAVP were observed in two patients and all were reversible. Laboratory examinations of one patient included in treatment group 3 (1.25 μg/kg) showed hyponatremia (serum sodium levels \<120 mEq/L, grade 4) 1 h after the first dDAVP dose. The patient also experienced nausea and mild dyspnea (grade 1). These events were considered non-serious as they were transient and reversible, and hyponatremia was spontaneously corrected 24 h later (137 mEq/L). Another patient of treatment group 4 (1.5 μg/kg) showed signs of a hypersensitive reaction early after starting the preoperative dDAVP infusion, manifesting hot flushing, skin rash and palpitations (grade 2). This event was considered serious and treatment was interrupted before completion of the first dose. Patient was medicated IV with diphenhydramine (20 mg) and dexamethasone (8 mg), showing complete resolution of symptoms within 45 min. Since treatment was interrupted, this patient was not evaluable for secondary endpoints. Reactions were adequately managed by slowing the infusion rate of dDAVP to 30--40 min in treatment group 5 (2 μg/kg). The maximum tolerated dose was not reached, and the individual dose of 1 μg/kg given preoperatively and postoperatively was then considered for further studies. Median follow-up was 24 months (range 17--39 months). None of the patients relapsed during follow-up.Table 2Descriptive characteristics of patients enrolled in the study (n = 20)Patient characteristicNo.Age, median (range)47 years (36--62)Tumor size, median (range)20 mm (5--40)Histopathology Ductal carcinoma in situ (DCIS)4 (20 %) Invasive ductal carcinoma15 (75 %) Invasive lobular carcinoma1 (5 %)Axillary involvement in invasive carcinoma, n = 167 (43 %)Molecular subtype of invasive carcinoma, n = 16 Luminal (A and B)11 (69 %) Her21 (6 %) Triple negative4 (25 %)V2R status known, n = 18 Positive tumor expression6 (33 %)Type of surgery Breast conserving surgery12 (60 %) Mastectomy8 (40 %)Postoperative adjuvant therapy None (DCIS)4 (20 %) Cyclophosphamide-based chemotherapy6 (30 %) Radiotherapy3 (15 %) Chemoradiotherapy7 (35 %) A reduced intraoperative bleeding of up to 50 % was noted with increasing doses of dDAVP, as measured by the number or weight of pads used during surgical procedure (Fig. [1](#Fig1){ref-type="fig"}a, b). A significant reduction was observed in the number of surgical pads used in patients receiving a preoperative first dose of dDAVP of 1 μg/kg (treatment groups 4 and 5, considered together) in comparison to lower doses (see also Fig. [1](#Fig1){ref-type="fig"}a). As expected, vWF:Ag plasma levels exhibited a mean increase of 50--100 % with respect to baseline after each preoperative and postoperative dDAVP infusion, and maximum levels were obtained in patients of group 5 treated with the highest total dose of 2 μg/kg (Fig. [1](#Fig1){ref-type="fig"}c). Similar results were found for vWF:RCo (Fig. [1](#Fig1){ref-type="fig"}d) and FVIII:C levels (data not shown).Fig. 1Hemostatic effects of perioperative dDAVP. **a** Number and **b** weight of surgical pads used during the surgical procedure, as a function of the preoperative first dose of dDAVP (treatment groups 4 and 5 are presented together, since in both cases received 1 µg/kg). \*p \< 0.05 (1.0 versus 0.25 µg/kg), ANOVA with Tukey post-test. **c** vWF antigen (vWF:Ag) and **d** functional vWF (vWF:RCo) levels in samples collected prior to surgery (*baseline*), and after the preoperative dose (1st dDAVP dose) and the postoperative dose (2nd dDAVP dose). ^\#^p \< 0.05 (2.0 µg/kg versus all other dose levels), two-way ANOVA with Tukey post-test. In all cases, data represent mean ± SEM. Evaluable samples were available for CTCs assessment from 16 of the 20 patients enrolled. A preliminary analysis indicated no significant differences between treatment groups, and thus data were pooled together due to the small number of patients. Detectable levels of CK-19 mRNA were found in several patients, and 9 of the 16 patients had high RQ values of \>0.05 before surgery at baseline (Fig. [2](#Fig2){ref-type="fig"}). Twenty-four hours after surgery, only 5 patients showed high RQ values and also the median levels of expression were reduced. Two weeks later, median values remained reduced, but returned to baseline 1 month after surgery.Fig. 2Detection of circulating tumor cells (CTCs) by qPCR. CTCc were assessed by means of expression of transcripts for CK-19 in whole blood, as described in detail in "[Patients and methods](#Sec2){ref-type="sec"}". Samples from 16 patients were obtained within 7 days prior to surgery (*baseline*), and 24 h, 2 weeks and 1 month after surgery. Data from all treatment groups were pooled. *Horizontal lines* indicate the median values. The cut-off RQ value was 0.00445 for healthy woman volunteers aged 25--61 years, based on ROC analysis (specificity: 100 %; sensibility: 81.25 %; area = 0.91). We examined the expression of V2R by immunohistochemistry in paraffin tumor samples available from 18 patients. In all cases, V2R was detected in endothelial cells of small vessels of tumor stroma or surrounding tissues. Six of the 18 cases evaluated revealed positive expression of V2R in breast carcinoma cells (Fig. [3](#Fig3){ref-type="fig"}; see also Table [2](#Tab2){ref-type="table"}). Expression pattern was cytoplasmic, either diffuse or focal, with membrane accentuation, and the intensity of staining ranged from moderate to strong.Fig. 3Immunohistochemical staining of vasopressin receptors. V2R expression was detected using polyclonal antibodies against the human receptor, as described in detail in "[Patients and methods](#Sec2){ref-type="sec"}". Representative pictures of tumor sections from patients enrolled in the trial and positive control tissue are depicted. **a** Breast carcinoma expressing V2R **b** V2R-negative breast carcinoma **c** Kidney tubules **d** MCF-7 human breast carcinoma xenograft. Arrowhead denotes positive staining of small vessels. Original magnification: **a**, **b**, **d** ×400; **c** ×100. Discussion {#Sec12} ========== Pioneer works by Mannucci et al. ([@CR16], [@CR17]) in healthy subjects and patients with hemophilia A and von Willebrand´s disease demonstrated a good tolerance and efficacy of dDAVP as a hemostatic agent at doses up to 0.5 μg/kg by the IV route. However, since single doses of 0.2--0.3 μg/kg seemed to produce a near-maximal response in healthy subjects a reduction in dosage was suggested, in order to reduce side effects such as tachycardia (Mannucci et al. [@CR17]). There are anecdotal case reports that document the satisfactory perioperative use of dDAVP in oncology patients with hemostatic disorders, including a case of a woman with Glanzmann thrombasthenia receiving 0.4 μg/kg of the compound during the resection of a breast tumor later diagnosed as fibroadenoma (Ohishi et al. [@CR21]). To our knowledge, this is the first dose-escalation trial of dDAVP as a perioperative adjunctive treatment in the management of operable cancers. The compound was well tolerated at the highest total dose level tested in this study (2 μg/kg) when administered divided in two slow IV infusions of 1 μg/kg, 30--60 min before and 24 h after surgery. Two patients developed adverse events, including hyponatremia and a hypersensitivity reaction that were completely reversible. It is known that the hemostatic dosage is higher than the dose used for antidiuresis. Maximal antidiuretic effect is already achieved with low doses, while duration of hemostatic effect tend to prolong with increasing doses (Lethagen et al. [@CR14]). Although water retention is not a prominent clinical problem, the risk of hyponatremia should be taken into account, particularly in elderly patients receiving hypotonic solutions or after frequently, repeated doses of dDAVP (Svensson et al. [@CR29]; Lethagen et al. [@CR14]). Intraoperative bleeding can be a major risk for gastrointestinal or urologic cancers, but it is not a serious problem in early-stage breast cancer patients as included in this study. However, dDAVP still significantly reduced blood loss at a preoperative dose of 1 μg/kg (treatment groups 4 and 5) as determined by surgical pads used during operation. In this sense, a significantly higher increase of vWF was also noted at the highest dose level with respect to the other treatment groups. Interestingly, vWF is now considered as a versatile multifunctional protein (Rauch et al. [@CR23]) given its potential role in different non-hemostatic processes, like metastasis resistance and tumor cell apoptosis (Terraube et al. [@CR30], [@CR31]). It is known that interaction of vWF with metastatic cells is mediated via integrin αVβ3, affecting their adhesion and survival. However, certain aggressive cancer cells are able to escape vWF-induced cell death through production of the protease ADAM28 that can counterbalance the pro-apoptotic function of vWF (Mochizuki et al. [@CR18]). Preclinical studies in aggressive mouse tumor models (Alonso et al. [@CR1]; Giron et al. [@CR7]) and veterinary clinical trials in dogs with locally-advanced mammary cancer (Hermo et al. [@CR9], [@CR10]) have demonstrated inhibition of metastatic progression and survival benefit, respectively, of perioperative dDAVP at doses in the range of 1--2 μg/kg. In the present clinical trial, CTCs were evaluated at different times after perioperative dDAVP treatment, as measured by qPCR detection of CK-19 transcript in peripheral whole blood. An important proportion of blood samples from breast cancer patients were positive for CK-19 preoperatively at baseline, but median expression levels were reduced early postoperatively and also 2 weeks later. One month after surgery, CTCs returned to baseline levels. Even though no placebo or control group underwent surgery was studied, it is noteworthy that surgical manipulation of breast cancer has been consistently associated with a postoperative increase of CK-19 mRNA-positive cells in peripheral blood (Daskalakis et al. [@CR3]; Galan et al. [@CR4]). Thus, it seems that administration of dDAVP during the perioperative period not only improves hemostatic control but also appear to minimize shedding and/or survival of breast carcinoma cells. Recent findings indicated that dDAVP is able to reduce tumor angiogenesis by inducing the formation of angiostatin (Ripoll et al. [@CR26]), a naturally occurring inhibitor of angiogenesis generated by limited proteolysis of plasminogen. V2R-expressing breast cancer cells are stimulated by dDAVP to secrete plasminogen activators such as urokinase, thus excising angiostatin from plasminogen. Biological effects of the peptide on both tumor and endothelial cells appear complex and required further investigations. Notwithstanding, perioperative administration of dDAVP seems to induce a dual angiostatic and antimetastatic effect, breaking cooperative tumor-endothelium interactions in incipient metastatic lesions (Garona and Alonso [@CR5]). Here we explored the expression of V2R in breast cancer tissues by immunohistochemistry, finding one-third of cases were positive and thus may respond with this full dual action. The negative cases, however, still would benefit from dDAVP treatment through endothelial vWF secretion with consequent hemostatic and antimetastatic effects. Furthermore, experimental evidence has suggested a direct role of vWF in the modulation of angiogenesis. Inhibition of vWF by short interfering RNA in endothelial cells caused increased in vitro angiogenesis and an enhanced vascularization response was observed in vWF-deficient mice (Starke et al. [@CR28]). In conclusion, at the highest dose level evaluated perioperative dDAVP appeared to be safe when administered in two slow IV infusions of 1 μg/kg, before and after the surgical procedure. The results of our study suggest that treatment is associated with reduction of intraoperative bleeding, higher circulating vWF levels and a drop in CTC counts after surgery. Perioperative or early postoperative therapies should target not only circulating or residual cancer cells, but also the wound healing mechanisms usurped by these cells to survive and metastasize (Harless [@CR8]). In this regard, the perioperative period is an underutilized window of opportunity, where tumor-host interactions can be modulated to reduce the risk of local relapses and metastases (Coffey et al. [@CR2]). We believe that the present study provides promising evidence to improve the outcome of breast cancer surgery using a well-known hemostatic agent with good tolerance. Clinical trials to establish the effectiveness of administering adjunctive perioperative dDAVP therapy are warranted. Study design: RSW, ASL, ES, GNC, REG, DFA. Study investigator: RSW, FJT. Enrolled patients: RSW, MOG, GSF, FJT. Collection and assembly of data: DJS, NVP, ASL, EM, RSM, IF, MDG, DDL, JG, MP, AVT, DFA. Data analysis and interpretation: RSW, MP, DEG, GVR, REG, IAD, DFA. Manuscript preparation: RSW, JG, DFA. Manuscript review and revisions: RSW, MOG, FJT, ASL, DEG, GVR, REG, IAD, DFA. All authors read and approved the final manuscript. Acknowledgements {#d30e1059} ================ This trial was sponsored by Elea Laboratories S.A.C.I.F. y A. (Argentina). The support of Chemo-Romikin S.A. and the National Cancer Institute, Argentinean Ministry of Health through a clinical research grant to DFA is also acknowledged. JG and MP are postgraduate research fellows and ASL, AVT, DEG, GVR and DFA are members of the National Council of Scientific and Technical Research (CONICET). Compliance with ethical guidelines {#d30e1064} ================================== **Competing interests** MDG, DDL, ES and REG are employed by Elea Laboratories S.A.C.I.F. y A. and GNC and IAD are employed by Chemo-Romikin S.A. We also acknowledge that RSW, FJT, DEG, GVR and DFA serve in a consultant/advisory role for Elea Laboratories S.A.C.I.F. y A. and Chemo-Romikin S.A. All other authors have no conflicts of interest to declare.

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The coronavirus disease (COVID-19) has become a major public health problem since the beginning of 2020. The elderly population and people with chronic diseases are categorized as the major risk groups because of the weakened immune system compared with healthy individuals.^[@r1]^ A strong immune system is formed by our lifestyle parameters and environmental factors such as dietary habits, smoking, physical activity, chronic stress, air pollution, population growth, socioeconomic status, and industrialization. The immune system develops by age, nutrition, and antigen stimulation after birth and among them, nutrition plays a key role in the immunomodulation. Dietary habit is affected by several factors such as socioeconomic status, cultural traditions, employment, and habits, including smoking and alcohol. Being exposed to the poor socioeconomic status causes imbalanced nutrition in the individuals that impair the immune system. On the other hand, smoking, excessive alcohol consumption, and physical inactivity are known to contribute to the adverse effects on the immune system of humans.^[@r2]^ Environmental factors, including air pollution, environmental pollution, rapid industrialization, and smoking, have adverse effects on the respiratory system of the individuals, besides their negative impact on the immune system. Therefore, people with smoking habits or living close to the industrial zones may become more vulnerable to the COVID-19 infection. Extra precautions can be taken in the epidemic areas with high levels of air pollution or fields of work with high possibility of lung damage, such as mining.^[@r3]^ Additionally, chronic stress is one of the major negative effects that is brought by the demands of modern life into our lives. Persistent exposure to chronic stress impairs the immune system, endocrine system, and behavioral responses. Currently, millions of people are under stress because of COVID-19 and its consequences, such as economic crisis, unemployment, debts, and restricted social life.^[@r4]^ Essential jobs have become another parameter affecting human health during the COVID-19 pandemic. Millions of people are not able to apply self-isolation or protections for themselves, because they have to work in different fields, including markets, cargo companies, banks, mine workers, farmers, public transportation drivers, sanitation workers, health workers, and construction workers. Thus, employment is also another parameter that should be considered as a factor during the COVID-19 pandemic.^[@r5]^ In conclusion, this pandemic reminds everyone of the importance of strengthening the immune system and the adverse health effects of the global changes on public health. People with bad dietary habits, living close to industrial zones, having risky jobs, having a chronic disease, smoking, or experiencing chronic stress should take extra precautions to avoid a weakened immune system. Conflict of Interest Statement {#s1} ============================== The authors have no conflicts of interest to declare.

Introduction {#sec1} ============ Misfolding and aggregation of human islet amyloid polypeptide (hIAPP, also known as amylin) into amyloid fibrils, followed by subsequent deposition of these amyloid fibrils into pancreatic islets, are the neuropathological hallmark of type 2 diabetes (T2D).^[@ref1]^ hIAPP is a 37-residue peptide hormone synthesized by the pancreatic β-cells with physiological circulating concentrations ranging from 1.6 to 20 pM in nondiabetic people.^[@ref2]^ Amyloid formation by hIAPP is believed to be associated with β-cell death and dysfunction, the failure of islet transplantation, and the development of T2D. The hIAPP fibrillization process usually exhibits a typical three-stage sigmoidal kinetics,^[@ref3]−[@ref5]^ starting with a lag phase where hIAPP monomers slowly accumulate into small seeds (commonly termed as small nucleus), followed by a growth phase where the small seeds act as catalysts to interact with monomers and rapidly grow into larger aggregates via peptide addition, and finally reaching an equilibrium phase where most of the aggregates convert into mature fibrils.^[@ref6]−[@ref8]^ hIAPP aggregates not only increase their sizes and change their morphologies with time but also undergo the complex structural transition from random coil → α-helix → β-sheet with an increase in the β-sheet content.^[@ref9],[@ref10]^ Because the formation of nucleus seeds is a rate-determining step for hIAPP aggregation, the hIAPP fibrillization process is modeled using a seeding-dependent aggregation mechanism. This mechanism is also a general feature of amyloid formation by other amyloid peptides (e.g., Aβ, α-synuclein, and tau protein).^[@ref11]^ Moreover, small hIAPP seeds are often found to be highly toxic to cultured pancreatic islet β-cells and to islets.^[@ref12],[@ref13]^ Therefore, the study of seeding-induced amyloid aggregation and toxicity mechanisms is fundamentally and (pre)clinically important for therapeutic and prevention strategies against T2D. In general, seeding a protein/peptide solution with preformed homogeneous aggregates can dramatically change (usually enhance) the growth rate of amyloids. Jarrett et al.^[@ref12],[@ref13]^ conducted comparative kinetic studies of Aβ aggregation to demonstrate their nucleation-dependent polymerization mechanisms. They found that the addition of Aβ preformed fibrils into initially soluble Aβ solutions eliminated the nucleation time and thus led to a rapid aggregation. Kayed et al.^[@ref14]^ utilized a variety of biochemical methods to study the nucleation--polymerization process for hIAPP amyloid formation in the presence of the preformed hIAPP seeds. Come et al.^[@ref15]^ studied the aggregation of a fragment of the prion protein (PrP) containing residues 96--111 in the absence and presence of PrP~96--111~ seeds. The seeded groups significantly reduced the lag-phase time and promoted fibril formation. All of these in vitro studies showed that the preformed homologous seeds indeed accelerate amyloid formation through bypassing the lag phase.^[@ref16]^ In vivo studies further confirmed that amyloid proteins/peptides can spread the pathology between cells and tissues, and in some cases, they stimulate the disorder features when implemented into animal models through homogeneous seeding.^[@ref17]^ Luk et al.^[@ref18],[@ref19]^ found that an inoculation of synthetic α-synuclein fibrils into wild-type mice can elicit a massive formation and subsequent cell-to-cell transmission of pathological α-synuclein via the murine central nervous system. Consequently, such amyloid pathology accumulation generated a progressive loss of neurons and culminated in motor deficit in originally healthy mice. Kane et al.^[@ref20]^ performed the seeding experiment by injecting Alzheimer brain extracts intracerebrally into the Aβ precursor protein (APP) transgenic mice. A sharp contrast between the profuse presence of Aβ plaques in a tissue-injected mice group and the nonexistence of Aβ deposits in an uninjected mice group showed that Aβ can be seeded in vivo. Holmes et al.^[@ref21]^ developed a Fluorescence resonance energy transfer (FRET)-based biosensor to study the onset and progression of tau pathology using tau seeds in transgenic mice. They found that tau seeds can transmit from cell to cell via neural connections. All of these in vivo data indicated that seeding likely acted as an infectious agent to self-propagate different amyloid diseases including Alzheimer's disease (AD),^[@ref20]^ Parkinson disease,^[@ref18]^ and tauopathies.^[@ref21]^ In a broader view, the seeding process could be homologous or heterologous. Several studies^[@ref22]−[@ref24]^ reported the binding and coaggregation of different amyloid peptides, a process known as cross-seeding. Because most of the amyloid peptides share similar aggregation kinetics and structures, it is possible that amyloid seeding induces both homologous and heterologous amyloid formation. For example, α-synuclein fibrils could induce tau aggregation^[@ref25],[@ref26]^ in AD^[@ref22],[@ref23]^ and huntingtin aggregation in Huntington disease.^[@ref27]^ Similarly, intravenous injection of preformed fibrils of hIAPP or Aβ into hIAPP transgenic mice can act as seeds to stimulate hIAPP amyloids in the islet of Langerhans, and this finding supports that both seeding and cross-seeding can occur at local islets via blood.^[@ref26]^ To clarify the molecular requirements for peptide compatibility and the mechanisms behind them, a range of amyloid peptides were paired to study their cross-seeding behaviors in vitro. Kapurniotu and co-workers^[@ref28],[@ref29]^ studied the aggregation kinetics of amyloid fibrils formed by pure Aβ~40~, pure hIAPP, and mixtures of both peptides at a molar ratio of 1:1. They found that both nucleation and fibrillization of Aβ--hIAPP mixtures were delayed as compared with the aggregation kinetics of pure Aβ or pure hIAPP. They concluded that the cross-seeding of Aβ--hIAPP and the homoseeding of Aβ and hIAPP likely occur in a competitive manner. Mandal et al.^[@ref30]^ performed multidimensional NMR to study the interaction between Aβ and α-synuclein in a membrane-mimic environment. Aβ and α-synuclein appeared to strongly interact with each other and mutually promote their respective amyloid fibrillization. Giasson et al.^[@ref31]^ also observed that the coincubation of α-synuclein and tau led to a synergistic fibrillization promotion of both peptides. Growing evidence from clinical studies^[@ref32],[@ref33]^ also showed the coexistence of different amyloid protein aggregates in one disease and of different amyloid diseases in the same individual, suggesting a direct interaction between different amyloid peptides.^[@ref34]^ On the other hand, not any two different amyloid peptides can cross-seed each other. Aβ fibrils efficiently cross-seeded hIAPP in solution, whereas hIAPP fibrils did not cross-seed Aβ effectively, with only 2% of cross-seeding efficiency.^[@ref35]^ Similarly, transthyretin decreased Aβ deposition and suppressed cognitive deficits in AD mouse models.^[@ref36],[@ref37]^ It seems that the cross-seeding efficiency depends on the structural similarity between seeds and the other amyloid aggregates. It is likely that targeting peptides adopt a structure, at least partially identical to seeds, for amyloid growth. Although the results collected from literature point to the more complex mechanisms for amyloid cross-seeding, which remain to be answered, they also suggest that the cross-seeding is generally specific, and some cross-seeding barriers could exist because of the mismatch of sequences and structures between different amyloid peptides. Completely different from hIAPP, rIAPP does not form amyloid peptides and is nontoxic to β-cells, although rIAPP differs from hIAPP only at six residues (H18R, F23L, A25P, I26V, S28P, and S29P)^[@ref38]^ ([Scheme [1](#sch1){ref-type="scheme"}](#sch1){ref-type="scheme"}). Our previous molecular dynamics simulation^[@ref39],[@ref40]^ showed that hIAPP and rIAPP can interact with each other to form hybrid structures via peptide elongation and lateral association. An exploration into this contrast amyloidogenic property between two nearly same sequences could be a big step toward a better understanding of amyloidosis and finding potential amyloid-prevention methods. In our previous work,^[@ref41]^ we studied the cross-sequence interaction between full-length hIAPP~37~ and rIAPP~37~. Our previous data showed that when coincubating rIAPP~37~ with hIAPP~37~, both in freshly prepared monomer states, rIAPP initially inhibited hIAPP aggregation at both lag and growth phases, but once the aggregation-promoting hIAPP nuclei or oligomers were formed, they could recruit and cross-seed rIAPP to promote final fibril formation. However, little is known about how different hIAPP seeds interact with rIAPP and about the toxicity of hIAPP/rIAPP complexes. Different from our previous work, here, we studied the homoseeding of hIAPP and the cross-seeding of rIAPP with preformed hIAPP seeds at different aggregation stages using combined experimental methods. The results showed that hIAPP seeds formed at different stages can not only seed hIAPP but can also cross-seed rIAPP, but the homologous seeding of hIAPP itself was more effective than the heterologous seeding of hIAPP with rIAPP. The different seeding and cross-seeding efficiencies also reflect a fact that there exists a cross-seeding barrier probably due to the mismatch of cross-seeding structures. {#sch1} Results and Discussion {#sec2} ====================== Cross-Seeding of rIAPP and Homoseeding of hIAPP by Different hIAPP Seeds {#sec2-1} ------------------------------------------------------------------------ Thioflavin T (ThT) fluorescence aggregation assays were used to assess the seeding of hIAPP and the cross-seeding of rIAPP in the presence of different preformed hIAPP aggregates. The assays were performed by adding freshly prepared hIAPP (25 μM) or rIAPP (25 μM) to the ongoing incubation (preaggregated) hIAPP solution (25 μM) at different time points (0, 2, 6, 10, and 14 h). This design is equivalent to using different hIAPP seeds formed at three different aggregation stages to seed the same species of hIAPP or cross-seed different species of rIAPP. ThT fluorescence intensities (excitation at 450 nm and emission at 490 ± 10 nm) were recorded every 2 h to monitor aggregate formation. As a control, pure hIAPP aggregation exhibited a typical sigmoidal nucleation--polymerization curve, starting with a lag phase of 0--4 h, followed by a rapid growth phase from 4--20 h, and ending at a stable plateau with a maximum ThT intensity of ∼52 after 20 h. Pure rIAPP under the same incubation conditions did not aggregate in solution, as evidenced by the absence of any ThT signal.^[@ref41]^ First, we studied the cross-seeding behavior of rIAPP in the presence of different hIAPP seeds obtained from the nucleation, growth, and final equilibrium phases. [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}a (red line) shows that when coincubating both freshly prepared hIAPP (25 μM) and rIAPP (25 μM) together at 0 h, the lag time was prolonged to ∼6 h, whereas the growth rate was almost unchanged between 6 and 10 h and then increased after 10 h. Finally, the maximal ThT intensity reached a stable plateau of ∼61, which is ∼22.2% higher than that of pure hIAPP without adding rIAPP. Similarly, adding rIAPP to a seeded hIAPP solution at 2 h also increased the lag time to 5 h and promoted fibril formation with the maximum ThT intensity of ∼64 ([Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}b). This result indicates that the introduction of rIAPP to the hIAPP solution at the nucleation stage slows down the seed formation and early aggregation of hIAPP, but once hIAPP seeds are formed, they can cross-seed rIAPP to form more fibrils. Then, we examined the cross-seeding of rIAPP using the hIAPP seed solution at the growth phase. In [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}c, 15 min after the addition of rIAPP to the 6-h-seeded hIAPP solutions, the aggregation rate was accelerated, as indicated by immediate ThT signal enhancement followed by a deeper slope at the growth phase. A similar burst of cross-seeding was also observed when adding rIAPP to a 10-h-seeded hIAPP solution, but the growth rate seemed not to change much ([Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}d). Finally, when adding rIAPP to the preformed hIAPP fibrils at a final plateau stage of 14 h, the cross-seeding between rIAPP and hIAPP still occurred, but the acceleration of aggregation was much less than those found for preformed hIAPP aggregates at the nucleation and growth phases ([Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}e), indicating that hIAPP aggregated states before the formation of large hIAPP fibrils are the major causative agent of cross-seeding. Moreover, in all cross-seeding tests containing the same amount of hIAPP and rIAPP (25 μM), because rIAPP alone does not aggregate and form amyloid fibrils, during the cross-seeding process, any increase in the final ThT intensity does not result from hIAPP fibrils alone but instead results from new hybrid hIAPP/rIAPP fibrils. {#fig1} To further quantitatively compare the cross-seeding efficiency induced by different hIAPP seeds that were preformed at different aggregation stages, we summarized the increase in final ThT intensities for all cross-seeding tests relative to the final ThT intensity of pure hIAPP alone. As shown in [Table [1](#tbl1){ref-type="other"}](#tbl1){ref-type="other"}, different preformed hIAPP aggregates can all cross-seed rIAPP to promote final fibril formation but they also exhibited different cross-seeding efficiencies. hIAPP aggregates, which were preformed at a growth phase of ∼6 h, showed the strongest cross-seeding potential to rIAPP. Evidently, the coaggregation and the cofibrillization of hIAPP and rIAPP were accelerated, leading to a 36.2% increase in amyloid fibrils, as reflected by the final ThT intensity enhancement. The cross-seeding between freshly prepared rIAPP and hIAPP aggregates preformed at the nucleation phase still led to 22--29% increase in the total amount of amyloid fibrils. Early hIAPP aggregates may contain more disordered structures, so the cross-seeding activities were reduced because of a greater extent of structural mismatch between hIAPP seeds and rIAPP. This is also confirmed by the delay of lag phase due to cross-seeding, where the introduction of rIAPP clearly interferes with the nuclei formation of hIAPP. Using hIAPP (proto)fibrils as the seeds where β-sheet structures dominate, the cross-seeding activity was not as effective as the optimal one. It is possible that hIAPP protofibrils or fibrils offer fewer active surface sites, particularly hydrophobic aggregation sites, to interact with rIAPP because these active sites have already been preoccupied by hIAPP. ###### Summary of the Aggregation Kinetics for the Cross-Seeding of hIAPP and rIAPP[a](#t1fn1){ref-type="table-fn"}   lag phase time change caused by monomer addition[c](#t1fn3){ref-type="table-fn"} final fibril change caused by monomer addition[d](#t1fn4){ref-type="table-fn"} ------------------ ---------------------------------------------------------------------------------- -------------------------------------------------------------------------------- ------- -------- addition at 0 h +2 h --2 h 22.20 99.50 addition at 2 h +1 h --1.5 h 28.90 98.90 addition at 6 h N/A N/A 36.20 100.10 addition at 10 h N/A N/A 21.50 99.80 addition at 14 h N/A N/A 14.10 99.50 Data were extracted from the ThT curves in [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}. Freshly prepared rIAPP or hIAPP monomers (25 μM) were added to the pure hIAPP solution (25 μM) seeded at different time points of 0--14 h. Using the 0--4 h of pure hIAPP aggregation as a lag phase, the addition of rIAPP (hIAPP) monomer to the seeded hIAPP solution induces the increase (decrease) of the lag phase. Using the final ThT plateau of pure hIAPP aggregation as an indicator of fibrils being formed, the addition of rIAPP (hIAPP) monomer to all seeded hIAPP solution induces the increase in final fibril formation. In parallel, we also examined the seeding behavior of hIAPP for comparison using the same cross-seeding protocols and conditions. We added freshly prepared hIAPP monomers to hIAPP solutions seeded at different times and then monitored the aggregation kinetics changes before and after adding hIAPP monomers by recording ThT signals. In [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}a, the addition of 25 μM hIAPP to another 25 μM hIAPP solution at 0 h is equivalent to the incubation of freshly prepared 50 μM hIAPP monomers at 0 h, whose aggregation curve should be different from that of 25 μM hIAPP itself, as shown in [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}a. As compared to a control group of 25 μM hIAPP, the double concentration group of 25 hIAPP + 25 hIAPP (50 μM) exhibited faster nucleation at the lag phase and more fibrils being formed at the final phase. Moreover, homoseeding in the 25 hIAPP + 25 hIAPP group is more efficient than cross-seeding in the 25 hIAPP + 25 rIAPP group. In both cases of the homoseeding initiated at the nucleation phase, the hIAPP aggregation showed faster kinetics with a short lag phase of 2 h. This is different from cross-seeding that leads to a prolonged lag phase. In the other cases of mixing hIAPP monomers with preformed hIAPP seeds at the growth and equilibrium phases, there were immediate burst aggregations, followed by a faster aggregation to achieve higher ThT plateaus. These home-seeding data showed trends similar to the cross-seeding data, but the final ThT intensities of homoseeding mixtures were always higher than those of cross-seeding mixtures at the same peptide concentrations. It is also possible that both coaggregation and homoseeding can occur at the same time, leading to a faster aggregation process. Moreover, we also found that there were no statistical differences in the final ThT intensity for all homoseeding groups, which is another distinctive feature between hIAPP homoseeding and hIAPP/rIAPP cross-seeding. The homoseeding efficiency of hIAPP itself appears not to be necessarily relied on the preaggregated state of homoseeds, although its seeding-induced aggregation rate still does. By contrast, the cross-seeding process involves a critical step for rIAPP being converted into an amyloidlike structure, and this transformation is believed to largely depend on the hIAPP-seed condition. From the sequence perspective, the high amyloidogenic property of the hIAPP (20--29) region has been demonstrated. Peptide fragments from this hIAPP (20-29), for example, ~22~NFGAILSS~29~,^[@ref42]^~22~NFGAIL~27~,^[@ref43]^ and ~24~GAILSS~29~^[@ref44]^ can independently assemble into amyloid fibrils similar to full-length hIAPP fibrils. Differently, for the rIAPP peptide, the presence of three proline residues located in the 24--29 region (GPVLPP) is believed to disrupt the amyloidogenic property and reduce the β-sheet formation.^[@ref45],[@ref46]^ The presence of hIAPP seeds may alter the folding pathway of rIAPP and drive rIAPP to be incorporated into hIAPP seeds. It is also possible that the N-terminal β-sheet of hIAPP aggregates could serve as a template interface either to recruit and accommodate rIAPP with a conformationally similar N-terminal β-sheet (not the C-terminal 20--29 region) or to facilitate the structural transition of rIAPP to partially fold into compatible β-sheet structures. A comparison of homoseeding and cross-seeding ThT data reveals some similarities and differences. First, hIAPP at different aggregation phases can always seed hIAPP monomers and cross-seed rIAPP monomers, but the homoseeding/cross-seeding efficacies seem to be more dependent on the aggregation-prone intermediate species. The population of hIAPP intermediate species, not those species at the initial nucleation and final equilibrate phases, is more critical to achieve high coaggregation and homo-/cross-seeding. These hIAPP intermediate species are largely partially folded with some solvent-exposed hydrophobic moieties that promote intermolecular interactions with hIAPP or rIAPP. Second, homoseeding is more efficient in initiating and promoting aggregation than cross-seeding. This is not surprising because mismatch sequences between hIAPP and rIAPP increases energy barriers for efficient cross-interactions. Cross-Seeding Induces Structural Changes in Amyloid Aggregates {#sec2-2} -------------------------------------------------------------- To gain further insights into cross-seeding, we monitored the structural changes of cross-seeded hIAPP/rIAPP aggregates using atomic force microscopy (AFM) and circular dichroism (CD) under the exact same conditions used in ThT tests. [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"} shows the representative AFM images of the cross-seeding of fresh rIAPP monomers with hIAPP solutions seeded for different times. As a control, AFM images of pure hIAPP aggregation (25 μM) showed typical amyloid morphologies at different aggregation phases, confirming the conversion of small oligomers into higher-order amyloid fibrils. The widths of most hIAPP fibrils were similar and ranged between 8 and 12 nm. CD spectroscopy images in [Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"} also showed that pure hIAPP experienced a typical structural transition from the initial random coil to the β-sheet structure, as indicated by the appearance of the two peaks at 195 and 215 nm, both of which corresponds to the β-sheet structure. For comparison, when coincubating equimolar hIAPP and rIAPP monomers at 0 h, it is clear that the lag phase was prolonged to 4 h, during which a large amount of spherical aggregates of 1--2 nm diameters were predominated. Therefore, the prolonged lag phase indicates the occurrence of coaggregation between hIAPP and rIAPP. After that, short, thin protofibrils and long, thicker mature fibrils were observed at 8 and 16 h, respectively. Their morphologies were almost identical to those of pure hIAPP (proto)fibrils. Moreover, upon cross-seeding rIAPP by preformed hIAPP seeds at different time points of 2--14 h, AFM images in all cases consistently showed the morphological changes from small aggregates, low density protofibrils, to highly dense fibrils, and the final morphologies of cross-seeded fibrils were found to be similar to those of homoseeded fibrils. Height profiles obtained from the AFM images showed that all amyloid fibrils exhibited similar heights of 5--15 nm. The AFM results were also supported by our recent simulation work^[@ref39]^ that hIAPP/rIAPP assemblies reflected a polymorphic nature of cross-seeding species, that is, hIAPP can cross interact with rIAPP to form hybrid amyloid aggregates and fibrils via two pathways of peptide elongation and lateral association. {#fig2} {#fig3} Additionally, we conducted CD experiments for secondary structure characterization of the fibrils generated through cross-seeding. Considering that rIAPP has the same concentration of hIAPP and rIAPP itself mainly adopts random coil conformations, in the cross-seeding cases studied, we found that the adsorption spectra of cross-seeding samples were largely dominated by a strong negative peak at ∼197 nm, corresponding to major random coil conformations. This makes the other combined secondary structures difficult to characterize, particularly a characteristic peak of β-sheet at ∼195 nm. To address this issue, all raw CD curves of cross-seeding samples recorded at the end of the reaction (24 h) were corrected by subtracting a control curve of pure rIAPP. In this way, the corrected curves should present a linear superposition of hIAPP conformations and transformed rIAPP conformations. In [Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}, the CD spectrum of the final hIAPP fibrils showed a positive peak at ∼192 nm and a negative valley at ∼210 nm, confirming the β-sheet-rich structure of pure hIAPP. Upon cross-seeding with rIAPP, all corrected CD curves not only shifted their positive peaks to ∼195 nm with enhanced magnitude but also deepened the negative peaks. The continuous shift and the increase in the peaks indicate that cross-seeding continues to develop β-sheet-rich fibrils. On the basis of the entire CD spectrum, we performed CDpro analysis to obtain the final secondary structure content of pure hIAPP and cross-seeded hIAPP/rIAPP samples using the CDSSTR (Circular dichroism standardized stepwise treatment regimen) method ([Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}b). As compared with the pure hIAPP group containing ∼46% of α-helix and ∼30% of β-sheet after 20 h incubation, all cross-seedings of hIAPP/rIAPP groups incubated at different time points resulted in higher β-sheet of 37--51% and lower α-helix of ∼31--37%. This suggests that the cross-seeding of hIAPP/rIAPP induces an increase in β-sheet content at the expense of structural conversion of α-helical or random structures. Moreover, considering the two facts that (1) pure rIAPP peptides do not produce any ThT signal and (2) the same amount of hIAPP (25 μM) was used in all tested cross-seeding cases, any increase in the final ThT intensity actually does not result from hIAPP fibrils but instead results from new hybrid hIAPP/rIAPP fibrils ([Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}). Second, because rIAPP always retains its random coil conformation, the difference in CD spectrum between the hIAPP/rIAPP mixtures and pure hIAPP is likely induced by the incorporated rIAPP ([Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}). Taken together, collective data from ThT, AFM, and CD analyses confirm the occurrence of cross-seeding between hIAPP and rIAPP. Cross-Seeding Increases Cell Toxicity {#sec2-3} ------------------------------------- To examine whether the cross-seeding aggregates are innocuous, we conducted a cell viability experiment using the MTT assay with the RIN-m5f cell line ([Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}). To establish a baseline, the absorbance of the RIN-m5f cell media alone was measured, and the value was set as 100% of cells being viable. When incubating pure hIAPP (25 μM) with cell culture media for 48 h, the cell viability decreased to 65% of that of the control, confirming that hIAPP aggregates are toxic to cells. By contrast, pure rIAPP (25 μM) presented very low cytotoxicity to cells, as evidenced by the 97.5% cell viability during 48 h of cell culture. In all cross-seeding tests, the cell viability was reduced as compared with that of the control, but the extent of cell viability showed an increasing trend as a function of time point for adding rIAPP to the seeded hIAPP solution. Specifically, when introducing rIAPP to the hIAPP solution at early aggregation time points of 0, 2, and 6 h, the cross-seeding aggregates ultimately led to a high toxicity ranging from 58 to 52%. As confirmed by ThT and AFM, the cross-seeding of rIAPP with hIAPP at the lag phase (0--2 h) extends the nucleation stage and thus produces the more predominant oligomers that are highly toxic and also prolongs their lifetime. In comparison, when using the hIAPP fibrillar aggregates preformed at the later aggregation stages (14 h) to cross-seed rIAPP, the cell viability was ∼60%, which was slightly lower than the 65% cell viability induced by pure hIAPP. It is generally accepted that small, soluble amyloid oligomers are the most toxic species as compared with insoluble final amyloid fibrils.^[@ref47]^ When adding rIAPP monomers to the preformed hIAPP protofibrils, hIAPP/rIAPP oligomers are unlikely to form because of the absence of hIAPP oligomers. Instead, hIAPP protofibrils will recruit rIAPP monomers to form large hIAPP/rIAPP protofibrils. Such a cross-seeding effect would reduce the formation of the potential toxic hIAPP/rIAPP oligomers but promote the formation of less toxic hybrid hIAPP/rIAPP fibrils, both of which lead to the increase in cell viability. {#fig4} Conclusions {#sec3} =========== hIAPP and rIAPP have completely opposite aggregation propensities, with only six residue differences in their sequences. hIAPP is known as the causal agent to induce T2D via its strong aggregation and toxic properties, whereas rIAPP is not. The cross-seeding of these two different peptides is fundamentally important for understanding the mechanism of hIAPP aggregation linked to T2D but remains elusive. To address this unexplored fundamental issue, here, we have studied the effect of cross-seeding between rIAPP monomers and different hIAPP seeds on aggregation kinetics, structure, and toxicity in vitro. In all cases of cross-seeding, the hIAPP solution containing different preformed seeds can indeed cross-seed rIAPP to promote final amyloid fibril formation. But, the cross-seeding activity was strongly depended on hIAPP seeds. Specifically, hIAPP seeds formed at the growth phase exhibited the best cross-seeding capacity for rIAPP where the coaggregation and cofibrillization of hIAPP and rIAPP were accelerated, whereas hIAPP fibrillar seeds exhibited poor cross-seeding capacity. For the toxicity tests, all cross-seedings of rIAPP with different hIAPP seeds induced a higher cell toxicity than pure hIAPP. Particularly, in the case of using hIAPP seeds formed at the nucleation stage to cross-seed rIAPP, the lag phase was retarded, which elevated the production of more toxic intermediates and thus caused the highest cell toxicity. Moreover, the comparison of homo- and cross-seeding aggregation kinetics showed that the homoseeding of hIAPP is more efficient to promote amyloid aggregation than the cross-seeding of hIAPP and rIAPP. This study demonstrates the cross-seeding between strong-aggregation hIAPP and nonaggregation rIAPP, which may provide some clues to better understand the mechanisms of amyloidogenesis. Methods and Materials {#sec4} ===================== Reagents {#sec4-1} -------- 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP, ≥99.9%), dimethyl sulfoxide (DMSO, ≥99.9%), 10 mM phosphate-buffered saline (PBS) (pH = 7.4), 10 mM NaOH, and ThT (98%) were purchased from Sigma-Aldrich (St. Louis, MO). hIAPP (1-37) (≥95.0%) and rIAPP (1-37) (≥95.0%) were purchased from American Peptide Inc. (Sunnyvale, CA). All other chemicals were of the highest grade available. Peptide Purification and Preparation {#sec4-2} ------------------------------------ Both IAPP peptides were obtained in a lyophilized form and stored at −20 °C as arrived. To prepare the monomeric peptide solution, 1.0 mg of each preaggregated peptide was dissolved in HFIP for 2 h, sonicated for 30 min to remove any preexisting aggregates or seeds, and centrifuged at 14 000 rpm for 30 min at 4 °C. The top peptide (80%) solution was then extracted, subpackaged, frozen with liquid nitrogen, and then dried using a freeze-dryer. The dry peptide powder was lyophilized at −80 °C and used within 1 week. Purified hIAPP powder (0.2 mg) was aliquoted in 30 μL of 10 mM NaOH solution and sonicated for 1 min to obtain a homogenous solution. The initiation of hIAPP (25 μM) aggregation in solution was accomplished by adding 30 μL of the obtained NaOH--hIAPP solution to 2 mL of 10 mM PBS buffer. Then, for the hIAPP/rIAPP solution mixed in different stages, we used the same protocol to prepare initial pure hIAPP solutions at the beginning and added 30 μL of fresh NaOH--rIAPP solutions to each pure hIAPP solution at 0, 2, 6, 10, and 14 h. All solutions were incubated at 37 °C. ThT Fluorescence Assay {#sec4-3} ---------------------- ThT fluorescence assay is considered to be a standard method to detect the formation of amyloid fibrils because ThT can specifically bind to the β-sheet structure of protein fibrils and gives a strong fluorescence emission. A ThT solution (2 mM) was prepared by adding 0.033 g of ThT powder into 50 mL of deionized (DI) water. The resultant 250 μL of the 2 mM ThT solution was further diluted in 50 mL of Tris buffer (pH = 7.4) to a final concentration of 10 μM. The peptide solution (60 μL) was added into the 10 μM ThT--Tris solution (3 mL) at each time point. Fluorescence spectra were recorded using an LS-55 fluorescence spectrometer (Perkin-Elmer Corp., Waltham, MA). All measurements were carried out in aqueous solution using a 1 × 1 cm^2^ quartz cuvette. The ThT fluorescence emission wavelengths were recorded between 470 and 500 nm with an excitation wavelength of 450 nm. Each experiment was repeated at least three times, and each sample was tested in quintuplicates. CD Spectroscopy {#sec4-4} --------------- The secondary structures of hIAPP and rIAPP in solution were examined by CD spectroscopy using a J-1500 spectropolarimeter (Jasco Inc., Japan) in the continuous scanning mode at room temperature. Peptide solutions incubated for 20 h (160 μL for each time point) were placed into a rectangular quartz cuvette of a 1 mm pathlength without dilution. The spectra were recorded between 250 and 190 nm at a 0.5 nm resolution and a 50 nm/min scan rate. All spectra were corrected by subtracting the baseline and averaged by three successive scans for each sample. Tapping-Mode AFM {#sec4-5} ---------------- The morphology changes of peptides during fibrillization were monitored by tapping-mode AFM. The sample (20 μL) used in both ThT fluorescence assay and CD spectrum test was taken for the AFM measurement at different time points to correlate the hIAPP or rIAPP morphology changes with their growth kinetics. A peptide solution was deposited onto a freshly cleaved mica substrate for 1 min, rinsed three times with 50 mL of DI water to remove the salts and loosely bound peptide, and dried with compressed air for 5 min before AFM imaging. Tapping-mode AFM imaging was performed in air using a Nanoscope III multimode scanning probe microscope (Veeco Corp., Santa Barbara, CA) equipped with a 15 μm E scanner. Commercial Si cantilevers (Nanoscience) with an elastic modulus of 40 N m^--1^ were used. All images were acquired as 512 × 512 pixel images at a typical scan rate of 1.0--2.0 Hz with a vertical-tip oscillation frequency of ∼160 kHz. Representative AFM images were obtained by scanning at least six different locations of different samples. Cell Culture {#sec4-6} ------------ Rat insulinoma (RIN-m5F) cells (ATCC, Manassas, VA) were used as model pancreatic β-cells and cultured in 75 cm^2^ T-flasks in sterile-filtered RPMI-1640 medium (ATCC, Manassas, VA) containing 10% fetal bovine serum (ATCC, Manassas, VA) and 1% penicillin/streptomycin (ATCC, Manassas, VA). The flasks were incubated in a humidified incubator with 5% CO~2~ at 37 °C. Cells were then cultured to confluence and harvested using a 0.25% Trypsin--EDTA (1×) solution (Lonza, Walkersville, MD). The cells were counted using a hemocytometer and plated in a 96-well tissue culture plate at 50 000 cells per well in 100 μL of medium, which allow them to attach inside of the incubator for 24 h. MTT Toxicity Assay {#sec4-7} ------------------ MTT-based cell toxicity assays were performed to assess the cytotoxicity of hIAPP and rIAPP assemblies. A 96-well plate with cells was split into seven groups, with each group containing 12 replicates. The first group containing cells only in the medium was used as a positive control. NaOH--hIAPP solutions diluted by the cell medium were added to groups 2--7 to achieve the 25 μM final concentrations. The cells were then incubated for another 48 h. During the first 24 h, we used the same protocol to prepare NaOH--rIAPP solutions and added them to each pure hIAPP incubation solution (groups 3--7) at 0, 2, 6, 10, and 14 h. MTT (5 mg) was dissolved in sterile PBS solution (1 mL). Then, we mixed this MTT--PBS solution with 10 mL of the cell medium. The original cell medium was removed, and 100 μL of this MTT--PBS-medium solution was added to each well. The cells were incubated for 4 h at 37 °C to convert MTT to formazan crystals. After that, the entire 100 μL of the MTT--PBS-medium solution was removed from each well. Formazan crystals formed at the bottom of each well were dissolved by adding 100 μL of DMSO per well and were thoroughly mixed. The cells were incubated for an additional 10 min at 37 °C and mixed again to ensure that the formazan was fully dissolved. The plates were placed in a Synergy H1 microplate reader (BioTek, Winooski, VT), and the absorbance was read at 540 nm to determine the formazan content. The sample absorbance was then compared with the control groups to determine cell viability. All statistical data were expressed as mean ± standard deviation. Statistical analysis of all data was performed using one-way analysis of variance and Tukey's test. *p* values \<0.05 were considered to be statistically significant. ^⊥^ R.H. and B.R. contributed equally to this work. R.H., B.R., M.Z., H.C., Y.L., B.J., and J.M. carried out the experiments. All authors designed the experiments, interpreted the results, and prepared the manuscript. All authors have given approval to the final version of the manuscript. The authors declare no competing financial interest. This work is financial supported by the NSF (CBET-1510099), Alzheimer Association New Investigator Research Grant (2015-NIRG-341372), and National Natural Science Foundation of China (NSFC-21528601) and partially supported by the NSF (DMR-1607475).

Canine SRSF2 DNA sequence has been submitted to a public repository (NCBI GenBank database accession number KT072629). Introduction {#sec005} ============ In dogs, cutaneous mast cell tumor (MCT) is the most common skin tumor, and it accounts for up to 10--30% of all cases. MCTs occur mostly in the dermis and subcutaneous tissue but some visceral forms can also be located in other sites e.g. gastrointestinal tract and spine bone marrow as well as liver, oral cavity, urethra, salivary gland, nasopharynx and spleen \[[@pone.0142450.ref001]--[@pone.0142450.ref003]\]. It is commonly identified as a solitary neoplastic mass in the skin and/or subcutaneous tissue of older dogs, with a mean age of onset of approximately 9 years of age. Some dog breeds, such as Boxers, Labrador Retrievers and Shar Pei, are more prone to develop MCTs \[[@pone.0142450.ref004],[@pone.0142450.ref005]\]. Activating mutations of the tyrosine kinase receptor c-KIT, which binds to stem cell factor (SCF), a known hematopoietic cytokine, have been described in canine MCTs. Mutations in c-KIT occur in 15--50% of MCTs, and have been associated with a more aggressive tumoral phenotype \[[@pone.0142450.ref006]\], possibly due to an increased proliferation and a resistance to apoptosis \[[@pone.0142450.ref007],[@pone.0142450.ref008]\]. The most common type of mutations identified in canine MCTs are internal tandem duplications (ITD) involving exon 11 \[[@pone.0142450.ref006],[@pone.0142450.ref009]\] but also deletions and point mutations in exons 8, 9 and 11 can occur \[[@pone.0142450.ref002],[@pone.0142450.ref010]\]. Human mastocytosis is a rare and clonal hematopoietic disease described as the proliferation and the accumulation of abnormal mast cells in the bone marrow and organs \[[@pone.0142450.ref011]\]. Mastocytosis is schematically divided into cutaneous mastocytosis (CM) and systemic mastocytosis (SM). Localized mast cell tumors as mastocytomas and mast cell sarcoma are very rare. CM is usually diagnosed at birth or in childhood and spontaneously regress over time. However, some types are locally invasive, clinically very severe and, consequently, hard to treat. In most adult patients, the disease is systemic, although also the skin is often affected. Most cases of SM are associated with the presence of activating mutations in the c-KIT proto-oncogene. The most frequent KIT genetic alteration is the substitution of aspartic acid to valine at position 816 (KIT D816V), that leads to the constitutive activation of the kinase domain of the receptor \[[@pone.0142450.ref012]\]. It has been recently discovered as further cooperating events may contribute to the phenotype and/or the pathogenesis of SM \[[@pone.0142450.ref013],[@pone.0142450.ref014]\] e.g. mutations in tet methylcytosine dioxygenase 2 (TET2) which have been reported in 40% of KIT D816V-positive SM cases \[[@pone.0142450.ref015]\]. The enzyme TET2 regulates gene methylation and expression, catalyzing the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) \[[@pone.0142450.ref016]\]. In SM, it has recently been reported a more aggressive disease and an overall worse prognosis when there is the coexistence of KIT D816V and TET2 mutations \[[@pone.0142450.ref017]\]. Other mutations were identified in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2, respectively). They affect both histone modifications and DNA methylation, catalyzing the decarboxylation of isocitrate to alpha-ketoglutarate (or 2-oxoglutarate, 2-OG). Hotspot mutation sites are represented by heterozygous substitution clusters in conserved arginines R132 of IDH1 and R140 and R172 of IDH2 \[[@pone.0142450.ref018]\]. Further additional mutations were found in genes encoding for components of the splicing machinery involved in the intron splicing during pre-mRNA maturation, in particular the serine/arginine-rich splicing factor 2 and the splicing factor 3b, subunit 1 (respectively SRSF2 and SF3B1). Overall, recent data are suggestive of a specific hierarchy, where TET2 gene alterations arise in early progenitor cells, while SRSF2 mutation can occur relatively later during the ontogeny but both prior to KIT mutation during the disease progression \[[@pone.0142450.ref011]\]. Likewise, neuroblastoma RAS Viral (V-Ras) oncogene homolog (NRAS) mutations have also been reported in SM, having the potential to precede KITD816V in clonal development \[[@pone.0142450.ref019]\]. Besides SM, loss-of-function mutations in TET2 as well as alterations in other genes mentioned above have been also reported in a variety of hematological malignancies, including acute myeloid leukemias (AMLs), chronic myelomonocytic leukemia (CMML), myeloproliferative neoplasms (MPNs), myelodysplastic syndromes (MDS) and lymphoid malignancies \[[@pone.0142450.ref020]--[@pone.0142450.ref024]\]. To the best of our knowledge, no data on mutational status of these genes are available for canine MCTs. In the present study, hypothesizing analogies in molecular mechanisms and gene dysfunctions with human SM and hematopoietic diseases, the mutation profile of genes commonly mutated in myeloid malignancies has been evaluated in a cohort of 75 MCTs, most of them previously screened for c-KIT mutations \[[@pone.0142450.ref008]\]. Materials and methods {#sec006} ===================== Samples and ethical statement {#sec007} ----------------------------- All tissue biopsies and blood samples were not specifically taken for the purposes of this study; they were part of authors *in-house* collections and were already used in previous studies \[[@pone.0142450.ref008], [@pone.0142450.ref025], [@pone.0142450.ref026]\]. Tissue biopsies were originally collected as part of routine treatment procedures from dogs affected by at least one histologically-confirmed MCT (Patnaik grade II or III) \[[@pone.0142450.ref027]\], recurrent after surgery (as standard of care) and/or nonresectable. Female and male dogs, regardless of breed, were previously recruited with owner consent from veterinary clinics in France and in United States. Blood samples were collected in Italy from 39 healthy random-source adult kennel dogs undergoing routine examination as described in details previously \[[@pone.0142450.ref026]\]. An Institutional Animal Care and Use Committee approval number was not requested because of an agreement between the Faculty of Veterinary Medicine of University of Padua (Italy) and the kennel for the execution of routinary clinical checkups as described in details previously \[[@pone.0142450.ref026]\]. Animal care was carried out in accordance with good veterinary practices. DNA extraction, PCR and sequence analysis {#sec008} ----------------------------------------- Genomic DNA was extracted from 75 frozen canine MCT tissue samples using QIAamp DNA Mini Kit (Qiagen France, Paris, France), according to manufacturer's protocol. In the 23% of the cohort samples, different c-KIT mutations were previously identified in exons 8, 9 and 11\[[@pone.0142450.ref008]\]. Among them, internal tandem duplications of exon 11 represented 36% of total mutations registered. In the present study, PCR amplifications of all TET2 coding exons and the hot-spot regions of IDH1 (exon 2), IDH2 (exon 1), SF3B1 (exons 13, 14, 15, 16), SRSF2 (exon 1), NRAS (exons 1, 2) and KRAS (exon 1) were executed. Primer oligonucleotide sequences were designed using the AmplifX software (<http://crn2m.univ-mrs.fr/AmplifX>) and CanFam3.1 genome sequences available <http://www.ncbi.nlm.nih.gov/>. Primer sequences, are reported in [Table 1](#pone.0142450.t001){ref-type="table"}. For every exon analyzed, the extreme parts of the flanking introns were also sequenced (around 100 bp upstream the 5´-end and downstream its 3´-end) to check for the presence of alternative splicing sites. All the detected variations were analyzed by using the tool Berkeley Drosophila Genome Project (BDGP, <http://www.fruitfly.org>) that computed splice sites predictions. Genes were amplified using Taq Phire^®^ Hot Start II DNA Polymerase (Thermo Fisher Scientific, Walthman, MA, USA). The reaction mix contained the following reagents: 1X Phire^®^ Reaction Buffer, 200 μM dNTPs, 0.5 μM of each primer and 0.15 μL of the enzyme (in a final volume of 22 μL). Approximately, 30 ng of genomic DNA were added to each PCR reaction and amplified through the following thermal protocol: an initial denaturation step at 98°C for 30 sec, an amplification step of 35--40 cycles (denaturation at 98°C for 5 sec, annealing at the primer-specific temperature for 5 sec and elongation at 72°C for 5--10 sec depending on the length of the PCR product) and a final elongation step at 72°C for 1 min. PCR products were purified and sequenced in an ABI 3730 sequencer. Sequence PCR reactions were performed with both primer forward (F) and reverse (R) using the Big Dye Terminator V1.1. (Applied Biosystem, Life Technologies, Carlsbad, USA) and the mix included: 3.2 pmol of oligo F or R, 1μL of Big Dye Terminator V1.1, 1X reaction buffer and water (in a final volume of 10 μL). The thermal protocol consisted in: an initial denaturation (1 min at 96°C) followed by 25 cycles of 10 sec at 96°C, 5 sec at 50°C and 2 min at 60°C. 10.1371/journal.pone.0142450.t001 ###### Forward (F) and reverse (R) primer sequences of canine genes included in the present study and used for polymerase chain reaction with the corresponding annealing temperature and product length. {#pone.0142450.t001g} GENE AND PRIMER SEQUENCES (5´-3´) EXON TEMP. ANNEALING PRODUCT LENGHT ----------------------------------- -------- ----------------- ---------------- **KRAS** F: `CTCATCTGTGGTCAACTGAA` 1 60°C 466 bp R: `AGCCAATGGAACCCAAGTA` **IDH1** F: `TGGCACTGTCTTCAGGGAAGCTAT` 2 70°C 163 bp R: `TGGGCAACCAAGGACAGGAAAA` **IDH2** F: `CTCCATCTCTGTCCTCGTAGAGT` 4 67°C 343 bp R: `TTAGCACCGCTGCCATCCTTT` **NRAS** F: `TCTCTAGTTGTGGCTCGCCCATTA` 1 65°C 223 bp R:`CAAAAGCCAGAGGTAGGGTCAGT` F:`GCTAGGAGCTTATCTAACCTTGGC` 2 60°C 367 bp R: `TGCGGTATCCTCATTTCCTGTTCC` **SF3B1** F: `ACTGGAGGATCAAGAGCGTCAT` 13 67°C 1101 bp R: `GCTGTCGTGTTACGGACATACT` F: `ATGCTAGAGTGGAAGGTCGAGA` 14 67°C 855 bp R: `TGTGTTGGCGGATACCCTT` F: `GACCATTAGCGCTTTGGCCATT` 15--16 67°C 529 bp R: `GTTCCACAACACTGCTTCACCA` **TET2** F: `AGCCTGATGGAACAGGATAGA` 3 60°C 782 bp R: `GCCTGACTGTTAATGGCA` F:`CAAGAAAGTAATCCAGGCAAAGGC` 3 60°C 718 bp R: `AATACCGTTCAGAGCTGCCA` F: `CCTGTCCCTTCCAGAAACCAGAAA` 3 60°C 605 bp R: `TGTTGGGTTATGCTTGAGGTGTTC` F: `CCCCAACCAAAGTAACACAGCTCT` 3 60°C 702 bp R: `GCTTTGGATGAAGGGTCTGTCTTG` F: `GGCATCACTGCGGTCAGTTCTT` 3 60°C 715 bp R: `ATTCTGTCCTTGCTCCAATCCCA` F: `TCCCAAGGCAACAATGATCAGC` 3 60°C 760 bp R: `GGGGTGGAATCTCTTGCTTAGTTG` F: `CTCCCCAGAAGGACATTCAAAAG` 3 60°C 784 bp R: `CTCTCTTGCACAGCACAAGCAT` F: `GGATAAGCTTTGTGGATGTAGCCT` 4 60°C 371 bp R: `GCTCGCAGACTATTAGTCCTGT` F: `TCCAGTTTGCTTGGCTTAGAC` 5 60°C 380 bp R: `GAGCAACGTTCATTTCAACTAGC` F: `AATGCCCTAGTTGTGACCCAG` 6 60°C 421 bp R: `AAATGTCGGTTCAACTCCCTTCCC` F: `CCAGAATCCAAGATTGGTAGCC` 7 60°C 295 bp R: `GACTGCTTACTTCATCTGTACTCA` F: `TCATTTGGATCTAGGCTGTAGGGG` 8 65°C 336 bp R: `AACAGAACACTGTGGCTTCACT` F: `CGAGAGTCTTTCTGACCTGTTC` 9 60°C 398 bp R: `AAGGTCACCTTTGCAACAGC` F: `AGGCATGTCACTAATCTGGTCCAA` 10 60°C 638 bp R:`GGGACTTCAGGGAAGATTCTGGTA` F: `GGGGTTCTCACATACATTTAAGCA` 11 65°C 920 bp R: `GAGCTGTTGAACATGCCTGG` F: `ACTTCATGGGAGCCACCTCTAGAT` 11 60°C 853 bp R: `AGACAGGTTGGTTGGTTGGTTGTG` Blood samples of 39 healthy dogs were collected and DNA extraction was performed as previously reported \[[@pone.0142450.ref026]\]. Around 30 ng of genomic DNA were used in PCR reaction to amplify TET2 exon 11 and the products obtained were subsequently sequenced as described above. Sequences were analyzed and aligned by using the SeqScape software v3.0 (Life Technologies, Carlsbad, USA) and identity percentage between dog protein sequences and mouse, rat, dog and cat were assessed through BLAST (<https://blast.ncbi.nlm.nih.gov/Blast>). Statistical analysis {#sec009} -------------------- To evaluate the possible relationship between the presence of glutamine repetitions in canine TET2 exon 11 and c-KIT mutations or the tendency to develop MCT, a Pearson χ^2^ correlation test was performed by GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, USA). A value of P\< 0.05 was considered significant. Results {#sec010} ======= Gene sequences are conserved among canine and human species {#sec011} ----------------------------------------------------------- In myeloproliferative disorders and particularly in SM, the majority of the genes considered in this study possess hot spot sites for mutations; therefore, in the first part of the study, a comparison between human and canine genomic sequences was performed to verify the potential conservation of the same mutations sites in dog and, subsequently, their localization. To give a general overview, the percentages of protein sequence identities in target genes between the canine and the other most commonly studied species (human, cat, mouse and rat) are reported in [Table 2](#pone.0142450.t002){ref-type="table"}. In general, a high degree of conservation was noticed among them and, for our purposes, the CanFam3.1 genome sequence proved to be definitely complete and reliable. 10.1371/journal.pone.0142450.t002 ###### List of target genes and percentage of protein sequence identity between dog and other reference species (*Homo sapiens*, *Felis catus*, *Mus musculus*, *Rattus norvegicus*). {#pone.0142450.t002g} **Gene** **Human** **Cat** **Mouse** **Rat** ---------- ----------- --------- ----------- --------- TET2 84% 91% 58% 60% IDH1 97% 99% 95% 96% IDH2 96% 99% 97% 96% NRAS 100% 100% 100% 100% KRAS 99% 97% 96% 96% SF3B1 100% 100% 100% 100% SRSF2 100% NA 100% 100% NA: sequence not available in the databases. The amino acids residues considered hot spot sites for mutations in humans as R132 for IDH1, R140 and R170 for IDH2, G12 and Q61 for NRAS and G12 for KRAS were recognized in dog. Since in humans TET2 mutations occur almost all over the sequence, all the corresponding canine coding exons were amplified; the two sequences shared the 84% of amino acid identity ([Table 2](#pone.0142450.t002){ref-type="table"}). On the other hand, the canine SRSF2 partial sequence obtained in this study differed, either in exons and introns, from NCBI release. Anyway, the analog of human hot spot site (P95) was conserved also in dog. The updated partial sequence was submitted to NCBI with the following accession number: KT072629. Target gene mutational status in MCTs samples {#sec012} --------------------------------------------- All genes were successfully amplified in all the 75 samples except for SRSF2, that was amplifiable in only 37 samples cause of its complexity and GC-richness. In our MCT cohort, surprisingly, no mutations were ever found analyzing sequencing results of IDH1, IDH2, NRAS, KRAS, SF3B1 and SRSF2 genes (data not shown). Among samples, some intronic variants not related with splicing sites were detected in the target genes. These alterations, with the relative allelic frequencies and population distribution, are collected in [Table 3](#pone.0142450.t003){ref-type="table"}. 10.1371/journal.pone.0142450.t003 ###### List of genetic variations grouped for gene, relative population frequency and allelic frequencies in the MCT cohort of samples. {#pone.0142450.t003g} Gene Intron/Exon Variation Population Frequency Allelic frequencies ----------- ------------- --------------------------- ---------------------- ------------------------ **TET2** exon 3 c.732G\>A p. = 3/75 (4%) G: 0.98, A: 0.02 **TET2** exon 3 c\. 2315G\>A: p.Gly772Asp 4/75 (5.3%) G: 0.97, A: 0.03 **TET2** intron 3 c.3439+75del 6/75 (8%) T: 0.96, delT: 0.04 **TET2** intron 8 c.4075-38del 5/75 (6.67%) T: 0.97, delT: 0.03 **TET2** intron 10 c.4212+63_4212+65insCAG 62/75 (82.7%) WT: 0.31, insCAG: 0.69 **TET2** intron 10 c.4568-65C\>T 6/75 (8%) C: 0.95, T: 0.05 **TET2** exon 11 c.4914T\>C:p = 58/75 (77.3%) T: 0.41, C: 0.59 **TET2** exon 11 c.5213A\>G: p.Asn1728Ser 57/75 (76%) G: 0.41, A: 0.59 **TET2** exon 11 c.5278G\>A: p.Ala1760Thr 1/75 (1.33%) G: 0.01, A: 0.99 **IDH1** intron 2 c\. 292+37T\>C 3/75 (4%) T: 0.98, C: 0.02 **NRAS** intron 2 c\. 290+44C\>T 18/75 (24%) C: 0.87, T: 0.13 **KRAS** intron 1 c\. 93+104A\>T 8/75 (10.67%) A: 0.95, T: 0.05 **KRAS** intron 1 c\. 93+139T\>C 8/75 (10.67%) T: 0.95, C: 0.05 **SRSF2** intron 1 362+59_362+62dup 21/37 (58%) WT: 0.64, Dup: 0.36 TET2 mutational status in dog MCT {#sec013} --------------------------------- In canine TET2 only two samples evidenced the presence of mutation in their coding sequence: one sample showed a heterozygous non-synonymous substitution (c.491A\>G: p. Asp164Gly) in exon 3 while another sample was homozygous for a complete codon deletion (c.2226-2228del: p. His742del) always in exon 3. Both MCTs were histologically classified as Patnaik grade II. As regards to *c-KIT* mutations, the former had a wild-type sequence, while the second one had an internal tandem duplication occurring in exon 11 (ITD^572-583^). Since the frequency of TET2 mutations was low (2.7%), a correlation between TET2 and c-KIT mutational status and/or MCT histological grading, was not possible. Also for TET2 gene, some intronic variants not related with splicing sites, and single nucleotide polymorphisms (SNPs) in the coding sequence were detected in the samples. These alterations, with the relative allelic frequencies and population distribution, are collected in [Table 3](#pone.0142450.t003){ref-type="table"}. Deepening in sequence analysis, other genetic variations were detected, in two specific regions of the gene. The protein database Uniprot (<http://www.uniprot.org/>) recognized them as polyglutamine rich-regions because they are rich in glutamine residues: one is located in exon 3 and another one in exon 11. The alignment of canine sequence with the same human, cat, mouse and rat sequence portion showed that poly-glutamines residues were quite conserved among species and dog possessed the highest number of glutamine repetitions ([Fig 1](#pone.0142450.g001){ref-type="fig"}). In details, in canine exon 3, 65 out of 75 MCTs (86,66%) showed the deletion of glutamine 753 (c. 2250_2252del; p.Gln753del). In exon 11, different rearrangements in the number of glutamine repetitions were detected among samples. All variations observed in our cohort of samples with relative frequencies in the group are listed in [Table 4](#pone.0142450.t004){ref-type="table"}. Performing a Pearson χ^2^ analysis between the number of glutamine repetitions in each sample and the presence of c-KIT mutations, no statistical correlations were evident (P = 0.3427). Furthermore, the number of glutamine repetitions in exon 3 and 11 did not correlate with the histologic grade (Fisher exact test, P = 0.5808 and Pearson χ^2^, P = 0.2308, respectively). {#pone.0142450.g001} 10.1371/journal.pone.0142450.t004 ###### List of genetic variations detected in the glutamine rich region of TET2 exon 11 with relative population frequency and total glutamine residues number in the 75 MCT samples. {#pone.0142450.t004g} Variation Population Frequency Glutamine repetitions --------------------------------- ---------------------- ----------------------- Wild-type sequence 59/75 12 c\. 4682insGCA; p. 1562insQ 5/75 13 c.4686_4697del; p. 1564_1567del 4/75 8 c.4686_4694del; p. 1564_1566del 3/75 9 c.4698_4700del; p.1568del 4/75 11 Afterwards, to better understand if these sequence rearrangements might have a correlation with the onset of the disease, we screened the DNA from 39 healthy dogs for the same polyglutamine region in TET2 exon 11. Subsequently, considering as the wild-type phenotype the 12 glutamine repetitions presented in the reference NCBI sequence (XM_535678--4) we categorized all samples (healthy and pathologic) in three groups: samples that evidenced less than 12 glutamines (Q\<12), wild-type dogs and samples with more than 12 glutamines (Q\>12). From the contingency table and the Pearson χ^2^ test, no significant correlation emerged between the number of glutamine repetitions and the risk to develop mast cell tumor ([Fig 2](#pone.0142450.g002){ref-type="fig"}). {#pone.0142450.g002} Discussion {#sec014} ========== Mast cells (MCs) neoplastic disorders occur in both canine and human species sharing many but not all biological and clinical features. Spontaneous MCT has been proposed as a model to study biological and therapeutic approach for human neoplastic MCs diseases, i. e. mastocytosis \[[@pone.0142450.ref028]\]. At the same time, due to the implications of c-KIT aberrations in the development of MCs tumors, canine MCT could represent a useful model to study human c-KIT driven malignancies and TKIs, targeting c-kit. Therefore, comparative studies of MCs disorders may represent an opportunity to improve our knowledge on both mastocytosis and c-KIT driven tumors for diagnosis in case of c-KIT wild type state and/or with the aim to develop novel treatment options that can be translated in human patients. In this respect, starting from a list of genes that showed recurrent somatic mutations in human myeloproliferative diseases and mastocytosis, we screened a cohort of 75 canine MCTs for hot-spot mutations sites. No mutations were identified in IDH1 and IDH2 genes in our cohort of MCTs while, in SM, IDH2 mutations occurred for 6.9% of cases \[[@pone.0142450.ref011]\]. To the best of our knowledge, only one study has been published in dog \[[@pone.0142450.ref029]\] where no mutations in both these genes were found in canine gliomas. Considering the high percentage of mutations in the human analog tumor (\~ 80% in grades II-III) these results were surprising and might suggest a minor role of these genes in the pathogenesis of canine gliomas and MCT. In SM, two genes involved in spliceosome machinery, SRSF2 and SF3B1, showed a mutation occurrence of 24% and 5.6% respectively \[[@pone.0142450.ref011]\]. However, no mutations were detected in canine MCT. No data about the relevance of these genes and their mutational status in canine oncology are actually available; therefore, present results, are the first data ever published so far. On the other hand, more information are available about NRAS and KRAS, in dog cancer. Present results showing the absence of mutations obtained in our MCTs samples are consistent with a number of previously published studies in which RAS mutations have been shown to be extremely rare in the most common types of canine tumor such as mammary tumors, soft-tissue tumors (included MCTs), melanomas and lymphoproliferative disorders \[[@pone.0142450.ref030],[@pone.0142450.ref031]\]. In contrast, higher mutational frequencies of RAS genes have been obtained in human lung, pancreatic, gastrointestinal, brain and liver tumor, in acute myelogenous leukemia, in follicular and undifferentiated papillary thyroid tumors \[[@pone.0142450.ref032]\]. Therefore and likewise to IDH1/2 we might hypothesized that RAS mutations do not play a major role in the pathogenesis of canine MCT and this supposition is in line with previously published data \[[@pone.0142450.ref030]\]. About TET2, the high percentage of mutations found in aggressive form of human mastocytosis (20.8%) was not confirmed in canine MCT (2.7%). Moreover, the typical frame-shift mutations observed in humans and coding for a truncated protein with consequently loss of function, was never observed. These results surprised the authors and, until this moment, represented the first attempt, in veterinary medicine, to investigate the role of TET2 mutations in a canine tumor since no information are available in previous published studies. Noteworthy, a frequent rearrangement was observed in a glutamine-rich region of TET2 exon 11, resulting in variations of the number of glutamine repetitions (from 8 to 13) among cases. Very little information are actually published in human oncology about a possible relationship between length of polyglutamine regions in some genes and the risk to develop cancer. The number of CAG repetitions in androgen receptor seems to be correlated with the risk of occurrence of prostate cancer; furthermore polymorphisms in glutamine regions of nuclear receptor coactivator 3 (NCOA3 also known as AIB1) seems to play a role in the susceptibility of some type of breast cancer \[[@pone.0142450.ref033]--[@pone.0142450.ref035]\]. The sequencing analysis conducted in a little group of healthy dogs and matched statistically with results of MCT samples did not reveal any significant relationship between number of glutamine repetitions and the risk of MCT development. In conclusion, this preliminary study aimed to investigate, in canine MCT, the mutational status of genes known to be involved in human myeloproliferative disorders. The study was undertaken in a relatively small cohort of canine samples, and only human analogue hot-spot sites for mutation were took into consideration. Further investigations are needed to better characterize the pathogenic pathways involved in both diseases. Among these ones, to sequence the entire IDH1, IDH2, NRAS, KRAS, SRSF2 and SF3B1 genes and, subsequently, to analyze interesting genes that were excluded from this preliminary study (i. e. Additional Sex Combs Like 1 alias ASXL1, Janus Kinase 2 alias JAK2). Clearly, the advent of deep sequencing methods might be more useful in this sense, giving a more wide observation on genome modifications and allowing the identification of new hot-spot mutation sites in canine genes. This approach will permit to clarify the possible value of canine MCT as a comparative animal model for human SM. Authors would like to thank Prof. Antonio Frangipane di Regalbono and Dott.ssa Cinzia Centelleghe for providing healthy dog blood samples. This work has been presented at the ESVONC Annual Congress 2015 (Krakow, Poland, 28th-- 30th May 2015). [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: MD PD. Performed the experiments: EZ KH. Analyzed the data: EZ KH. Contributed reagents/materials/analysis tools: MG MD PD. Wrote the paper: EZ MG MD PD. Collected samples: MG MD PD.

New neurons are continually born throughout adulthood in the subventricular zone of the lateral ventricle and in the subgranular zone of the dentate gyrus in the hippocampus. The sequential synaptic integration of adult-born neurons has been widely examined in rodents, but the mechanisms regulating the integration remain largely unknown. The primary cilium, a microtubule--based signaling center, plays essential roles in vertebrate development, including the development of the central nervous system. We examined the assembly and function of the primary cilium in the synaptic integration of adult-born hippocampal neurons. Strikingly, primary cilia are absent in young adult-born neurons but assemble precisely at the stage when newborn neurons approach their final destination, further extend dendrites and form synapses with entorhinal cortical projections. Conditional cilia deletion from adult-born neurons induced severe defects in dendritic refinement and synapse formation. Primary cilia deletion leads to enhanced Wnt/beta-catenin signaling which may account for these developmental defects. Taken together, our study identifies the assembly of primary cilia as a critical regulatory event in the dendritic refinement and synaptic integration of adult-born neurons.

Introduction {#s1} ============ We define the distal anterior cerebral artery as the distal portion after the anterior communicating artery. Aneurysms in the distal anterior cerebral artery have low occurrence rates with 2.1--9.2% including both the pericallosal and callosomarginal arteries.^[@b1],\ [@b2]^ High surgical morbidity is found in the surgical clipping of distal anterior cerebral artery (DACA) aneurysms, when it is done using an interhemispheric approach due to the narrowing of operative space and the necessary sacrifice of bridging veins.^[@b3],\ [@b4]^ To this end, an endovascular approach is increasingly being used for the treatment of both ruptured and unruptured aneurysms as a primary treatment option when dealing with intracranial aneurysms after the success of the International Subarachnoid Aneurysmal Trial. That said, it is still challenging to treat distal anterior cerebral artery aneurysms using endovascular approach with morbidity results similar to that of clipping.^[@b5]^ Case Report {#s2} =========== A 17-year-old girl was admitted after experiencing sudden onset of thunderclap headache preceded by acute loss of consciousness at her home. There was no past history of hypertension, seizures, drug abuse, trauma or infection. On admission, the initial clinical examination showed an unconscious patient GCS 10/15 Hunt & Hess classification Grade IV. After stabilization, a CT scan was performed ([Figure 1](#f1){ref-type="fig"}) showing diffuse subarachnoid haemorrhage and cisternal clot at interpeduncular cistern (Modified Fisher I). Moderate dilatation of temporal horns of lateral ventricle also seen. Angiography ([Figure 2](#f2){ref-type="fig"}) revealed a ruptured saccular aneurysm of less than 3 mm in diameter, with a neck of 1.5 mm at the origin of left frontopolar artery (FPA). Considering the small size of aneurysm and ruptured nature, we decided to manage it conservatively. After 2 weeks of initial subarachnoid haemorrhage, the aneurysm bled once again. Another CT scan ([Figure 3](#f3){ref-type="fig"}) was obtained showing superior interhemispheric frontal haematoma opened to ventricular system (Modified Fisher IV). Coiling done on Day 15 after second haemorrhage. After selective microcatheteration of the left FPA aneurysm, it was completely obliterated with a single coil maintaining the distal arterial lumen totally permeable ([Figure 4](#f4){ref-type="fig"}). Follow-up magnetic resonant angiogram was performed 6 months after coil embolization and showed stable occlusion. ([Figure 5](#f5){ref-type="fig"}). The patient never lost follow up, after 1 year of coiling she had an episode of GTCS, immediate CT scan done showing chronic infarct left frontal lobe ([Figure 6](#f6){ref-type="fig"}). {#f1} {#f2} {#f3} {#f4} {#f5} {#f6} Discussion {#s3} ========== According to researchmost ACA aneurysms occur at the mean age of 55 years. Only 1200 cases have been reported between 1939 and 2011. The majority of these are proximal internal carotid artery aneurysms and distal anterior cerebral artery's are even rarer. The ultimate reason behind this is unknown; however, it has been postulated that distal anterior cerebral artery are associated with anatomical variations at this site. It is further posited that supreme anterior communicating artery is a communication between the two pericallosal arteries at the major branches during embryonic life. This connection may be a remnant and could be the aetiology of aneurysms at this site.^[@b6]^ Conflicting accounts of literature put the occurrence of DACA aneurysms between 2.1 and 9.2% of all intracranial aneurysms.^[@b7],\ [@b8]^ Of all the aneurysms in the DACA region, those in the A3 segment are most common with an incidence rate between 69 and 82%, mostly concentrated at the CMA-PCA (callosomarginal artery-pericallosal artery) junction.^[@b9]^ In the A2 segment, incidence reports are between 5 and 22% of all DACA aneurysms. These happen mainly at the A2 trunk or its frontonasal braches having the highest concentration at A2A on A2 truck at the origin of the FPA.^[@b10]^ DACA aneurysms due to their rarity, complex morphology, small size and depth inside the brain have always been a challenge for surgeons with high morbidity rates. Open surgery, when using clip reconstruction or *in situ* bypass with aneurysm trapping receives high occlusion rates but the periprocedural morbidity and mortality rates are still quite high.^[@b11],\ [@b12]^ Endovascular coil embolization allows for a complete to near complete aneurysm occlusion in 80% of cases. Since the advent of modern research and development of newer more suitable microcatheters, guidewires and coils DACA aneurysms can be treated with far higher success rates. It is very important to differentiate between saccular and dissecting aneurysms when choosing modality of treatment and as such we must first correctly identify the aneurysm. Dissecting aneurysms rarely develop near secondary to dissection of the intracranial arteries and are mostly found in the posterior circulation of the vertebrobasilar system with a close association to subarachnoid haemorrhage. Whereas, those of the carotid system are usually presented with cerebral infarctions caused by stenosis and occlusion. Only 14% of anterior circulation aneurysms are dissecting in nature and present as cerebral infarction and not as haemorrhage. Lastly, dissecting aneurysms tend to occur proximally in the anterior circulation because there is a stronger arterial pressure present there compared with the distal branches. Saccular aneurysms are almost always the result of hereditary weakness in blood vessels and typically occur in arteries of *"circle of Willis"*. They lack a tunica media and elastic lamina around their dilated location with wall of sac made up of thickened hyalinized intima and adventitia. These are mostly found around the circle of Willis at bifurcations where small vessels link to main vessels because the brain vasculature is inherently weak at these points. Making them particularly susceptible to saccular aneurysms. When choosing treatment modality, the patient and aneurysm characteristics need to be taken into account to formulate the best treatment option varying on a case to case basis and giving preference to coiling rather than surgery. [Table 1](#t1){ref-type="table"} offers a complete breakdown of the key deciding factors and a guide on how to choose preferred treatment modality. ([Figure 7](#f7){ref-type="fig"}) outlines a flow-chart for how the treatment modality should best be carried out. In this case, the aneurysm was successfully obliterated using coiling and gives a prime example of successful treatment of DACA aneurysms using an endovascular approach. ###### Factors to decide whether to clip or coil ---------------------------------------------------------------------------------------------------- **Factor** **Details** **Coiling **\ **Clipping **\ **Preferred** **possible** **possible** ------------- ---------------------- ------------------------------ ---------------- --------------- Age Young age Yes Yes Clipping Old age Yes No Coiling GCS 10 and below Yes No Coiling Above 10 Yes Yes Clipping Co-\ Present Yes No Coiling morbidities Absent Yes Yes Clipping Location Anterior \ Yes Yes Clipping circulation Posterior \ Yes No (possible with great \ Coiling circulation difficulty and poor outcome) Size Small Yes Yes Coiling Large Coiling with \ No Coiling assistant techniques Type Dissecting Yes Yes Coiling Saccular Yes Yes Coiling Finances Present Yes No Coiling Absent No Yes Clipping ---------------------------------------------------------------------------------------------------- {#f7} Conclusion {#s4} ========== Despite the anatomical difficulty of access and pathological hindrance of small aneurysms that result in relatively high intraprocedural events, the outcome still remains favourable in the present case. Endovascular technical evolution and the skill of an experienced neurointerventionalist make endovascular management of small DACA aneurysms a feasible alternative. Learning Points {#s5} =============== 1. DACA aneurysms are very rare with only 1200 cases reported since 1939. While there is debate on their true cause, it is posited that they are a result of embryonic remnants of supreme anterior communicating artery. 2. Dissecting aneurysms are mainly found near secondary to dissection of the intracranial arteries and in the posterior circulation of the vertebrobasilar system with stenosis and occlusion. Saccular aneurysms are mainly found around the circle of Willis at the bifurcation of small vessels because the brain vasculature is weakest at those points. 3. There is a huge risk in the surgical approach to such cases, as it delves too deep into the brain often resulting in a poor outcome, especially in third world countries; thus, an endovascular approach should be taken given that it offers better results^[@b13]^ .  **Acknowledgements:** I would like to thank Mian Faizan and Team Online for their continued support and hard efforts that go into the fine-tuning and technical development of my articles. **Informed consent:** Written informed consent for the case to be published (including images, case history and data) was obtained from the patient(s) for publication of this case report, including accompanying images. **Ethics approval:** The Institutional Review Board of the institute gave thier approval and blessing for this research to be conducted and published. **Patient consent:** Patient informed consent was taken both verbally and in writing.

1. Introduction {#sec1} =============== The left ventricle pseudoaneurysm (LVPA) is a complication related to the rupture of the free wall of left ventricle, which communicates through a defect with newly formed saccular pocket \[[@B1]\]. It is often secondary to a myocardial infarction, chest trauma, and a surgical or instrumental cardiac procedure \[[@B2]--[@B5]\]. Sometimes LVPA is discovered during a complication, the etiology being unseen \[[@B4], [@B6]\]. This is an unexpected anomaly that may cause sudden cardiac death \[[@B6], [@B7]\]. The others complications are heart failure, arrhythmias, and embolism \[[@B8]\]. In all cases, the surgical treatment is required \[[@B7]\]. This anomaly is rare in Africa. Six cases have been collected 18 years ago in Tunisia \[[@B7]\]. In this clinical case, the authors report a LVPA discovered incidentally. 2. Observation {#sec2} ============== A 64-year-old black man, retired teacher, resident at Brazzaville (Republic of Congo), had consulted in pneumology examination for cough, mucopurulent sputum, and low-grade fever. The usual blood tests were unremarkable. The patient was treated with antibiotics and mucolytic for the diagnosis of acute bronchitis. The evolution was unremarkable. However, a chest radiograph had objectified an angulation of the lower left arc of the heart ([Figure 1(a)](#fig1){ref-type="fig"}); this was the reason for cardiology consultation. Family and private medical history was not particular. The patient was asymptomatic and in a good condition. His measurements were weight 70 Kg, height 1.76 m, and BMI 22.3. In cardiac examination, the heart rate was 95 beats per minute, with no murmur cardiac in auscultation; systolic blood pressure was 132 mmHg and 74 mmHg for diastolic blood pressure. In blood chemistry, the hemoglobin was 15.2 g/dL, the blood sedimentation rate was 13 mm in 1st hour, the creatinine was 12.4 mg/L, and the glycaemia was 1.04 g/L. The other analysis suggests that transaminases, troponin, CPK-MB, blood lipids, were normal. The EKG in sinus rhythm had objectified negative T waves in the lateral area ([Figure 1(b)](#fig1){ref-type="fig"}). In echocardiography, heart dimensions were normal. The ejection fraction in Simpson four cavities was 57% ([Figure 2(a)](#fig2){ref-type="fig"}). We visualized a neocavity connected with the apex of left ventricle ([Figure 2(b)](#fig2){ref-type="fig"}) by a collar of 0.92 cm of diameter ([Figure 2(c)](#fig2){ref-type="fig"}). These aspects suggested a pseudoaneurysm. In addition, a poorly limited rounded mass was hyperechoic and pendant at the bottom of pseudoaneurysm, suggesting a thrombus ([Figure 2(d)](#fig2){ref-type="fig"}). The precise boundaries of the pocket could not be specified in ultrasound apical and subcostal. The thoracic CT scan with injection of contrast material had showed in cross-sectional ([Figure 3(a)](#fig3){ref-type="fig"}) the dimensions of LVPA: 45.9 mm × 68.4 mm. In the same section, a communication between the pseudoaneurysm and left ventricle is noted ([Figure 3(b)](#fig3){ref-type="fig"}). In front section ([Figure 3(c)](#fig3){ref-type="fig"}), the longest diameter of LVPA was 71 mm. A reconstruction of LVPA was performed in 3D imaging ([Figure 3(d)](#fig3){ref-type="fig"}). The treatment included atenolol and acenocoumarol. The international normalized ratio (INR) was 3.2 at last medical checkup. Invasive cardiac investigation (coronary angiography) and cardiac surgery are not feasible in Congo. For this reason, the medical evacuation abroad is required for full support and to determine the aetiology. 3. Discussion {#sec3} ============= The LVPA is an anomaly rarely described in the literature \[[@B4]\]. Few cases have been reported in North Africa and sub-Saharan Africa \[[@B5], [@B7], [@B9]\]. Before the advent of ultrasound and CT scan, only angiography allowed the diagnosis \[[@B7]\]. This anomaly is secondary to myocardial infarction or cardiac trauma \[[@B3], [@B10]\]. Therefore, an array of heart failure or chest pain is the reason of consultation. Sometimes, the event goes unnoticed and LVPA is discovered long later \[[@B6]\]. The diagnosis of LVPA is placed in the imaging. The deformation of the cardiac shadow is noted in standard radiography \[[@B9]\]. However, the heart may have a normal appearance \[[@B9]\]. Echocardiography leads to diagnosis, objectifying communication between the left ventricle and the pocket \[[@B8]\], as in the case we described. The delimitation of contours can be complicated by the configuration of the pocket. Indeed, ultrasound appeared limited in our case, because the pseudoaneurysm communicated with the left ventricle at the tip, and went back under the ribs forward. Transesophageal ultrasound would have a better analysis of the pseudoaneurysm. CT scan has proven to be an indispensable tool in our case, to define the limits of pseudoaneurysm. Other authors have also used this exploration for a comprehensive assessment of the lesions \[[@B8]\]. Aside from the mediastinal rupture and sudden death, the other complications of LVPA are heart failure, arrhythmias, and vascular embolism \[[@B6], [@B10]\]. In the absence of major complications, the treatment is comprised of beta-blockers and/or anticoagulants. In our case, an indication of acenocoumarol was justified after viewing an intra-anevrismal thrombus in echocardiography \[[@B9]\]. The cure is the open heart surgery in which a resection of the bag is made \[[@B7]\]. Surgery is indicated even in asymptomatic patients as outlined by Mecheche et al. \[[@B7]\]. Surgical treatment can prevent sudden death. In patients with high surgical risk, an instrumental closure may be achieved \[[@B5]\]. An indication of aneurysmectomy was accepted in our case. The heart surgery was not available in Congo; the complete treatment will be performed abroad. The postoperative course is usually simple. After cardiac surgery, there is no recurrence of pseudoaneurysm. Through this clinical case, we have shown the limits of treatment despite an accurate diagnosis in our country and in most countries of sub-Saharan Africa. In absence of cardiac surgery, the patient will be exposed to a sudden death. 4. Conclusion {#sec4} ============= The pseudoaneurysm of left ventricle is an anomaly shortly described. It is rarely asymptomatic idiopathic and puts the patients at risk of sudden death. Diagnosis involves several imaging modalities that can guide the surgeon. Conflict of Interests ===================== The authors declare that they have no conflict of interests regarding the publication of this paper. {#fig1} {#fig2} {#fig3} [^1]: Academic Editor: Yoshiro Naito

Introduction {#sec1} ============ Lung cancer is the leading cause of cancer-related death worldwide and causes one-quarter of all cancer deaths.[@bib1] Non-small-cell lung cancer (NSCLC) accounts for 80% of all lung cancer cases.[@bib2] Recently, with the development of molecular targeted therapy and immunotherapy, the prognosis of advanced NSCLC has improved considerably.[@bib3]^,^[@bib4] In contrast, only a limited breakthrough has been achieved in the management of early-stage NSCLC. Patients with early-stage NSCLC mainly undergo surgery with or without adjuvant therapy; however, approximately 30%--40% of stage I NSCLC patients ultimately die of postoperative local or metastatic recurrence.[@bib5] Lung adenocarcinoma (LUAD) is the major pathological subtype of NSCLC. Unfortunately, similar to other subtypes of NSCLC, there are no biomarkers that can predict recurrence and no specific treatment methods or drugs that can effectively prevent or inhibit the recurrence of stage I LUAD. Additionally, the mechanism of stage I LUAD recurrence remains largely unknown. MicroRNAs (miRNAs) are small noncoding RNAs that play important roles in many normal biological processes, including cell proliferation, differentiation, and apoptosis.[@bib6] miRNAs function through direct binding to the 3′ UTR of a target gene mRNA, which induces target gene mRNA cleavage or translational repression.[@bib7]^,^[@bib8] Accumulated evidence shows that deregulation of miRNAs has been implicated in many cancers, including LUAD, and dysregulated miRNA is an important factor leading to tumorigenesis and progression.[@bib9], [@bib10], [@bib11] Importantly, studies have shown that even one aberrantly expressed miRNA is sufficient to cause tumor initiation and progression and that restoration or inhibition of that aberrantly regulated miRNA can dramatically suppress cancer progression, indicating that miRNAs have the potential to be an effective therapeutic target.[@bib12]^,^[@bib13] Thus, investigating the role of each miRNA that is dysregulated in cancer is very important for understanding cancer development, progression, and therapy.[@bib12] Additionally, certain miRNAs have been indicated to be diagnostic and prognostic biomarkers in cancer clinical studies.[@bib13]^,^[@bib14] In this study, we used our sample cohort and The Cancer Genome Atlas (TCGA) dataset to show that increased expression levels of miR-134-5p and decreased expression levels of disabled-2 (DAB2) are closely correlated with recurrence in patients with stage I LUAD. Additionally, we demonstrated that overexpression of miR-134-5p or silencing of DAB2 stimulates LUAD cell metastasis and chemoresistance, whereas inhibition of miR-134-5p or overexpression of DAB2 suppresses LUAD cell metastasis and chemoresistance. Furthermore, we demonstrated that miR-134-5p exerts its oncogenic role by directly targeting DAB2 in LUAD. Our findings suggest that the level of miR-134-5p and DAB2 has potential as a biomarker for predicting recurrence in patients with stage I LUAD and that inhibition of miR-134-5p or restoration of DAB2 expression may be a novel strategy for inhibiting early-stage LUAD progression. Results {#sec2} ======= High Expression Levels of miR-134-5p Are Closely Correlated with Early Recurrence in Stage I LUAD Patients {#sec2.1} ---------------------------------------------------------------------------------------------------------- To screen miRNAs involved in early recurrence of stage I LUAD, we performed miRNA array analysis on specimens from stage I LUAD patients who had relapsed within 40 months after resection (n = 8) and who had not relapsed within 40 months after resection (n = 11)[@bib5] because 80% of stage I LUAD recurrences occur within 40 months after surgery.[@bib15] Through miRNA array analysis, we identified five miRNAs whose expression increased more than 2-fold in the early recurrence groups relative to that in the nonrecurrence groups ([Figure 1](#fig1){ref-type="fig"}A). Furthermore, this result was confirmed using a stage I LUAD dataset from TCGA. TCGA data analysis showed that the expression levels of the five miRNAs identified by array analysis were significantly increased in the group of stage I LUAD patients who relapsed within 40 months relative to those in the nonrelapse groups and that miR-134-5p was the miRNA with the greatest increase in the recurrence group ([Figure 1](#fig1){ref-type="fig"}A), suggesting that miR-134-5p may be associated with early recurrence of stage I LUAD. Thus, we further investigated the correlation between the miR-134-5p levels and recurrence in patients with stage I LUAD. Our data show that the stage I LUAD patient group with high miR-134-5p expression had a higher recurrence rate ([Figure 1](#fig1){ref-type="fig"}B) and a shorter median recurrence-free survival time than did the miR-134-5p-low group ([Figure 1](#fig1){ref-type="fig"}C). Such clinical results were confirmed using the TCGA dataset, and similar results were observed with our clinical data ([Figures 1](#fig1){ref-type="fig"}D and 1E). Taken together, these findings indicate that increased expression levels of miR-134-5p are closely associated with early recurrence of stage I LUAD.Figure 1miR-134-5p Was Associated with Early Recurrence in Stage I LUAD Patients(A) miRNAs that significantly increased in primary tumors from stage I LUAD patients who had relapsed within 40 months relative to that in those who had not relapsed within 40 months. (B) The miR-134-5p expression level was significantly associated with recurrence in stage I LUAD patients from the Daping Hospital cohort. (C) The miR-134-5p expression level was negatively correlated with the recurrence-free survival (RFS) rate and median time of RFS in stage I LUAD patients from the Daping Hospital cohort. (D) The miR-134-5p expression level was significantly associated with recurrence in stage I LUAD patients from the TCGA cohort. (E) The miR-134-5p expression level was negatively correlated with the RFS rate and median time of RFS in stage I LUAD patients from the TCGA cohort. \*p \< 0.05; \*\*p \< 0.01. miR-134-5p Significantly Stimulates LUAD Cell Metastasis {#sec2.2} -------------------------------------------------------- Metastasis and chemoresistance are the most common causes of cancer recurrence.[@bib16] Thus, we investigated the effects of miR-134-5p on LUAD cell metastasis. To investigate whether miR-134-5p is directly involved in the regulation of LUAD cell metastasis, the LUAD cell lines A549 and H1299 were transfected with miR-134-5p mimics or a miR-134-5p inhibitor ([Figure 2](#fig2){ref-type="fig"}A) and were then subjected to western blot and Transwell assays. Our results showed that overexpression of miR-134-5p significantly inhibited epithelial-to-mesenchymal tranzition (EMT) marker protein E-cadherin (EMT negative regulator) ([Figure 2](#fig2){ref-type="fig"}B) expression and stimulated both the migration and invasion of H1299 and A549 cells ([Figure 2](#fig2){ref-type="fig"}C). In contrast, inhibition of miR-134-5p strongly stimulated E-cadherin expression ([Figure 2](#fig2){ref-type="fig"}B) and inhibited migration and invasion in both the H1299 and A549 cell lines ([Figure 2](#fig2){ref-type="fig"}C). Furthermore, to confirm the *in vitro* experimental results *in vivo*, we generated A549 cell lines stably expressing miR-134-5p or the miR-134-5p inhibitor ([Figure 2](#fig2){ref-type="fig"}A). We then administered these cells to nude mice by tail vein injection. As shown in [Figure 2](#fig2){ref-type="fig"}D, overexpression of miR-134-5p significantly increased the formation of A549 cell-induced metastatic lesions in the lungs of mice compared to that induced by vector control cells. In contrast, inhibition of miR-134-5p strongly suppressed the formation of A549 cell-induced metastatic lesions in the lungs of mice compared to that induced by vector control cells ([Figure 2](#fig2){ref-type="fig"}E). These findings clearly indicate that miR-134-5p positively regulates LUAD cell metastasis.Figure 2miR-134-5p Positively Regulated LUAD Cell Metastasis(A) The miR-134-5p expression level was measured using qRT-PCR in the indicated cells. The indicated cells were transfected with the indicated oligonucleotides or infected with the indicated lentivirus. After 72 h, cells were subjected to qRT-PCR analysis. (B) miR-134-5p negatively regulated E-cadherin expression in LUAD cells. The indicated cells were transfected with miR-134-5p mimics or inhibitors. After 72 h of transfection, cells were subjected to western blot analysis. (C) miR-134-5p positively regulated LUAD cell invasion and migration. The indicated LUAD cells were transfected with negative control oligonucleotides, miR-134-5p mimics, or the miR-134-5p inhibitor. After 24 h of transfection, cells were subjected to invasion and migration assays. (D) Overexpression of miR-134-5p stimulated LUAD cell metastasis *in vivo*. A549 cells were infected with lentivirus expressing miR-134-5p or empty vector and injected into the tail vein of 6-week-old nude mice (n = 5 per group). The mice were sacrificed 3 weeks after cell injection, and nodules on the lung surface and in tissue sections were counted under a microscope. (E) Inhibition of miR-134-5p strongly suppressed LUAD cell metastasis *in vivo*. A549 cells were infected with lentivirus expressing the miR-134-5p inhibitor or control vector and injected into the tail vein of 6-week-old nude mice (n = 5 per group). Mice were sacrificed 3 weeks after cell injection, and nodules on the lung surface and in tissue sections were counted under a microscope. CTR, control; mimics, miR-134-5p; inhibitor, miR-134-5p antisense; LV miR-134-5p, lentivirus expressing miR-134-5p; LV miR-134-5p inhibitor, lentivirus expressing miR-134-5p antisense. \*p \< 0.05; \*\*p \< 0.01; \*\*\*p \< 0.001. miR-134-5p Contributes to the Development of Chemoresistance in LUAD Cells {#sec2.3} -------------------------------------------------------------------------- Next, we investigated the effects of miR-134-5p on the chemosensitivity of LUAD cells. First, we investigated the correlation between the miR-134-5p expression level and postoperative progression in stage I LUAD patients who received chemotherapy. Our clinical data showed that the stage I LUAD patient group with high miR-134-5p expression had a higher recurrence rate than did the group with low miR-134-5p expression ([Figure 3](#fig3){ref-type="fig"}A). In addition, our *in vitro* experiments showed that overexpression of miR-134-5p significantly suppressed CDDP-induced cell growth inhibition ([Figure 3](#fig3){ref-type="fig"}B) and apoptosis ([Figure 3](#fig3){ref-type="fig"}C; [Figure S1](#mmc1){ref-type="supplementary-material"}) in both the A549 and H1299 LUAD cell lines. Consistent with the results of the *in vitro* experiment, the results of the A549 xenograft model experiment showed that overexpression of miR-134-5p significantly suppresses CDDP-induced LUAD growth inhibition ([Figures 3](#fig3){ref-type="fig"}D and 3E) and apoptosis ([Figure 3](#fig3){ref-type="fig"}F). Taken together, our findings suggest that aberrantly overexpressed miR-134-5p significantly contributes to chemoresistance development in LUAD.Figure 3Overexpression of miR-134-5p Contributes to Stage I LUAD Chemoresistance(A) Analysis of the Daping Hospital cohort showed that the miR-134-5p expression level was significantly correlated with recurrence in stage I LUAD patients who received chemotherapy (n = 60). (B) Overexpression of miR-134-5p suppressed CDDP-induced cell growth inhibition in both A549 and H1299 cells. After 48 h of transfection with the indicated oligonucleotides, cells were plated in a 96-well plate. Twelve hours after seeding, cells were incubated with or without the indicated concentration of CDDP for 48 h and were then subjected to CCK8 assay. (C) Overexpression of miR-134-5p suppressed CDDP-induced cell apoptosis in both A549 and H1299 cells. After 48 h of transfection with the indicated oligonucleotides, cells were plated in a six-well plate. Twelve hours after seeding, cells were incubated with or without the indicated concentration of CDDP for 24 h and were then subjected to apoptosis analysis. (D) miR-134-5p overexpression suppressed CDDP-induced tumor growth inhibition *in vivo*. miR-134-5p-overexpressing A549 cells and the corresponding control cells were used to generate xenograft tumors. After the mean tumor volume reached 100 mm^3^, mice were started on treatment with either PBS or CDDP (2 mg/kg body weight), and the tumor volumes were measured every 2 days. (E) Mice were sacrificed after 2 weeks of CDDP treatment, and the tumors were weighed. (F) Apoptotic cells were detected via a TUNEL assay in the indicated xenograft tumor samples. \*p \< 0.05; \*\*p \< 0.01; \*\*\*p \< 0.001. The above data show that overexpression of miR-134-5p stimulates chemoresistance, prompting us to investigate whether miR-134-5p inhibition could overcome chemoresistance in LUAD. Here, we used the CDDP-resistant LUAD cell line A549/CDDP ([Figure 4](#fig4){ref-type="fig"}A) to examine the effects of miR-134-5p inhibition on LUAD chemoresistance, because A549/CDDP cells show higher expression levels of miR-134-5p than do the parental A549 cells ([Figure 4](#fig4){ref-type="fig"}B). As shown in [Figure 4](#fig4){ref-type="fig"}C, our cell viability assay showed that inhibition of miR-134-5p restores the sensitivity of A549/CDDP cells to CDDP treatment. Consistent with this finding, the flow cytometry analysis results showed that compared to CDDP treatment alone, inhibition of miR-134-5p significantly stimulated CDDP-induced A549/CDDP cell apoptosis ([Figure 4](#fig4){ref-type="fig"}D). Furthermore, these *in vitro* experiment results were confirmed *in vivo* using xenograft models that were generated by CDDP-resistant A549 cells. Our *in vivo* experiment show that combination of miR-134-5p inhibition and CDDP treatment significantly inhibited CDDP-resistant tumor growth compared to single treatment groups ([Figure 4](#fig4){ref-type="fig"}E). Taken together, our findings demonstrate that inhibition of miR-134-5p can overcome the insensitivity of chemoresistant LUAD cells to chemotherapy.Figure 4Inhibition of miR-134-5p Overcomes the Insensitivity of CDDP-Resistant A549 Cells to CDDP Treatment(A) A549/CDDP cells exhibit resistance to CDDP treatment compared to their parental cells. The indicated cells were treated with the indicated concentration of CDDP for 48 h and were then subjected to a cell viability assay. (B) miR-134-5p expression was measured by qRT-PCR in A549/CDDP cells and their parental cells. (C) Inhibition of miR-134-5p significantly enhanced the sensitivity of A549/CDDP cells to CDDP treatment. A549/CDDP cells were transfected with the miR-134-5p inhibitor, treated with the indicated concentration of CDDP for 48 h, and subjected to a cell viability assay. (D) Inhibition of miR-134-5p significantly stimulated CDDP-induced apoptosis in A549/CDDP cells. A549/CDDP cells were transfected with the miR-134-5p inhibitor, treated with the indicated concentration of CDDP for 48 h, and subjected to apoptosis analysis. (E) Inhibition of miR-134-5p enhanced the sensitivity of CDDP-resistant tumors to CDDP treatment inhibition *in vivo*. Expressing miR-134-5p-inhibitor A549/CDDP cells and the corresponding control cells were used to generate xenograft tumors. After the mean tumor volume reached 100 mm^3^, mice were started on treatment with either PBS or CDDP (2 mg/kg body weight). Mice were sacrificed after 2 weeks of CDDP treatment, and the tumors were weighed. \*p \< 0.05; \*\*p \< 0.01; \*\*\*p \< 0.001; ns, not significant. DAB2 Is a Target of miR-134-5p in Stage I LUAD {#sec2.4} ---------------------------------------------- To investigate the mechanism underlying the effect of miR-134-5p on LUAD cells, we performed mRNA sequencing analysis using miR-134-5p-overexpressing A549 cells and the corresponding parental cells. Through mRNA sequencing analysis, we identified 144 genes that were downregulated in miR-134-5p-overexpressing A549 cells by more than 2-fold relative to their expression in control cells ([Figure 5](#fig5){ref-type="fig"}A). Among them, through a search of the miRNA target prediction databases [microRNA.org](http://microRNA.org){#intref0010} and TargetScan, we demonstrated six genes for which the 3′ UTR contained miR-134-5p binding sites ([Figure 5](#fig5){ref-type="fig"}A). Furthermore, by clinical samples analysis, we investigated the association between these six genes and miR-134-5p expression levels in stage I LUAD. Our clinical data show that the DAB2 expression level was negatively correlated with the miR-134-5p level in stage I LUAD ([Figure 5](#fig5){ref-type="fig"}B). In addition, previous studies have shown that cancer suppressor gene DAB2 downregulation induces EMT progression, thereby stimulating cancer metastasis.[@bib17]^,^[@bib18] These findings suggesting that DAB2 may be a target gene of miR-134-5p involved in the regulation of miR-134-5p on stage I LUAD metastasis. In fact, our *in vitro* experiments showed that overexpression of miR-134-5p significantly inhibited DAB2 expression in both A549 and H1299 LUAD cells ([Figure 5](#fig5){ref-type="fig"}C). Consistent results were also observed in xenograft tumors generated by miR-134-5p-overexpressing A549 cells and vector control cells ([Figure 5](#fig5){ref-type="fig"}D). In addition, our luciferase activity assay showed that overexpression of miR-134-5p significantly inhibited luciferase expression driven by the wild-type 3′ UTR of DAB2; however, it did not affect luciferase expression driven by the mutant 3′ UTR of DAB2 ([Figures 5](#fig5){ref-type="fig"}E and 5F), indicating that DAB2 is a target of miR-134-5p and that miR-134-5p inhibits DAB2 expression through directly binding to the 3′ UTR of DAB2 in LUAD cells.Figure 5DAB2 Is a Target of miR-134-5p in Stage I LUAD(A) Candidate targets of miR-134-5p in LUAD cells. mRNA sequencing identified 144 genes (heatmap) that were downregulated more than 2-fold in miR-134-5p-overexpressed A549 cells relative to that in control cells. Then, through a search of the miRNA databases TargetScan and [microRNA.org](http://microRNA.org){#intref0030}, six candidate target genes of miR-134-5p were identified. (B) The level of miR-134-5p was negatively correlated with DAB2 expression level in primary tumors of stage I LUAD. The miR-134-5p and DAB2 expression levels were measured by qRT-PCR and immunohistochemistry, respectively, in stage I LUAD tissues (n = 54). (C) Overexpression of miR-134-5p significantly suppressed DAB2 expression in both A549 and H1299 cells. The indicated cells were transfected with miR-134-5p (mimic) or control oligonucleotides (CTR). After 72 h of transfection, cells were subjected to western blot analysis. (D) Immunohistochemical analysis showed that DAB2 expression was strongly inhibited in xenograft tumors generated by miR-134-5p-overexpressing A549 cells. (E) Predicted binding sites of miR-134-5p in the wild-type 3′ UTR of DAB2. Mutations in this 3′ UTR are highlighted in red. (F) Luciferase activity of the reporter driven by the wild-type or mutant 3′ UTRs of DAB2 in A549 cells cotransfected with control oligonucleotides (CTR) or miR-134-5p mimics. Luciferase reporter constructs containing the wild-type (DAB2-WT) or mutant 3′ UTR of DAB2 (DAB2-MT) were transfected into A549 cells with or without miR-134-5p mimics. After 48 h of transfection, luciferase intensity was assessed. \*p \< 0.05; \*\*\*p \< 0.001; ns, not significant. DAB2 Is Negatively Correlated with Early Recurrence in Stage I LUAD and Stimulates LUAD Cell Metastasis and Chemoresistance {#sec2.5} --------------------------------------------------------------------------------------------------------------------------- The above results indicate that miR-134-5p plays an important role in the early recurrence of stage I LUAD metastasis and chemoresistance and that DAB2 is a target of miR-134-5p in stage I LUAD. These data further suggest that DAB2 may negatively regulate metastasis and chemosensitivity in stage I LUAD. In fact, the TCGA dataset analysis results show that stage I LUAD patients in the high-DAB2 group had longer median recurrence-free survival times and lower recurrence rate than did those in the low-DAB2 group ([Figures 6](#fig6){ref-type="fig"}A and 6B). Additionally, we found significantly decreased expression of DAB2 in CDDP-resistant A549/CDDP cells compared to that in the parental cells ([Figure 6](#fig6){ref-type="fig"}C). In addition, *in vitro* experiments showed that silencing DAB2 significantly stimulated LUAD cell migration, whereas overexpression of DAB2 strongly reduced LUAD cell migration ([Figures 6](#fig6){ref-type="fig"}D and 6E). Silencing DAB2 also suppressed the CDDP-induced inhibition of LUAD cell growth, whereas overexpression of DAB2 enhanced the CDDP-induced inhibition of LUAD cell growth ([Figures 6](#fig6){ref-type="fig"}F and 6G). More importantly, overexpression of DAB2 overcame the insensitivity of CDDP-resistant A549/CDDP cells to CDDP treatment ([Figure 6](#fig6){ref-type="fig"}H). These data suggest that DAB2 is an important regulator of metastasis and chemosensitivity in early-stage LUAD.Figure 6DAB2 Expression Level Was Negatively Correlated with Stage I LUAD Progression(A) Analysis of the stage I LUAD TCGA dataset showed that the DAB2 expression level was significantly associated with the median recurrence-free survival time in patients with stage I LUAD (n = 137). (B) Analysis of the stage I LUAD TCGA dataset showed that the DAB2 expression level was negatively correlated with the recurrence rate in stage I LUAD patients (n = 137). (C) DAB2 expression was significantly decreased in A549/CDDP cells relative to that in the parental A549 cells. (D) The indicated cells were infected with the indicated lentivirus. After 72 h of infection, cells were subjected to western blotting. (E) DAB2 negatively regulated the migration of both A549 and H1299 cells. Cells were infected with the indicated lentivirus. After 48 h of infection, cells were subjected to a migration assay. (F) Knockdown of DAB2 suppressed CDDP-induced cell growth inhibition in both A549 and H1299 cells. After 48 h of infection with the indicated lentivirus, cells were plated in a 96-well plate. Twelve hours after seeding, cells were incubated with or without the indicated concentration of CDDP for 48 h and were then subjected to a CCK8 assay. (G) Overexpression of DAB2 enhanced CDDP-induced cell growth inhibition in both A549 and H1299 cells. After 48 h of infection with the indicated lentivirus, cells were plated in a 96-well plate. Twelve h after seeding, cells were incubated with or without the indicated concentration of CDDP for 48 h and were then subjected to a CCK8 assay. (H) Restoration of DAB2 enhanced the sensitivity of the CDDP-resistant cell line A549/CDDP to CDDP treatment. A549/CDDP cells were infected with the indicated lentivirus and plated in a 96-well plate. Twelve hours after seeding, cells were incubated with or without the indicated concentration of CDDP for 48 h and were then subjected to a CCK8 assay. \*p \< 0.05; \*\*p \< 0.01; \*\*\*p \< 0.001. miR-134-5p Plays an Oncogenic Role through DAB2 in LUAD {#sec2.6} ------------------------------------------------------- Next, we investigated whether DAB2 directly contributes to miR-134-5p-induced metastasis and chemoresistance in LUAD. Our data showed that overexpression of DAB2 blocked the miR-134-5p-induced inhibition of E-cadherin expression ([Figure 7](#fig7){ref-type="fig"}A) and stimulation of migration ([Figure 7](#fig7){ref-type="fig"}B) and chemoresistance in H1299 cells ([Figure 7](#fig7){ref-type="fig"}C). In contrast, DAB2 knockdown suppressed the inhibition of miR-134-5p-induced stimulation of E-cadherin expression ([Figure 7](#fig7){ref-type="fig"}D) and inhibition of migration ([Figure 7](#fig7){ref-type="fig"}E) and chemoresistance ([Figure 7](#fig7){ref-type="fig"}F). Taken together, these findings suggest that DAB2 is an important player controlling the effects of miR-134-5p on LUAD metastasis and chemoresistance.Figure 7miR-134-5p Stimulates LUAD Cell Metastasis and Chemoresistance through DAB2(A) Restoration of DAB2 expression suppressed the miR-134-5p-induced inhibition of E-cadherin expression in H1299 cells. miR-134-5p mimics were transfected into H1299 cells with or without the DAB2 expression vector. After 72 h of transfection, cells were subjected to western blot analysis. (B) Restoration of DAB2 expression inhibited the miR-134-5p-induced stimulation of H1299 cell invasion. miR-134-5p mimics were transfected into H1299 cells with or without the DAB2 expression vector. After 48 h of transfection, cells were subjected to an invasion assay. (C) Restoration of DAB2 expression overcame miR-134-5p-induced chemoresistance in H1299 cells. miR-134-5p mimics were transfected into H1299 cells with or without the DAB2 expression vector. After 48 h of transfection, cells were incubated with the indicated concentration of CDDP for 48 h and were then subjected to a cell viability assay. (D) Silencing DAB2 inhibited the miR-134-5p inhibition-induced upregulation of E-cadherin expression in H1299 cells. miR-134-5p inhibitors were transfected into H1299 cells with or without the DAB2 shRNA expression vector. After 72 h of transfection, cells were subjected to western blot analysis. (E) Silencing DAB2 inhibited miR-134-5p inhibition-suppressed H1299 cell invasion. H1299 cells were transfected with the miR-134-5p inhibitor and/or DAB2 shRNA-expressing lentivirus. After 48 h of transfection, cells were subjected to a cell invasion assay. (F) Silencing DAB2 suppressed the miR-134-5p inhibition-enhanced sensitivity of H1299 cells to CDDP treatment. H1299 cells were transfected with the miR-134-5p inhibitor and/or DAB2 shRNA-expressing lentivirus. After 48 h of transfection, cells were subjected to a cell viability assay. In addition, we evaluated the correlation between the expression levels of miR-134-5p and several other genes in stage I LUAD, because contrary to our findings, previous studies have shown that miR-134-5p plays an anti-metastasis and anti-chemoresistance role by targeting ABCC1, TAB1, PAK2, DPYD, ITGB1, EGFR, CCND1, and FOXM1.[@bib16]^,^[@bib19], [@bib20], [@bib21], [@bib22], [@bib23], [@bib24], [@bib25] However, we did not observe a correlation between the expression levels of these genes and miR-134-5p in LUAD ([Figure S2](#mmc1){ref-type="supplementary-material"}), suggesting that these genes are not major targets in stage I LUAD. Discussion {#sec3} ========== Identification of prognostic factors that can stratify patients according to clinical and biological markers may help in selecting adequate treatment strategies.[@bib26] In this study, by using our clinical sample cohort and TCGA dataset, we found that increased expression levels of miR-134-5p in primary tumors were significantly correlated with early recurrence in stage I LUAD patients. In addition, we used the TCGA dataset to demonstrate that the expression level of the miR-134-5p target gene DAB2 was negatively correlated with recurrence in I LUAD patients. These findings suggest that the expression levels of miR-134-5p and DAB2 have potential as a biomarker for predicting early recurrence in stage I LUAD patients. However, this result needs to be further confirmed in larger samples. In general, stage I LUAD is treated with surgery combined with radiation or chemotherapy. However, treatment for stage I LUAD often fails due to local or metastatic recurrence,[@bib27] suggesting that metastasis and chemoresistance significantly contribute to recurrence. Recent studies have shown that aberrantly expressed miRNAs plays a key role in the metastasis and therapeutic resistance of a variety of cancers. For example, Gullà et al.[@bib28] reported that increased expression of miR-221 was associated with drug resistance in multiple myeloma and that inhibition of miR-221 can overcome drug resistance of multiple myeloma; Wang et al.[@bib29] reported that overexpression of miR-96 promotes cellular sensitivity to CDDP and poly ADP-ribose polymerase (PARP) inhibition in several type cancers; Gasparini et al.[@bib30] reported that overexpression of miR-155 enhances the sensitivity of breast cancer cells to irradiation; and Xu et al.[@bib12] reported that decreased expression of miR-382 was significantly associated with osteosarcoma lung metastasis and that overexpression of miR-382 can inhibit osteosarcoma lung metastasis. In this study, we used a series of *in vitro* and *in vivo* experiments to provide evidence that increased expression of miR-134-5p significantly contributes to LUAD metastasis and chemoresistance development. The oncogenic role of miR-134-5p was also identified by other research groups previously in lung cancer. Zhang et al.[@bib31] reported that miR-134-5p stimulates the proliferation of lung cancer cells. Chen et al.[@bib32] also reported that increased expression of miR-134-5p promotes cell proliferation and inhibits apoptosis in lung cancer. More importantly, our data clearly show that inhibition of miR-134-5p significantly inhibits LUAD cell metastasis and overcomes chemoresistance *in vitro* and *in vivo*. Taken together, these findings suggest that miR-134-5p functions as a tumor stimulator in LUAD and contributes to early recurrence of stage I LUAD through stimulating metastasis and chemoresistance. Additionally, they suggest that inhibition of miR-134-5p may be a useful strategy for the treatment or prevention of stage I LUAD metastasis and chemoresistance. In contrast to our results, several reports show that the expression of miR-134-5p is significantly downregulated in NSCLC and negatively associated with NSCLC progression.[@bib22] This is possible because some miRNAs exhibit different expression patterns in different types of cancers and play different roles.[@bib9]^,^[@bib33] In contrast to a previous NSCLC study, we investigated the effects of miR-134-5p on LUAD, a subtype of NSCLC, and focused on stage I LUAD. miRNAs play their roles through target genes, and a single miRNA can target many different genes while functioning as both a tumor suppressor and tumor stimulator.[@bib34] Even the same miRNA in the same tumor plays opposite roles at different stages by targeting different genes.[@bib35] These findings suggest that the role of a miRNA depends on its target gene. Here, we used a stage I LUAD dataset from TCGA to investigated the correlation between the expression levels of miR-134-5p and some oncogenes that were previously reported as target genes of miR-134-5p.[@bib16]^,^[@bib19], [@bib20], [@bib21], [@bib22], [@bib23], [@bib24], [@bib25] However, we observed no correlation between the expression levels of miR-134-5p and these oncogenes in stage I LUAD, suggesting that these oncogenes may not be primary targets for miR-134-5p in stage I LUAD. Here, we also clarified the cancer stimulation mechanism of miR-134-5p in stage I LUAD. Previous studies have shown that DAB2 is a tumor suppressor gene, which is significantly downregulated in lung cancer, and that reduced DAB2 expression is closely associated with lung cancer development and progression.[@bib36]^,^[@bib37] Studies also show that downregulated DAB2 is associated with recurrence in esophageal squamous cell carcinoma[@bib38] and promotes EMT in breast cancer through upregulating transforming growth factor β (TGF-β).[@bib17] Previous studies show that TGF-β is a stimulator of EMT and causes E-cadherin downregulation and N-cadherin upregulation.[@bib39] Our data also show that downregulated DAB2 is closely associated with recurrence of stage I LUAD, and that DAB2 positively regulates E-cadherin expression and negatively regulates metastasis of LUAD cells. In addition, we identified DAB2 as a novel target gene of miR-134-5p in stage I LUAD. Our clinical data showed that DAB2 expression is inversely correlated with miR-134-5p expression in stage I LUAD specimens and that overexpression of miR-134-5p expression in LUAD cells leads to suppression of DAB2 expression. In addition, the luciferase reporter assay showed that miR-134-5p directly targets the 3′ UTR of DAB2. Furthermore, our data indicate that restoration of DAB2 expression blocked miR-134-5p overexpression-induced LUAD cell metastasis and chemosensitivity, whereas DAB2 silencing suppressed the inhibition of miR-134-5p-induced LUAD cell metastasis and chemoresistance. These findings clearly suggest that DAB2 is a key downstream effector of miR-134-5p that mediates miR-134-5p-induced LUAD cell metastasis and chemoresistance. In summary, we combined clinical and experimental studies to determine the clinical significance and role of miR-134-5p and DAB2 in the early recurrence of stage I LUAD. Our work provides new insight into the mechanisms of miR-134-5p in early-stage LUAD metastasis and chemoresistance. Our findings may also aid in the development of potential therapeutics for the treatment of early-stage LUAD recurrence. Materials and Methods {#sec4} ===================== Reagents {#sec4.1} -------- Fetal bovine serum (FBS) and cell culture medium were purchased from Biological Industries (Cromwell, CT, USA). Glutaraldehyde, puromycin, and cisplatin (CDDP) were purchased from Sigma (St. Louis, MO, USA). A miRNeasy FFPE (formaldehyde-fixed, paraffin-embedded) kit was obtained from QIAGEN (Hilden, Germany). The miR-134-5p mimics, miR-134-5p inhibitor, negative control oligonucleotides, miR-134-5p primer set, and U6 small nuclear RNA (RNU6) primer set were obtained from RiboBio (Guangzhou, China). A High-Capacity cDNA Reverse Transcription Kit and a QuantiTect SYBR Green PCR kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Lentiviral vectors expressing miR-134-5p or inhibitor of miR-134-5p were obtained from GeneChem (Shanghai, China). Lentiviral vectors expressing DAB2 or short hairpin RNA (shRNA) targeting DAB2 were obtained from GenePharma (Shanghai, China). A cell counting kit-8 (CCK8) kit and an apoptosis assay kit were purchased from Biosharp (Wuhan, China) and GeneCopoeia (Guangzhou, China), respectively. Migration and invasion assay kits were obtained from BD Biosciences (Franklin Lakes, NJ, USA). Anti-β-actin and anti-β-tubulin antibodies were obtained from Abcam (Cambridge, MA, USA). An anti-E-cadherin antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). TRIzol and an anti-DAB2 antibody were obtained from Invitrogen (Carlsbad, CA, USA). A dual-luciferase assay system and terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay kit were purchased from Beyotime (Shanghai, China). A miRNAs array was obtained from Affymetrix (Santa Clara, CA, USA). Cell Culture and Human Specimens {#sec4.2} -------------------------------- The CDDP-resistant LUAD cell line A549/CDDP was obtained from the Cancer Research Institute of XinQiao Hospital, Third Military Medical University. Other LUAD cell lines used in this study were purchased from the American Type Culture Collection. All LUAD cells were grown in RPMI 1640 medium supplemented with 10% FBS at 37°C in an atmosphere of 95% air and 5% CO~2~. LUAD specimens were obtained from 81 stage I LUAD patients during surgery at Daping Hospital and Research Institute of Surgery. This study was conducted in accordance with the guidelines of the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Daping Hospital and Research Institute of Surgery, Third Military Medical University. Clinical data of the patients enrolled in this study were acquired from medical records and telephone follow-up interviews. The patients' characteristics from the Daping Hospital cohort are summarized in [Table S1](#mmc1){ref-type="supplementary-material"}. The TCGA dataset for stage I LUAD patients was obtained from <http://gdac.broadinstitute.org> and <https://xena.ucsc.edu>, and the patients' characteristics are summarized in [Table S2](#mmc1){ref-type="supplementary-material"}. miRNAs Array {#sec4.3} ------------ For the miRNA array analysis, total RNA was extracted from FFPE tissue samples using the RNA Prep Pure FFPE Kit according to the manufacturer's instructions. Only tissues containing more than 70% tumor cells were used for RNA isolation. The patients' characteristics are summarized in [Table S3](#mmc1){ref-type="supplementary-material"}. Gene expression profiling was performed using Affymetrix GeneChip miRNA 4.0 arrays according to the manufacturer's instructions. Real-Time qRT-PCR Analysis {#sec4.4} -------------------------- Total RNA was isolated from cells using TRIzol reagent according to the manufacturer's protocol, and reverse transcription (RT) was performed using the High-Capacity cDNA Reverse Transcription Kit. PCR amplification of miR-134-5p and RNU6 was performed with a specific primer set. The relative expression of miR-134-5p was normalized to RNU6 expression. PCR amplification of DAB2 and GAPDH was performed with the QuantiTect SYBR Green PCR kit. The primers used for DAB2 amplification were 5′-GTAGAAACAAGTGCAACCAATGG-3′ and 5′-GCCTTTGAACCTTGCTAAGAGA-3′; the primers used for GAPDH amplification were 5′-GGAGCGAGATCCCTCCAAAAT-3′ and 5′-GGCTGTTGTCATACTTCTCATGG-3′. Transwell and Cell Viability Assays {#sec4.5} ----------------------------------- Indicated cells were transfected with the indicated oligonucleotides and plasmid for 48 h. Then, 1$\times$10^4^ cells in serum-free growth medium were seeded in the upper wells of Transwell chambers. The lower wells contained the same medium supplemented with 10% serum. After 24 h, cells that had invaded to the lower side of the chamber were fixed with 2.5% glutaraldehyde, stained with 0.1% crystal violet, dried, and counted. For the cell viability assay, indicated cells were plated in 96-well plates at density 1$\times$10^4^ per well. After 12 h of seeding, cells were treated with indicated drugs for 48 h. Then, cell viability was determined using CCK8 reagent according to the manufacturer's instructions. Western Blot and Immunohistochemical Assays {#sec4.6} ------------------------------------------- Western blotting and immunohistochemical assays were performed as described previously.[@bib40] Apoptosis Analysis {#sec4.7} ------------------ The indicated cells were seeded in six-well cell culture plates. Twelve hours after seeding, cells were treated with the indicated concentration of CDDP. Twenty-four hours later, cells were harvested and stained with annexin V and 7-aminoactinomycin D (7-AAD) according to the manufacturer's protocol, followed by flow cytometric analysis. Apoptotic cells in tumor tissues were detected using an *In Situ* Cell Death Detection Kit according to the manufacturer's instructions. Luciferase Reporter Assay {#sec4.8} ------------------------- In brief, fragments of the DAB2 3′ UTR that were predicted to interact with miR-134-5p were amplified by PCR from human genomic DNA and inserted into the *Mlu*I and *Hind*III sites of the miRNA expression reporter vector. For the luciferase reporter experiments, the indicated cells were seeded into 24-well cell culture plates at a concentration of 1$\times$10^4^ cells per well. The next day, the cells were transfected with the indicated reporter plasmids expressing firefly luciferase. The Renilla luciferase plasmid was cotransfected as the transfection control. Cells were lysed 48 h after transfection, and luciferase activity was measured by the Dual-Luciferase assay system according to the manufacturer's protocol. Firefly luciferase activity was normalized to Renilla luciferase activity. Animal Experiments {#sec4.9} ------------------ Xenograft models were generated using A549 cells infected with expressing miR-134-5p or miR-134-5p inhibitor lentivirus. For the tumor growth assay, 2$\times$10^6^ of the indicated cells in 0.1 mL of PBS were subcutaneously injected into 6-week-old female nude mice (five mice per group). When the tumors reached a size of approximately 100 mm^3^, the mice were started on treatment with either PBS or CDDP (2 mg/kg body weight). The treatment was administered every other day. After 2 weeks, the mice were sacrificed and the tumors were weighed. For the lung metastasis experiments, a total of 1.5$\times$10^6^ of the indicated cells were suspended in 0.1 mL of PBS and injected into the lateral tail vein of 6-week-old female nude mice (six mice per group). Three weeks after injection, all mice were sacrificed, the lungs were embedded with paraffin and sliced for H&E staining, and tumor foci in the sections were counted. The mice were obtained from Shanghai Laboratory Animal Center, and all animal care and experimentation procedures were conducted according to the guidelines of the Institutional Animal Care and Use Committee of the Army Military Medical University. Statistical Analysis {#sec4.10} -------------------- All data are presented as the means ± SD, and significant differences between treatment groups were analyzed by a Student's t test or one-way ANOVA using GraphPad Prism. The recurrence-free survival rate of patients with stage I LUAD was calculated using a Kaplan-Meier survival analysis. Differences were considered statistically significant at p \< 0.05. Author Contributions {#sec5} ==================== C.-X.X. and D.W. designed the experiments; L.Z., P.H., and Q.L. performed experiments; all authors analyzed data; and C.-X.X wrote the paper. Conflicts of Interest {#sec6} ===================== The authors declare no competing interests. Supplemental Information {#appsec2} ======================== Document S1. Tables S1--S3 and Figures S1 and S2Document S2. Article plus Supplemental Information This study was supported by the Startup Fund for Talented Scholars of Daping Hospital and Research Institute of Surgery, Third Military Medical University (to C.-X.X) and the National Natural Science Foundation of China (81772495, to D.W). Supplemental Information can be found online at <https://doi.org/10.1016/j.omtn.2019.09.025>.

{#sp1 .508}

Background ========== Antifilaggrin autoantibodies (AFAs) are highly specific for rheumatoid arthritis and are probably involved in its pathophysiology. We showed that they are synthesised in the rheumatoid synovial membrane and that their target antigens in the tissue correspond to variants of the α and β chains of fibrin. The variants are generated by deimination, i.e. transformation of their arginine into citrulline residues. Deimination, mediated by a peptidylarginine deiminase (PAD) activity, generates the epitopes recognised by AFA/antifibrin autoantibodies. Four PAD isoforms (or types), have been identified and cloned in humans and rodents (mouse and rat). Expression of one or several of these isoforms has been reported in numerous tissues, but their targets are still poorly known. Objective ========= Since fibrin deimination occurs in the rheumatoid synovial tissue, we undertook to identify which PAD types are expressed in the tissue. Methods ======= By immunising rabbits with peptides situated in the most variable regions of the otherwise highly conserved PAD type sequences (three synthetic peptides per PAD), we produced antisera specific for each of the four PAD isoforms. The antisera were affinity-purified on the corresponding peptides. Each set of anti-peptide antibodies was confirmed to be specific for one isoform by immunoblotting on recombinant or purified PADs. Additional antisera or purified antibodies to whole human PADs II, III and V were used to confirm the results obtained with the anti-peptide antibodies. The synovial tissue from seven patients with rheumatoid arthritis was analysed. In all the tissues, the presence of deiminated proteins and particularly of deiminated fibrin was demonstrated by immunoblotting and/or immunohistology. Then low-salt extracts of the tissues were analysed by immunoblotting with all the immunological tools to PADs. Results ======= Expression of PAD type V was clearly detected in all seven patients. Expression of PAD type II was observed in six patients. No reactivities were observed with antibodies specific for PAD types I and III. Conclusion ========== Of the four PAD isoforms, only the types II and V are significantly expressed in the synovial tissue of patients with rheumatoid arthritis. Their respective roles in deimination of fibrin in the tissue remain to be determined.

Background {#Sec1} ========== Chronic obstructive pulmonary disease (COPD), one of the most prevalent health conditions, is the fourth leading cause of death worldwide \[[@CR1]\]. COPD morbidity and mortality have been increasing in many countries, in part due to an aging population world-wide \[[@CR2]\]. By the mid-21st century, the chance of living beyond 60 years will be 98 % in Japan/Oceania, 82 % in Western Europe, and 69 % in China \[[@CR3]\]. Relative to the 2011 world population, recent United Nations projections estimate that by 2100 the number of people aged \>60 years will triple, with an eightfold increase in those \>80 \[[@CR1]\]. However, although increases in life expectancy and the size of the elderly population during the past several decades might explain the current increase in COPD, the relationship may be more complex, including factors such as differential susceptibility to tobacco, anatomic and systemic differences, behavioral differences, and differences in response to available therapeutic modalities. Japan is a super-aged society, ranked first in the world. The late elderly (aged ≥75 years) accounted for 11.6 % of Japan's population in the 2010 national population census. In recent years pulmonary and primary care physicians in Japan have been more ready to diagnose and treat COPD patients of advanced age, and although it is known that COPD currently mostly affects middle-aged and elderly people, few studies have focused on how the features of COPD differ by age. In the past, COPD severity was simply classified based upon the % forced expiratory volume in 1 s (FEV~1~), because it was believed that the majority of patients followed a path of disease progression in which the severity of the disease tracked the severity of the airflow limitation \[[@CR4]\]. However, it has become clear in recent years that comprehensive assessment requires more than FEV~1~ measurement. The Global Initiative for Chronic Obstructive Lung Disease (GOLD) has proposed an assessment for COPD treatment based on the patient's level of symptoms and future risk of exacerbation, in addition to the severity of spirometric abnormality \[[@CR5]\]. Very elderly patients with COPD may present differently from younger ones; they may have a different pattern of comorbidities, and a different survival rate after acute exacerbations. Physicians may also have an age bias that may affect both diagnosis and treatment. The purpose of this study was thus to examine the characteristics of COPD patients aged 75 and over compared with those aged below 75. Methods {#Sec2} ======= Study populations {#Sec3} ----------------- Keio University and affiliated hospitals have established an observational COPD cohort, registered with the University Hospital Medical Information Network (UMIN000003470), for investigations of the management of COPD comorbidities \[[@CR6]\]. All patients were clinically stable without exacerbations for at least 1 month prior to study. The protocol was approved by the Ethics Committees of Keio University on July 29, 2009 (No. 20090008) and the affiliated hospitals, and written informed consent was obtained from each patient. Measurement of pulmonary functions {#Sec4} ---------------------------------- All participants underwent spirometry when in a stable condition during the baseline examination, according to ATS protocols using an electronic spirometer \[[@CR7]\]. Predicted values were derived from the guidelines for pulmonary function tests issued by the Japanese Respiratory Society \[[@CR8]\]. The classification of disease severity was based on GOLD spirometric grading \[[@CR2]\], Grade I: mild (FEV~1~/forced vital capacity (FVC) \<0.70, %FEV~1~ ≥80 %), Grade II: moderate (FEV~1~/FVC \<0.70, %FEV~1~ 50--80 %), Grade III: severe (FEV~1~/FVC \<0.70, %FEV~1~ 30--50 %) and Grade IV: very severe COPD (FEV~1~/FVC \<0.70, %FEV~1~ \<30 %). Assessment of clinical parameters {#Sec5} --------------------------------- At enrollment, a full medical and smoking history and current pharmacological treatment were obtained and clinical examinations were performed. Comorbid diagnoses were established using clinical history and examination findings, supported by a review of available medical records. The Hospital Anxiety and Depression Scale (HADS) was used, with a cut-off score of 11 points each for a probable status of anxiety or depression \[[@CR9]\]. Gastro-esophageal reflux disease (GERD) symptoms were evaluated using a self-reported Frequency Scale for the Symptoms of GERD (FSSG) questionnaire, consisting of 12 items, with a cut-off score of 8 points for GERD \[[@CR10]\]. Questionnaires on health-related quality of life (QOL) {#Sec6} ------------------------------------------------------ Each patient's health-related QOL was evaluated using three questionnaires. Two were disease-specific: the COPD Assessment Test (CAT) and St George's Respiratory Questionnaire (SGRQ) \[[@CR11]--[@CR14]\]. The CAT has been validated in Japan using the same population enrolled in the present study \[[@CR6]\]. The Medical Outcomes Study Short-Form 36-Item (SF-36) version 2 was also used to measure the patients' general health status \[[@CR15], [@CR16]\], and it was reported that COPD patients have SF-36 lower scores, representing worse health-related QOL \[[@CR17]\]. All of the questionnaires were completed by the patients themselves, at home, in the stable state. Evaluation of emphysema on CT scan {#Sec7} ---------------------------------- Quantitative High Resolution computed tomography (HRCT) analyses of emphysema were performed. Low-attenuation areas (LAAs) using a threshold level of −950 HU were determined using a Discovery CT 750HD CT system (GE Healthcare, Tokyo), adjusting the threshold on each model of CT scan using a CT scanner test object, the Multipurpose Chest Phantom N1 "Lungman" (Kyoto Kagaku, Kyoto, Japan) \[[@CR18]\] and calculated its percentage relative to the entire lung area (LAA%) using the workstation Lexus 64^®^ (AZE Ltd., Tokyo) \[[@CR19]\]. Echocardiographic evaluation {#Sec8} ---------------------------- Echocardiograms were obtained using two commercially available echocardiography systems (GE Vivid7/Vivid9, GE Healthcare, Horten, Norway and iE33/Sonos7500, Philips; Amsterdam, Netherlands). A 2.5-MHz transducer was used to obtain the images in the parasternal and apical views, corresponding to the standard long-axis, and two-chamber and four-chamber images, respectively. Standard two-dimensional and color Doppler data were collected. The estimated systolic pulmonary artery pressure (eSPAP) was calculated \[[@CR20]\], and pulmonary arterial hypertension was defined by an eSPAP ≥35 mmHg \[[@CR21]\]. Statistical analysis {#Sec9} -------------------- All data are expressed as mean, unless otherwise stated. Student's *t* test was performed to compare mean values between the two groups. Comparisons of data among the four patient groups were performed using analysis of variance (ANOVA), followed by a Tukey--Kramer post hoc analysis. Comorbidities were included as a categorical variable. A χ^2^ analysis was conducted to compare the frequencies between two groups. Relationships between quantitative data were examined using Spearman tests. Analysis of covariance (ANCOVA) were conducted to examine whether the slope of the relationship between two parameters differed between the patients aged \<75 and ≥75 years. P values less than 0.05 were considered significant. All data were analyzed using the JMP version 9.0.2 software for Windows. Results {#Sec10} ======= Demographic measures {#Sec11} -------------------- The mean age of the 443 COPD patients was 72.6 ± 8.2 SD years (range 43--91 years), and 92 % were males. The clinical characteristics of the subgroups of late-elderly patients (aged ≥75 years) and relatively younger patients (aged \<75 years) are tabulated (Table [1](#Tab1){ref-type="table"}). There was no significant difference between the two subgroups in smoking amount, BMI, proportion of the GOLD grades, and their treatment with medication. Current smokers were more prevalent in the younger patients.Table 1Demographic data of the COPD patients (n = 443)Age \<75 yearsAge ≥75 years*p* valueNo. of subjects252191Age (mean, years)66.980.0Sex (male/female)231/21175/16n.s.Smoking amount (mean, pack-year)56.955.6n.s.Current smoker (%)17.37.70.004BMI (mean, kg/m^2^)22.522.3n.s.VC (mean, ml)33652934\<0.001VC (mean, % predicted)93.893.5n.s.FEV~1~ (mean, ml)17171465\<0.001FEV~1~ (mean, % predicted)60.562.1n.s.GOLD grade (I/II/III/IV)52/114/67/1941/88/48/14n.s.LTOT (%)10.715.9n.s.CAT (mean)12.512.7n.s.SGRQ symptom (mean)36.537.9n.s.SGRQ activity (mean)39.747.50.001SGRQ impact (mean)18.322.30.04SGRQ total (mean)27.432.20.02Oral corticosteroids (%)3.23.6n.s.Inhaled corticosteroids (%)35.032.8n.s.Long-acting β2 agonists (%)42.048.8n.s.Long-acting muscarinic antagonists (%)64.058.3n.s.*BMI* body mass index, *VC* vital capacity, *FEV* ~*1*~ forced expiratory volume in 1 s, *GOLD* Global Initiative for Obstructive Lung Disease, *LTOT* long-term oxygen therapy, *CAT* COPD assessment test, *SGRQ* St George's Respiratory Questionnaire, *n.s.* not significant Numbers, mean (SD), and median (range) of age in GOLD grades I to IV were as follows; I: n = 93, 71.6 (8.2), 73 (49--89), II: n = 202, 72.9 (8.6), 73 (43--91), III: n = 115, 72.9 (7.7), 74 (51--89), IV: n = 33, 72.3 (7.9), 74 (54--89). There was no significant difference in age among the GOLD grades. Vital capacity (VC) and FEV~1~ by GOLD grade {#Sec12} -------------------------------------------- An individual's VC and FEV~1~ are expected to fall with increasing age, premised on the repeated findings that respiratory function worsens with age. In GOLD grades I--III, the VC and FEV~1~ values were significantly lower in the late-elderly patients compared to the younger patients, but among the GOLD grade IV patients there was no difference between the two groups (Fig. [1](#Fig1){ref-type="fig"}a, c). In contrast, the mean VC % predicted and FEV~1~ % predicted were not lower in the late-elderly patients compared to the younger patients when grouped by GOLD grade (Fig. [1](#Fig1){ref-type="fig"}b, d).Fig. 1Comparisons of VC and FEV~1~ values between COPD patients aged \<75 and ≥75 years with different grades of COPD. **a** VC, **b** %VC, **c** FEV~1~, **d** %FEV~1.~ *Gray columns* \<75 years old, *Black columns* ≥75 years old. Data are presented as mean ± standard deviation (SD). \*p \< 0.05 Age-related difference in the relationship between emphysema on CT scan and FEV~1~ {#Sec13} ---------------------------------------------------------------------------------- CT scans were performed on 246 patients enrolled at Keio University Hospital. Overall, there was a significant correlation between the degree of emphysema and %FEV~1~ (r = −0.453, p \< 0.0001), but in the patients aged \<75 years, the slope of the relationship between these two variables was significantly steeper than in patients aged \>75 (ANCOVA, *p* = 0.002) (Fig. [2](#Fig2){ref-type="fig"}).Fig. 2Correlation between %FEV~1~ and LAA % in each age group. **a** *Gray circles* \<75 years old, **b** *black circles* ≥75 years old. \*p = 0.002 comparison of the slopes between two different age groups Age-related difference in the prevalence of pulmonary hypertension {#Sec14} ------------------------------------------------------------------ Echocardiography was performed on 265 patients enrolled at Keio University Hospital, and the eSPAP was measurable for 179 of these patients (72.8 %). Among the GOLD grade II patients, the prevalence of possible pulmonary hypertension was significantly higher in the late-elderly patients compared to the younger patients. (28.2 vs. 7.5 %, *p* = 0.02) (Fig. [3](#Fig3){ref-type="fig"}a). After controlling for differences in LAA % between the two groups of patients, patients ≥ 75 years had a higher level of eSPAP (31.9 vs. 27.1 mmHg, *p* = 0.001) (Fig. [3](#Fig3){ref-type="fig"}b).Fig. 3Relationships between pulmonary hypertension and age. **a** Comparisons of prevalence of possible pulmonary hypertension (eSPAP ≥35 mmHg) between COPD patients aged \<75 and ≥75 years in different stages of COPD, \*p = 0.02, **b** comparisons of eSPAP between COPD patients aged \<75 and ≥75 years after controlling for differences in LAA %. Data are presented as mean ± SD. \*p = 0.001 Age-related difference in health-related QOL {#Sec15} -------------------------------------------- The CAT score increased (worsened) with worsening GOLD grade in the younger COPD patients (*p* \< 0.0001); this was largely due to the very high score in the younger patients in GOLD Grade IV. There was no overall significant difference in CAT score between GOLD grades in the late-elderly COPD patients (*p* = 0.15) (Fig. [4](#Fig4){ref-type="fig"}). Differences between elderly and younger patients were inconsistent across the GOLD grades. The same pattern seen with the CAT were also seen in with SGRQ scores (compare Fig. [4](#Fig4){ref-type="fig"} with Fig. [5](#Fig5){ref-type="fig"}), and with the generic questionnaire, the SF-36 (Additional file [1](#MOESM1){ref-type="media"}: Figure S1, Additional file [2](#MOESM2){ref-type="media"}: Figure S2).Fig. 4Total CAT scores of COPD patients aged \<75 and ≥75 years with different GOLD grades of COPD. *Gray columns* \<75 years old, *black columns* ≥75 years old. Data are presented as mean ± SD. \*p \< 0.05 between two different age groups. p \< 0.0001 comparison between four GOLD grade groups of \<75 years old, and ^†^p \< 0.05 by post hoc analysis between Grade IV vs. I, II, and III groupsFig. 5Comparisons of SGRQ scores between COPD patients aged \<75 and ≥75 years in different grades of COPD. **a** Symptom scores, **b** activity scores, **c** impact scores, **d** total scores. Data are presented as mean ± SD. \*p \< 0.05. p \< 0.001 comparison between four GOLD grade groups of \<75 years old, and ^†^p \< 0.05 by post hoc analysis between Grade IV vs. I, II, and III groups (**a**--**d**). p \< 0.001 comparison between four GOLD grade groups of ≥75 years old, and †p \< 0.05 by post hoc analysis between Grade IV vs. I and II groups (**b**), Grade IV vs. I group (**c**, **d**) Age-related variability in the introduction of long-term oxygen therapy (LTOT) {#Sec16} ------------------------------------------------------------------------------ There was no significant difference in the ratio of patients receiving LTOT between the younger and late-elderly COPD patients as a whole (Table [1](#Tab1){ref-type="table"}), although there was a pattern of more LTOT use in the late-elderly patients in GOLD Grades I-III, but significantly less in GOLD IV patients (Table [2](#Tab2){ref-type="table"}), perhaps because of the combined mortality impact of age, severe airflow limitation and severe hypoxia.Table 2Prevalence of LTOT among COPD patients \< 75 and ≥ 75 years oldPrevalence (%)*p* value\<75 years old≥75 years oldI1/52 (1.9 %)3/41 (7.3 %)n.s.II4/114 (3.5 %)13/88 (14.8 %)0.0043III7/67 (10.4 %)11/48 (22.9 %)n.s.IV14/19 (73.7 %)2/14 (14.3 %)0.002*n.s.* not significant Differences in the frequency of comorbidities by age {#Sec17} ---------------------------------------------------- Some comorbidities including hypertension (46.5 vs. 29.6 %, *p* \< 0.01), aortic aneurysm (6.6 vs. 1.7 %, *p* \< 0.05), prostatic hypertrophy (19.7 vs. 7.7 %, *p* \< 0.01), anemia (37.2 vs. 15.7 %, *p* \< 0.01), and cataract (64.1 vs. 34.8 %, *p* \< 0.01) were significantly more prevalent in the late-elderly compared to the younger patients (Table [3](#Tab3){ref-type="table"}).Table 3Prevalence of comorbiditiesPrevalence (%)*p* value\<75 years≥75 yearsTotalAsthma21.123.022.0n.s.Interstitial pneumonia5.410.97.8n.s.Lung cancer5.26.05.5n.s.Other malignancies17.625.721.20.05Anxiety8.35.67.1n.s.Depression7.913.510.20.07Hypertension29.646.537.0\<0.01Coronary artery disease9.414.211.5n.s.Arrhythmia9.912.010.8n.s.Chronic heart failure4.38.26.0n.s.Aortic aneurysm1.76.63.90.01Diabetes mellitus15.014.814.9n.s.Dyslipidemia17.615.916.8n.s.Hyperuricemia7.310.48.7n.s.Cerebral infarction5.27.76.3n.s.Chronic renal failure1.23.22.0n.s.Gastro-esophageal reflux disease34.931.133.3n.s.Peptic ulcer7.312.69.60.09Chronic sinusitis12.88.811.1n.s.Prostatic hypertrophy7.719.713.0\<0.01Liver dysfunction/Liver cirrhosis8.78.88.7n.s.Collagen disease2.11.71.9n.s.Anemia15.737.225.0\<0.01Osteoporosis15.223.718.90.06Cataract34.864.147.9\<0.01*n.s.* not significant Discussion {#Sec18} ========== Japan became a super-aged society before other countries, but this trend is present in all developed and in developing countries \[[@CR1]\]. The results of the present study suggest that COPD patients surviving to become late-elderly have a different pattern of lung function disturbance and emphysema to those who are younger. Although a strong correlation of emphysema on CT scan with spirometry was found in a large-scale cohort \[[@CR22]\], the severity of emphysema varies widely even among patients with the same grade of COPD \[[@CR19]\], our data suggests that age may be a factor in that variation. One of the problems in comparing different ages of COPD patients is the reliability of estimates of normal ranges for FEV~1~ in the late-elderly. VC and FEV~1~ are thought to decline linearly with age; this being the basis of equations for calculating predicted values based on age. In the present study, late-elderly COPD patients showed lower VC and FEV~1~ values compared to those in the younger patients, but they were classified in the same grade of COPD based on percentage of predicted values. These observations imply that VC and FEV~1~ were similarly reduced by age across the grades of airflow limitation in patients with COPD to those in the Japanese general population. The less steep slope of the relationship between emphysema and FEV~1~ shown in Fig. [2](#Fig2){ref-type="fig"} may be due to a healthy survivor effect (late elderly patients with severe airflow limitation being more likely to die), but relationships between the severities of airflow limitation and emphysema can be different in the late-elderly and younger patients with COPD. We compared health-related QOL using the CAT, SGRQ and SF-36 between the two groups at different levels of airflow limitation. Overall there was a weak but generally consistent trend towards patients with more severe airflow limitation having worse QOL. To the authors' best knowledge, no previous studies have focused on the age-related differences of health-related QOL in patients with COPD. Age negatively affects physical function, physical role limitations and general health \[[@CR23]\] and our study reports similar findings, although we have shown that late-elderly patients in GOLD IV have better health status than younger patients. This may reflect a healthy survival effect, since the proportion of late elderly patients GOLD IV who were on LTOT was much lower than in the younger patients with the same degree of airflow limitation. This conclusion was supported by the data on pulmonary hypertension, since for any given degree of airflow limitation, the late-elderly patients were more likely to have pulmonary hypertension, except in GOLD IV. We have previously reported that comorbid diseases such as depression, anxiety, and GERD may increase CAT scores \[[@CR3]\]. However, there was no difference in the prevalence of such comorbidities between the late-elderly and younger patients. In contrast age-related comorbid diseases including hypertension, aortic aneurysm, prostatic hypertrophy, anemia, and cataract were not associated with CAT scores in the present study (data not shown). One of the strengths of our cohort study is that the study population is a community-living sample. The patients enrolled provide a representative range of the socio-economic characteristics of COPD patients in Japan, registered at a general clinical practice, a university hospital or a related facility. Coincidentally, other COPD cohort studies conducted in different Japanese locality investigated participants with a similar average age \[[@CR24], [@CR25]\]. Indeed, COPD patients in Japan are typically elderly, and there might be a "healthy survivor effect" in their late seventies and beyond. We speculate that these older-aged COPD cohorts contain a higher proportion of people who, for whatever reasons, did not experience such serious or life-threatening comorbid problems as their younger peers. We have no reason to believe that the pattern seen in these COPD patients is a particularly Japanese phenomenon. As COPD is more prevalent at older ages, it represents an increasing problem for public healthcare worldwide. Few studies have reported the clinical features of COPD in very elderly patients \[[@CR26], [@CR27]\], and the need for more research into the impact of age on this growing subpopulation of COPD patients is urgent \[[@CR28]\]. Our study identified late-elderly COPD patients with moderate airflow limitation who manifested severe emphysema and/or pulmonary hypertension, and disabling dyspnea that were not usually complicating features of COPD patients with moderate airflow limitation for the younger patients. In part this may be because the conventional %FEV~1~ staging scheme may be misleading as a measure of severity in late-elderly patients \[[@CR29]\]. An alternative staging strategy that accounted for age-related changes in pulmonary function and variability in spirometric performance, i.e., the lambda-mu-sigma method, demonstrated that 28.1 % of patients with severe COPD were classified as moderate using the standard GOLD grades \[[@CR30]\]. One limitation of this study is the lack of age-matched healthy controls for comparison, although that is complicated by the fact that the elderly often live with one or more chronic conditions \[[@CR31]\]. Another limitation is that the objective quantification of emphysema severity and pulmonary hypertension was not assessed for all of the enrolled subjects, because we had to use different types of CT scanners and echocardiography systems in each affiliated hospital. Finally, this study mainly consisted of male patients (91.6 %), and further investigations are necessary to test whether our observations are applicable to female COPD patients. Conclusion {#Sec19} ========== The present study demonstrates that very elderly individuals present a different pattern of COPD than younger patients. This is possibly the result of different pathways of disease coupled with a healthy survivor effect. Assessments of COPD severity from a multidimensional perspective are essential for the appropriate attribution of symptoms and use of COPD-directed therapies. The results of the present study further strengthen the need for a better assessment of airflow limitation in late-elderly patients than current FEV~1~ prediction values and late-elderly COPD patients warrant specific attention in clinical practice. Additional files {#Sec20} ================ 10.1186/s13104-015-1810-8 Comparisons of SF-36 components between COPD patients aged \< 75 and ≥ 75 years in different stages of COPD. Data are presented as mean ± standard deviation (SD). \*p \< 0.05. a: Vitality, b: Physical functioning, c: Bodily pain, d: General health perceptions.10.1186/s13104-015-1810-8 Comparisons of SF-36 components between COPD patients aged \< 75 and ≥ 75 years in different stages of COPD. Data are presented as mean ± standard deviation (SD). \*p \< 0.05. a: Physical role functioning, b: Emotional role functioning, c: Social role functioning, d: Mental health. K-CCR : Keio COPD Comorbidity Research COPD : chronic obstructive pulmonary disease CT : computed tomography CAT : COPD assessment test SGRQ : St. George's Respiratory Questionnaire FEV~1~ : forced expiratory volume in 1 s UMIN : University Hospital Medical Information Network GOLD : Global Initiative for Chronic Obstructive Lung Disease FVC : forced vital capacity HADS : Hospital Anxiety and Depression Scale GERD : gastro-esophageal reflux disease FSSG : frequency scale for the symptoms of GERD QOL : quality of life SF-36 : Medical Outcomes Study Short-Form 36-Item HRCT : high resolution computed tomography LAAs : low-attenuation areas eSPAP : estimated systolic pulmonary artery pressure ANOVA : analysis of variance ANCOVA : analysis of covariance LTOT : long-term oxygen therapy MH participated in the design of the study and performed the statistical analysis, and was a major contributor in writing the manuscript. HN planned the study design and contributed to the interpretation of the results. TB conceived of the study and participated in its design and coordination and helped to draft the manuscript. MS, MM, and SC contributed to the collection of data and interpretation of results. ST and KA conceived of the study and participated in its design and coordination. PWJ contributed to the data analysis, interpretation of data, and editing of the manuscript. All authors read and approved the final manuscript. Acknowledgements {#FPar1} ================ We thank Chiyomi Uemura for her contribution to the collection of data. This study was funded by GlaxoSmithKline. We also thank the following members for their participation in the Keio COPD Comorbidity Research (K-CCR) group: Hidefumi Koh (Saiseikai Utsunomiya Hospital), Fumio Sakamaki (Tokyo Saiseikai Central Hospital), Takeshi Terashima and Tatsu Matsuzaki (Department of Respiratory Medicine, Tokyo Dental College), Morio Nakamura and Keishi Tsuduki (Eiju General Hospital), Naoto Minematsu (Hino City Hospital), Koichi Sayama (Kawasaki City Hospital), Takashi Inoue (Sano Public Welfare General Hospital), Naoki Miyao (Nihon Kokan Hospital), Kazumi Nishio (Kawasaki City Ida Hospital), Hiroki Tateno (Saitama City Hospital), Yoshitaka Oyamada and Shuichi Yoshida (Tokyo Medical Center), Toru Shirahata (Saitama Medical Center), Akira Umeda (International Medical Welfare College Shioya Hospital). Competing interests {#FPar2} =================== TB discloses having received honoraria/paid expert testimony and her university having received research grants from GlaxoSmithKline. PWJ discloses that his university has received honoraria and research grants from GlaxoSmithKline. The other authors declare that they have no competing interests.

Introduction {#s1} ============ Dehydration of lactic acid remains a challenge because of the technical and chemical hurdles it represents (Bonnotte et al., [@B6]). While some progress has been achieved in terms of selectivity, especially with phosphate catalysts, the various mechanisms leading to the formation of undesired by-products such as acetaldehyde remain a subject of debate (Holmen, [@B22]). Generally, whether studying zeolites or phosphates as catalytic systems, it is admitted that, unlike conventional dehydration (catalyzed in the gas phase) often carried out on relatively acidic catalysts, dehydration of the lactic acid requires a very fine association between acidic and basic sites (acid-base pairs) (Holmen, [@B22]; Bonnotte et al., [@B6]). Catalytic dehydration reaction of lactic acid to acrylic acid was thoroughly studied not only in the gas phase, but also in water under supercritical and subcritical conditions (Mok et al., [@B30]; Hatada et al., [@B20], [@B19]; Aida et al., [@B1]) or even indirectly (acetoxylation of the lactic acid to *2*-acetoxypropionic acid which is then pyrolyzed to acrylic acid) (Beerthuis et al., [@B3]). However, whether in the gas or liquid phase, several parallel, and secondary reactions limit the selectivity to acrylic acid. Among them, decarboxylation or decarbonylation of lactic acid to acetaldehyde are particularly limiting. In addition, hydrogen from the decarboxylation reaction of lactic acid can induce side reactions such as reduction of lactic acid or acrylic acid to propionic acid. Formation of acetaldehyde is favored over the formation of acrylic acid with activation energies of 115 and 137 kJ.mol^−1^, respectively, according to the calculations of Wadley et al. ([@B47]). The formation of acetaldehyde is often attributed to the presence of medium and strong acid sites on the catalyst (Katryniok et al., [@B23]). The other products (*2,3*-pentanedione, acetic acid, and sometimes lactide and hydroxyacetone) are generally formed in smaller amounts. As aforementioned, due to the parallel or secondary reactions, it is thus difficult to obtain high yields of acrylic acid, and the catalyst therefore plays a key role in the orientation of the selectivities. Several types of catalysts such as zeolites or basic oxides have been largely tested in the catalytic gas-phase dehydration of lactic acid (Bonnotte et al., [@B6]). However, while hydroxyapatites (HAPs) behave both acid and basic sites, they have not been so much studied in this reaction. HAPs are a class of solids apart from phosphates, because of their structure and versatility in composition. As a main constituent of bones and teeth, this material has also been studied for a long time in the field of medicine for several applications such as bone integration (Zhou and Lee, [@B53]), dental implants (Kurashina et al., [@B24]), or drugs (Zhang et al., [@B51]). It also quickly became a subject of study for their application in basic or bi-functional catalysis, in particular in the synthesis of heavy alcohols with the Guerbet reaction (Tsuchida et al., [@B43]), the Knoevenagel condensation reaction (Sebti et al., [@B38]), the Michael addition (Gruselle et al., [@B16]), or for dehydration (Lan and Zhang, [@B26]), oxidation (Zhao et al., [@B52]) or dehydrogenation reactions (Hara et al., [@B18]). HAPs have the general chemical formula Ca~5~(PO~4~)~3~OH, but are generally described by the Ca~10~(PO~4~)~6~(OH)~2~ formula, which actually represents two molecules contained in the crystalline pattern (crystalline symmetry, space group 6/m) (Charlton et al., [@B7]). The composition of the hydroxyapatites is extremely variable, because not only they are rarely stoichiometric (Ca/P = 1.67), but also because each constitutive element can be, to a certain extent, substituted without losing the crystalline structure (Ben Osman, [@B4]): Ca^2+^ ions can be replaced by mono and divalent cations such as Sr^2+^, Ba^+^, Pb^2+^, Mg^2+^, Zn^2+^, and Na^+^. $\text{PO}_{4}^{3 -}$ groups may be substituted with $\text{CO}_{3}^{2 -}$, $\text{AsO}_{4}^{3 -}$, $\text{SiO}_{4}^{4 -}$, $\text{VO}_{4}^{3 -}$, $\text{SO}_{4}^{2 -}$, and $\text{HPO}_{4}^{2 -}$. Similarly, the OH^−^ anions can be replaced by halogenic anions: F^−^, Cl^−^, Br^−^, or I^−^. These compositional variations obviously bring a very strong impact on the acidic and basic properties of solids. They are mainly responsible for modifications of sites\' density but a modification of their strength is also observed, though to a lesser extent. Therefore, the main objective of this work was to study the influence of the OH^−^ ions substitution by F^−^ on the chemical, physical and catalytic properties of hydroxyapatites. The synthetized materials were characterized using different physical and chemical techniques and tested in the gas phase dehydration of lactic acid to acrylic acid. Experimental {#s2} ============ Materials --------- Several solids were synthesized. The protocols for the preparation and synthesis of hydroxyapatites are described in details in the next sub-section. The reagents used for each synthesis were calcium nitrate hexahydrate (\[Ca(NO~3~)~2~·6H~2~O\], Sigma Aldrich), diammonium phosphate (\[(NH~4~)~2~HPO~4~\], WVR), an aqueous solution of ammonia (28 wt.%, NH~4~OH, Sigma Aldrich). The various substituents used were: ammonium fluoride and chloride (\[NH~4~F\], \[NH~4~Cl\], Sigma Aldrich), zinc, potassium and calcium nitrates (\[Zn(NO~3~)~2~, 6H~2~O\], KNO~3~, NaNO~3~, Sigma Aldrich). Lactic acid, acrylic acid, acetaldehyde and propionic acid were purchased from Sigma Aldrich. All reactants were of analytical grade and used as received. Catalysts Preparation --------------------- Among the different possible synthetic routes, the co-precipitation method was used in the present study. Depending on the desired composition of the hydroxyapatite, various adaptations of pH, temperature, reagents concentrations and maturation times were used. Two aqueous solutions containing the main reagents: the first solution (between 300 mL and 1 L) containing diammonium phosphate (and optionally an anionic substituent) and the second solution containing calcium nitrate (and optionally a cationic substituent) were prepared. The first solution was heated to the given temperature (65°C ≤ *T* ≤ 80°C) under magnetic stirring (600 rpm), and the pH was adjusted at a value of 9 or 10 with a peristaltic pump for injecting the aqueous solution of ammonia. The second solution was then added drop wise to the first solution (which took between 45 min and 2 hours), using a dropping funnel. During this process, the pH was maintained at the initial value set by adding controlled volume of the aqueous solution of ammonia. The solution was then kept under stirring at a given temperature for a time ranging from 2 to 6 h. After maturation, the formed solid was recovered by filtration on a vacuum flask equipped with a Buchner filter. After evacuation of the solution, the wet solid was washed several times with two liters of hot distilled water. As-obtained solid was then placed in an oven at 100°C overnight. Finally, the solid was grounded before calcination between 400 and 600°C, in a muffle furnace under static air for 2 h. Characterization ---------------- ICP-OES analyzes were carried out on an AGILENT 700 ICP-OES within the REALCAT platform applied to high throughput screening. The carbon content analyzes were carried out at the CNRS Central Analysis Service in Villeurbanne using a MITSUBISHI oxidizing combustion device coupled to DIONEX ion exchange chromatography. The specific surface area (SA \[m^2^.g^−1^\]) of the catalysts was evaluated using adsorption/desorption of nitrogen on a MICROMERITICS ASAP 2000 analyzer after degassing during 3 h at 300°C. SA was determined using the Brunauer-Emett-Teller (BET) calculation method using the isotherm obtained at 77 K. The crystalline phases were identified thanks to the X-Ray Diffraction (XRD) technique on a BRUKER AXS D8 Advance diffractometer configured in Bragg-Brentano geometry, equipped with a LynxEye Super linear detector and a CuKα X-ray source (λ = 1.5406 Å). The patterns were recorded at room temperature, with values of 2θ between 10 and 80°, and with a pitch of 0.02° and an acquisition time of 0.5 s. The lattice parameters were determined using the Le Bail method and JANA2006 Software (Petricek et al., [@B35]). The surface composition (about 8 nm in depth) was determined using the X-ray Photoelectron Spectrometry (XPS) technique using a KRATOS ANALYTICAL Axis Ultra DLD spectrometer equipped with a monochromatic AlKα source (*h*υ = 1486.6 eV; 10 mA, 12 kV). The acquisition of the spectra was carried out with charge compensation, in a 10^−9^ mbar analysis chamber with a 40 eV passing energy for the high resolution and 160 eV for over-flights. The spectra were recalibrated using as a reference the C-C/C-H component of the C1s level set at 284.8 eV. The acidity of the catalysts was evaluated using the ammonia-programmed temperature desorption (TPD-NH~3~) technique. The analysis was carried out thanks to a device allowing successive injection of calibrated loops of ammonia until saturation of the sample (*ca*. 50 mg) pretreated under helium at its initial calcination temperature (2 h, 10°C.min^−1^) in a quartz reactor. The adsorption was carried out between 40 and 130°C and desorption up to 600°C with a ramp of 10°C.min^−1^. The quantity of ammonia at the outlet of the reactor was evaluated by means of an ALPHAMOS gas chromatography (GC) equipped with a thermal conductivity detector (TCD). The study of the adsorption of propyne followed by Fourier transform infrared transmission allowed us to study the acid and basic surface properties of our samples. Prior to analysis, these latter were finely ground and then pressed into self-supporting pellets using a hydraulic pressure press operating at 10^6^ Pascals. The as-obtained pellets were then transferred to the device described in [Figure S1](#SM1){ref-type="supplementary-material"}. After treatment at 400°C (samples\' calcination temperature, 2 h, 5°C.min^−1^), the pellet was returned to room temperature and the cell was evacuated at a pressure of about 5.10^−2^ mbar. A reference spectrum was then recorded on a THERMO Nicolet 490 Fourier transform transmission infrared spectrometer between 400 and 4,000 cm^−1^ (resolution of 1 cm^−1^). Before lowering the pellet to the KBr window, a so-called "*background"* spectrum was acquired and subtracted from the recorded spectra. A volumetric volume section (2.124 cm^3^) enabled controlled pressure calibrated additions (\~1.3--13 mbar) of propyne. When the pellet was saturated (spectrum shape no longer evolving), the cell was evacuated for 10 min and a last spectrum was recorded. Catalytic Tests --------------- The catalytic dehydration of lactic acid was performed in a typical fixed bed reactor using the procedure described hereafter. The reactor was loaded with the catalyst and heated to the reaction temperature during the night before the test. The outlet lines were also heated to 190°C. The stabilization of the flow at the reactor inlet required at least 7 h; the "by-pass" section was placed under helium flow (between 15 and 30 mL.min^−1^), and the lactic acid solution was injected (between 20 and 50 wt.% in lactic acid, with a flow rate between 0.025 and 0.05 mL.min^−1^). Analysis of the products was carried out off-line, with a column adapted to all products resulting from the reaction: a semi-capillary column ZB-WAX-Plus (ZEBRON, PHENOMENEX) of 30 m, 0.53 mm external diameter and 1 μm film thickness (polyethylene glycol). The temperature and time of the reaction were fixed depending on the performed analysis. Results and Discussion {#s3} ====================== Physical and Chemical Properties -------------------------------- In this study, 4 different hydroxyapatites were considered. In the case of the standard stoichiometric hydroxyapatite ([Table 1](#T1){ref-type="table"}, Entry 1) the final ICP Ca/P ratio is relatively close to the ratio imposed by the quantities of reagents used during the with a value of 1.62, thus slightly lower than the theoretical one of 1.67. In the case of fluorapatites, the theoretical fluorine substitution rates of 0.5, 1 and 2 correspond to fluorapatite Ca-XAP-F-OH ([Table 1](#T1){ref-type="table"}, Entry 2), Ca-XAP-F2 ([Table 1](#T1){ref-type="table"}, Entry 3) and Ca-XAP-F4 ([Table 1](#T1){ref-type="table"}, Entry 4), respectively. The Ca/P ratio increased with the increase of the F content in the materials and was equal or higher than the theoretical one. ###### Catalytic materials. **Entry** **Catalysts[^\*^](#TN1){ref-type="table-fn"}** **Preparation conditions** **ICP Ca/P ratio** **XPS Ca/P ratio** **BET SA (m^**2**^.g^**−1**^)** **%F** ----------- ------------------------------------------------ ---------------------------- -------------------- -------------------- --------------------------------- -------- 1 Ca-HAP-S pH 10, *T* = 65°C 1.62 \- 94.5 \- 2 Ca-XAP-F-OH pH 9, *T* = 65°C 1.66 1.50 79.4 1.60 3 Ca-XAP-F2 pH 9, *T* = 65°C 1.70 1.49 64.6 2.97 4 Ca-XAP-F4 pH 9, *T* = 65°C 1.73 1.54 53.7 4.15 *Theoretical Ca/P ratio was 1.67 in all cases*. Nitrogen adsorption/desorption isotherms of all the samples (not shown here) were of the IIb type (H3 type hysteresis) according to the IUPAC classification. This type of isotherm is characteristic of non-porous or macroporous materials. The SAs are listed in [Table 1](#T1){ref-type="table"}. Such relatively low SAs (\<100 m^2^.g^−1^) are due to relatively low porosities of these materials because of the presence of rod aggregates or irregular grains that represent the most common morphologies observed for HAPs prepared by precipitation (Rodríguez-Lorenzo et al., [@B37]; Roche and Stanton, [@B36]). It is generally observed that SA tends to decrease with the increase of the fluorine substitution extent when the samples are calcined at 900°C (Rodríguez-Lorenzo et al., [@B37]; Roche and Stanton, [@B36]). In the present work a calcination temperature of 400°C was used and we cannot thus directly compare the values between both studies. However, they are consistent with other data reported in the literature even if the methods of preparation significantly differ (Matsuura et al., [@B28]; Silvester et al., [@B40]). We observed a clear---and progressive---SA drop, with a SA of 94.5 m^2^.g^−1^ between the reference hydroxyapatite ([Table 1](#T1){ref-type="table"}, Entry 1) and 53.7 m^2^.g^−1^ for fluorapatite containing the highest fluorine content (57%, [Table 1](#T1){ref-type="table"}, Entry 4). It is interesting to note that these observations go against those observed upon evolution of SA according to the Ca/P ratio (Tsuchida et al., [@B44]; Lamonier et al., [@B25]). Hydroxyapatites crystallize generally in a system very close to the hexagonal bipyramidal one (P63/m group) (Sudarsanan et al., [@B41]; Elliott et al., [@B13]). The fluorapatites can thus be represented in a compact hexagonal structure, alternating layers of PO~4~-tetrahedrals comparable to spheres of radius \~ 2.6 Å alternating in a ABABABAB feature as shown in [Figure 1](#F1){ref-type="fig"} (each blue or gray sphere represents an $\text{PO}_{4}^{3 -}$ ion). ![**(A)** Representation of ABABAB alternating layer stacks in a compact hexagonal arrangement. **(B)** Representation along the *c*-axis of a compact hexagonal structure. **(C)** Representation along the *c* axis of an elementary cell of a compact hexagonal system (Elliott et al., [@B13]).](fchem-08-00421-g0001){#F1} Depending on their size, ions like F^−^, Cl^−^, or OH^−^ occupy different positions along the *z*-axis passing through the center of triangles formed by Ca(II), or in the center of the triangle itself (*z* = 1/4 or *z* = 3/4) either when the size of the ion increases to an intermediate position between *z* = 1/4 and *z* = 1/2 or *z* = 3/4 and *z* = 1. The obtained diffractograms allowed to validate the formation of the apatite phase. The series of diffractograms ([Figure 2](#F2){ref-type="fig"}) has well-defined peaks demonstrating the presence of a single apatite phase, and not an agglomeration of hydroxyl and fluorapatite phases. On the other hand, concerning the fluorapatite Ca-XAP-F4 ([Figure 2](#F2){ref-type="fig"}) the XRD evidenced the presence of a mixture of fluorapatite and calcium fluoride (fluorite). {#F2} Moreover, the cell parameter values reported in [Table 2](#T2){ref-type="table"} are consistent with those in the literature, particularly considering the work of Sudarsanan et al. who established that the cell parameters for a natural or synthetic fluorapatite are: *a* = 9.367 Å and *c* = 6.884 Å (Sudarsanan et al., [@B41]). These parameters are very close to those of Ca-XAP-F2 (although not all OH^−^ ions were substituted in that case). Moreover, the trend observed in [Table 2](#T2){ref-type="table"} of a decrease in the *a* parameter is also in good agreement with the observation of other teams (Rodríguez-Lorenzo et al., [@B37]; Yao et al., [@B49]). Indeed, progressive substitution of OH^−^ ions by F^−^ ions causes a decrease in the *a* value directly correlated to the introduced amount of F^−^: as the phosphate atoms are not bonded to the Ca(I) atoms organized along the *z* axis, there will be no (or little) influence on the size of the cell in this direction. On the other hand, the F^−^ species located at the centers of the triangles formed by the Ca(II) ions will interact more strongly, by their electronegativity on these calcium ions than on the OH^−^ ions, inducing a "*compaction"* and thus a decrease in the size of the cell. ###### Cell parameters determined from XRD. **Catalyst** ***a* (A)** ***c* (A)** -------------- ------------- ------------- Ca-HAP-S 9.417 6.884 Ca-HAP-F-OH 9.396 6.889 Ca-XAP-F~2~ 9.368 6.889 Ca-XAP-F~4~ 9.359 6.885 NH~3~ desorption profiles showed in [Figure 3](#F3){ref-type="fig"}. They illustrate the decrease in the amount of acidic sites of fluorapatites with the gradual incorporation of fluorine. The total amount of NH~3~ desorbed clearly depends on the quantity of F incorporated to the catalyst ([Table 3](#T3){ref-type="table"}). The highest the amount of F, the lowest the quantity of desorbed NH~3~. This decrease can also be linked to the increase in the Ca/P ratio following the introduction of F. Indeed, it was observed very often in the literature that the acid/base properties of HAPs are highly, if not exclusively, depending on the Ca/P ratio (Ghantani et al., [@B15]; Bonnotte et al., [@B6]). It is quite obvious from [Figure 4](#F4){ref-type="fig"} that one cannot distinguish which element of composition actually induces this change in the acidic properties of the fluorapatites, as the Ca/P surface ratio is directly linked with the surface atomic percentage of F. {#F3} ###### Quantities of NH~3~ desorbed during the TPD-NH~3~ study. **Catalyst** **NH~**3**~ \[μmol\*g^**−1**^\]** **NH~**3**~ \[μmol\*m^**2**^\]** -------------- ----------------------------------- ---------------------------------- Ca-HAP-S 360 4.18 Ca-HAP-F-OH 394 4.20 Ca-XAP-F~2~ 233 3.29 Ca-XAP-F~4~ 198 3.65 {#F4} In addition, since ammonia adsorbs indiscriminately on Lewis and Brønsted acid sites, the above experiment does not able to determine which population of these sites is actually involved. The main acid sites present in hydroxyapatites are believed to be calcium ions (Lewis acid sites). The Ca/P ratios in the studied catalysts present an over-stoichiometry, it can be assumed that, for reasons of balance of charges, the solid incorporates type B carbonates. The final formula of the fluorapatites would then be Ca~10−*x*~(PO~4~)~6−*x*~(CO~3~)~*x*~(OH)~2−y−0.5*x*~F~y−0.5*x*~, which can explain the increase in the Ca/P ratio with the substitution of $\text{PO}_{4}^{3 -}$ groups by $\text{CO}_{3}^{2 -}$ ions and the creation of OH^−^ and/or F^−^ vacancies. These latters may be considered as Lewis sites, as previously suggested by a work published by some of the present authors (Silvester et al., [@B40]). As the materials were synthetized under room atmosphere, it is therefore highly possible that carbonate ions are integrated into fluorapatites thanks to the dissolution of atmospheric CO~2~ in the basic solution. Thus, several acid sites can be distinguished: 1. -- Ca^2+^: Although some authors identify calcium as a potential Lewis acid site such as Silvester ([@B39]) and Silvester et al. ([@B40]), who identified by them using XPS after adsorption of a probe molecule, *2*-phenylethylamine, other teams struggle to identify it by adsorption of other molecules such as CO (Diallo-Garcia et al., [@B10],[@B11]). More recently, the team of Hill et al. studied adsorption followed by DRIFTS of probes, with CO~2~ for basicity, pyridine and CO for acidity, and acetylene, and glycine for amphoteric properties on a stoichiometric hydroxyapatite. They showed the presence of very weak Lewis acid sites, and attributed this low Lewis acidity to Ca^2+^ species (Hill et al., [@B21]). Moreover, several authors demonstrated the adsorption of lactic acid on Ca^2+^ of HAPs, like authors specifically studying lactic acid dehydration to acrylic acid (Ghantani et al., [@B15]; Yan et al., [@B48]) or authors studying lactic acid adsorption on hydroxyapatites containing zinc (Turki et al., [@B45]); 2. -- $\text{HPO}_{4}^{2 -}$: These Brønsted acid sites are generated when the Ca/P ratio is over 1.67 \[Ca~10−*x*~(HPO~4~)~*x*~(PO~4~)~6−*x*~(OH)~2−*x*~, *n*H~2~O\]. In the presence of fluorine, the Ca/P ratio increasing, one would decrease the quantity of these sites, alongside with the quantity of vacancies \[^δ+^\]; 3. -- OH^−^/F^−^ \[^δ+^\] deficiencies: These deficiencies are also generated during under-stoichiometric calcium conditions and their presence is therefore linked to the presence of $\text{HPO}_{4}^{2 -}$. The literature on propyne adsorption followed by infrared is relatively scarce. A few studies dealing with propyne (Thomasson et al., [@B42]; Hackler et al., [@B17]; Mordenti et al., [@B31]; Chizallet et al., [@B8]; Michalska et al., [@B29]; Valange et al., [@B46]; Moulin et al., [@B32]) or acetylene (Thomasson et al., [@B42]; Hill et al., [@B21]) adsorption on MgO, La~2~O~3~, ZrO~2~, CaHAP, CaO on MOF(X) (Metal-Organic Framework containing halogens). Propyne has a C≡ C triple bond able of interacting with an A^LB+^ (Lewis or Brønsted acid site) and a ≡C-H acetylenic proton able of interacting with a basic B^LB−^ site (Lewis or Brønsted basic site). This allows several adsorption modes, including a dissociative one. The various adsorption modes are summarized in [Figure 5](#F5){ref-type="fig"}. ![Non-dissociative (1 to 3) and dissociative (4) adsorption modes of propyne (Mordenti et al., [@B31]; Chizallet et al., [@B8]; Moulin et al., [@B32]).](fchem-08-00421-g0005){#F5} [Figure S2](#SM1){ref-type="supplementary-material"} shows the gas phase propyne FTIR spectrum. It presents stretching bands of C≡C and ≡C-H bonds reported in [Table S1](#SM1){ref-type="supplementary-material"}. In addition, [Figure S3](#SM1){ref-type="supplementary-material"} shows all the spectra obtained after the addition of 6.5 μmol of propyne as well as the spectra after evacuation under vacuum for 10 min. Each spectrum presented results from the subtraction between the measured spectrum and the reference spectrum of the pellet recorded before propyne addition. The presence of fairly intense bands in the υ~(≡C−H)~ region and low intensity bands in the υ~(C≡C)~ region can be observed. Such bands almost completely disappeared after evacuation. On the other hand, it is also worth to note the appearance of wide bands centered around 1,640 cm^−1^ and bands in the OH region around 3,700 cm^−1^ which are exalted after evacuation. These bands could be due to the presence of water adsorbed on the surface, as also evidenced by the shoulder in the 2,800--3,700 cm^−1^ range ([Figure S3](#SM1){ref-type="supplementary-material"}). A glance at [Figure S4](#SM1){ref-type="supplementary-material"} and [Table S3](#SM1){ref-type="supplementary-material"} suggests that the υ~(≡C−H)~ band is moving less and less strongly toward red with the presence of fluoride ("Δ" shift of −48 cm^−1^ for Ca-XAP-F4 to −54 cm^−1^ for Ca-XAP-F-OH). In addition, the intensity of the bands after evacuation decreases as the amount of fluorine increases, and the gas phase propyne band υ~(≡C−H)~ can also be observed for Ca-XAP-F2 and Ca-XAP-F4. However, we must take into account the smaller specific surface areas of these samples compared to Ca-HAP-S and Ca-XAP-F-OH. It should also be noted that the significant increase in the intensity of the band to 2,978 cm^−1^ attributed to υ~(−CH3)~ is difficult to explain. Two groups of samples can be distinguished in [Figure S5](#SM1){ref-type="supplementary-material"} and corresponding [Table S4](#SM1){ref-type="supplementary-material"}: Ca-HAP-S and Ca-XAP-F-OH on one side, and Ca-XAP-F2 and Ca-XAP-F4 on the other side. The first group of samples mainly shows a band between 2,108 and 2,112 cm^−1^, which represents an average shift of −31 cm^−1^, which further increases in intensity. Again, under evacuation, the intensity of this band decreases sharply, suggesting a weak interaction with the surface. It is relatively difficult to decide on the value of the displacement of this band and its associated adsorption mode when referring to the literature. For example, Chizallet et al. who studied propyne adsorption on dehydroxylated and bare magnesium oxide surfaces (pellets treated at 750°C) observed a displacement of υ~(C≡C)~ of Δ = −50 cm^−1^ (Chizallet et al., [@B8]) while Huber *et al*. who also studied magnesium oxide surfaces (pellets treated at 750°C) observed a Δ shift of −19 cm^−1^ (Bailly et al., [@B2]). The first team assumed the presence of species adsorbed in associative mode 3 ([Figure 5](#F5){ref-type="fig"}), but they mentioned that propyne is probably dissociated at these sites, based on the formation of a new band in the OH zone (3,441 cm^−1^), which is disturbed by additional increments in the amount of introduced propyne. On the other hand, the second team assigned the two observed bands at 2,123 cm^−1^ (Δ = −19 cm^−1^) and 2,094 cm^−1^ (Δ = −48 cm^−1^), respectively, to modes 1 and 2 ([Figure 5](#F5){ref-type="fig"}). For mode 1, the ≡C-H bond is highly perturbed and the authors associate it with a band located at 3,280 cm^−1^, a band that is also observed in the present study. On the other hand, the shift observed in the C≡C zone being higher than that observed by Huber et al., it suggests a type 3 adsorption mode. In addition, the team of Valange et al. obtained results quite similar to ours, by studying the adsorption of propyne on lanthanum oxides with carbonates on their surface, like in our case. They propose the adsorption scheme of [Figure 6](#F6){ref-type="fig"} (Valange et al., [@B46]). To this type of adsorption (mode 3), they combine a υ~(≡C−H)~ band at 3,250 cm^−1^ (Δ = −84 cm^−1^) and a υ~(C≡C)~ band at 2,115 cm^−1^ (Δ = −27 cm^−1^). It was therefore assumed that this was also the main mode of adsorption of propyne present in this work. However, the presence of a shoulder at 2,082 cm^−1^ (in case of Ca-HAP-S) revealing a band as the amount of fluorine increases (2,086 cm^−1^ band for Ca-XAP-F4), suggests the generation of more acidic adsorption sites for the C≡C bond. Note also the presence of a low intensity band at 2,054 cm^−1^, close to that observed by Chizallet et al. at 2,045 cm^−1^ (Chizallet et al., [@B8]). The presence of these sites could lead to propyne dissociation, stabilized in propynide ion and OH^−^ ion (adsorption mode 4), as reported for magnesium oxide (Chizallet et al., [@B8]) or zinc oxide (Nakajima et al., [@B33]). Therefore, a close study of the OH^−^ zone in order to identify the possible presence of new bands, potentially resulting from the dissociative adsorption of propyne was carried out ([Figure S6](#SM1){ref-type="supplementary-material"}, [Table S5](#SM1){ref-type="supplementary-material"}). New bands appeared in the OH^−^ zone between 3,667 and 3,720 cm^−1^, thus in an area identified by Diallo-Garcia et al. as P-OH bands for hydroxyapatite (Diallo-Garcia et al., [@B10]). These bands increased in intensity with the addition of propyne and the presence of fluorine in the material. The most intense and best-defined bands are thus visible for the Ca-XAP-F4 spectrum. As propyne additions progress, these bands showed a red shift until they are evacuated where they return to their starting position as when propyne was first added. These bands are also very intense and, unlike the bands attributed to propyne, they are at their maximum intensity after evacuation for each apatite. Finally, the other bands associated with OH^−^, as described in [Figure 7](#F7){ref-type="fig"}, are not---or only slightly---affected by propyne successive additions. ![Adsorption scheme based on IR band allocations for propyne adsorption on lanthanum oxides (Valange et al., [@B46]).](fchem-08-00421-g0006){#F6} {#F7} It could therefore be assumed that propyne dissociation occurs with successive additions of propyne, resulting in the formation of different POH species on the apatite surface. It is also possible to highlight the disruption of the bands associated with phosphates, which implies them as at least one of the adsorption sites of propyne ([Figure S7](#SM1){ref-type="supplementary-material"}). However, the possible dissociation of propyne can be still a matter of debates, as it has never been reported on apatites so far. In addition, in similar studies with acetylene, this dissociation has not been observed (Diallo-Garcia et al., [@B11]; Hill et al., [@B21]). Finally, there remains an area of the spectrum around 1650 cm^−1^ for which an evolution of the bands similar to that observed in the OH region is observed. The formation of these OHs would be linked to the formation of another adsorbed species, evidenced by a wide and intense band ([Figure S8](#SM1){ref-type="supplementary-material"}, [Table S6](#SM1){ref-type="supplementary-material"}). The bands in [Figure S8](#SM1){ref-type="supplementary-material"} increase in intensity with successive additions of propyne before evacuation. During the addition of propyne, only the band centered around 1,635 to 1,645 cm^−1^ is visible. After evacuation, new bands appear only for Ca-HAP-S, at 1,697, 1,599, and 1,576 cm^−1^. It is difficult to interpret the origin of these bands because they do not correspond to any specific or expected bands. Two hypotheses can be formulated: 1. -- Formation of carbonates or hydrogenocarbonates: In their work on the identification of acid-base pairs, Diallo-Garcia et al. studied CO~2~ adsorption followed by infrared and observed CO~2~ reactive adsorption (Diallo-Garcia et al., [@B10]). This latter indeed reacts with the surface OH^−^ to form $\text{CO}_{3}^{2 -}$ or $\text{HCO}_{3}^{-}$ ions and water according to the following reaction: CO~2~ + 2OH^−^ = $\text{CO}_{3}^{2 -}$ + H~2~O. Note that some CO~2~ was identified in the present work in the gas phase propyne IR spectrum (see [Figure S2](#SM1){ref-type="supplementary-material"} and [Tables S1, S2](#SM1){ref-type="supplementary-material"}). The formation of water could also explain the disturbance remaining after evacuation between 3800 and 2500 cm^−1^ as well as the intense and wide band centered around 1640 cm^−1^, as identified by Diallo-Garcia ([@B9]) by FTIR with non-heat treated apatites. However, one can then wonder why the formation of water would be more important on Ca-XAP-F4, which has by far the most intense band around 1640 cm^−1^, whereas this fluorapatite is not supposed to contain OH^−^ or very little. The bands observed for Ca-HAP-S at 1697, 1599 and 1576 cm^−1^ are comparable to those identified by Diallo-Garcia et al. for hydrogencarbonates (Lauron-Pernot et al., [@B27]; Diallo-Garcia et al., [@B12]). The presence of water and carbonates is not impossible, but it is difficult to understand how their presence would increase when the presence of OH^−^ ions decreases. Nevertheless, one could think that the presence of water could be at the origin of these new bands in the OH area, as the evolution of the bands in the two respective regions seems similar. 2. -- The formation of propargylic ions following propyne dissociation on acid-base pairs: It is very difficult to verify this hypothesis, as data on the formation of such species are scarce, and even non-existent on our materials. However, some data are available from Nakajima et al.\'s work on butyne adsorption on ZnO (Nakajima et al., [@B33]). They attribute to propargylic species the bands located at 1,880 and 1,866 cm^−1^, which however remains far from our values. These species would gradually form during adsorption and some species would dissociate during evacuation. However, this hypothesis seems unlikely due to the observed very high intensities but especially due to the fact that apatites generally do not possess high strength acid or basic properties. Catalytic Tests --------------- Concerning the catalytic properties, Ca-XAP-F2 catalyst was tested under various experimental conditions for sake of operating parameters optimization ([Figure 8](#F8){ref-type="fig"}). {#F8} By varying the lactic acid concentration, gas and liquid flows, and reaction temperature, it can be seen that the selectivity and distribution of products is roughly unaffected. On the other hand, as expected, a slight decrease in conversion can be observed when the contact time decreases (increase in GHSV). However, while higher global performances could not be achieved, acrylic acid productivity increased. Indeed, when the lactic acid concentration increased by 250% or the GHSV by 200%, the acrylic acid yield decreased by 7% (from 37.3 to 34.7%) and 5.3% (from 34.7 to 32.82%), respectively. In terms of performance, Ca-XAP-F2 catalyst stands out with a yield of acrylic acid of 37.3%, compared to 27.1% for the best performance obtained with HAP-S ([Figure 9](#F9){ref-type="fig"}). A real effect of the fluoride ions on the catalytic performances is thus observed, with a total conversion and selectivity to acrylic acid significantly higher. Other fluorapatites were also tested. Results are given in [Figure 9](#F9){ref-type="fig"}. It is difficult to draw clear trend from these catalytic tests\' results ([Figure 8](#F8){ref-type="fig"}) regarding the impact of the substitution extent of OH^−^ by F^−^ ions. Indeed, the largest acrylic acid yield (46.1%) is obtained with Ca-XAP-F4 while the minimum yield (31.1%) is obtained with Ca-XAP-F2. An intermediate value (38.2%) is obtained with Ca-XAP-FOH. The selectivity to acrylic acid is remarkably high, with a maximum of nearly 50% reached for Ca-XAP-F4. {#F9} It is quite difficult to explain why the yield drops in the presence of Ca-XAP-F2 compared to the other two fluorapatite compounds. One of the plausible explanations is the difference in catalyst preparation conditions. Indeed, Ca-XAP-F2 was prepared with different quantities and concentrations of precursors than the other Ca-XAPs, although the main parameters (molar ratios, reaction temperature, pH) are the same. [Figure 10](#F10){ref-type="fig"} presents the selectivities to acrylic acid and acetaldehyde obtained during the tests presented in [Figure 9](#F9){ref-type="fig"} as a function of the substitution ratio of OH^−^ by F^−^. The substitution ratio is 0 for Ca-HAP-S and 1 for Ca-XAP-F2. {#F10} An increase in acrylic acid selectivity, which can be directly correlated with that of the substitution rate of OH^−^ ions by F^−^ ions can be clearly seen in [Figure 10](#F10){ref-type="fig"}. On the other hand, the changes in conversion and selectivity to acetaldehyde are similar, with a sharp drop above a substitution rate of 1 (from Ca-XAP-F2). There is therefore theoretically an optimal amount of fluorine that corresponds to a substitution rate between 1 and 2, beyond which the conversion of lactic acid falls. The best acrylic acid yield was obtained with Ca-XAP-F2 with more than 38%. On the other hand, the presence of fluoride ions not only improves selectivity to acrylic acid, but also reduces selectivity to acetaldehyde. The formation of acetaldehyde in dehydration reaction in the gas phase is generally attributed to the presence of strong acid sites (Katryniok et al., [@B23]). This is also an important parameter in the conversion of lactic acid in the liquid phase under supercritical conditions, that are generally very acidic (Aida et al., [@B1]). In these conditions, lactic acid could be easily converted to acetaldehyde. It is difficult to obtain high selectivity to acrylic acid due to the various parallel or secondary reactions. Thus, the nature of the catalysts and their chemical compositions play a crucial role in the orientation of the reaction pathways. Several research group worked to understand the reaction mechanism. This could, without any doubt, enable further development of efficient catalysts and processes. Paparizos et al. studied aluminum phosphate catalyst for dehydration of lactic acid. They obtained an acrylic acid yield of 43.3% (Paparizos et al., [@B34]). Zhang et al. reported excellent catalytic performances with a mixture of potassium and barium phosphates catalysts (K:Ba = 40:60). They reported selectivities to acryclic acid of 92% with also a high conversion of lactic acid (91%) (Zhang et al., [@B50]). Matsuura et al. ([@B28]) and Ghantani et al. ([@B15]) studied also the dehydration of lactic acid over hydroxyapatites. Matsuura et al. obtained excellent yields (in the order of 70%) of acrylic acid depending on the Ca/P ratio. The authors concluded similarly to Blanco et al. ([@B5]) that the acrylic acid formation depends on the balance between surface acid and base sites with a relatively moderate strengths and the absence of strong sites. On the other hand, Umbarkar et al. reported a total conversion of lactic acid associated with a 70% selectivity to acrylic acid (Ghantani et al., [@B14]). Very recently, the team of Yan et al. studied the effect of calcination temperature (from 360 to 700°C) on the catalytic performance of HAPs (Yan et al., [@B48]). They obtained the highest yield of acrylic acid of 62% at 360°C with a HAP (Ca/P: 1.62) calcined at 360°C. Phosphates are clearly an interesting solution for the development of efficient catalysts for the gase phase dehydration of lactic acid. However, it appears relatively difficult to identify which are the most active phases and which active sites are involved in the given mechanisms of the dehydration reaction on these catalysts by comparison with the litterature, as the reaction conditions in the various works are actually quite different. Conclusions {#s4} =========== During the study of the dehydration reaction of lactic acid, large variations in the catalytic performances of the different hydroxyapatites were observed. Although the emphasis is often placed on the Ca/P ratio as a paramount parameter for controlling the acidic and basic properties of hydroxyapatites, we have shown that substitution of OH^−^ by a halogen, fluorine, has a beneficial impact on the dehydration reaction. Indeed, a stoichiometric replacement of these OH^−^ ions by F^−^ ions not only improves the selectivity to acrylic acid but also significantly reduces the selectivity to acetaldehyde. Compositional changes with respect to the non-substituted reference hydroxyapatite strongly impacted the specific surface area of the samples. Increase in the Ca/P ratio alone cannot explain the decrease in specific surface area upon F^−^ introduction, which should be attributed, at least in part, to the change in the density of the material, following a compaction of the triangles of Ca (II). Acid-base properties of the samples were assessed by IR characterization of propyne adsorption. Some correlations could be observed between the presence of fluorine and the red shift of the propyne υ~(≡C−H)~ and υ~(C≡C)~ bands. It is possible that the presence of fluorine modifies both the distance between the acid site and the basic site (acid-base pair as adsorption site) while modifying the strength of the sites, in particular the potential acid site identified, as the Ca^2+^ cation. Finally, the F^−^-substituted samples showed superior catalytic performances compared to the native non-substituted sample, which could be linked to the structural/strength modification of the surface acid-base pairs suggested by FTIR-followed propyne adsorption experiments. Data Availability Statement {#s5} =========================== The original contributions presented in the study are included in the article/[Supplementary Material](#SM1){ref-type="supplementary-material"}, further inquiries can be directed to the corresponding author/s. Author Contributions {#s6} ==================== All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. Conflict of Interest {#s7} ==================== The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors acknowledge the support from the French National Research Agency (ANR-2010-CD2I-011- 01). The REALCAT platform is benefiting from a Governmental subvention administrated by the French National Research Agency (ANR) within the frame of the Future Investments program (PIA), with the contractual reference ANR-11-EQPX-0037. Chevreul Institute (FR 2638), Ministère de l\'Enseignement Supérieur, de la Recherche et de l\'Innovation, Région Hauts-de-France and FEDER are acknowledged for supporting and funding partially this work. Supplementary Material {#s8} ====================== The Supplementary Material for this article can be found online at: <https://www.frontiersin.org/articles/10.3389/fchem.2020.00421/full#supplementary-material> ###### Click here for additional data file. [^1]: Edited by: Pedro Maireles-Torres, University of Malaga, Spain [^2]: Reviewed by: Tomoo Mizugaki, Osaka University, Japan; Rafael Mariscal, Institute of Catalysis and Petrochemistry (CSIC), Spain [^3]: This article was submitted to Catalysis and Photocatalysis, a section of the journal Frontiers in Chemistry

Introduction ============ The leaves of *Pereskia bleo* (Kunth) DC. (Cactaceae) are used traditionally in Malaysia for the treatment of cancer, high blood pressure, diabetes and diseases associated with rheumatism and inflammation. They are also used as remedy for the relief of gastric pain, ulcers and for revitalizing the body \[[@B1-molecules-14-01713]\]. The leaves are generally consumed by the locals either raw or taken as a concoction brewed from fresh leaves. Chemical investigations on *Pereskia bleo* are rare in comparison to other *Pereskia* species, as there were only three phytochemical and biological studies reported for this plant. The earliest phytochemical study was by Doetsch *et al.* \[[@B4-molecules-14-01713]\], who reported the isolation of four alkaloids, namely 3,4-dimethoxy-β-phenethylamine, mescaline, 3-methoxytyramine and tyramine. An investigation by Tan *et al.* \[[@B2-molecules-14-01713]\] reported that the methanol extract of *Pereskia bleo* possessed cytotoxic effects against T-47D cells and the cell death was found to be apoptotic in nature, mainly *via* the activation of the caspase-3 and c-myc pathways. A more recent investigation by Er *et al*. \[[@B3-molecules-14-01713]\] indicated the anti-proliferative and mutagenic activities of aqueous and methanol extracts of *Pereskia bleo* leaves against mouse mammary cancer cells (4T1) or normal mouse fibroblast cells (NIH/3T3). In our previous cytotoxicity screenings on *Pereskia bleo* \[[@B5-molecules-14-01713]\], the EtOAc fraction possessed notably high cytotoxic effects against selected human carcinoma cell lines, but exerted no damage to a non-cancer human fibroblast cell line (MRC-5). The active EtOAc fraction was found to contain β-sitosterol (**4**), 2,4-di-tert-butylphenol (**5**), α-tocopherol (**6**) and phytol (**7**) \[[@B5-molecules-14-01713]\]. As part of our ongoing research on *Pereskia bleo*, a pure compound and a mixture of sterols were also isolated from the leaves of *Pereskia bleo*. In the present study, we report further progress in ongoing research on *Pereskia bleo*, which led to the isolation and identification of dihydroactinidiolide (**1**) and a mixture of sterols \[campesterol (**2**), stigmasterol (**3**) and β-sitosterol (**4**)\] and cytotoxic investigation on all isolated compounds against five human carcinoma cell lines, namely the human nasopharyngeal epidermoid carcinoma cell line (KB), human cervical carcinoma cell line (CasKi), human colon carcinoma cell line (HCT 116), hormone-dependent breast carcinoma cell line (MCF7) and human lung carcinoma cell line (A549) and non-cancer human fibroblast cell line (MRC-5). Results and Discussion ====================== Extraction and isolation of pure compounds and the sterol mixture ----------------------------------------------------------------- β-Sitosterol (**4**), 2,4-di-tert-butylphenol (**5**), α-tocopherol (**6**) and phytol (**7**) were obtained from *Pereskia bleo* as previously described by Sri Nurestri *et al.* \[[@B5-molecules-14-01713]\]. On repeated chromatographic purification of the active EtOAc fraction, a red viscous oil and white colored needles were obtained and identified as dihydroactinidiolide and a mixture of sterols. Dihydroactinidiolide (**1**), red viscous oil; EI-MS *m/z* (%): 180 \[M\]^+^ (15), 137 (8), 111 (100), 109, 67. Compound **1**was identified by comparison of its mass spectral data with NIST mass-spectral library \[[@B21-molecules-14-01713]\] and other reported spectroscopic data \[[@B6-molecules-14-01713],[@B7-molecules-14-01713],[@B8-molecules-14-01713]\]. The mixture of sterols appeared as white colored needles that according to GC-MS analyses consisted of campesterol (**2**, 14.33%), stigmasterol (**3**, 4.95%) and β-sitosterol (**4**, 70.21%). Compound **2**(campesterol); EI-MS *m/z* (%): 400 (42, \[M^+^\]), 382 (34), 367 (20), 315 (30), 289 (30), 55 (100). The mass spectral data was also in agreement with reported data \[[@B9-molecules-14-01713]\]. Stigmasterol (**3**) was identified by GC-MS analysis and by comparison of its mass spectral data \[EI-MS *m/z* (%): 412 (16, \[M^+^\]), 394 (4), 369 (2), 351 (6), 271 (16), 255 (22), 229 (5), 55 (100)\] with reported data \[[@B9-molecules-14-01713]\]. Compound **4**(ß-sitosterol); EI-MS *m/z* (%): 414 (100, M^+^), 396 (57), 381 (43). ß-sitosterol (**4**) was identified by GC-MS analysis as well as comparison of its mass spectral data with reported data \[[@B10-molecules-14-01713]\]. The structures of compounds **1**-**7** are illustrated in [Figure 1](#molecules-14-01713-f001){ref-type="fig"}. {#molecules-14-01713-f001} In vitro Neutral Red cytotoxicity assay --------------------------------------- The *in vitro* cytotoxicity assay was carried out using a Neutral Red cytotoxicity assay as previously described by Borenfreund and Puerner \[[@B11-molecules-14-01713]\] with some modifications; this test determines the accumulation of the Neutral Red dye in the lysosomes of viable and uninjured cells. The results of cytotoxicity screening of the components are summarized in [Table 1](#molecules-14-01713-t001){ref-type="table"}. It is generally known that ethnomedical data substantially increases the chances of finding active plants relative to a random approach \[[@B2-molecules-14-01713]\]. The consequence is that, once having found activity in the plant from the ethopharmacological observation (e.g. raw or concoction brewed from the plant leaves shows effect for cancer treatment), there is a desire to determine the chemical structures of the compounds that are responsible for the activity, as not all the compounds in the extracts have the same activity. However, the observed activity might be due to synergism between compounds present in the plant extract. The synergism among these compounds which contribute to the cytotoxic activity, is not only dependent on the concentration of the compounds, but also on the structure and interaction(s) between the compounds \[[@B27-molecules-14-01713]\]. This can explain the differences in the cytotoxic effect between crude extracts and isolated compounds against the same cell lines, as shown in our earlier report \[[@B5-molecules-14-01713]\]. For example, the cytotoxic effect of the crude methanol extract on the KB cell lines showed an IC~50~ of 6.5 µg/mL and such impressive activity was supported by some of the isolated compounds \[dihydroactinidiolide (**1**), 2,4-di-tert-butylphenol (**5),**α-tocopherol (**6**) and phytol (**7**)\]. In contrast, the cytotoxic effect of the crude methanol extract on the MCF7 cell line gave IC~50~ of 39.0 µg/mL (mild) whilst two isolated compounds 2,4-di-tert-butylphenol (**5)** and α-tocopherol (**6**), showed good inhibitory activities with IC~50~ values of 5.75 and 7.5 µg/mL, respectively. molecules-14-01713-t001_Table 1 ###### Cytotoxic activity (IC~50~values) of compounds **1**-**5** and mixture of sterols against KB, CasKi, HCT 116, MCF7, A549 and MRC5 cell lines. Compound Cytotoxicity (IC~50~) in µg/mL (µM) --------------------------------- ------------------------------------- ------------ ------------ ------------ ------------ ------------ Dihydroactinidiolide (**1**) 6.7 30 40 5 97 91.3 (37.22) (166.67) (222.22) (27.78) (538.89) (507.22) β -sitosterol (**4**) \>100 72 62 \>100 78 \>100 (\>241.55) (173.91) (149.76) (\>241.55) (188.41) (\>241.55) 2,4-di tert butylphenol (**5**) 0.81 5.75 4.5 29 6 20 (3.93) (27.91) (21.84) (140.78) (29.13) (97.09) α-tocopherol (**6**) 8 7.5 6 31 6 30.5 (18.6) (17.44) (13.95) (72.09) (13.95) (70.93) Phytol (**7**) 7.1 34 18 100 31 74.3 (23.99) (114.86) (60.81) (337.84) (104.73) (251.01) Mixture \>100 \>100 \>100 \>100 \>100 \>100 Doxorubicin^a^ 1.3x10^-2^ 7.6x10^-2^ 6.0x10^-3^ 3.6x10^-1^ 2.2x10^-1^ 5.5x10^-1^ (0.023) (0.139) (0.011) (0.663) (0.401) (1.01) ^a^Doxorubicin was used as the reference compound. 2,4-Di-tert-butylphenol (**5)** displayed very remarkable cytotoxic activity against KB cells with an IC~50~value of 0.81 µg/mL and strong cytotoxicity against MCF7 (IC~50~ 5.75 µg/mL), A549 (IC~50~ 6 µg/mL) and CasKi cells (IC~50~4.5 µg/mL). This *in vitro* data of 2,4-di-tert-butylphenol (**5**) support the findings that phenolic antioxidants exert cytoctoxic activity against cancer cells \[[@B14-molecules-14-01713],[@B15-molecules-14-01713]\]. 2,4-Di-tert-butylphenol (**5**) is an antioxidant widely used in the plastics industries, and its presence in plants cannot readily be explained biogenetically. It is more probable that the plant accumulated this compound from the soil it grew in, that might have contained the compound. In our experience, this compound has also been detected in other plants like *Termitomyces heimi*, *Pleurotus sajor-caju* and *Hericium erinaceus* collected from different locations to where the *Pereskia bleo* leaves were obtained (unpublished data from our group of researchers working on *Termitomyces heimi*, *Pleurotus sajor-caju* and *Hericium erinaceus*). The observation of 2,4-di-tert-butylphenol (**5**) in our study is not an isolated case, as it has also been reported to exist in natural sources by other researchers \[[@B29-molecules-14-01713],[@B30-molecules-14-01713],[@B31-molecules-14-01713]\]. To support our finding that 2,4-di-tert-butylphenol (**5**) is not an artifact, an extraction on *Pereskia bleo* was repeated using redistilled methanol and ethyl acetate. GC-MS analysis on the ethyl acetate extract still showed the presence of 2,4-di-tert-butylphenol (**5**) representing the major component of the total ethyl acetate extract. This shows that 2,4-di-tert-butylphenol (**5**) is present in the extract itself and not a solvent artifact. Other constituents in the plant also contribute to its cytotoxic activity as shown by α-tocopherol (**6**), phytol (**7**) and dihydroactinidiolide (**1**). In the present study, α-tocopherol (**6**), which is a dietary antioxidant, displayed pronounced cytotoxicity against CasKi (IC~50~ 6 µg/mL) and A549 (IC~50~ 6 µg/mL). The result obtained here is consistent with other reports \[[@B37-molecules-14-01713],[@B38-molecules-14-01713],[@B39-molecules-14-01713],[@B40-molecules-14-01713],[@B47-molecules-14-01713],[@B48-molecules-14-01713],[@B49-molecules-14-01713],[@B50-molecules-14-01713]\] on cytotoxic activities in other cell lines. Lesser number of investigations described an opposite effect \[[@B44-molecules-14-01713],[@B45-molecules-14-01713],[@B46-molecules-14-01713]\]. There was no report on the cell lines that were used in this study. According to [Table 1](#molecules-14-01713-t001){ref-type="table"}, phytol (**7**) demonstrated strong activity against KB cells (IC~50~ 7.1 µg/mL). The cytotoxicity data showed in this report thus supports our hypothesis in our previous report \[[@B5-molecules-14-01713]\] that phytol might be responsible for the remarkable cytotoxic effect of the EtOAc fraction against the KB cancer cell lines. In this study, dihydroactinidiolide (**1**) demonstrated strong cytotoxic effect against HCT116 with IC~50~ 5.0 µg/mL. Dihydroactinidiolide (**1**) is structurally similar to the C11-terpene lactones that arise from the biological or oxidative degradation of carotenoids and has been isolated from various plants and insect sources. It has also been identified as the flavor molecule in tea and tobacco \[[@B6-molecules-14-01713],[@B7-molecules-14-01713],[@B8-molecules-14-01713]\]. Sterols are important constituents of all eukaryotes and play a vital role in plant cell membranes. In addition to their cholesterol lowering effect, plant sterols may possess anti-atherosclerosis \[[@B32-molecules-14-01713],[@B33-molecules-14-01713]\], antibacterial \[[@B36-molecules-14-01713]\], anti-inflammation \[[@B34-molecules-14-01713]\] and anti-oxidation activities \[[@B35-molecules-14-01713]\]. In the present study, β-sitosterol (**4**) and the mixture of sterols \[campesterol (**2**), stigmasterol (**3**) and β-sitosterol (**4**)\] did not display cytotoxic effects against the tested cell lines. The results obtained here were in agreement with published data \[[@B16-molecules-14-01713],[@B17-molecules-14-01713],[@B18-molecules-14-01713],[@B19-molecules-14-01713],[@B20-molecules-14-01713]\]. There have been reports that plant sterols are able to stimulate estrogen dependent cancer cells *in vitro* (e.g. Ju *et al*. \[[@B42-molecules-14-01713]\]). The MCF7 cell line used in this study was purchased from ATCC. It was reported that MCF7 cells from ATCC were unaffected by estrogen or antiestrogen \[[@B43-molecules-14-01713]\]. Thus, the result showed that the sterols do inhibit the growth of MCF7 cells. Doxorubicin which is clinically used for the treatment of a great variety of cancer disease \[[@B24-molecules-14-01713],[@B25-molecules-14-01713],[@B26-molecules-14-01713]\] was used as the positive control in present study. Based on the result, it can be concluded that doxorubicin is not only cytotoxic against all the human cancer cell lines tested, but also the non-cancer human cell line. This result supports the statement that doxorubicin is a potent cytostatic drug which is applied for the treatment of cancer diseases but the routine use of this drug could bring major adverse effect \[[@B24-molecules-14-01713]\]. Although the cytotoxicity of the isolated compounds and mixture of *Pereskia bleo* are not as effective as doxorubicin, they however have low toxicity against normal MRC5 cell line in comparison to doxorubicin. The use of the isolated compounds as single anticancer agents would not merit consideration. However, their use in combination with cytotoxic therapeutic drugs might reduce the adverse effects of some of these drugs. Support for this suggestion is provided by Amir *et al.* \[[@B41-molecules-14-01713]\], who reported that in addition to having potent antitumor properties as single agents, natural products have also demonstrated potential synergy with established cytotoxic therapeutic drugs in pre-clinical studies. At this stage, it is not possible to justify the use of isolated compounds in comparison to doxorubicin in the treatment of cancer. A more comprehensive investigation is required. Experimental ============ General ------- GCMS analysis was performed using a Agilent Technologies 6980N gas chromatography equipped with a 5979 Mass Selective Detector (70 eV direct inlet); a HP-5ms (5% phenylmethylsiloxane) capillary column (30.0 m x 250 µm x 0.25 µm) initially set at 60°C for 10 minutes, then programmed to 230°C at 3°C min^-1^and held for 1 min at 230°C using helium as the carrier gas at a flow rate of 1 mL min^-1^. The total ion chromatogram obtained was auto integrated by ChemStation and the components were identified by comparison with an accompanying mass spectral database \[[@B21-molecules-14-01713]\]. Thin layer chromatography (TLC) analyses were carried out using precoated TLC plates 60 F~254~ (20.25 mm thickness) purchased from Merck and were visualized in UV light (254 and/or 343 nm) and/or iodine vapour. Plant sample collection and identification ------------------------------------------ The fresh leaves of *Pereskia bleo* were collected from Petaling Jaya, Selangor, Malaysia in September 2006. They were identified by Professor Dr. Halijah Ibrahim of Institute of Biological Sciences, Faculty of Science, University of Malaya, Malaysia and a voucher specimen (SN01-06) was deposited at the herbarium of the Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia. Extraction and isolation of pure compound and mixture ----------------------------------------------------- β-Sitosterol (**4**), 2,4-Di-tert-butylphenol (**5**), α-tocopherol (**6**) and phytol (**7**) were isolated from *Pereskia bleo* as previously described by Sri Nurestri *et al.* \[[@B5-molecules-14-01713]\]. Compound **1** and mixture of sterols were obtained according to the following procedure. Dried, ground leaves (1,050.56 g) of *Pereskia bleo* were extracted with MeOH (3x 1.5 L). The MeOH-containing extract obtained was initially treated with charcoal, then filtered over Celite^®^ and the filtrate was evaporated under reduced pressure to give a crude MeOH extract (99.44 g). Treatment with charcoal was necessary to remove the high amounts of chlorophyll present in the extract, which interfered with chromatographic separation efforts. The crude MeOH extract was then further partitioned between EtOAc and H~2~O in a separating funnel. The EtOAc-soluble layer was concentrated *in vacuo* giving an 18.34 g EtOAc fraction, which was subjected to flash silica gel column chromatography (Si-gel CC) eluting with CHCl~3~ (10 L), and then with CHCl~3~-MeOH \[9:1 (9 L)\] and finally MeOH (7.6 L). The CHCl~3~ fraction was concentrated to give a dark brown residue (3.47 g). The brown residue was subjected to a Si-gel CC initially eluting with a gradient of hexane followed by hexane enriched with increasing percentages of CH~2~Cl~2~, monitoring with TLC. The volume of each fraction was 25 mL. The mixture of sterols (20.5 mg) was obtained from the fraction upon elution with CH~2~Cl~2~-hexane (3.5: 6.5). Further elution with CH~2~Cl~2~ yielded a mixture (206.7 mg) containing **1**. Purification of **1**was obtained through preparative-TLC using CHCl~3~ as the developing solvent to yield pure compound **1**(5.4 mg). Cell lines and culture medium ----------------------------- Human nasopharyngeal epidermoid carcinoma cell line (KB), human cervical carcinoma cell line (CasKi), human colon carcinoma cell line (HCT 116), human hormone-dependent breast carcinoma cell line (MCF7), human lung carcinoma cell line (A549) and non-cancer human fibroblast cell line (MRC-5) were purchased from the American Tissue Culture Collection (ATCC, USA). KB cells were maintained in Medium 199 (Sigma), CasKi, A549 and MCF7 cells in RPMI 1640 medium (Sigma), HCT 116 in McCOY'S 5A Medium (Sigma) and MRC-5 cells in EMEM (Eagle Minimum Essential Medium) (Sigma), supplemented with 10% foetal bovine serum (FBS, PAA Lab, Austria), 100 µg/mL penicillin or streptomycin (PAA Lab, Austria) and 50 µg/mL of fungizone (PAA Lab, Austria). The cells were cultured in a 5% CO~2~ incubator (Shel Lab water-jacketed) kept at 37°C in a humidified atmosphere. In vitro Neutral Red cytotoxicity assay --------------------------------------- The Neutral Red cytotoxicity assay is based on the initial protocol described by Borenfreund and Puerner \[[@B11-molecules-14-01713]\] with some modifications. Briefly, the cells (1x10^4^/well) were seeded in 96-well microtiter plates (Nunc) and allowed to grow for 24 hours before treatment. After 24 hours of incubation, the cells were treated with six different concentrations (0.1-100 µg/mL) of test compounds, in three replicates. The plates were further incubated for 72 h at 37°C in a 5% CO~2~ incubator. A stock solution was initially obtained by dissolving the test compounds in DMSO. Further dilution to different tested concentrations were then carried out ensuring that the final concentration of DMSO in the test and control wells was not in excess of 1% (v/v). No effect due to the DMSO was observed. Doxorubicin was used as the positive control. The well containing untreated cells was the negative control. At the end of the incubation period, the media were replaced with medium containing 50 µg/mL of Neutral Red. The plates were incubated for another 3 hours to allow for uptake of the vital dye into the lysosomes of viable and injured cells. After the incubation period, the media were removed and cells were washed with the neutral red washing solution. The dye was eluted from the cells by adding 200 µL of Neutral Red resorb solution and incubated for 30 minutes at room temperature with rapid agitation on a microtiter plate shaker. Dye absorbance was measured at 540 nm using a spectrophotometer ELISA plate reader. The average data from triplicates were expressed in terms of killing percentage relative to negative control. The percentage of inhibition (%) of each of the test samples was calculated according to the following formula: where OD control: Optical Density of negative control; OD sample: Optical Density of sample Cytotoxicity of each sample is expressed as IC~50~ value. The IC~50~value is the concentration of test compounds that cause 50 % inhibition or cell death, averaged from the three experiments, and was obtained by plotting the percentage inhibition versus concentration of test compounds. According to US NCI plant screening program, a plant extract is generally considered to have active cytotoxic effect if the IC~50~ value, following incubation between 48 to 72 hours, is 20 μg/mL or less, while it is 4 μg/mL or less for pure compounds \[[@B12-molecules-14-01713],[@B13-molecules-14-01713],[@B22-molecules-14-01713],[@B23-molecules-14-01713]\]. However, we recognized that whether an IC~50~ value corresponds to a significant or non-significant cytotoxicity depends on the sensitivity of the cell line. Conclusions =========== In conclusion, and depending on the cell lines used, the cytotoxic activities observed for *Pereskia bleo* \[[@B5-molecules-14-01713]\] are ascribable to the presence of the active compounds **1**, **5**, **6** and **7**. Although the cytotoxicity of these compounds and mixture are not as effective as doxorubicin, in comparison to the latter they have low toxicity against normal MRC5 cell line. The cytotoxicity assay used in the present study could only provide important preliminary data to help select plant extracts or isolated compounds with potential antineoplastic properties for future work. A detailed investigation on the mechanism of cell death would provide more convincing evidence. An investigation into this phenomenon is now underway and will be reported in due course. The resulting information will certainly contribute to a better understanding of the anti-carcinogenic activity of the natural constituents in *Pereskia bleo*. *Pereskia bleo* has been traditionally used for the treatment of cancer and the findings of the current study thus provide scientific validation on the use of the leaves of *Pereskia bleo*. In view of the increasing popular consumption of medicinal plants as alternative therapy, it is therefore necessary to conduct serious research to support the therapeutic claims and also to ensure that the plants are indeed safe for human consumption. This work was supported by a research fund from the University of Malaya (Vote F PS056/2007C) and the Ministry of Science, Technology and Innovation (MOSTI) (E-sciencefund 1202032026). We are also grateful to Prof A. Hamid A Hadi for use of his laboratory space. *Sample Availability:* Samples are available from the authors.

Aims and Scope ============== *Cancer Medicine* is a peer-reviewed, open access, interdisciplinary journal providing rapid publication of cutting-edge research from global biomedical researchers across the cancer sciences. 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Editor-in-Chief =============== Qingyi Wei, M.D., Ph.D. The University of Texas MD Anderson Cancer Center, Houston, USA Editorial Board =============== Cancer Biology -------------- Richard J. Ablin University of Arizona College of Medicine, Tucson, USA Rosalind Eeles Institute of Cancer Research, Sutton, UK Elizabeth A. Grimm The University of Texas MD Anderson Cancer Center, Houston, USA Giseli Klassen Federal University of Parana, Curitiba, Brazil Gang Li University of British Columbia, Vancouver, Canada Youyong Lu Beijing Institute for Cancer Research, Beijing, China Kenneth Offi t Memorial Sloan-Kettering Cancer Center, New York, USA Athanasios G. Papavassiliou University of Athens Medical School, Athens, Greece Kenneth Pienta University of Michigan, An Arbor, USA Jack A. Schalken Radboud University Medical Centre, Nijmegen, Netherlands Kazu Ushijima National Cancer Center, Tokyo, Japan David Vaux Walter and Eliza Hall Institute of Medical Research, Parkville, Australia Cun-Yu Wang UCLA School of Dentistry, Los Angeles, USA Hongyang Wang Eastern Hepatobiliary Surgery Institute / Hospital, Shanghai, China Momiao Xiong The University of Texas Health Science Center, Houston, USA Li Zhang The University of Texas MD Anderson Cancer Center, Houston, USA Clinical Cancer Research ------------------------ Yung-Jue Bang Seoul National University College of Medicine, Seoul, Korea Anthony Chan The Chinese University of Hong Kong, China Jiuwei Cui The First Hospital of Jilin University, Jilin, China Peter J. Fuller Prince Henry Institute, Victoria, Australia Maura L. Gillison Ohio State University Comprehensive Cancer Center, Columbus, USA Guido Kroemer University of Paris Descartes, Paris, France Lalit Kumar All India Institute of Medical Sciences, New Delhi, India Razelle Kurzrock The University of Texas MD Anderson Cancer Center, Houston, USA Geoffrey Liu Princess Margaret Hospital, Toronto, Canada Jiade J. Lu National University of Singapore, Singapore Li Mao University of Maryland, Baltimore, USA Akira Nakagawara Chiba University, Chiba, Japan Pamela L. Paris University of California, San Francisco, USA Dimitrios H. Roukos Ioannina University School of Medicine, Ioannina, Greece Frank A. Sinicrope Mayo clinic, Rochester, USA Jun Wang BGI-Shenzhen, Shenzhen, China Glen J. Weiss Translational Genomics Research Institute, Phoenix, USA Senming Wang Southern Medical University, Guangzhou, China Eric T. Wong Beth Israel Deaconess Medical Center, Boston, USA Xianglin Yuan Huazhong University of Science and Technology, Wuhan, China Cancer Prevention ----------------- Marianne Berwick University of New Mexico, Albuquerque, USA Paolo Boffetta The Tisch Cancer Institute, New York, USA Eduardo Cazap Latin-American & Caribbean Society of Medical Oncology, Buenos Aires, Argentina Brock C. Christensen Dartmouth Medical School, Hanover, USA Steve Dubinett David Geffen School of Medicine at UCLA, Los Angeles, USA Kari Hemminki German Cancer Research Center, Heidelberg, Germany Zhibin Hu Nanjing Medical University, Nanjing, China Stephen D. Hursting The University of Texas, Austin, USA Daehee Kang Seoul National University College of Medicine, Seoul, Republic of Korea Lambertus A. Kiemeney Radboud University Medical Centre, Nijmegen, Netherlands Donghui Li The University of Texas MD Anderson Cancer Center, Houston, USA Dongxin Lin Chinese Academy of Medical Sciences, Beijing, China Somdat Mahabir National Cancer Institute, Bethesda, USA Girish Maru ACTREC Tata Memorial Centre, Navi Mumbai, India Andrew Olshan University of North Carolina, Chapel Hill, USA Jong Y. Park H. Lee Moffi tt Cancer Center & Research Institute, Tampa, USA Susan K. Peterson The University of Texas MD Anderson Cancer Center, Houston, USA Thomas E. Rohan Albert Einstein College of Medicine, Bronx, USA Xifeng Wu The University of Texas MD Anderson Cancer Center, Houston, USA Editorial Advisory Board ------------------------ Lawrence Baker University of Michigan, Ann Arbor, USA David Christiani Harvard School of Public Health, Boston, USA Robert B. Diasio Mayo Clinic, Rochester, USA B. Mark Evers Lucille P. Markey Cancer Center, Lexington, USA Waun Ki Hong The Univeristy of Texas MD Anderson Cancer Center, Houston, USA Ernest Hawk The University of Texas MD Anderson Cancer Center, Houston, USA Leroy Hood Institute for Systems Biology, Seattle, USA James Mulé H. Lee Moffi tt Cancer Center & Research Institute, Tampa, USA Philip Agop Philip Wayne State University, Detroit, USA Nicholas Vogelzang Comprehensive Cancer Centers of Nevada, Vegas, USA

1. Introduction =============== The beach chair position (BCP) is commonly used for arthroscopic shoulder surgery. It has several advantages compared with a lateral decubitus position, such as good visualization of the surgical field, reduced traction neuropathy, and ease of conversion to an open surgery.^\[[@R1]\]^ However, the BCP is frequently associated with increased hemodynamic instability, such as hypotension, bradycardia,^\[[@R1]\]^ and cerebral hypoperfusion.^\[[@R2]\]^ There are many challenges in preventing intraoperative hypotension during the BCP. Application of compression stocking^\[[@R3]\]^ and vasopressor infusion^\[[@R4],[@R5]\]^ can be helpful to prevent hypotension during the BCP. Arginine vasopressin, a strong vasoconstrictor, is effective in preventing hypotension during the BCP, but it causes a significant decrease of brain oxygenation.^\[[@R4]\]^ Phenylephrine, an α-adrenergic agonist, is widely used for treatment of hypotension during perioperative periods.^\[[@R6]\]^ It is more commonly used as a first treatment of choice of intraoperative hypotension than vasopressin in our institution. Intravenous infusion of phenylephrine (1.5 μg/kg/min) before being placed in the BCP was effective in reducing the incidence of hypotension for shoulder surgery.^\[[@R7]\]^ Cerebral oxygen saturation, however, decreased after phenylephrine infusion, possibly caused by cerebral vasoconstriction. To our knowledge, there is no previous research describing the hemodynamic effects of lower doses of intravenous phenylephrine infusion during the BCP. In this study, we evaluated the hemodynamic effect of 2 doses of intravenous phenylephrine infusion (0.5 and 1.0 μg/kg/min) during the BCP for arthroscopic surgery. The primary outcome was incidence of hypotension after the BCP. The secondary outcome was the difference in hemodynamic effects among a control group and infusion groups. 2. Materials and methods ======================== This study was approved by the Institutional Review Board of Inje University Haeundae Paik Hospital (IRB number: 129792-2015-096) and was registered as a clinical trial (clinical trial number: NCT02585570). Written informed consent was obtained from all patients. Sixty-six American Society of Anesthesiologists physical status 1 to 3 patients who were scheduled to undergo elective arthroscopic shoulder surgery in the BCP were enrolled. Patients were excluded if there was a history of significant cerebrovascular disease, cardiac disease (NYHA class ≥3), uncontrolled hypertension, and age \<20 years. Patients were premedicated with intravenous midazolam (1 mg) before being transported to the operating room. Patients underwent standard monitoring in the operating room in the supine position, including electrocardiogram, pulse oximetry, non-invasive blood pressure, capnography, and bispectral index (BIS). Hartmann\'s solution (6 mL/kg/h) was administered intravenously. The right or left radial artery was cannulated with a 20-guage angio-catheter to monitor invasive arterial blood pressure. The pressure transducer was placed at the level of external auditory meatus.^\[[@R8]\]^ Mean arterial pressure (MAP), stroke volume variance (SVV), and cardiac index (CI) were measured using a Vigileo/Flotrac system (Edward Lifesciences, Irvine, CA). We calculated the total systemic vascular resistance index (TSVRi).^\[[@R9],[@R10]\]^ Pre-induction hemodynamic parameters were measured, and then general anesthesia was induced with intravenous (IV) propofol 2 mg/kg, remifentanil 0.1 μg/kg/min, and sevoflurane 2 to 3 vol%. Rocuronium (0.6 mg/kg) was administered intravenously to facilitate endotracheal intubation. When vital signs were stabilized after endotracheal intubation, hemodynamic values were measured as a post-induction baseline (PIB). After endotracheal intubation, the patient\'s lungs were mechanically ventilated with an oxygen/air mixture (fraction of inspired oxygen of 50%) to maintain the end-tidal carbon dioxide between 35 and 40 mm Hg. Sevoflurane was adjusted to maintain a BIS between 40 and 60. The patients were allocated into 3 groups (group NS, group LP, and group HP) using a single blinded randomization method. The blocked randomization was performed by using computer software generation. Group NS received normal saline IV infusion (0.3 ml/kg/hr), and Group LP and Group HP received phenylephrine IV infusion of 0.5 or 1.0 μg/kg/min, respectively, for 5 minutes before being placed in the BCP. Five minutes after the start of the IV phenylephrine infusion, patients were placed into the BCP. MAP, heart rate (HR), oxygen saturation, CI, SVV, and BIS were continuously monitored during intraoperative period, and all measurements were recorded every minute for first 15 minute after BCP under no surgical stimulation. When hypotension (MAP \<60 mm Hg) occurred, ephedrine (5 mg) was administered intravenously to the patients. When severe hypotension occurred with a decreased BIS value (\<20) or increased SVV value (\>30), the patient was removed from the study. When bradycardia (HR \<50 bpm) occurred, atropine (0.5 mg) was administered intravenously. 2.1. Statistical analysis ------------------------- The sample size was calculated using nQuery Advisor 7.0 program. When effect size was set at 0.3, and power was set at 0.95, the required number of patients for each group was 18. Assuming a drop-out rate of 20%, the final sample size was set at 22 patients per group. Patients' baseline and clinical characteristics were summarized by subgroups using descriptive statistics. Chi-squared test or Fisher exact test was used to compare categorical variables between groups, while analysis of variance (ANOVA) or Kruskal--Wallis test was used to compare continuous variables between groups. Shapiro--Wilk test was employed for test of normality assumption. Repeated measurement analysis of variance was used to compare repeated measurements in groups and within each group, and the Bonferroni procedure was applied in post-hoc analyses. ANOVA with repeated measures with a Huynh-Feldt correction was used when sphericity was not assumed. Mean ± the standard error of the mean plots were also graphically presented. All statistical analyses were carried out using SPSS version 25.0 statistical software (SPSS Inc, Chicago, IL) and Medcalc (Medcalc Software bvba, Ostend, Belgium). A *P*-value of \<.05 was considered statistically significant. 3. Results ========== A total of 66 patients were enrolled in this study. A total of 3 patients, however, were excluded because of mechanical malfunction of the Vigileo/Flotrac system (n = 1, group LP) and severe hypotension (n = 2, group NS) after induction of general anesthesia (Fig. [1](#F1){ref-type="fig"}). {#F1} Patient baseline demographic characteristics and hemodynamic parameters before being placed into the BCP are described in Table [1](#T1){ref-type="table"}. There were no significant differences in these parameters among the 3 groups. ###### Patient baseline demographic characteristics and hemodynamic parameters between groups.  The incidence of hypotension was not significantly different between groups (Table [2](#T2){ref-type="table"}). The total incidence of hypotension after placement into the BCP was 93.65% in all patients. The incidence of bradycardia in group HP (66.7%) was relatively high compared with the other groups (10% in group NS and 4.8% in group LP) but was not statistically significant (*P* = .0869). The rescue dose of ephedrine (23.25 ± 15.75, 27.5 ± 13.25, and 23.33 ± 13.90 mg) was not statistically different in group HP, group LP, and group NS, respectively (*P* = .541). ###### Incidence of hypotension and bradycardia in patients after the beach chair position.  Hypotension occurred in the majority of patients within 5 minutes after being placed in the BCP (84.7%, 50/59). The MAP of each group at 1-minute intervals within the first 5 minutes after being positioned in the BCP is described in Table [3](#T3){ref-type="table"}. There was a significant difference in the trends between groups (*P* = .028). The changes between the MAP at 1 minute compared to at the PIB between groups was statistically different (*P* = .014), especially in Group HP (--20.09 ± 21.79 mm Hg) and Group NS (--41.75 ± 18.24 mm Hg), which were different based on post-hoc Dunn test analysis. ###### Mean arterial pressure at each time point by group.  The HR of each group at 1-minute intervals 5 minutes after the BCP are described in Table [4](#T4){ref-type="table"}. There was significant difference in the HR between group HP and group NS 1 minute after BCP by post-hoc Dunn test (*P* = .031). The change of HR at 1 minute compared to PIB was statistically different (*P* = .047), especially in group HP (--14.59 ± 10.05 bpm) and group NS (--9.35 ± 17.08 bpm), which were different based on post-hoc analysis. ###### Heart rate comparison between groups in the 5-minute interval after the beach chair position.  There were no statistically significant differences in CI, SVV, and TSVRi between groups (*P* \> .05). However, when we compared the TSVRi between group HP and group NS, there was a significant difference in the trend (time-group interaction *P* = .0496) (Table [5](#T5){ref-type="table"}). This significance comes from difference in the TSVRi at 1 minute compared to PIB (P = .035). Group HP (--681.5 ± 712.8) and Group NS (--1188.9 ± 794.7) were different by post-hoc analysis. The TSVRi at 1 minute after being placed into the BCP was significantly different between group HP and group NS (*P* = .012). ###### Total systemic vascular resistance index values between group HP and group NS.  PIB MAP was significantly lower (*P* = .0259) in the hypotension group \[n = 59, 96 mm Hg (82.5--106.75)\] than in the non-hypotension group \[n = 4, 122 mm Hg (110.5-- 123.0)\]. There were no postoperative neurological complications in the 3 groups. 4. Discussion ============= Low dose IV infusion of phenylephrine (0.5 or 1.0 μg/kg/h) did not decrease the incidence of hypotension after placement into the BCP. In this study, hypotension occurred in 93.65% of all patients within 1 minute after being placed in the BCP. Hemodynamic instability associated with the BCP is mainly caused by a reduction in cardiac preload during general anesthesia.^\[[@R11]\]^ Buhre et al^\[[@R11]\]^ found that 14% of blood volume shifted from the intra- to the extra-thoracic space after raising patients to the sitting position. In the sitting position, venous pooling in the lower extremities decreases the central blood volume. The systemic vascular resistance index is increased after the BCP when the patients are awake, but the TSVRi and blood pressure are decreased after a position change when the patients are under anesthesia. Thus, it is important to increase cardiac preload and the TSVRi. Application of a sequential compression device^\[[@R12]\]^ or stocking^\[[@R3]\]^ can reduce venous pooling in the lower extremities, and reduce hemodynamic derangements after a positional change. Unlike in previous studies,^\[[@R13],[@R14]\]^ the incidence of hypotension was very high in this study. A reason for this may be that blood pressure was assessed at the external auditory meatus instead of the right atrium, and the incidence of hypotension would be lower if MAP was measured at the level of right atrium as the estimated height between the meatus and right atrium would be 20 cm, resulting in an increase of 14.5 mm Hg in MAP at the right atrium. The MAP, measured at the level of the external auditory meatus, not at the level of the heart, was significantly correlated with the left or right regional cerebral oxygen saturation (rSO2) measured by near-infrared spectroscopy in seated patients for shoulder surgery.^\[[@R13]\]^ This study was performed in a clinical field, so we placed the transducer in the external auditory meatus to prevent cerebral ischemia because we could not measure rSO2. It is not clear that hypotension at the external meatus definitely reflects poor rSO2, however it gives a higher risk of low cerebral perfusion pressure and long-term hypoperfusion can lead to a decrease in rSO2 and subsequent complications. Therefore, the ABP transducer should be placed at the level of the external auditory meatus and not at the level of the heart upon insertion of an invasive percutaneous arterial catheter.^\[[@R2],[@R8]\]^ Further studies should be required for the clinical correlation between MAP at external meatus and rSO2. A second reason is that patients with hypertension or diabetes mellitus were included in this study. Preoperative use of antihypertensive medication was associated with an increased intraoperative hypotension episode.^\[[@R15]\]^ Furthermore, hypotension after induction of general anesthesia is associated with the presence of diabetes mellitus.^\[[@R16]\]^ Third, the dose of phenylephrine in this study is small. As the dose of phenylephrine increases, MAP increases.^\[[@R17]\]^ Phenylephrine, an α1-adrenergic receptor agonist, causes a direct increase in systemic vascular resistances, arterial pressure, and left ventricular afterload.^\[[@R17]\]^ Hypotension occurred most frequently within 1 minute after placement into the BCP. In addition, the cardiac index 1 minute after BCP placement tended to increase compared with baseline CI. This is thought to be due to the increase in cardiac output and decrease in MAP associated with preload-dependency after the BCP.^\[[@R17]\]^ Further evaluation of appropriate fluid loading is necessary. Although phenylephrine infusion did not reduce the incidence of hypotension, group HP had a decrease in the severity of hypotension. The TSVRi was also higher in group HP compared to group NS 1 minute after placed in the BCP. It seems to effect of afterload due to the continuous infusion of 1 μg/kg/min of phenylephrine. There was a difference in the post-induction MAP between the hemodynamic variables of the hypotensive group (59 patients) and the non-hypotensive group (4 patients). If the post-induction MAP is less than 96 mm Hg, severe hypotension may occur after postural conversion. In another study, pre-induction CI, SVI, and post-induction SVV before a positional change were potential important prediction factors for the development of hypotension after the BCP.^\[[@R14]\]^ Further research is needed to determine whether post-induction MAP is an indicator of postural hypotension. There are some limitations of this study. First, many studies have shown that during the BCP cerebral desaturation is mostly involved. Although cerebral oxygen saturation can provide a valuable endpoint when evaluating the effect of vasopressor therapy on cerebral perfusion,^\[[@R7]\]^ we did not measure cerebral oxygen saturation. Cerebral oximetry monitoring of the BCP is difficult to use because it is not covered by insurance in South Korea, and we did not know if these doses (1.0 and 0.5 μg/kg/min) of phenylephrine would preserve cerebral oxygen saturation at an appropriate level during surgery. Second, we did not measure TSVRI using the Flotrac system in this study. To measure SVRI with the Flotrac system, central venous pressure should be measured. However, there was no indication for central venous pressure monitoring in the elective arthroscopic shoulder surgery in this study. Therefore, the TSVRi was calculated in this study.^\[[@R10]\]^ Third, the remifentanil used for anesthesia may have had hemodynamic effects. BP were significantly decreased compared to baseline BP after induction of general anesthesia in normotensive and hypertensive patients.^\[[@R18]\]^ This study has shown that phenylephrine (1 μg/kg/min) can reduce the severity of hypotension but cannot prevent intraoperative hypotension after the BCP. Despite phenylephrine infusion, the incidence of hypotension after the BCP was very high. Careful observation and strict management of blood pressure are still needed in these patients. Author contributions ==================== **Conceptualization:** Ki Hwa Lee. **Data curation:** Hyun-seong Lee, Jin-Young Bang. **Formal analysis:** Myoung Jin Ko, Yei Heum Park. **Investigation:** Hyun-seong Lee. **Methodology:** Hyojoong Kim. **Project administration:** Hyojoong Kim. **Supervision:** Ki Hwa Lee. **Visualization:** Hyun-seong Lee, Soo Jee Lee. **Writing -- original draft:** Myoung Jin Ko, Hyojoong Kim. **Writing -- review & editing:** Soo Jee Lee, Yei Heum Park, Ki Hwa Lee. Abbreviations: ANOVA = analysis of variance, BCP = beach chair position, BIS = bispectral index, CI = cardiac index, HR = heart rate, IV = intravenous, MAP = mean arterial pressure, PIB = post-induction baseline, SVV = stroke volume variation, TSVRi = total systemic vascular resistance index. How to cite this article: Ko MJ, Kim H, Lee H-s, Lee SJ, Park YH, Bang J-Y, Lee KH. Effect of phenylephrine infusion on hypotension induced by the beach chair position: a prospective randomized trial. *Medicine*. 2020;99:28(e20946). Statistical analysis in this study was supported by the Inje University Haeundae Paik Hospital and ACE Statistical Consulting. The authors have no conflicts of interest to disclose. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

ACC : acetyl‐CoA carboxylase AFP : alpha fetoprotein ALDH1A1 : aldehyde dehydrogenase isoform 1A1 AMP : adenosine monophosphate AMPK : AMP‐activated kinase CAP : capsaicin CSC : cancer stem cell GFAP : glial fibrillary acidic protein HCC : hepatocellular carcinoma HIF‐1α : hypoxia‐inducible factor 1α Oct4 : octamer‐binding transcription factor 4 PGC1α : proliferator‐activated receptor‐γ coactivator 1‐α PPARγ : proliferator‐activated receptor‐γ SF : sorafenib 1. Introduction {#mol212488-sec-0001} =============== Cancer continues to be the second leading cause of death in Western countries. More than 2 million new cases of cancer and 610 000 cancer deaths are predicted to occur only in the United States during 2018 (Siegel *et al*., [2018](#mol212488-bib-0050){ref-type="ref"}). Hepatocellular carcinoma (HCC) is the most common liver malignancy in adults and the third leading cause of cancer mortality worldwide (Beal *et al*., [2017](#mol212488-bib-0002){ref-type="ref"}). The prognosis for patients with advanced HCC remains extremely poor due to the high resistance to cytotoxic therapy. For the past decade, the multikinase inhibitor sorafenib has been the only approved standard treatment for patients with advanced HCC (Kudo, [2017](#mol212488-bib-0028){ref-type="ref"}), albeit it only extends life expectancy by 2--3 months (Cheng *et al*., [2009](#mol212488-bib-0010){ref-type="ref"}; Llovet *et al*., [2008](#mol212488-bib-0034){ref-type="ref"}; Tovoli *et al*., [2018](#mol212488-bib-0054){ref-type="ref"}). This can be attributed to the acquisition of resistance, predominantly due to the upregulation of certain survival pathways that may cover up the death signals induced by sorafenib (Zhu *et al*., [2017](#mol212488-bib-0068){ref-type="ref"}). Several clinical trials have investigated other multitargeted kinase inhibitors, but none have shown any benefits over single‐agent sorafenib. In August 2018, the Food and Drug Administration approved lenvatinib for first‐line treatment of patients with unresectable hepatocellular carcinoma based on overall survival results obtained in clinical trials (Kudo, [2018](#mol212488-bib-0029){ref-type="ref"}; Spallanzani *et al*., [2018](#mol212488-bib-0051){ref-type="ref"}). Nevertheless, it is still too early to know whether this drug produces long‐term secondary effects or resistance. Hence, the elucidation of the underlying mechanisms of evasive resistance is required to overcome unwanted tumour recurrence and, consequently, to improve the beneficial effects of chemotherapy. Drug resistance to conventional chemotherapeutic agents has been closely related to many intrinsic or acquired properties associated with the presence of cancer stem cells (CSCs), which possess higher proliferative output and the ability to self‐renew. CSCs overexpress drug resistance genes, such as drug efflux transporters or detoxifying enzymes, which confer protection from the adverse effects of chemotherapeutic insult (Begicevic and Falasca, [2017](#mol212488-bib-0003){ref-type="ref"}). Therefore, to prevent drug resistance and tumour recurrence, it is imperative to gain a better understanding of the mechanisms involved in the resistance of stem cells to chemotherapy, which could lead to the discovery of new targets that can be exploited with a therapeutic purpose. Previous research has shown that the isolation of hepatocellular CSC (HCC‐CSC) can be performed using cell surface stemness‐associated markers, such as the transmembrane glycoprotein CD133, alpha fetoprotein (AFP), or aldehyde dehydrogenase isoform 1A1 (ALDH1A1), which have been used for further HCC‐CSC classification into different prognostic subtypes (Dai *et al*., [2018](#mol212488-bib-0013){ref-type="ref"}; Ma *et al*., [2008a](#mol212488-bib-0036){ref-type="ref"}). In particular, HCC‐CSCs expressing CD133 were associated with a poor prognosis because they bear great tumourigenic potential, possess greater colony‐forming efficiency and display chemoresistance to the classical anticancer drugs doxorubicin and fluorouracil (Ma *et al*., [2008b](#mol212488-bib-0037){ref-type="ref"}). Although the underlying molecular mechanisms of chemoresistance in these cells are not completely clear, the data indicate sustained activation of the PI3K/Akt cascade (Ma *et al*., [2008b](#mol212488-bib-0037){ref-type="ref"}). In line with this, signalling pathways involved in the acquired resistance to sorafenib include activation of the PI3K/Akt and JAK/STAT axes, the induction of HIF‐1α‐mediated adaptation to hypoxia or the upregulation of FGF signalling pathways (Zhu *et al*., [2017](#mol212488-bib-0068){ref-type="ref"}). Similarly, recent findings showed that the sensitivity to sorafenib after developing resistance can be restored with PI3K/Akt inhibitors (Wu *et al*., [2016](#mol212488-bib-0059){ref-type="ref"}; Yi *et al*., [2017](#mol212488-bib-0060){ref-type="ref"}). Our recent research on HCC resistance demonstrated that activation of the enzyme AMP‐activated kinase (AMPK) inhibits the PI3K/Akt pathway and sensitizes HCC cells to sorafenib (Bort *et al*., [2017](#mol212488-bib-0004){ref-type="ref"}). In addition, we demonstrated that the activation of AMPK by cannabinoids induces autophagy and suppresses HCC cell proliferation (Vara *et al*., [2011](#mol212488-bib-0056){ref-type="ref"}). Moreover, emerging evidence suggests that metformin, an AMPK activator used as an antidiabetic drug, resensitizes resistant cancer cells to chemotherapy by targeting several signalling pathways, including AMPK (Ling *et al*., [2014](#mol212488-bib-0033){ref-type="ref"}; You *et al*., [2016](#mol212488-bib-0061){ref-type="ref"}). In this regard, recent evidence demonstrates that metformin inhibits CSC proliferation (Finley, [2017](#mol212488-bib-0017){ref-type="ref"}; Saini and Yang, [2018](#mol212488-bib-0046){ref-type="ref"}) and reduces the expression of stemness markers (Paiva‐Oliveira *et al*., [2018](#mol212488-bib-0041){ref-type="ref"}). Therefore, AMPK emerges as a new target that could be involved in drug resistance and the development of CSCs. In this study, we analysed the role of AMPK in the development of stem‐like cells and in the sorafenib resistance of HCC cells. We show that the downregulation of AMPK plays a key role in mediating an increase in the expression of stemness markers and that the pharmacological activation of AMPK or AMPK overexpression overcomes this stem cell‐like phenotype. Furthermore, sorafenib resistance was associated with stemness and was abrogated by AMPK transfection or activation. Our results expand the role of AMPK in cancer and highlight novel treatment options for resistant cancer. 2. Materials and methods {#mol212488-sec-0002} ======================== 2.1. Reagents and antibodies {#mol212488-sec-0003} ---------------------------- Sorafenib was purchased from Sigma‐Aldrich (St. Louis, MO, USA). The compound A‐769662 and capsaicin were purchased from Tocris Bioscience (Bristol, UK). The primary antibodies anti‐CD133, anti‐ALDH1A1, anti‐pAkt‐ser473, p‐mTOR, pAMPKα1‐thr172, pACC‐ser79, anti‐cyclin D1, anti‐PGC1α, and anti‐PPARγ and the antibodies against the corresponding total forms were obtained from Cell Signaling Technology (Danvers, MA, USA). The primary antibody anti‐β‐catenin was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). The primary antibody anti‐Hif‐1α was purchased from Novus (St. Louis, MO, USA). Anti‐alpha fetoprotein and peroxidase‐labelled secondary anti‐mouse IgG were purchased from Sigma‐Aldrich, and anti‐rabbit IgG was purchased from Calbiochem (San Diego, USA). 2.2. Cell lines and cell culture {#mol212488-sec-0004} -------------------------------- The human hepatocellular carcinoma HepG2 cell line was purchased from the American Type Culture Collection (ATCC HB‐8065, Rockville, MD, USA). The human hepatoma cell line Huh7 was kindly provided by L. Boscá (Instituto de Investigaciones Biomédicas Alberto Sols, Madrid). Cell lines were incubated at 37 °C in a humidified atmosphere with 5% CO~2~ and cultured in DMEM/10% FBS supplemented with 1% nonessential amino acids, 100 IU·mL^−1^ penicillin G sodium, 100 μg·mL^−1^ streptomycin sulphate, and 0.25 μg·mL^−1^ amphotericin B (Invitrogen, Paisley, UK). To generate cancer stem‐like cells, HepG2 and Huh7 cells were cultured continuously for 12 months with a step‐wise increase in the sorafenib concentration (starting at 0.75 μ[m]{.smallcaps} and increasing the concentration by 0.15 μ[m]{.smallcaps} at each passage up to a final concentration of 8 μ[m]{.smallcaps}). Surviving cells were selected and designated as HepG2SF1 and Huh7SF1 cells. HepG2 and Huh7 parental cells were cultured in parallel without sorafenib and served as controls. 2.3. Cell proliferation assay {#mol212488-sec-0005} ----------------------------- Cell proliferation was analysed using the MTT assay. Briefly, 5 × 10^3^ cells/well was seeded into 12‐well plates and allowed to attach and grow for 24 h. After treatment with sorafenib for 24 h, 200 μL of MTT (3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide) dye solution (Sigma‐Aldrich) was added to each well and incubated at 37 °C for 4 h. Subsequently, the cells were lysed with 2‐propanol to dissolve the formazan crystals. Then, the optical density of each well was measured using a microplate reader (iMARK, Bio‐Rad Laboratories, Inc., Hercules, CA, USA) at a wavelength of 595 nm, and the nonspecific absorbance measured at 650 nm was subtracted. Each experiment was performed in triplicate. Cell viability was calculated as the percentage compared to the control cells, which were arbitrarily assigned 100% viability. The half‐maximal inhibitory concentration (IC50) values, defined as the concentration that inhibited 50% cell growth relative to control cells, were calculated using [graphpad]{.smallcaps} 6.0 (La Jolla, CA, USA) software. Cell viability was also determined by counting viable and dead cells by Trypan blue staining. Trypan blue‐positive and blue‐negative cells were counted using a Countess automated cell counter (Invitrogen, Carlsbad, CA, USA). The results are expressed in relation to the total number of cells counted. 2.4. Colony forming assay {#mol212488-sec-0006} ------------------------- Cells were seeded in flat‐bottom six‐well plates at different densities. Two weeks later, the medium was removed. Colonies were fixed in methanol, stained with 0.05% (w/v) crystal violet solution for 5 min, washed with PBS and counted. 2.5. Differentiation assay {#mol212488-sec-0007} -------------------------- For the *in vitro* differentiation of stem‐like cells into neurons, stem‐like cells (1 × 10^5^ cells/well) were seeded into 6‐well plates and incubated in phenol red‐free neurobasal medium (Invitrogen) supplemented with 2% B‐27 serum‐free supplement (Invitrogen), 2% CSS, and 2 m[m l]{.smallcaps}‐glutamine (Invitrogen) for 15 days. For glial redifferentiation, stem‐like cells were incubated in phenol red‐free DMEM (Sigma‐Aldrich) with 1% N‐2 supplement (Invitrogen), 2% CSS and 2 m[m l]{.smallcaps}‐glutamine (Invitrogen). 2.6. Western blot analysis {#mol212488-sec-0008} -------------------------- After treatment or transfection for 48 h, cells were harvested, and proteins were extracted using lysis buffer (50 m[m]{.smallcaps} Tris, pH 7.4, 0.8 [m]{.smallcaps} NaCl, 5 m[m]{.smallcaps} MgCl~2~, 0.1% Triton X‐100) containing protease inhibitor and phosphatase inhibitor cocktail (Roche, Diagnostics; Mannheim, Germany), incubated on ice for 15 min and cleared by microcentrifugation. Protein concentrations were measured using the Bio‐Rad™ protein assay kit (Richmond, CA, USA). The cell protein extracts (20 μg) were boiled for 5 min in loading buffer and then separated on 8--15% SDS/PAGE gels depending on the protein to be analysed. The separated protein bands were transferred onto a PVDF membrane and incubated with the primary antibodies diluted 1 : 1000 overnight at 4 °C. Horseradish peroxidase‐conjugated goat anti‐mouse and goat anti‐rabbit IgG secondary antibodies were then added at a dilution ratio of 1 : 2000, and the membranes were incubated at room temperature for 2 h. The immune complex was visualized with an ECL system (Cell Signaling Technology). 2.7. Flow cytometry {#mol212488-sec-0009} ------------------- A total of 5 × 10^5^ HCC cells were seeded into 6‐well plates and treated according to the experiment. The cells were then harvested in 0.35% trypsin, collected and centrifuged at 1500 ***g*** for 5 min at 4 °C. Subsequently, the cells were washed in 1 mL ice‐cold PBS and then centrifuged at 1500 g for 5 min at 4 °C. The cells were then incubated with an anti‐human CD133 antibody Alexa Fluor^®^ 488 conjugate (Cell Signaling Technology) at room temperature for 1 h. The cells were then washed twice with wash buffer to remove excess antibody and analysed on a FACSCalibur flow cytometry system (BD Biosciences, San Jose, CA, USA) using [cyflogic]{.smallcaps} software V1.2.1 (Perttu Terho, Mika Korkeamaki, CyFlo Ltd., Turku, Finland). A total of 10^4^ events were collected for each sample. 2.8. Confocal microscopy {#mol212488-sec-0010} ------------------------ The cells were fixed in 4% paraformaldehyde in PBS and incubated with 0.1% Triton X‐100 for permeabilization. Immunolabelling with an anti‐βIII tubulin polyclonal antibody (Covance, Princeton, NJ, USA) or an anti‐GFAP (glial fibrillary acidic protein) monoclonal antibody (Thermo Scientific, Waltham, MA, USA) was performed by incubation at room temperature for 1 h. Secondary labelling was performed with Alexa Fluor 488‐conjugated secondary antibodies (Invitrogen). Coverslips were then mounted with DAPI‐containing Mowiol mounting medium (Sigma‐Aldrich). Imaging was performed with a Leica TCS SP5 laser scanning confocal microscope with [las‐af imaging]{.smallcaps} software using a 40X oil objective. The quantification of images was performed with [imagej]{.smallcaps} v1.8.0 software (NIH Image, Bethesda, MD, USA). 2.9. RNA extraction and reverse transcription quantitative polymerase chain reaction {#mol212488-sec-0011} ------------------------------------------------------------------------------------ Total cellular RNA was extracted from sensitive and resistant cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer\'s protocol. Total RNA (2--4 μg) underwent cDNA synthesis using SuperScript™ RT (Roche, Basel, Switzerland) according to the manufacturer\'s protocol. qPCR was performed in a 10 μL volume using SYBR‐Green PCR Master Mix (Takara Bio, Inc., Kusatsu, Japan) on a 7500 Real‐Time PCR System (Applied Biosystems Inc., Foster City, CA, USA) according to the manufacturer\'s protocols. PCR amplification was carried out using the following primer sequences: Nanog‐F 5′‐TTTGTGGGCCTGAAGAAAACT‐3′, Nanog‐R 5′‐AGGGCTGTCCTGAATAAGCAG‐3′; Oct4‐F 5′‐GACAGGGGGAGGGGAGGAGCTAGG‐3′, Oct4‐R 5′‐CTTCCCTCCAACCAGTTGCCCCAAAC‐3′; and ABCB1A‐F 5′‐TTGCTGCTTACATTCAGGTTTCA‐3′, ABCB1A‐R 5′‐AGCCTATCTCCTGTCGCATTA‐3′. 2.10. siRNA transfections {#mol212488-sec-0012} ------------------------- Cells were transfected in 1 mL OPTIMEM containing 4 μg Lipofectamine iMax (Invitrogen) with 100 n[m]{.smallcaps} AMPK‐specific small interfering RNA (siRNA) duplexes (Ambion‐Life Technologies, Carlsbad, CA, USA) or scrambled RNA (control) according to the manufacturer\'s protocols (Invitrogen). At 48 h after transfection, the medium was removed and replaced with DMEM. At the indicated time points after transfection, cells were used for MTT cell viability assays or western blot analysis. 2.11. Transient transfections {#mol212488-sec-0013} ----------------------------- Plasmids encoding the full‐length human AMPK‐α1, AMPK‐β1 and AMPK‐γ1 were kindly provided by G. Hardie (University of Dundee, UK). Sensitive and resistant HepG2 and Huh7 cells were cotransfected with 5 μg recombinant α1 (pcDNA5‐FRT α1‐Flag), β1 (pCMV β1‐untagged) and γ1 WT (pcDNA5‐Flpln‐T10 γ1 WT‐Flag) plasmids using 5 μL Lipofectamine 3000 (Thermo Fisher) in antibiotic‐free medium and seeded into 12‐well plates. After 48 h of transfection, the transfection medium was replaced with another medium without serum, and the cells were maintained for 24 h and then assayed for cell viability. Protein expression was assayed by western blotting using anti‐FLAG antibodies (anti‐Flag M2 antibody, Sigma‐Aldrich). For tetracycline‐inducible plasmids, Flag‐tagged pcDNA5‐Flpln‐T10 protein expression was induced by the addition of 1 μg·mL^−1^ tetracycline in the transfection medium. 2.12. Animal studies {#mol212488-sec-0014} -------------------- All animal experiments followed the ARRIVE guidelines and were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986, and associated guidelines, EU Directive 2010/63/EU for animal experiments. The procedure was approved by the Alcalá University Ethical Commission and by the Ethical Commission of the Comunidad de Madrid (procedure PROEX 241/15). All animal studies were conducted in accordance with the Spanish institutional regulation (RD 53/2013) for the housing, care and use of experimental animals and met the European Community directives regulating animal research. Recommendations made by the United Kingdom Coordinating Committee on Cancer Research (UKCCCR) were followed carefully. To assess the welfare of animals, a panel of 10 indicators were recorded each day. When adverse effects, pain or distress was observed in the animals (score of 15 of 40), a humane endpoint was applied. Athymic nude‐Foxn1 (nu/nu) four‐week‐old mice were purchased from Envigo RMS (Barcelona, Spain) and housed in a laminar air flow cabinet under pathogen‐free conditions on a 12‐h light/dark schedule at 21--23 °C and 40‐60% humidity with access to food pellets and tap water ad libitum. Four animals were housed per cage. G Power analysis was used to calculate the sample size (Charan and Kantharia, [2013](#mol212488-bib-0006){ref-type="ref"}) according to our previous data and experience and considering a two‐tail effect and a significance level of 5%. Hepatocarcinoma tumours were induced in athymic mice by subcutaneous injection of 5 × 10^6^ HepG2 or HepG2SF1 cells. When tumours reached a volume of 70 mm^3^, mice were then randomly divided into experimental groups of 6 animals each, which received i.p. injections of 30 mg·kg^−1^ sorafenib (SF), 5 mg·kg^−1^ capsaicin or vehicle (DMSO) daily. Tumour size was measured daily and calculated using the formula V(mm^3^) = 1/2(length × width^2^). At the end of the study, the mice were sacrificed by placing them in a CO~2~ gas‐filled chamber, and the excised tumours were recovered and weighed. For the tumourigenic assay, hepatocarcinoma tumours were induced by subcutaneous injection of 2.5 × 10^6^, 5 × 10^6^, 10 × 10^6^ or 15 × 10^6^ HepG2 or HepG2SF1 cells on both sides of the mice. Tumour development was examined daily, and tumour volumes were calculated as indicated above. 2.13. Statistical analysis {#mol212488-sec-0015} -------------------------- Statistical significance was estimated with [graphpad]{.smallcaps} 6.0 (La Jolla, CA, USA) software using 1‐way or 2‐way ANOVA and Tukey\'s multiple comparison test or the unpaired Student\'s t‐test when indicated. Data are presented as the mean ± SD. 3. Results {#mol212488-sec-0017} ========== 3.1. Sorafenib resistance in HCC cells induces a stem‐like phenotype {#mol212488-sec-0018} -------------------------------------------------------------------- Drug resistance has been frequently associated with the emergence of cancer stem cells (Phi *et al*., [2018](#mol212488-bib-0042){ref-type="ref"}). To study the resistance mechanisms in HCC cells, we induced sorafenib‐resistant cells by long‐term incubation of HepG2 and Huh7 HCC cell lines with step‐wise increasing concentrations of sorafenib (0.75--8 μ[m]{.smallcaps}). After 12 months of exposure, the surviving cells that had routinely grown in the presence of 8 μM sorafenib were selected and designated as HepG2SF1 and Huh7SF1 cells. To investigate whether long‐term sorafenib treatment induced CSC differentiation in HCC cultures, we analysed the expression of the cell membrane protein CD133, which was recently identified as a stem cell marker in HCC (Castelli *et al*., [2017](#mol212488-bib-0005){ref-type="ref"}). According to previous data (Suetsugu *et al*., [2006](#mol212488-bib-0052){ref-type="ref"}), parental HepG2 cells do not express CD133 (Fig. [1](#mol212488-fig-0001){ref-type="fig"}A). Likewise, in Huh7 cells, CD133 is barely expressed (Fig. [1](#mol212488-fig-0001){ref-type="fig"}A). Interestingly, in HepG2SF1 cells treated with sorafenib long term, there was a slight increase in CD133 expression, whereas in Huh7SF1 cells, a notable increase in CD133 expression was observed (Fig. [1](#mol212488-fig-0001){ref-type="fig"}A). Flow cytometry determination of CD133 confirmed its increase in HepG2SF1 and Huh7SF1 cells (Figs [1](#mol212488-fig-0001){ref-type="fig"}A and S1). As CD133 was poorly detected in HepG2 cells, we examined the embryonic protein alpha fetoprotein (ΑFP), which has also been proposed as a stem cell marker and is clearly detected in HepG2 cells (but not in Huh7 cells). As shown in Fig. [1](#mol212488-fig-0001){ref-type="fig"}A, ΑFP expression increased in HepG2SF1 cells treated with sorafenib long term, indicating the acquisition of a stem‐like phenotype. To further investigate this hypothesis, we examined the expression of other stem cell‐associated genes, such as aldehyde dehydrogenase isoform 1A1 (ALDH1A1), a cytosolic enzyme that metabolizes reactive aldehydes and reactive oxygen species. Due to its role in metabolism, this detoxifying enzyme can confer cellular protection against cytotoxic drugs such as sorafenib. ALDH1A1 expression was increased in HepG2SF1 and Huh7SF1 cells (Fig. [1](#mol212488-fig-0001){ref-type="fig"}A). To examine the time course of the appearance of these stem markers, we analysed their expression 6 months after sorafenib treatment, and we observed that although A and CD133 in HepG2SF1 cells and CD133 in Huh7SF1 cells could be appreciated at 6 months, their expression was much lower than that at 12 months (Fig. S1). The enzyme ALDH1A1 was increased only after 12 months of sorafenib treatment (Fig. S1). Therefore, we continued the study with cells treated for 12 months with sorafenib. ![Long‐term treatment of HCC cells with sorafenib induces a stem‐like phenotype. (A) HepG2 and Huh7 cells were incubated with increasing concentrations of sorafenib for 12 months. The levels of stem cell markers were determined by western blot, qPCR and flow cytometry in parental HepG2 and Huh7 cells as well as in HepG2SF1 and Huh7SF1 cells treated long term with sorafenib. The qPCR data show the relative mRNA expression of actin, which was used as a housekeeping gene. Data represent the mean ± SD of seven independent experiments. (B) Colony‐forming capability (represented as the number of colonies formed/plated cells) of cells in the presence of vehicle (C) or 8 μ[m]{.smallcaps} sorafenib (SF). (C) Cell proliferation determined by the MTT assay and cell counting of parental HepG2 and Huh7 and HepG2SF1 and Huh7SF1 cells treated long term with sorafenib. (D) Differentiation capability of HepG2SF1 and Huh7SF1 cells. Cells were grown in neuron differentiation media for 21 days, after which the expression of βIII tubulin was determined by immunofluorescence or western blot. Alternatively, cells were grown in glial differentiation media, and the expression of glial acidic fibrillary protein (GFAP) was determined by immunofluorescence or western blot. Scale bar of confocal images indicates 25 μm. Data are the mean ± SD of three independent experiments. (E) Tumourigenic potential of HepG2SF1 and Huh7SF1 cells. Nude mice were inoculated with 2.5, 5 or 10 million HepG2 or HepG2SF1 cells, and tumour size was examined daily. Figure represents the frequency of tumour development (upper left panel) and the growth curves of the tumours over 15 days (mean ± SEM,*n *=* *6). \**P* ≤ 0.01 compared by Student\'s *t*‐test.](MOL2-13-1311-g001){#mol212488-fig-0001} We next analysed the expression of ABCB1A or p‐glycoprotein, a member of the multidrug resistant transporters that actively extrudes a variety of hydrophobic amphipathic drugs from the cell. This transporter was only analysed in Huh7 cells because we were not able to detect ABCB1A in HepG2 cells, which is probably due to the low expression of this gene in this cell line. Finally, we examined the transcription factors Nanog and octamer‐binding transcription factor 4 (Oct4), which are usually expressed in embryonic stem cells, where they are involved in pluripotency regulation, proliferation and renewal. These transcription factors have also been used to detect cancer stem cell subpopulations (Iv Santaliz‐Ruiz *et al*., [2014](#mol212488-bib-0021){ref-type="ref"}; van Schaijik *et al*., [2018](#mol212488-bib-0047){ref-type="ref"}; Zheng *et al*., [2013](#mol212488-bib-0067){ref-type="ref"}). The increased expression of all the genes analysed in sorafenib‐resistant cells (Fig. [1](#mol212488-fig-0001){ref-type="fig"}A) indicated that long‐term treatment of HCC cells with sorafenib induced the expression of proteins involved in drug resistance and pluripotency, characteristic of cancer stem cells. Then, we investigated whether long‐term sorafenib treatment led to other features of cancer stem cells. Cancer stem cells are capable of forming large colonies through clonal expansion from a single cell, and clonogenic activity has been considered an important indicator of undifferentiation (Rajendran and Jain, [2018](#mol212488-bib-0043){ref-type="ref"}). In agreement with this notion, HepG2SF1 and Huh7SF1 cells had a higher capacity of forming colonies than their parental cells (Fig. [1](#mol212488-fig-0001){ref-type="fig"}B). Notably, HepG2SF1 and Huh7SF1 cells formed colonies even when treated with sorafenib, whereas their parental cells exhibited decreased numbers of colonies in the presence of this drug (Fig. [1](#mol212488-fig-0001){ref-type="fig"}B). In addition, HepG2SF1 and Huh7SF1 cells showed a higher proliferation rate than HepG2 and Huh7 cells (Fig. [1](#mol212488-fig-0001){ref-type="fig"}C). One of the critical properties of CSCs is their potential to differentiate into unlimited heterogeneous populations of cancer cells (Desai *et al*., [2018](#mol212488-bib-0014){ref-type="ref"}). To investigate the differentiation potential of the long‐term sorafenib‐treated cells, HepG2, Huh7, HepG2SF1 and Huh7SF1 cells were incubated in neuronal differentiation or glial differentiation media for 15 days. Afterwards, differentiation into neurons was assessed by determining the expression of the specific neuronal marker βIII tubulin and differentiation into astrocytes by the expression of glial fibrillary acidic protein (GFAP). As shown in Fig. [1](#mol212488-fig-0001){ref-type="fig"}D, the incubation of cells with neuronal differentiation media induced a notorious increase in the expression of βIII tubulin in HepG2SF1 and Huh7SF1 cells compared with their parental cells (Fig. [1](#mol212488-fig-0001){ref-type="fig"}D). Likewise, the expression of GFAP increased in HepG2SF1 and Huh7SF1 cells when incubated in glial differentiation media (Fig. [1](#mol212488-fig-0001){ref-type="fig"}D). We then examined the tumourigenic potential of the stem‐like cells. According to the European normative on the reduction of animals in research (3 Rs principle), *in vivo* experiments were carried out only in the HepG2 cell line. To this end, HepG2 or HepG2SF1 cells were transplanted to generate xenograft‐derived tumours in mice. The frequencies of tumour formation were measured after the inoculation of nude mice with either 2.5, 5 or 10 million cells. As shown in Fig. [1](#mol212488-fig-0001){ref-type="fig"}E, tumours derived from HepG2SF1 cells appeared earlier than those derived from HepG2 cells, confirming that the stem‐like cells had an enhanced capacity to form xenograft‐derived tumours compared to their parental cells. Likewise, tumours derived from stem‐like cells grew faster than tumours derived from parental cells (Fig. [1](#mol212488-fig-0001){ref-type="fig"}E). Taken together, these results indicate that HepG2SF1 and Huh7SF1 cells exhibit phenotypic characteristics of cancer stem cells. To investigate whether these stem‐like cells were resistant to sorafenib, cells were treated in the absence of serum with 0, 1, 2, 3, 5 and 7 μ[m]{.smallcaps} sorafenib for 24 h. The results in Fig. [2](#mol212488-fig-0002){ref-type="fig"}A show that HepG2SF1 and Huh7SF1 cells could tolerate higher concentrations of sorafenib in the absence of serum compared to parental cells, with an IC50 fivefold and threefold higher than that of HepG2 and Huh7 cells, respectively (Fig. [2](#mol212488-fig-0002){ref-type="fig"}B). {#mol212488-fig-0002} Dysregulated signalling of the PI3K/Akt/mTOR pathway has been associated with resistance to several different chemotherapeutic agents, including sorafenib (Zhu *et al*., [2017](#mol212488-bib-0068){ref-type="ref"}). To address the status of this pathway, we examined the total and phosphorylation levels of Akt and mTOR in sensitive and sorafenib‐resistant HCC cells. As shown in Fig. [2](#mol212488-fig-0002){ref-type="fig"}B, HepG2SF1 and Huh7SF1 stem‐like cells had higher Akt and mTOR phosphorylation levels compared to sensitive cells, which is in good agreement with previous reports demonstrating the activation of this signalling pathway in chemotherapy‐resistant cells (Dong *et al*., [2017](#mol212488-bib-0015){ref-type="ref"}; Lindblad *et al*., [2016](#mol212488-bib-0032){ref-type="ref"}). 3.2. AMPK inhibition induces the acquisition of a stem‐like phenotype {#mol212488-sec-0019} --------------------------------------------------------------------- We recently showed that AMPK activation increases HCC cell sensitivity to sorafenib (Bort *et al*., [2017](#mol212488-bib-0004){ref-type="ref"}). Consequently, we wondered whether AMPK inhibition was involved in sorafenib‐acquired resistance and in the stem‐like cell phenotype of HepG2SF1 and Huh7SF1 cells. To this end, we first determined the expression levels of AMPK and its phosphorylation at the activation site Thr172, as well as the phosphorylation and levels of its downstream target, acetyl‐CoA carboxylase (ACC), in HepG2SF1 and Huh7SF1 cells. Decreased phosphorylation and expression of both AMPK and ACC were clearly observed in sorafenib‐resistant cells compared with their parental cells (Fig. [3](#mol212488-fig-0003){ref-type="fig"}A). Therefore, we examined the effect of AMPK downregulation on the expression of stem cell markers in HepG2SF1 and Huh7SF1 cells. To this end, cells were either treated with the AMPK inhibitor dorsomorphin or transfected with an siRNA targeting AMPK. As shown in Fig. [3](#mol212488-fig-0003){ref-type="fig"}B, AMPK inhibition or knockdown increased the expression of ΑFP, CD133 and ALDH1A1 in sensitive HepG2 cells and of CD133 and ALDH1A1 in Huh7 cells. The increase in CD133 in AMPK‐depleted cells was further confirmed by flow cytometry (Figs [3](#mol212488-fig-0003){ref-type="fig"}C and S2). These results suggest a negative role for AMPK in the stemness of HCC cells. {#mol212488-fig-0003} To address the relevance of AMPK downregulation in the resistance to sorafenib, we treated sensitive cells with the AMPK inhibitor dorsomorphin and examined cell viability. The inhibition of AMPK reduced sorafenib‐induced cell death in sensitive HepG2 and Huh7 cells (Fig. [4](#mol212488-fig-0004){ref-type="fig"}A) but had no effect on stem‐like sorafenib‐resistant cells (Fig. S3). Likewise, AMPK knockdown with an siRNA completely abolished the antiproliferative effect of sorafenib in sensitive parental cells, with no effect on stem‐like cells (Figs [4](#mol212488-fig-0004){ref-type="fig"}B and S3). These findings suggest that AMPK activity is necessary for sorafenib‐induced cell death and that AMPK downregulation makes cells refractory to sorafenib. ![The AMPK inhibition induces sorafenib resistance in HCC cells. (A) Effect of dorsomorphin on the viability of HepG2 and Huh7 cells treated with sorafenib. Cells were treated with or without 5 μ[m]{.smallcaps} dorsomorphin and sorafenib at the indicated concentrations for 24 h. Cell viability was determined by the MTT assay and is expressed as the percentage of the control (DMSO treatment). (B) Effect of AMPK knockdown on the viability of HepG2 and Huh7 cells treated with sorafenib. Cells were transfected with either siRNA‐control or an AMPK‐selective siRNA for 24 h and then treated with sorafenib at the indicated concentrations for 24 h. Cell viability was determined by the MTT assay and is expressed as the percentage of the control (DMSO treatment). Experiments were run in triplicate and carried out three times on separate occasions (data are the mean ± SD). \**P *\<* *0.005 significant difference between resistant and control cells and ^\#^ *P *\<* *0.005 between treated and nontreated cells by two‐way ANOVA and Tukey\'s multiple comparisons test.](MOL2-13-1311-g004){#mol212488-fig-0004} 3.3. AMPK upregulation decreases the expression of stem cell markers in HepG2SF1 and Huh7SF1 cells {#mol212488-sec-0020} -------------------------------------------------------------------------------------------------- To further explore the functions of AMPK as a drug resistance suppressor, we investigated whether AMPK upregulation could modify the expression of drug resistance and stem cell markers in sorafenib‐refractory cells. For this purpose, we first induced AMPK expression by transient transfection with plasmids containing the AMPK subunits α1, β1 and γ1 (Fig. S4). As shown in Fig. [5](#mol212488-fig-0005){ref-type="fig"}A, AMPK transfection notably decreased the expression of CD133, ALDH1A1 and alpha fetoprotein in sorafenib‐resistant cells, as determined by western blot. The determination of CD133 by flow cytometry confirmed the decrease in this stem cell marker in HepG2SF1 cells transfected with AMPK (Figs [5](#mol212488-fig-0005){ref-type="fig"}A and S2). Likewise, the levels of the drug efflux transporter ABCB1A and the embryonic genes OCT4 and NANOG decreased in AMPK‐transfected cells (Fig. [5](#mol212488-fig-0005){ref-type="fig"}A). {#mol212488-fig-0005} We then treated resistant cells with the well‐known AMPK activator A‐769662 and capsaicin, which, as we previously showed, activates AMPK in HepG2 cells (Bort *et al*., [2017](#mol212488-bib-0004){ref-type="ref"}). As shown in Fig. [5](#mol212488-fig-0005){ref-type="fig"}B, A‐769662 and capsaicin activated AMPK in both sensitive and resistant cells. Treatment of HCC cells with A‐769662 markedly decreased the expression of ΑFP, CD133 and ALDH1A1 in HepG2SF1 cells as well as the levels of CD133 and ALDH1A1 in Huh7SF1 cells. The effect of capsaicin on CD133 levels was similar, although not as strong. Similarly, A‐769662 and capsaicin decreased the expression of Nanog, OCT4 and ABCB1A in the stem‐like cells (Fig. [5](#mol212488-fig-0005){ref-type="fig"}B). 3.4. AMPK overexpression or activation restores the sensitivity to sorafenib in stem‐like cells {#mol212488-sec-0021} ----------------------------------------------------------------------------------------------- We next examined whether AMPK overexpression could resensitize stem‐like cells to sorafenib. Interestingly, the overexpression of AMPK in HepG2SF1 and Huh7SF1 cells resulted in sorafenib‐induced cell death at levels comparable with those of sensitive cells (Fig. [6](#mol212488-fig-0006){ref-type="fig"}A). The overexpression of AMPK in sensitive cells did not modify the effect of sorafenib (Fig. S4). In agreement with these results, the treatment of stem‐like cells with compound A‐769662 or capsaicin resensitized cells to sorafenib (Fig. [6](#mol212488-fig-0006){ref-type="fig"}B), whereas it had little effect on sensitive cells (Fig. S4). {#mol212488-fig-0006} 3.5. Potential mechanisms involved in the AMPK effect {#mol212488-sec-0022} ----------------------------------------------------- To elucidate the potential mechanisms underlying the AMPK effect on stem‐like cells, we analysed some of the signalling pathways associated with development and differentiation in HCC cells transfected with AMPK. Previous studies have reported that the Wnt/β‐catenin signalling pathway is associated with the control of stem cell differentiation (Valkenburg *et al*., [2011](#mol212488-bib-0055){ref-type="ref"}). The activation of this pathway causes the accumulation of β‐catenin in the cytosol, which is translocated to the nucleus, where it binds to LEF/TCF transcription factors and regulates the expression of target genes, including genes involved in differentiation and the cell cycle. To investigate whether the Wnt/β‐catenin pathway was activated in HCC stem‐like cells, we determined, by western blot, the levels of β‐catenin and cyclin D1, a downstream target of β‐catenin. The results in Fig. [7](#mol212488-fig-0007){ref-type="fig"} show that β‐catenin and cyclin D1 levels were decreased in HepG2SF1 and Huh7SF1 cells compared to their parental cells, while they were increased in AMPK‐transfected cells. It has been demonstrated that β‐catenin, through repression of the transcription factor TCF7L2, upregulates the expression of peroxisome proliferator‐activated receptor gamma coactivator‐1 alpha (PGC‐1α), which has been shown to be involved in hepatocyte differentiation (Jiao *et al*., [2017](#mol212488-bib-0024){ref-type="ref"}; Wanet *et al*., [2017](#mol212488-bib-0058){ref-type="ref"}). In contrast, AMPK can directly phosphorylate and activate PGC‐1α and regulate its expression (Colombo and Moncada, [2009](#mol212488-bib-0011){ref-type="ref"}). Therefore, we analysed PGC1α levels in HCC cells and observed that this coactivator was downregulated in stem‐like HepG2SF1 and Huh7SF1 cells (Fig. [7](#mol212488-fig-0007){ref-type="fig"}), in concordance with AMPK levels in those cells. In line with this, the levels of peroxisome proliferator‐activated receptor gamma (PPARγ) were decreased in stem‐like cells (Fig. [7](#mol212488-fig-0007){ref-type="fig"}). Interestingly, the overexpression of AMPK by transient transfection increased the levels of PGC1α and PPARγ (Fig. [7](#mol212488-fig-0007){ref-type="fig"}). {#mol212488-fig-0007} Recent findings have indicated that the hypoxia‐inducible factors HIF‐1α and HIF‐2α play critical roles in the gain of more malignant phenotypes by cancer stem cells (Mimeault and Batra, [2013](#mol212488-bib-0040){ref-type="ref"}). More specifically, it has been observed that hypoxia and enhanced HIF‐1α and HIF‐2α expression and activity, which frequently occur during tumour progression, may result in the upregulation of different stemness gene products that increase tumourigenicity (Heddleston *et al*., [2009](#mol212488-bib-0020){ref-type="ref"}; Ma *et al*., [2011](#mol212488-bib-0038){ref-type="ref"}; Mimeault and Batra, [2013](#mol212488-bib-0040){ref-type="ref"}). Importantly, it has been described that the downregulation of AMPK under normoxia induces a sustained increase in HIF‐1α and cooperates with c‐myc to increase tumourigenesis (Faubert *et al*., [2013](#mol212488-bib-0016){ref-type="ref"}). Thus, we determined the levels of HIF‐1α in HepG2SF1 and Huh7SF1 cells transfected with AMPK. As shown in Fig. [7](#mol212488-fig-0007){ref-type="fig"}, the stem‐like cells HepG2SF1 and Huh7SF1 had increased levels of HIF‐1α, suggesting that the depletion of AMPK could reverse the inhibition of this transcription factor, which might be involved in the upregulation of stem cell markers observed in these cells (Fig. S5). Notably, the levels of HIF‐1α decreased in AMPK‐transfected cells, in agreement with the observed decrease in stem cell markers under these conditions. All these results indicate that AMPK, by downregulating PGC‐1α and PPARγ and by upregulating HIF‐1α, could induce HCC stem cell reprogramming and restore sorafenib sensitivity. 3.6. AMPK activation reduces the tumour growth of stem‐like cells *in vivo* {#mol212488-sec-0023} --------------------------------------------------------------------------- To further assess the effects of sorafenib resistance on tumour growth *in vivo*, HepG2 or HepG2SF1 cells were inoculated in mice. Once the tumour size reached 70 mm^3^, the animals were randomly divided into groups and treated with vehicle, 30 mg·kg^−1^ sorafenib, 5 mg·kg^−1^ capsaicin or both compounds simultaneously for 12 days. We treated the mice with capsaicin since compound A769662 has shown poor bioavailability *in vivo* (Cool *et al*., [2006](#mol212488-bib-0012){ref-type="ref"}), and we have previous data on the *in vivo* AMPK activation by capsaicin (Bort *et al*., [2017](#mol212488-bib-0004){ref-type="ref"}). Tumour volumes were calculated daily. After sacrifice, the size and weight of the tumour were measured. The stem‐like HepG2SF1 cells had greater tumourigenicity than their parental HepG2 cells, since the tumours reached 70 mm^3^ twice as fast (data not shown). As shown in Fig. [8](#mol212488-fig-0008){ref-type="fig"}A, HepG2SF1 tumours grew clearly faster, especially in the last 5 days, when tumours grew exponentially. Moreover, sorafenib‐treated HepG2SF1 tumours grew at a similar rate as vehicle‐treated HepG2SF1 cells, further confirming the drug resistance developed by these cells. However, as expected, sorafenib reduced tumour growth in HepG2‐sensitive tumours (Fig. [8](#mol212488-fig-0008){ref-type="fig"}A). Notably, capsaicin was very efficient in reducing the tumour growth of stem‐like cells, which grew at a similar rate as that of parental cells (Fig. [8](#mol212488-fig-0008){ref-type="fig"}A). Likewise, stem‐like tumour weight at the end of the treatment was not significantly different between vehicle‐ and sorafenib‐treated mice, whereas it was significantly reduced by capsaicin (Fig. [8](#mol212488-fig-0008){ref-type="fig"}B). By contrast, the weights of sensitive tumours treated with sorafenib were significantly reduced compared with the vehicle‐treated animals. {#mol212488-fig-0008} Molecular analyses of sorafenib‐treated xenografts corroborated our findings in cell cultures. Immunoblot analysis of tumour lysates showed that tumours developed from stem‐like HepG2SF1 cells had increased expression of AFP, CD133 and ALDH1A1 as well as upregulation of the Akt/mTOR axis and lower expression of pAMPK, AMPK, pACC and ACC (Fig. [8](#mol212488-fig-0008){ref-type="fig"}C). Sorafenib treatment of HepG2 tumours slightly increased the expression of ΑFP, while sorafenib treatment of stem‐like tumours reduced the expression of CD133 but did not induce a significant modification of AFP or ALDH1A1. Interestingly, treatment of mice with capsaicin and especially with the combination of capsaicin and sorafenib activated AMPK and markedly reduced the expression of AFP, CD133 and ALDH1A1 (Fig. [8](#mol212488-fig-0008){ref-type="fig"}C), suggesting AMPK activation as a strategy to reduce stem‐like properties and resistance *in vivo*. 4. Discussion {#mol212488-sec-0024} ============= Sorafenib resistance remains a major problem for the effective treatment of hepatocellular carcinoma because sorafenib is the only standard clinical treatment against the advanced form of this disease (Chen *et al*., [2015a](#mol212488-bib-0009){ref-type="ref"}). The therapeutic benefit of this compound is limited, and invariably, tumour progression reappears (Le Grazie *et al*., [2017](#mol212488-bib-0030){ref-type="ref"}). Therefore, it is necessary to identify signalling pathways that promote drug resistance and to explore potential strategies to overcome resistance to find more effective therapies. In this study, we analysed the role of AMP‐activated kinase in the development of the cancer stem cell phenotype in two HCC cell lines. HepG2 and Huh7 cells grown in the presence of sorafenib for 12 months showed higher expression of drug resistance (ALDH1A1, ABCB1A) and stem‐related (CD133, OCT4, Nanog, alpha fetoprotein) genes and had higher clonogenic capacity, a higher proliferation rate and differentiation ability and increased tumourigenic potential, suggesting that sorafenib resistance induced or selected stem‐like cells. Although the biological function of CD133 is not well understood, it currently serves as a useful marker for liver cancer stem cells. Studies by Ma *et al*. ([2008a](#mol212488-bib-0036){ref-type="ref"}) found that CD133‐positive HCC cells possessed greater colony‐forming efficiency, a higher proliferative output and a greater ability to form tumours *in vivo*. The tumourigenic potential of those HCC cells correlated with the expression of both CD133 and ALDH (Ma *et al*., [2008a](#mol212488-bib-0036){ref-type="ref"}). Moreover, the overexpression of CD133 in 127 HCC specimens was associated with a poor prognosis (Dai *et al*., [2018](#mol212488-bib-0013){ref-type="ref"}). Recent results have shown that CD133 may interact with the regulatory subunit of PI3K, stabilizing AKT signalling and conferring CSC‐like properties to hepatocellular carcinoma (Jang *et al*., [2017a](#mol212488-bib-0023){ref-type="ref"},[2017b](#mol212488-bib-0022){ref-type="ref"}). Our results are in good agreement with these observations, as CD133‐positive cells presented higher proliferative and tumourigenic potential. In addition, we show that long‐term treatment of HCC cells with increasing doses of sorafenib induces the expression of pluripotency genes and converts them into highly aggressive and resistant cells. In agreement with our results, previous data showed that Nanog^+^ Huh7 cells exhibited increased chemoresistance, self‐renewal, sphere formation ability and *in vivo* tumour incidence compared to Nanog^−^ Huh 7 cells (Shan *et al*., [2012](#mol212488-bib-0049){ref-type="ref"}) when considering the Oct4 and Nanog transcription factors as gatekeepers of pluripotency (Lee *et al*., [2017](#mol212488-bib-0031){ref-type="ref"}; Seymour *et al*., [2015](#mol212488-bib-0048){ref-type="ref"}). In our study, the elevated expression of stem cell genes in HepG2SF1 and Huh7SF1 cells indicated that sorafenib resistance in HCC cells was associated with an increase in the CSC subpopulation and suggests the enrichment in CSCs as the underlying cause of resistance to sorafenib treatment in HCC. Interestingly, these cells were refractory to sorafenib‐induced cell death and showed overactivation of the Akt/mTOR pathway. Our finding that sorafenib resistance was associated with Akt/mTOR upregulation is in line with other previous studies (Jilkova *et al*., [2018](#mol212488-bib-0025){ref-type="ref"}; Zhai *et al*., [2014](#mol212488-bib-0064){ref-type="ref"}; Zhu *et al*., [2017](#mol212488-bib-0068){ref-type="ref"}) and reveals the importance of this pathway in drug resistance. The AMPK signalling pathway is a master regulator of cellular energy homeostasis and metabolism whose role in cancer is beginning to be revealed. There is clinical evidence that the regular use of pharmacological activators of AMPK to treat type 2 diabetes provides protection against the development of cancer (Hardie, [2015](#mol212488-bib-0018){ref-type="ref"}). Metformin impairs oncogenic signalling pathways such as Akt/mTOR and receptor tyrosine kinase (Saini and Yang, [2018](#mol212488-bib-0046){ref-type="ref"}), providing a proof of concept about the inhibitory role of activated AMPK in cell proliferation. In this study, we explored whether AMPK was involved in the drug resistance of HCC. Our results showed that in sorafenib‐resistant HCC cells, there was a decrease in the phosphorylation, expression and activation of AMPK. A recent study showed that sorafenib activated AMPK indirectly by inhibiting mitochondrial function in HEK293 cells (Ross *et al*., [2017](#mol212488-bib-0044){ref-type="ref"}). The sorafenib doses used in the previous study were higher than those used in the current study (10‐30 μ[m]{.smallcaps}), and the treatment times were shorter (1 h). Nevertheless, we observed a decrease in AMPK phosphorylation in our model when cells acquired resistance to sorafenib. Interestingly, AMPK upregulation, either by pharmacological activation or by transfection, was very successful not only in restoring sorafenib sensitivity but also in reducing the expression of most cancer stem markers analysed. Conversely, AMPK inhibition or AMPK knockdown induced sorafenib resistance in sensitive HCC cells. These results indicate that chemoresistance to sorafenib can be overcome by AMPK upregulation, suggesting a promising clinical therapeutic strategy to fight resistant HCC. This notion is in accordance with recent findings demonstrating that resistance to sorafenib may have a reversible phenotype (Kuczynski *et al*., [2015](#mol212488-bib-0027){ref-type="ref"}). Based on our aforementioned findings, we further confirmed the relationship between AMPK and sorafenib resistance in a mouse xenograft model. *In vivo* studies demonstrated that HepG2SF1 cells possessed higher tumourigenic potential and that tumours generated from these cells displayed stronger resistance to sorafenib. In addition, HepG2SF1 tumours had lower expression of AMPK and higher expression of stem cell markers. Although the role of AMPK in chemoresistance is unknown, Zeng *et al*. ([2018](#mol212488-bib-0063){ref-type="ref"}) recently showed that the inhibition of LKB1 and consequently AMPK induced docetaxel resistance in prostate cancer cells. Moreover, additional findings support the concept that AMPK activation could hinder the development of CSCs. For instance, AMPK activation inhibits the sphere‐forming ability of the CSC subpopulation in breast, pancreatic and ovarian cancer models (Saini and Yang, [2018](#mol212488-bib-0046){ref-type="ref"}). In line with this notion, a recent study demonstrated that CSCs from diabetic patients complicated with colorectal carcinoma and treated with metformin showed lower proliferation and higher rates of apoptosis than those of patients not pretreated with metformin (Zhang *et al*., [2013](#mol212488-bib-0065){ref-type="ref"}). In addition, metformin reduced the proliferation, migration, invasion, sphere formation and stemness characteristics of osteosarcoma cells *in vitro* (Chen *et al*., [2015c](#mol212488-bib-0008){ref-type="ref"}). In glioma cells, metformin suppressed spheroid formation and size and inhibited the expression of the glioma stemness‐related marker CD133 (Yuan *et al*., [2018](#mol212488-bib-0062){ref-type="ref"}). Although the role of AMPK in the development of cancer stem cells has not been established, there are recent reports demonstrating that HepG2 cells transfected with CD90 have higher expression of CD133 and lower levels of AMPK phosphorylation (Chen *et al*., [2015b](#mol212488-bib-0007){ref-type="ref"}), which is in line with our results. Likewise, recent data by Vazquez‐Martin *et al*. demonstrated that AMPK activation by metformin and A‐769662 notably prevented the transcriptional activation of Oct4 and impeded the reprogramming of mouse embryonic fibroblasts (Vazquez‐Martin *et al*., [2012](#mol212488-bib-0057){ref-type="ref"}), which is in good agreement with our results. The metformin‐induced attenuation of cancer stem cell features has also been reported by two other groups. Bao *et al*. ([2012](#mol212488-bib-0001){ref-type="ref"}) demonstrated that metformin significantly decreased cell survival, clonogenicity, sphere‐forming capacity and the expression of Oct4, Nanog and other CSC markers in pancreatic cells. Furthermore, Jung *et al*. ([2011](#mol212488-bib-0026){ref-type="ref"}) reported that metformin decreased the size and number of mammospheres and Oct4 expression in breast cancer MCF‐7 cells. These data are in accordance with our results and validate the notion that AMPK activation prevents CSC function. Recent data demonstrated that overexpression of the Wnt/β‐catenin signalling pathway induces rapid embryonic stem cell differentiation (Sun *et al*., [2017](#mol212488-bib-0053){ref-type="ref"}). However, the role of Wnt/β‐catenin signalling in stem cell reprogramming is controversial, as it seems that this pathway is involved in both the maintenance of potency and the induction of differentiation (Miki *et al*., [2011](#mol212488-bib-0039){ref-type="ref"}). Our data indicate that β‐catenin and cyclin D1 are downregulated in stem‐like cells and that AMPK overexpression increases the levels of both proteins, suggesting a role for this signalling pathway in the induction of HCC stem cell differentiation. The transcriptional coactivator PGC1α is a master regulator of mitochondrial biogenesis and energy expenditure. PGC‐1α directly activates multiple transcription factors to regulate the expression of a plethora of genes. Interestingly, AMPK can regulate PGC‐1α by increasing its expression and phosphorylation. PGC‐1α, in turn, has powerful transcriptional activity by interacting with many different transcription factors, such as PPARγ. Our data reveal that all these proteins are downregulated in sorafenib‐induced stem‐like HCC cells, consistent with their AMPK values and increase in AMPK‐transfected cells. In contrast, the hypoxia‐inducible factor HIF‐1α, which has been revealed as a master regulator of the stemness properties of cancer stem cells (Mimeault and Batra, [2013](#mol212488-bib-0040){ref-type="ref"}), is increased in HepG2SF1 and Huh7 cells compared with their parental cells and decreased in AMPK‐transfected cells. These results are in good agreement with previous results suggesting that the reduction in AMPK activity is sufficient to increase HIF‐1α protein levels in cancer cells under normoxic conditions (Faubert *et al*., [2013](#mol212488-bib-0016){ref-type="ref"}) and with the notion that HIF‐1α regulates the expression of stem cell markers such as Oct4, Sox2 and Nanog in cancer cells (Lu *et al*., [2015](#mol212488-bib-0035){ref-type="ref"}; Zhang *et al*., [2016](#mol212488-bib-0066){ref-type="ref"}). Likewise, in hepatospheres obtained from hepatocellular carcinoma cell lines by incubation in a defined medium, a marked increase in ABCB1A and HIF‐1α was observed and was associated with drug resistance (Hashimoto *et al*., [2014](#mol212488-bib-0019){ref-type="ref"}), concordant with our results. 5. Conclusions {#mol212488-sec-0025} ============== Our data demonstrate that the downregulation of AMPK by sorafenib treatment induces the enrichment of HCC cells in CSCs, which is probably mediated by HIF‐1α. These CSCs have enhanced tumorigenicity, clonogenic ability and differentiation capacity when cultured in specific media as well as enhanced expression of the stem cell markers Oct4, Nanog, ALDH1A1, ABCB1A and AFP. All these features can be reversed by AMPK activation or overexpression. In addition, AMPK upregulation restores sensibility to sorafenib. Our results suggest a novel role for AMPK in the chemotherapy resistance of hepatocellular carcinoma and provide a new therapeutic strategy for HCC. Conflict of interest {#mol212488-sec-0028} ==================== The authors declare no conflict of interest. Author contributions {#mol212488-sec-0027} ==================== AB designed and performed the experiments and validated and performed the formal analysis of data. BS designed and performed the experiments. PMG contributed with methodology and critical reading of the manuscript. DVC contributed with resources, methodology and critical reading of the manuscript. NRH contributed with resources, the development of methodology and revision of the manuscript and IDL designed the study, wrote the paper, supervised the research and acquired the financial support. All authors reviewed the manuscript. Supporting information ====================== ###### **Fig. S1.** Expression of stem cell markers in hepatocellular carcinoma cells treated with sorafenib for 6 and 12 months. HepG2 and Huh7 cells were cultured continuously for 12 months with a step‐wise increase in sorafenib concentrations (0.75--8 μ[m]{.smallcaps}). (A) The levels of AFP, CD133 and ALDH1A1 were determined by western blot at 6 (cells grown in 4 μ[m]{.smallcaps} sorafenib) and 12 (cells grown in 8 μ[m]{.smallcaps} sorafenib) months. β‐Actin is shown as a loading control. (B) Flow cytometry histograms of CD133 in HepG2 and Huh7 parental cells and in cells treated with sorafenib for 12 months (HepG2SF1 and Huh7SF1). Images are representative of two independent experiments. ###### Click here for additional data file. ###### **Fig. S2**. Flow cytometry histograms of CD133 in HepG2, Huh7, HepG2SF1 and Huh7SF1 cells transfected with an siRNA selective for AMPK or with a plasmid containing AMPK α1β1γ1. Images are representative of two independent experiments. ###### Click here for additional data file. ###### **Fig. S3.** Effect of AMPK inhibition on sorafenib sensitivity in stem‐like HCC cells. (A) Effect of the AMPK inhibitor dorsomorphin (Dorso) on HepG2SF1 and Huh7SF1 cell viability inhibition induced by sorafenib. Cells were treated with or without 5 μ[m]{.smallcaps} dorsomorphin and sorafenib at the indicated concentrations for 24 h. (B) Effect of AMPK knockdown with an siRNA on HepG2SF1 and Huh7SF1 cell viability inhibition induced by sorafenib. Cell viability was determined by the MTT assay and is expressed as the percentage of the control (DMSO treatment). (C) The levels of phosphorylated and total forms of AMPK and ACC in HCC cells were determined by western blot**.** β‐Tubulin (βTub) is shown as a loading control. A representative image of four different experiments is shown. Densitometric values (mean ± SD, *n *=* *3) relative to controls are shown below. \**P *\<* *0.005 significant difference between resistant and control cells and ^\#^ *P *\<* *0.005 between treated and nontreated cells by two‐way ANOVA and Tukey\'s multiple comparisons test. Experiments were run in triplicate and carried out at least two times on separate occasions. ###### Click here for additional data file. ###### **Fig. S4.** Effect of AMPK transfection or activation on sorafenib sensitivity in HepG2 and Huh7 cells. (A) The levels of pAMPK, AMPK, pACC and ACC in HepG2 and Huh7 cells transfected with AMPK α1β1γ1 were determined by western blot. Densitometric values (mean ± SD, *n *=* *4) relative to controls are shown on the right. (B) Effect of the transient expression of AMPK1β1γ1 on cell viability in HepG2 and Huh7 cells treated with increasing concentrations of sorafenib. Cells were treated with sorafenib at the indicated concentrations for 24 h. Cell viability was determined by the MTT assay and is expressed as the percentage of the control (DMSO treatment). (C) Effect of the AMPK activator A‐769662 and capsaicin on HepG2SF1 and Huh7SF1 cell viability following treatment with increasing concentrations of sorafenib. Cells were treated as described above. Experiments were run in triplicate and carried out at least three times on separate occasions. Data are the mean ± SD, *n *=* *3.\**P *\<* *0.005 significant difference between stem‐like and parental cells by two‐way ANOVA and Tukey\'s multiple comparisons test, \#*P *\<* *0.005 significant difference between AMPK‐transfected and nontransfected cells (panel A and B) or between control and treated cells (panels C). ###### Click here for additional data file. ###### **Fig. S5.** Putative mechanism involved in stemness in cells treated long term with sorafenib. In cells sensitive to sorafenib, high levels of AMPK inhibit HIF‐1α while enhancing PGC1α, PPARγ and β‐catenin. In sorafenib‐resistant cells, long‐term treatment with sorafenib induces a depletion of AMPK, releasing HIF‐1α inhibition, which regulates the transcription of stem‐related genes, such as Nanog and Oct4, promoting stemness. ###### Click here for additional data file. The authors would like to acknowledge D. Grahame Hardie for providing the AMPK plasmids and Professor Lilian Puebla for critically reading the manuscript. Authors acknowledge Wiley Editing Services for English language editing. This work was supported by the Tatiana Pérez de Guzmán Foundation \[grant number Patrocinio 2013‐01\]. BSG is a fellowship from the Spanish Education, Culture and Sports Ministry. 2.14. Data availability {#mol212488-sec-0016} ======================= The datasets generated and/or analysed during the current study are available at <https://doi.org/10.17632/ypw4d3j9wb.1>.

Introduction ============ Glioblastoma (GBM) is the most common central nervous tumor in adults. Despite therapeutic advances, the median survival time continues to be approximately 14 months.^[@B1]^ Younger age and higher Karnofsky Performance Status (KPS) scores are widely accepted independent predictors of prolonged survival.^[@B2]--[@B4]^ A significant association between the extent of resection and survival has also been shown in several retrospective studies.^[@B5]--[@B7]^ As several papers have reported, GBM is a histopathologically, radiographically, and genetically heterogeneous disease.^[@B8],[@B9]^ Recent studies have demonstrated that the heterogeneity of GBM may be related to the cell of origin, which has stem cell-like characteristics.^[@B10],[@B11]^ The adult human brain harbors neural stem cells within the subventricular zone (SVZ), which is located under the ependyma of the lateral ventricle.^[@B12]^ Recently, Lim et al. proposed a classification system based on the relationship of the contrast-enhanced lesion to the SVZ and the cortex on magnetic resonance imaging (MRI).^[@B13]^ They found that tumors contacting the SVZ and involving the cortex more often tended to be multifocal at diagnosis as well as recurrence. In addition, lower overall survival (OS) and progression-free survival have been reported in patients with GBM involving SVZ.^[@B14],[@B15]^ In cases of tumor recurrence, treatment options are individualized because no standard protocol has been developed. Till date, the benefits of repeat surgery for the treatment of recurrent GBM have not been fully established. Previous studies have retrospectively assessed patient outcomes after resection of recurrent GBM. The variables that were significantly associated with OS in at least one of these studies were preoperative KPS, extent of surgical resection, age, and time interval between the first and second surgeries.^[@B16]--[@B19]^ However, whether or not location of the recurrent lesion is associated with survival after repeat surgery remains unclear. We, therefore, aimed to determine whether SVZ involvement in patients with recurrent GBM is related to decreased survival after repeat surgery. Materials and Methods ===================== We retrospectively reviewed the medial records of 269 adult patients who had undergone surgical resection of a supratentorial GBM at the Tohoku University Hospital from January 1, 1997 to August 31, 2010. After initial surgery, patients had received involved-field external beam radiation therapy and either nitrosourea or temozolomide chemotherapy. Repeat surgery has been considered by the following points: (1) resectable tumor without severe morbidity and (2) younger patients or older patients with high KPS. Of the 269 patients, 61 received one or more additional resective surgeries for the treatment of histologically confirmed recurrent tumor. Medical charts were reviewed for information concerning patient age at the time of initial surgery, sex, additional therapy, KPS score at recurrence, and median survival time after repeat surgery. The degree of resection was retrospectively classified as follows on the basis of MRIs obtained \< 72 h after repeat surgery: gross-total resection (GTR) if no residual enhancement was noted on postoperative MRI or subtotal resection if any residual enhancement was noted on postoperative MRI.^[@B7]^ Perioperative mortality was defined as death within 30 days of repeat surgery. MRI sequences were acquired on a 1.5T scanner and typically included axial T~1~-weighted, T~2~-weighted fast spin-echo, and fluid-attenuated inversion-recovery sequences as well as gadopentetate dimeglumine-enhanced (Magnevist, Bayer Health Care, Leverkusen, Germany) axial and coronal T~1~-weighted images. As previously reported, tumors were classified as involving the SVZ (SVZ-positive) if the contrast-enhanced lesion contacted the lining of the lateral ventricle.^[@B2]^ Tumor recurrence was defined as the appearance or enlargement since prior imaging of a contrast-enhanced mass on T~1~-weighted MRI. The size of the contrast-enhanced lesion was approximated using the formula for the volume of an ellipsoid (4/3 × radius × radius × radius). Parametric data are expressed as mean ± standard deviation (SD). Nonparametric data were expressed as median \[interquaritile range (IQR)\]. Percentages were compared using the χ^2^ test. Continuous variables were compared using Student\'s *t*-test or the Mann-Whitney U test where appropriate. To determine the relative impact of multiple variables on OS and survival after repeat surgery, a Cox proportional hazards model was constructed. For the univariate analysis of potential prognostic factors, time-to-event distributions of the patients were estimated using Kaplan-Meier plots, and p values were obtained using log-rank tests. Variables with significance at the 0.20 level were selected for inclusion in the multivariate model and were entered in a forward stepwise fashion. Only variables with significance at the p = 0.05 level were accepted in the final model. All statistical tests were performed using SPSS version 21 (IBM, Chicago, Illinois, USA). Results ======= Among 269 patients with GBM, we obtained pre- and post operative MRI at initial surgery and follow-up MRIs from 223 patients. As shown in [Fig. 1](#F1){ref-type="fig"}, 102 GBMs were SVZ-positive and other 121 GBMs were SVZ-negative. During follow-up period, 66 of 102 SVZ-positive GBMs also recurred as SVZ-positive and 23 tumors recurred at locations noncontiguous with the recurrent lesion. In other 13 patients, 5 were still alive without recurrence and 8 were dead by other disease. Finally, 26 of 66 patients with SVZ-positive recurrent GBM received repeat surgery. On the other hand, repeat surgery was performed for 3 of 4 SVZ-positive and 32 of 85 SVZ negative recurrent tumors from primary SVZ-negative tumors. Twelve tumors recurred at locations noncontiguous with the recurrent lesion. In other 20 patients, 16 were still alive without recurrence and 4 were dead by other disease. There was no significant difference for frequency of repeat surgery between SVZ-positive and negative tumors at diagnosis \[22/102 (21.5%) vs. 35/121 (28.9%), p = 0.22, [Fig. 1](#F1){ref-type="fig"}\]. Median OS of SVZ-positive tumors was significantly shorter than that of SVZ-negative tumors (16 vs. 22 months, p = 0.005, [Fig. 2](#F2){ref-type="fig"}). The baseline demographic, clinical, and MRI characteristics of the patients evaluated and treated in this study are summarized in [Table 1](#T1){ref-type="table"}. The mean age (± SD) of the patients was 50.6 ± 14.6 years, and 38 patients (62%) were male. The median (IQR) KPS score at recurrence was 70 (60--80), while the mean tumor size (± SD) at recurrence was 15.6 ± 21.4 cm^3^. GTR was performed in 44 patients (72%). Of the 61 patients, SVZ-positive tumors were identified in 29 (48%) while SVZ-negative tumors were identified in 32 (52%). No significant difference in sex, age at recurrence, recurrent tumor size, or extent of resection at repeat surgery was observed between the two groups. However, preoperative KPS score in patients with SVZ-positive tumors was significantly lower than that in patients with SVZ-negative tumors. All 26 SVZ-positive tumors at initial diagnosis recurred as SVZ-positive tumors ([Fig. 3](#F3){ref-type="fig"}). Only three primary SVZ-negative tumors showed SVZ involvement at recurrence; the other primary SVZ-negative tumors were still SVZ-negative at recurrence ([Fig. 4](#F4){ref-type="fig"}). No perioperative mortality was observed in this study. All patients underwent follow-up MRIs for postoperative evaluation. Of the 61 patients, 54 (89%) received additional chemotherapy (temozolomide, ifosfamide + cisplatin + etoposide or intrathecal methotrexate) while 27 (44%) received stereotactic radiotherapy (SRT) following repeat surgery. During the follow-up period (24--206 months), second recurrence occurred in 57 patients and a third resective surgery was done in 17 (28%) of them. Of the 29 SVZ-negative tumors with second tumor recurrence, 23 (85%) re-recurred at locations contiguous with the recurrent lesion. Therefore, a third resective surgery was possible in 13 of the 23 patients (41%). On the other hand, in the 28 SVZ-positive GBMs with second recurrence, 21 tumors re-recurred at locations noncontiguous with the recurrent lesion \[cerebrospinal fluid (CSF) dissemination in 11 and contralateral invasion in 5\]. Therefore, only four patients (14%) received a third resection in this group. There was no significant difference in the number of patients who received chemotherapy after repeat surgery between the two groups. However, the number of patients who received SRT and/or underwent a third surgery was lower in the SVZ-positive group than in the SVZ-negative group ([Table 1](#T1){ref-type="table"}). The median OS and survival after repeat surgery was 25 months and 11 months, respectively, in this study. Patient age, sex, KPS score at recurrence, recurrent tumor size, resection rate at recurrence, SVZ involvement at initial and repeat surgery, and therapy after repeat surgery were examined as prognostic factors for survival using univariate analysis. The results are shown in [Table 2](#T2){ref-type="table"}. A significant difference in median survival after repeat surgery was noted between patients with SVZ-positive recurrence and patients with SVZ-negative recurrence (Kaplan-Meier estimates: 10 months vs. 14 months; p = 0.022; [Fig. 5](#F5){ref-type="fig"}). Median OS and survival after repeat surgery for patients with SVZ-positive recurrence of tumors that were SVZ-negative at diagnosis were 17 and 8 months, respectively. Only KPS at recurrence and SVZ involvement for survival from repeated surgery at the p = 0.20 level and were included in the multivariate model. Hazard ratios (HRs) from the multivariate results for each factor are shown in [Table 3](#T3){ref-type="table"}. When adjusting for all factors, only SVZ involvement at recurrence was a significant predictor of survival after repeat surgery (HR, 1.87; 95%CI, 1.06--3.28; p = 0.029). Discussion ========== Several papers in the past decade have emphasized the importance of surgical resection for primary GBM.^[@B5]--[@B7]^ However, the benefits of repeat surgery for recurrent GBM have not been completely determined. Previous papers have identified age, preoperative KPS score, and resection rate at recurrence as important prognostic factors.^[@B16]--[@B20]^ However, these factors were not identified as significant prognostic factors in our study, although the results of this study are subject to the limitations of a retrospective study, only SVZ involvement at recurrence was associated with decreased survival after repeat surgery. Previous papers have reported associations between SVZ involvement, aggressive tumor behavior, and shorter OS in patients with GBM.^[@B13]--[@B15]^ Lim et al. reported that contrast-enhanced lesions contacting both the cortex and SVZ were most likely to be multifocal at the time of initial diagnosis. In addition, recurrent tumors were more likely to develop at locations distant to the initial lesion in patients with SVZ involvement. In contrast, GBMs not involving the SVZ or cortex were not multifocal at initial diagnosis and always recurred within 2 cm of the resection margin.^[@B13]^ Chaichana et al. reported an association between periventricular tumor location (SVZ involvement) and poor survival.^[@B14]^ They proposed a classification system including periventricular involvement for the prediction of outcome in patients with primary GBM.^[@B21]^ Our study confirmed that SVZ involvement at diagnosis was an important predictor of OS. In our result, the frequency of repeat surgery in patients with SVZ-positive GBMs was lower than that in patients with SVZ-negative GBMs, however, there was no significant difference. Other factors such as invasion to eloquent lesions could be also important for indication of repeat surgery. In this study, SVZ involvement was identified at recurrence in all patients who had primary SVZ-positive GBMs. In addition, most of these tumors re-recurred at locations noncontiguous with the recurrent lesion (CSF dissemination or contralateral invasion). As a result, a third resection was possible in only four patients in this group. In contrast, except for a few cases, SVZ-negative GBMs recurred within the SVZ-negative region. In addition, the tumor location at the second recurrence was quite similar to that of the primary and first recurring lesions. Therefore, a third resection was possible in approximately half the patients with SVZ-negative recurrent GBMs. In addition, SVZ involvement was associated with survival after repeat surgery in patients with recurrent GBM. Although a third resection was not associated with survival after the second repeat surgery, more aggressive tumors with SVZ involvement may have been associated with poorer survival after repeat surgery. On the other hand, median OS and survival after repeat surgery for patients with SVZ-positive recurrence of tumors that were SVZ-negative at diagnosis were 17 and 8 months, respectively. These results suggest that OS for patients with SVZ-negative tumors at diagnosis was relatively favorable; however, if the tumors recurred with SVZ involvement, the chances of survival became low. Despite the limit ed availability of cases, these results may be of interest. However, what remains less well known is why SVZ involvement is associated with poorer survival. In basic science studies, Sanai et al. demonstrated that cells obtained from the lateral wall of the lateral ventricle, which is called the SVZ, harbors cells with stem cell-like features of self-renewal and multi-potentiality.^[@B12]^ While some GBMs may arise from transformed SVZ stem cells, other GBMs may be initiated by neoplastic transformation of astrocyte precursor cells or dedifferentiated mature astrocytes.^[@B22]^ The aggressive behavior of SVZ-positive GBMs may be related to the recruitment of neural stem cells from the SVZ that have a tendency toward invasive proliferation. However, Kappadakunnel et al. found no relationship between stem-cell gene expression and SVZ grade, but they did find an association between stem-cell gene expression and survival.^[@B23]^ As these researchers noted, more research is required to clarify the relationship among SVZ, cancer stem cells, and survival. Despite its retrospective design, this study is the first to report a possible association between recurrent GBM tumors adjacent to the SVZ and decreased survival after repeat surgery. Nonetheless, larger prospective studies may provide further relevant information. However, the findings of this study may be helpful to determine therapeutic strategies for recurrent GBM. With regard to recurrence, SVZ-negative recurrent GBMs may be good candidates for repeat surgery. The authors would like to thank Enago ([www.enago.jp](www.enago.jp)) for the English language review. ![Flowchart illustrating the subventricular zone (SVZ) involvement of glioblastoma at diagnosis and recurrence. Distant included the patients recurred at locations noncontiguous with the recurrent lesion \[cerebro-spinal fluid (CSF) dissemination or contralateral invasion\]. Others included the patients survived without recurrent lesion or died from other disease.](nmc-54-302-g1){#F1} {#F2} {#F3} {#F4} {#F5} ###### Summary of clinical, treatment, and magnetic resonance imaging characteristics of 61 patients with glioblastoma Parameter All patients SVZ-positive SVZ-negative p -------------------------------- -------------- -------------- -------------- --------------- Patients   No. (%) 61 29 (48) 32 (52) Sex 0.18   Male 38 21 17   Female 23 8 15 Age (years, mean + SD) 50.6 + 14.6 52.4 + 13.6 49.0 + 15.6 0.37 KPS at recurrence (IQR) 70 (60--80) 60 (50--70) 70 (60--90) **0.034** Tumor size (cm^3^) (mean + SD) 15.6 + 21.4 19.4 + 21.3 12.2 + 21.1 0.19 Extent of resection (%) 0.39   Gross total 44 (72) 19 (66) 25 (78)   Subtotal 17 (28) 10 (34) 7 (22) Primary lesion (%) **\< 0.0001**   SVZ-positive 26 (43) 26 (90) 0   SVZ-negative 35 (57) 3 (10) 32 (100) Therapy after repeat surgery   Chemotherapy (%) 54 (89) 25 (86) 29 (91) 0.69   SRT (%) 27 (44) 8 (28) 19 (59) **0.019**   3rd resective surgery (%) 17 (28) 4 (14) 13 (41) **0.024** GBM: glioblastoma, IQR: interquatile range, KPS: Karnofsky Performance Status, MRI: magnetic resonance imaging, SD: standard deviation, SRT: stereotactic radiotherapy, SVZ: subventricular zone. ###### Outcomes of 61 patients with GBM who underwent repeat surgery Parameters Survival from repeat surgery (months) p ------------------------------ --------------------------------------- ----------- Sex 0.23   Male (n = 38) 12   Female (n = 23) 11 Age 0.82   \< 50 (n = 27) 11   \> 50 (n = 34) 11 KPS at recurrence 0.11   70--100 (n = 37) 9   40--60 (n = 24) 12 Tumor size (cm^3^) 0.88   \< 10 cm^3^ (n = 38) 11   \> 10 cm^3^ (n = 23) 11 Resection rate 0.23   Total (n = 44) 11   Subtotal (n = 17) 11 Primary lesion **0.021**   SVZ-positive (n = 26) 9.5   SVZ-negative (n = 35) 13 Recurrent lesion **0.022**   SVZ-positive (n = 29) 10   SVZ-negative (n = 32) 14 Therapy after 2nd operation 0.87   SRT (+) (n = 19) 11   SRT (−) (n = 38) 11   3rd operation (+) (n = 20) 11   3rd operation (−) (n = 37) 11 KPS: Karnofsky Performance Scale, SRT: stereotactic radiotherapy, SVZ: subventricular zone. ###### Multivariate analysis of survival from repeat surgery Parameters HR 95%CI p ------------------------ ------ ------------ ----------- KPS at recurrence 0.13   KPS \< 70 1.54 0.88--2.68   KPS \> 70 1 SVZ (recurrent lesion) **0.029**   SVZ-positive 1.87 1.06--3.28   SVZ-negative 1 CI: confidence ratio, HR: hazard ratio, KPS: Karnofsky Performance Scale, SVZ: subventricular zone. [^1]: **Conflicts of Interest Disclosure** The authors report no conflict of interest. All authors who are members of The Japan Neurosurgical Society (JNS) have registered online Self-reported COI Disclosure Statement Forms through the website for JNS members.

{#sp1 .271}

Background ========== According to the Centers for Disease Control and Prevention (CDC), Lyme disease is the fastest growing vector-borne disease in the United States with over 40,000 cases reported during 2001--2002, representing a 40% annual increase in incidence \[[@B1]\]. Studies from the 1990\'s suggest that the actual number of cases may be as much as twelve times higher when factoring in underreporting \[[@B1]\]. Enzootic cycles of Lyme disease can be maintained in a wide range of ecologic conditions \[[@B2],[@B3]\]. Forty-nine of 50 states and the District of Columbia in the USA had reported Lyme disease cases by 1998 \[[@B4]\] The areas of the country endemic for Lyme disease have expanded \[[@B5]\]. In New York, over an 11 year period, cases have spread throughout the state from the original southeastern focus \[[@B6]\]. Increasing numbers have also been reported in the United Kingdom, France, and Sweden \[[@B3]\]. Lyme disease presents formidable challenges because of the high percentage of cases that become chronic in the absence of early treatment \[[@B7],[@B8]\] and the complexity and cost of managing the long-term use of antibiotics in treating chronic disease \[[@B9]\]. The number of Lyme disease cases that become chronic has been reported to be as low as 0.5% to 13% per year for patients treated at the time of an erythema migrans rash \[[@B10]-[@B13]\]. However, as many as 34% to 62% developed the chronic form of the disease in two studies \[[@B7],[@B8]\]. A third of Lyme disease patients in one population-based retrospective cohort treated in the late 1980\'s were found to have chronic Lyme disease an average of 6.2 years after treatment \[[@B7]\]. Two thirds of 215 Lyme disease patients diagnosed in Westchester County, New York, USA remained ill an average of 3.2 years after treatment \[[@B8]\]. Analysis ======== The Klempner et al. trials that appeared in the *New England Journal of Medicine*examined the benefits of treating chronic Lyme disease patients with one month of intravenous ceftriaxone followed by eight weeks of oral doxycycline \[[@B14]\]. Chronic Lyme disease patients who received antibiotics for 90 days were no more likely to improve than those receiving placebo. The high treatment failure rate of 60% was significantly greater than the 0--37% failure rates documented in previous studies of chronic Lyme disease \[[@B15]-[@B20]\] (Table [1](#T1){ref-type="table"}). For this reason, it seemed prudent to examine the validity of the authors\' conclusions regarding the treatment, in general, of chronic Lyme disease patients. ###### Diversity of chronic Lyme disease studies: populations, treatments, durations and outcomes. **Study** **Year** **Size** **Patient population** **Type of study** **Treatment** **Duration of Rx (days)** **Outcome** ------------------ ---------- ---------- ------------------------------------------------------------------------------------- ------------------- ----------------------------------------------------------------------------- --------------------------- -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Dattwyler \[20\] 1988 23 Late Lyme disease in the NorthEast (NE) USA Randomized trial IV Ceftriaxone vs IV penicillin 14 vs 10 92% responded to ceftriaxone, 50% improved with penicillin Logigian \[15\] 1990 27 Neurologic LD patients in NE USA Case series IV Ceftriaxone 14 63% improved, 22% improved but then relapsed, and 15% had no change in their condition Hassler \[19\] 1990 135 Stage three borreliosis manifestations of at least six months\' duration in Germany Randomized trial IV Cefotaxime vs IV penicillin G 10 87.9% vs 61.3% respectively Full or incomplete remission of symptoms in. Wahlberg \[17\] 1994 100 Consecutive LD patients in Finland Case series IV Ceftriaxone, oral amoxicillin plus probenecid and/or oral cefhadroxil.\* 14 to114 31% of 13 treatments successful with 14 days of IV ceftriaxone alone, 89% of 56 treatments with IV ceftriaxone followed by 100 days of amoxycillin plus probenecid successful, and 83% of 23 treatments with ceftriaxone followed by 100 days of cephadroxil successful. Donta \[18\] 1997 277 Chronic LD in NE USA Case series Oral tetracycline 30 to 330 20% of the patients were cured; 70% of the patients\' conditions improved. Logigian \[16\] 1999 18 Neurologic LD in NE USA Case series IV ceftriaxone 28 100% of 18 patients rated themselves as back to normal or improved. Klempner \[14\] 2001 107 LD with persistent symptoms in NE USA Randomized trial IV ceftriaxone + oral doxycycline 90 40% vs 36% for treatment vs placebo in improvement in quality of life (SF-36) Krupp \[46\] 2003 55 LD with disabling fatigue in NE USA Randomized trial IV ceftriaxone 30 69% rx vs 23% for treatment vs placebo in the primary outcome -- fatigue. No improvement on cognitive function or the clearance of Borrelia OspA antigen in the spinal fluid Donta \[25\] 2003 235 LD with fatigue, musculoskeletal pain, and neurocognitive dysfunction in NE USA Case series Oral macrolide + hydroxychloroquine 90 80% had self reported improvements of 50% or more Dattwyler \[27\] 2005 201 Late LD in NE USA Randomized trial IV ceftriaxone 14 vs. 28 76% vs 70% clinical cure rates for 14 and 28 days respectively Borg \[26\] 2005 65 Neurologic LD in Sweden Randomized trial IV ceftriaxone vs oral doxycycline 10 to 14 79% vs 72% completely recovered, the remaining improved. \* Patients treated with different combinations of oral, IM or IV antibiotics and variable durations of treatment. Internal validity of the Lyme disease trials conducted by Klempner et al ------------------------------------------------------------------------ Blinded, randomized controlled trials (RCT) are seen as the most reliable evidence in medicine if internal and external validity can be assumed \[[@B21],[@B22]\]. Klempner et al. enrolled a homogeneous patient population, used a randomized design, standardized treatment, placebo-controls, blinding, a validated quality of life outcome measure, and intent-to-treat analysis. The study suggested that treatment for 3 months was no better than placebo for a select population who remained ill an average of 4.7 years after an average of more than three courses of treatment \[[@B14]\]. Assessing the internal validity of these trials for estimating efficacy in the study population is beyond the scope of this paper; for present purposes, results of the trials are assumed to be internally valid, based on the RCT design. However, achieving internal validity does not imply generalizability. Generalizability of the Lyme disease trials conducted by Klempner et al ----------------------------------------------------------------------- Generalizability can be assessed by considering factors that may influence the outcome of an intervention across varied medical settings with diverse patient populations \[[@B23]\]. The authors of the trials did not discuss generalizability to the everyday medical setting. Investigators of other conditions have demonstrated the consequences when a study fails to address the broader group seen in everyday clinical practice. Jüni et al. cites the lack of effectiveness of fibrinolytic therapy for suspected acute myocardial infection when generalizing the results from a younger group to the elderly and when generalizing timely fibrinolytic therapy to patients presenting more than 12 hours after symptom onset \[[@B22]\]. This review examines whether the Lyme disease patients enrolled in the Klempner et al. trials represent those commonly seen in everyday practice. Subjects were eligible if they were at least 18 years old, had a history of Lyme disease acquired in the United States, and had at least one of the following: a history of a single or multiple erythema skin lesion, early neurologic or cardiac symptoms attributed to Lyme disease, radiculoneuropathy, or Lyme arthritis. Documentation by a physician of previous treatment of acute Lyme disease with a recommended antibiotic regimen was also required. At the time of enrollment, all patients had one or more of the following symptoms that interfered with their function: widespread musculoskeletal pain, cognitive impairment, radicular pain, paresthesias, or dysesthesias. Profound fatigue often accompanied one of these symptoms. The chronic symptoms had to have begun within 6 months after the initial infection with B. burgdorferi and had to have persisted for at least 6 months but less than 12 years. Patients were excluded if they had hypersensitivity to the study medication, had previously received parenteral antibiotic therapy for 60 days or more for their current symptoms, had active inflammatory synovitis, had a coexisting condition that could have accounted for their symptoms, or were unable to discontinue medication that could interfere with the evaluation of their response to the treatment regimen (e.g., narcotic analgesics or prednisone in a dose of 10 mg per day or more). Patients with a positive polymerase-chain-reaction (PCR) test for B. burgdorferi DNA in plasma or cerebrospinal fluid at baseline were also excluded. The results of the trials are not generalizable to patients receiving treatment for chronic Lyme disease within 6 months of their initial presentation, because the investigators excluded these patients from study. Chronic Lyme disease has been reported as early as 2--4 weeks after onset of acute disease \[[@B18],[@B24]\]. The impact of treatment delay is poorly understood. Asch et al. describe a retrospective cohort of 215 subjects with an average 6-week delay in getting treatment \[[@B8]\]. Sixty-two percent were ill an average of 3.2 years after initial treatment. The Klempner study did not consider the impact of treatment delay on long-term treatment failure. In the published report, the authors did not make it very clear that participants had already been ill with Lyme disease an average of 4.7 years at the time of their enrollment in the study \[[@B14]\]. Information about this potential selection factor is found only in the tables -- rather than in the results section or the abstract, where it should have been in order to avoid misinterpretation of the results \[[@B14]\]. The Klempner report\'s failure to take average duration of study participants\' illness into account when interpreting the results gives readers the potentially misleading impression that the Klempner et al. study can be generalized to the overall population of patients that present with persistent symptoms and a history of Lyme disease. Furthermore, the results of the trials may not be generalizable to chronic Lyme disease patients presenting for a first or second retreatment. The authors did not discuss the significance of the study participants\' average of 3 previous courses of antibiotics \[[@B14]\]. Again, this potential selection factor is only described in the tables, rather than, more appropriately, in the results section or the abstract \[[@B14]\]. Initial retreatment is both commonplace and successful in previous studies \[[@B15]-[@B20],[@B25]-[@B27]\]. By failing to enroll a sufficient number of patients who had received fewer than three previous courses of treatment, the researchers may have excluded the subset of Lyme disease patients most likely to benefit from retreatment. The investigators can only draw conclusions about the 3-month combination of oral and intravenous antibiotic treatment that was chosen for the study and not about longer treatments or simultaneous administration of multiple antibiotics. They dismissed the potential benefit of longer treatment or other combinations of antibiotics by saying \"Experience with other chronic infectious diseases caused by persistent bacteria (e.g., syphilis, tuberculosis, and helicobacter infection) suggests that it is unlikely that more prolonged antibiotic therapy or a different combination of antibiotics would result in greater improvement than was observed in this study\" \[\[[@B14]\], p.89\]. The authors did not provide references to support this statement; prolonged antibiotic use or simultaneous administration of multiple antibiotics *have*been effective for tuberculosis \[[@B28]\] and helicobacter infection \[[@B29]\]. The two trials cannot be generalized to chronic Lyme disease patients who have never received treatment, since these patients were excluded from study. Up to one third of patients never present with the classic erythema migrans rash, Bell\'s palsy, meningitis, heart block, and/or arthritis, which are indications of early Lyme disease \[[@B17],[@B18],[@B20],[@B27]\]. It is not clear that the same treatment that is effective for early Lyme will be equally effective for untreated chronic Lyme. Prolonged antibiotic treatment may be called for in cases of untreated chronic Lyme. The authors did not discuss the reasons two Klempner et al. trials failed whereas previous studies showed a benefit of retreatment with antibiotics \[[@B15]-[@B20]\]. Wells stressed the need for authors of clinical trials to keep informed about the results of other relevant studies \[[@B30]\]. There were six previous studies of chronic Lyme disease that described differing treatment regimens and durations, and broader populations than those included in the Klempner et al. trials (Table [1](#T1){ref-type="table"}). Both oral and intravenous antibiotics were effective for late, chronic, neurologic, and stage 3 Lyme disease in Europe and the USA. The treatment duration ranged from 10 to 330 days. Given the narrowly defined study population in the Klempner et al. trials, the results of previous treatment studies should not be ignored when drawing general conclusions about effects in a broader target population. Misinterpretation of the results -------------------------------- On June 12, 2001 the National Institutes of Health (NIH) issued a press release titled \"Chronic Lyme Disease Symptoms Not Helped by Intensive Antibiotic Treatment.\" The release quotes Klempner as follows, \"We think it is unlikely that a longer course of treatment or different antibiotic combination would result in greater improvement than what we found in these studies\" \[\[[@B31]\], p.1\]. The statement did not discuss the limited generalizability of the Klempner study \[[@B31]\]. Subsequent reviews of the trials have discouraged treatment for chronic Lyme disease without addressing their limitations. A 2002 review in *Arthritis Research*cited these trials as evidence that \"Prolonged antibiotic treatment for suspected \'chronic Lyme disease syndrome\' is therefore expensive, ineffective, burdened with side effects and should be avoided\" \[\[[@B32]\], p.23\]. Blacklow, in a summary and comment in the *Journal Watch Infectious Diseases*, stated \"it is unlikely that tinkering with antibiotic choices and durations of therapy will alter these findings\" \[\[[@B33]\], p.1\]. This conclusion restates Klempner et al.\'s dismissal of the value of additional antibiotic therapy or a different combination of antibiotics and even expands upon it in ways that were not suggested by the studies\' results. Several authors of other studies inappropriately cite the Klempner et al. trials to conclude that chronic Lyme disease is not infectious. Authors of a recently completed clinical trial on early Lyme disease \[[@B13]\] note that \"There is no scientific evidence to justify prolonged antibiotic therapy for patients with any manifestation of Lyme disease, and our study and that of others \[[@B34]\] should further help to discourage such practice. In addition, antibiotics are no better than placebo in treating patients who carry the label of \'chronic Lyme disease,\' probably because evidence indicates that this entity is not infectious\" \[\[[@B35]\], p.577\]. The authors make this statement without citing evidence that supports the notion that chronic Lyme disease is not infectious, other than the Klempner trials. In another published paper citing the Klempner et al. trials as evidence that post-Lyme syndrome is distinct from Lyme disease, patients were said to have \"developed a syndrome of diffuse arthralgia, myalgia, fatigue, and subjective cognitive difficulty during or soon after LD\" \[\[[@B36]\], p.385\]. However, these so-called \"post-Lyme\" symptoms are also typical of Lyme disease itself. The authors did not present clear evidence that \"post-Lyme syndrome\" was a distinct illness in patients who are demonstrably no longer infected with Lyme spirochetes. Another author inappropriately cites the two Klempner et al. trials to support a position that Lyme disease is neither infective nor inflammatory. In an editorial commentary in the *Journal of Infectious Diseases*, Radolf considered the two trials pivotal in supporting the position that \"the majority of physicians and scientists, the so-called mainstream camp, maintain that PTCLD (post-treatment chronic Lyme disease) is neither infectious nor inflammatory in nature\" \[\[[@B37]\], p.948\], and that \"researchers have failed to garner convincing and reproducible evidence for either persistent infection or ongoing inflammation\" \[\[[@B37]\], p.948\]. Radolf cites a second Klempner et al. paper \[[@B38]\] derived from the same two clinical trials, stating there was no evidence of persistent or viable infection by numerous measures including cultures and PCR, CSF pleocytosis, elevated white blood count, or increased erythrocyte sedimentation rate. Neither Radolf \[[@B37]\] nor Klempner et al. \[[@B38]\] discussed the poor sensitivities of these tests for chronic Lyme disease \[[@B15]\]. For example, only one of a series of twenty-seven cases of neurologic Lyme disease presented with a CSF pleocytosis and that case had only 7 cells \[[@B15]\]. Furthermore, PCR and culture tests may only be useful for subjects with early Lyme disease who have never been treated with antibiotics \[[@B39]\]. Neither increased white blood count nor erythrocyte sedimentation rate is elevated in acute or chronic Lyme disease \[[@B40]\]. A discussant in a clinician\'s corner published in JAMA \[[@B41]\] cited the Klempner et al. trials when advising against treatment of a 58-year-old man with chronic Lyme disease. The man, who lived in the Lyme endemic area of Martha\'s Vineyard, had been ill for 10 years. This man had a history of Bell\'s Palsy and in August 1992, \"he became less competent mentally. He could not do simple math and he became depressed. In 1994, he was diagnosed as having Lyme disease. At that time, he complained of neck pain radiating to his left shoulder and hand; numbness and tingling in his hand; back pain that radiated down his left leg; bilateral joint aches in both elbows and, to a lesser extent, his shoulders; bilateral tinnitus; and periodic blurred vision\" \[\[[@B41]\], p.1002\]. The man was treated with prednisone in 1992. Antibiotic treatment was delayed until 1994. The symptoms improved with repeated courses of oral tetracycline and clarithromycin, only to recur. The discussant cited the Klempner et al. trials as evidence against the value of further antibiotic treatment. Instead, he advised treatment for fibromyalgia even though he admitted that the patient did not meet the criteria for this condition. Fibromyalgia treatment has been disappointing for people with this kind of history \[[@B42]\]. Finally, two health insurance companies cite the Klempner et al. trials as justification for not covering treatment with intravenous antibiotics. One company policy states that they \"will not cover IV therapy beyond 28 days for Lyme Disease without review and input from a trained Infectious Disease Specialist approved by GHI-HMO.\" Furthermore, the company \"will not cover IV therapy for Lyme Disease for Chronic Lyme Disease or Post-Lyme Disease Syndrome without input from a trained Infectious Disease Specialist approved by GHI-HMO \" \[43\]. Citing the Klempner et al. trials, \[44\] Cigna does not cover any treatment for patients with persistent symptoms and a history of Lyme disease, unless recurrent arthritis, central nervous system (CNS), or peripheral nervous system involvement can be demonstrated. Treatment for chronic Lyme disease would otherwise be considered experimental, investigational, or unproven and therefore not covered, resulting in limited treatment options for many patients who might have benefited from additional antibiotics. Conclusion ========== Klempner et al. did not adequately critique the generalizability of their trials. This review argues that the study participants were not representative of the overall population of chronic Lyme disease patients that present with persistent symptoms and a history of disease. Limited generalizability has been a problem of other randomized trials \[[@B21]\] and it remains one here. This review argues that the poor treatment response in the Klempner et al. trials may be explained by having selected patients who had undergone delayed treatment or multiple treatments unsuccessfully. The quality of life of subjects enrolling in the Klempner et al. trials was worse than that of the average type II diabetic or patient recovering from a heart attack, and as poor as that of subjects suffering from congestive heart failure \[[@B14]\]. In other words, it may be an example of offering patients \"too little too late.\" Klempner himself described his concerns about the study population to the editors of *Science:*\"After a year of advertising, only 57 subjects had been enrolled. The goal is to get 260 by the time the study ends in 2 years. More than 1200 people have expressed interest, and 700 have come in for screening. But only 1 in 10 who appear in the clinic fits the study\'s strict criteria\" \[45, p.1431\]. Neither in the *Science*interview \[45\], nor when reporting the trials\' results in the New England Journal of Medicine \[[@B14]\], did Klempner discuss whether the strict criteria was a factor leading to the average 4.7 year onset of illness of subjects enrolled. Two additional randomized trials and one case series have been published since the 2001 Klempner et al. trials \[[@B25]-[@B27]\]. A fourth trial by Krupp supported antibiotic treatment for a subset of chronic Lyme disease patients with fatigue \[46\]. One month of intravenous ceftriaxone was effective at reducing the primary fatigue endpoint but not the secondary endpoints of cognitive function or OspA antigen \[46\]. These additional trials of chronic Lyme disease \[25, 26, 27, 46\] continue to suggest that treatment may be beneficial for some subgroups of patients who were not well represented in the Klempner et al. trials. In summary, this review exposes the limited generalizability of the findings of Klempner et al., and the overreaching impact these trials have had on influencing policies that affect unrepresented patient groups. In interpreting the results of these trials, physicians should consider the select group of patients that were chosen for study and whether the patients in their care might respond differently to treatment. Competing interests =================== The author is a clinician who treats Lyme diseaseand an advocate ofproviding moreaccurate information about treatment options. He has no interests that conflict with that goal.

{#sp1 .201}

After publication of this article \[[@CR1]\], the authors noticed Ali Edris's name was misspelled as Ali Edreis. The correct spelling of the author's name is included in the author list of this erratum and has been updated in the original article \[[@CR1]\]. The online version of the original article can be found under doi:10.1186/s13027-016-0104-7.

Influenza vaccination responses in older adults {#Sec1} =============================================== Influenza infection is associated with considerable morbidity annually and the elderly are amongst those at highest risk of serious outcomes \[[@CR1]--[@CR3]\]. Annual influenza vaccination is a strategy endorsed by the World Health Organization \[[@CR4]\], and is most effective when the vaccine strains closely match the circulating influenza viruses \[[@CR5]\]. However, the antibody response and protection elicited by the vaccine in the elderly is modest at best \[[@CR6]\], though recent studies suggest that increasing the dose of antigen in subunit vaccines significantly improves efficacy \[[@CR7]\]. Several factors have been proposed to explain hyporesponsiveness to influenza vaccination in the elderly, including host-related factors like genetics, immune status, health status, frailty and nutritional status \[[@CR8]\]. The complex changes in the immune system occurring with age collectively termed immunosenescence, affects innate and adaptive immunity and may contribute to the decreased efficacy of vaccines in the elderly \[[@CR9], [@CR10]\]. Recent data also suggest that age-associated decline in antibody responses could reflect the effect of repeated annual influenza vaccination rather than age or frailty \[[@CR11]\], however this hypothesis is being queried \[[@CR12]\]. Systems biology approaches have been utilized in recent studies to acquire a global picture of vaccine-induced immunity in humans, which has enabled the identification of early innate signatures predicting vaccine immunogenicity and the elucidation of novel mechanisms of immune regulation \[[@CR13]\]. Several reports have established age-dependent predictive signatures of influenza vaccine responses, however such studies typically considered the elderly as a homogeneous population, which is clearly not the case \[[@CR14]\]. We address this concern in the current study and show data confirming that a healthy elderly population, selected for lack of comorbidity and not just chronology, is likely to respond to influenza vaccination. Description of the study {#Sec2} ======================== The SENIEUR protocol clearly suggested that addressing chronological ageing alone was insufficient in immunogerontological studies \[[@CR15]\] as the elderly of the SENIEUR category (healthy elderly as defined by the SENIEUR protocol) displayed similar immune capacity than younger counterparts (in vitro). We took advantage of the Singapore Longitudinal Ageing Study (SLAS), which is an ongoing population-based cohort study of aging and health among Chinese adults aged 55 and above, to select presumed healthy elderly subjects. The SLAS design and description of the cohort demographics and health profile have been described previously \[[@CR16]\]. Individuals with no signs of frailty were selected using Fried's criteria, which assesses five dimensions hypothesized to reflect systems whose impaired regulation underlies frailty, namely unintentional weight loss, exhaustion, muscle weakness, slowness while walking, and low levels of activity (i.e. Fried's score of 0 to 5) \[[@CR17]\]. Additionally, we excluded individuals with a history of dementia, cancer or cardiovascular disease \[[@CR18]\], as well as those with any recent infection or any flu vaccine administration for at least 6 months before the study (Table [1](#Tab1){ref-type="table"}). The healthy elderly individuals (*n* = 22) were aged between 65 and 84 years (mean = 72.4 years) and the young group (*n* = 29) was represented by individuals of the same ethnicity (Chinese) aged between 23 and 33 years (mean = 29.1 years). The healthy elderly group showed a level of activity similar to the young group as measured by actigraphy, while significant differences were observed for biomarkers such as cytomegalovirus (CMV) seropositivity (100% in older adults and 45% in the young group, *p* \< 0.0001), the CD4/CD8 ratio (*p* \< 0.01) and the level of C-Reactive Protein (CRP, p \< 0.01) (Table [1](#Tab1){ref-type="table"}).Table 1Study subject characteristicsYoung cohort (*N* = 29)Healthy elderly cohort (*N* = 22)*p*Demography Age27.97 (23--33)72.41 (65--84)*\*\*\*\** Gender18 females (63.3%)10 females (45.5%)*ns* BMI22.8 (16.2--29.1)23.6 (17.4--32.6)*ns*Physical activity (% of time) Sedentary60.3 (46.5--98.9)60.2 (43.4--92.5)*ns* Light activity35.1 (0.9--47.9)35.2 (7.4--48.4)*ns* Moderate activity4.5 (0.1--11.1)3.8 (0.1--15.4)*ns* Vigorous activity0.1 (0--1.8)0.8 (0--12.1)*ns*Clinical CMV positivity13 (45%)22 (100%)*\*\*\*\** CD4/CD8 ratio1.33 (0.62--2.42)2.94 (0.52--14.71)*\*\** CRP (mg/L)1.2 (0.2--4.1)2.7 (0.3--9.3)*\*\** Comorbidities--0.36 (0--1)*--* MMSE/MoCA--28 (22--30) / 25.59 (18--30)*--* Pre/Post FEV1/FVC--0.73 (0.5--0.91)/0.71 (0.29--0.85)*--* Medications--1.05 (0--4)*--* Hospitalizations--0*--* MNA--12.8 (11--14)*--BMI* body mass index, *CMV* human cytomegalovirus, *CRP* C-reactive protein, *MMSE* mini mental state examination, *MoCA* Montreal cognitive assessment, *FEV* forced expiratory volume, *FVC* forced vital capacity, pre/post inhalation of a bronchodilator, *MNA* mini nutritional assessment score. Physical activity was measured during 2 weeks using actigraphy watches (Phillips Respironics). Comorbidities are expressed as the number of diagnosed conditions, medications are defined as the number of prescribed drugs and hospitalizations is the number of time the individual have been hospitalized in the past year Sustained antibody titers following influenza vaccination of healthy elderly {#Sec3} ============================================================================ We vaccinated the healthy elderly and the young healthy volunteers with Vaxigrip© (Sanofi-Pasteur) following 2014/2015 seasonal recommendations and subsequently collected fasting blood specimens on day 0 (baseline, D0), day 2 (D2), day 7 (D7) and day 28 (D28). We then determined the hemagglutination-inhibition (HAI) antibody titer at baseline and D28 post-vaccination as shown in Fig. [1a](#Fig1){ref-type="fig"}. Both groups responded to vaccination with a significant increase in HAI titers against all three strains tested at day 28 (Fig. [1a](#Fig1){ref-type="fig"}). Following the current international guidelines for seroconversion (\> 4-fold increase in HAI titers over baseline) and seroprotection (HAI titers ≥40) \[[@CR19], [@CR20]\], fewer young subjects had seroconverted compare with the elderly, which was primarily due to higher baseline titers in the young subjects (GMT values: Flu B: 411 ± 3 vs. 56 ± 4; H1N1: 149 ± 6 vs. 15 ± 5; and H3N2: 156 ± 4 vs. 38 ± 5 in young vs. elderly subjects). A high number of young individuals were already seroprotected at baseline against each of the three strains contained in the vaccine compared to the elderly (young versus old: 100% vs 63.6% against the B strain (*p* = 0.0005), 86.2% vs 40.9% against H3N2 (*p* = 0.001) and 72.4% vs 31.8% against H1N1 (*p* = 0.005)) (Fig. [1b](#Fig1){ref-type="fig"}). These high baseline titers may indicate previous exposure to influenza virus; subjects were excluded if they received an influenza vaccine within 6 months preceding the trial vaccination, however one cannot entirely rule out sustained protection from earlier vaccinations. A high baseline HAI titer has been reported to possibly interfere with TIV immunogenicity as measured by HAI titres \[[@CR21]\] indicating that one should be cautious with interpreting the results of the seroconversion. Post-vaccination, the seroprotection for all three strains increased to 100% in the young and close to 100% in the healthy elderly, and there was no significant difference between the young and healthy elderly (Fig. [1c](#Fig1){ref-type="fig"}) in terms of seroprotection for any of the 3 strains (for H1N1: Fisher's test *p* = 0.0739). Finally, when comparing the HAI titer endpoint at D28, older individuals display higher GMT for H3N2 (1159 vs. 610 with *p* = 0.0091), lower GMT for H1N1 (275 vs. 775 with *p* = 0.012) and no difference for FluB (1050 vs. 1726 with *p* \> 0.05), which indicates no gross overall hyporesponsiveness to influenza vaccination in this group of selected healthy elderly subjects. These HAI titres results were corroborated by the microneutralization assays (Fig. [1d](#Fig1){ref-type="fig"}) showing that the influenza-specific antibodies produced following vaccination have a similar neutralization capacity in healthy elderly and young individuals. Based on these two different analyses (HAI and microneutralization assays), we can conclude that carefully selected healthy older adults are able to mount an antibody response quantitatively and qualitatively similar to young individuals.Fig. 1Humoral immunity to influenza vaccination. **a** Graph showing the basal (D0) HAI titers and the response at day 28 (D28) in young (left) and old (right) vaccinees for each one of the influenza virus strains after vaccination; paired t-test applied. Seroprotection of subjects indicated by the dotted line for HAI titers ≥40. **b**, **c** % Seroprotection in young (Y) and healthy elderly adult (O) vaccinees at baseline (D0) and day 28 (D28) for each one of the three influenza virus strains; significances were calculated using Fisher's exact test on the number of subjects. **d** Results from the microneutralization assays performed on the same donors shown in A). The neutralization capacity is expressed as the reciprocal of the highest dilution of the donor' serum at which virus infection is blocked. **e** Gating strategy for the identification of plasmablasts (left panel) and paired analysis of the frequency of CD38^hi^CD27^hi^ plasmablasts in blood of young and healthy elderly individuals during the course of the response (D0-D28, right panel). Significant differences are expressed by \* *p* \< 0.05, \*\* *p* \< 0.01, \*\*\* *p* \< 0.001 and \*\*\*\* *p* \< 0.0001 Plasmablast dynamic is sustained in healthy elderly {#Sec4} =================================================== A major goal of this study was also to document the kinetics of cellular and humoral immune responses. Flow cytometry was used for immunophenotyping fresh whole blood to identify and monitor the recruitment of plasmablasts as well as assess their expansion and contraction phases from D0 to D28 (Fig. [1e](#Fig1){ref-type="fig"}). We reported the frequency of this populations as a ratio of the total immune cell fraction (CD45+). Firstly, in Fig. [1e](#Fig1){ref-type="fig"} we observed a significantly higher proportion of plasmablast cells (CD38^hi^CD27^hi^) in young subjects compared to elderly subjects at baseline (0.069% vs. 0.044%, *p* = 0.0063) that persists at D2 (*p* = 0.0013). This could be related to the higher baseline HAI titers in the young reported earlier (Fig. [1a](#Fig1){ref-type="fig"}), which suggests higher influenza virus/ vaccine exposure and/ or immunological memory. Thereafter, a significant increase in plasmablast frequencies from D2 to D7 (*p* \< 0.001) was observed with a peak at D7, but no significant difference between the two age groups (0.226% vs 0.254%, *p* = 0.8690) was detected. However, the frequency of CD38^hi^CD27^hi^ plasmablasts increased more in the elderly (5.8 times) than in the young (3.3 times) from D0 to D7, mirroring the sharper HAI titer increase in the elderly. The frequency of plasmablasts diminished to baseline levels at D28 (0.078% in young and 0.061% in healthy elderly individuals). Overall, there is a clear cellular dynamic response observed after vaccination, which is preserved in healthy elderly Chinese Singaporeans. Conclusion {#Sec5} ========== In our study, we demonstrate that overall, healthy elderly are fully capable to mount a robust humoral response via expansion of plasmablast, HAI response and neutralizing antibodies, comparable to that of young subjects. Herein, these data extends our previous findings \[[@CR22]\] with community-living elderly subjects from Singapore, and demonstrates that humoral immunity after influenza vaccination is preserved in healthy elderly. The studied healthy elderly group showed all signs of aging as previously described, including a higher prevalence of persistent infections (eg. CMV), high levels of pro-inflammatory molecules (eg. CRP) and features of immune aging (higher frequencies of CD28^−^ and CD27^−^ T cells, data not shown). The fact that plasmablast population dynamics is preserved in the healthy elderly from the SLAS cohort confirms earlier studies utilizing the SENIEUR protocol for immunogerontological studies. An important question to address in the near future will involve identifying which age-associated co-morbidity may interfere with optimal vaccine responses in the elderly. We recently ruled out type-2 diabetes as a condition that may interfere with influenza vaccine responses in a study of community-living elderly in Canada \[[@CR23]\]. Older adults commonly take statins to reduce cholesterol levels in order to manage cardiovascular risk, however recent published reports have raised concerns that statin use may impair vaccine-induced antibody responses, and reduce vaccine-induced protection, particularly for influenza A H3N2 \[[@CR24]\]. Whether subjects with hypercholesterolemia or those on statin medication should be considered at-risk and benefit from improved vaccines is debatable, and in our study such subjects were excluded. Other conditions such as frailty have been proposed to alter influenza or pneumococcal vaccine responses, however this has also recently been challenged \[[@CR25], [@CR26]\]. Discrepancies in study outcome may be explained by different mean ages of the subjects included into the studies, the definition of frailty used, and living conditions (community vs. institution). A recent review highlighted the need to consider several approaches to stratify the older population in order to provide with the best vaccine strategy \[[@CR27]\]. We are currently tackling these issues and other pertinent questions in an ongoing study aimed at understanding the associations between immunosenescence, hypo-responsiveness to vaccination and clinical phenotypes. CMV : Cytomegalovirus CRP : C-reactive protein HAI : Hemagglutination-inhibition HAI : Hemagglutination-inhibition SLAS : Singapore Longitudinal Ageing Study We acknowledge the support of the Flow Cytometry Platform and the Immunomonitoring Platform in SIgN, and the Investigational Medicine Unit in NUH. Funding {#FPar1} ======= This study was partly funded by Sanofi Pasteur, Sanofi and Nestec Ltd. Funding was also provided by the Agency for Science Technology and Research (BMRC IAF 311006 grant and BMRC transition funds \#H16/99/b0/011). Availability of data and materials {#FPar2} ================================== Please contact author for data requests: anis_larbi\@immunol.a-star.edu.sg AL, NTP, PT, and NBu conceived the study. MSZN and NTP recruited volunteers and performed clinical and frailty assessments. PT supervised the Vaxigrip clinical trial. XC and CTTY performed Luminex, immunophenotyping, microarray and serological assays. LV, NBu, CC, and SS analyzed microarray data and supervised the antibody titre assays performed at GCI and Sanofi Pasteur. VN, XC, MP, NBo, BA and AL performed data analysis, wrote and critically reviewed the manuscript. AL and NTP ensure the accuracy of the analysis for the biological and clinical data, respectively. All authors read and approved the final manuscript. Ethics approval and consent to participate {#FPar3} ========================================== This study was approved by the National Healthcare Group's Domain Specific Institutional Review Board and registered at [clinicaltrials.gov](http://clinicaltrials.gov) under the registration number NCT03266237. Consent for publication {#FPar4} ======================= "Not applicable". Competing interests {#FPar5} =================== The authors Christophe Carre, Nicolas Burdin,, Sanie Sesay and Lucian Visan were employed by Sanofi Pasteur, Marcy-l'Étoile, France, and the author Nabil Bosco was employed by Nestlé Research Centre, Singapore. All other authors declare no competing interests. Publisher's Note {#FPar6} ================ Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

1. Introduction {#sec1-nutrients-11-02011} =============== Phenylketonuria (PKU, OMIM 261600) and tyrosinemia type 1 (TT1, OMIM 276700) are both inborn errors affecting the phenylalanine-tyrosine degradation pathway necessitating a comparable dietary treatment. PKU affects approximately 1:18.000 newborns and is caused by a defect in the enzyme phenylalanine hydroxylase. Biochemically, this leads to high phenylalanine and low/normal tyrosine concentrations. Untreated, high phenylalanine concentrations will lead to severe mental impairments \[[@B1-nutrients-11-02011]\]. Treatment nowadays consists of dietary restriction of phenylalanine, amino acid supplements, and, if patients are responsive, the drug sapropterin dihydrochloride, which is the synthetic form of tetrahydrobiopterin (BH4) \[[@B2-nutrients-11-02011]\]. BH4 acts as a pharmacological chaperone of phenylalanine hydroxylase, improving the conversion of phenylalanine into tyrosine, thereby lowering the phenylalanine concentrations \[[@B3-nutrients-11-02011]\]. Various studies have been conducted on the effect of this dietary treatment and of BH4 on the nutritional and biochemical parameters in PKU, reporting that PKU patients are at risk for micronutrient deficiencies such as vitamin B12, folic acid, selenium, zinc and iron \[[@B4-nutrients-11-02011],[@B5-nutrients-11-02011],[@B6-nutrients-11-02011]\]. This also applies to PKU patients who are treated with BH4, in whom deficiencies may result from their liberalized diet \[[@B7-nutrients-11-02011],[@B8-nutrients-11-02011],[@B9-nutrients-11-02011]\]. Tyrosinemia type 1 is caused by a deficiency of the enzyme fumarylacetoacetate hydrolase and is much rarer than PKU, affecting approximately 1:100.000 newborns. The deficient enzyme leads to the accumulation of toxic metabolites, which in turn cause renal tubular dysfunction, neurological porphyria-like crises, liver failure and liver cancer \[[@B10-nutrients-11-02011]\]. In the past, the only definitive form of treatment was a liver transplantation, usually at a very young age, in order to survive. Since 1992, however, a new treatment option has become available called 2-(2-nitro-4-trifluoromethylbenyol)-1,3-cyclohexanedione (NTBC), which blocks the activity of 4-hydroxyphenylpyruvate dioxygenase, an enzyme upstream of the metabolic defect \[[@B11-nutrients-11-02011]\]. Treatment nowadays consists of NTBC paired with a dietary restriction of tyrosine and its precursor phenylalanine. On this treatment, outcome has improved tremendously. Since the NTBC-dietary treatment, the focus of research has shifted towards neurocognitive deficits that are observed and their possible cause \[[@B12-nutrients-11-02011],[@B13-nutrients-11-02011],[@B14-nutrients-11-02011],[@B15-nutrients-11-02011],[@B16-nutrients-11-02011]\]. At present, however, little research has been conducted on the nutritional and biochemical parameters in TT1. Although PKU and TT1 are different diseases, they are treated with a comparable protein-restricted diet and amino acid supplements. This raised the question whether TT1 patients may also be at risk for micronutrient deficiencies as in PKU patients. Therefore, the aim of this retrospective study was (1) to compare outcomes of regular biochemical follow-up measurements of TT1 and PKU patients (both with and without BH4 treatment); (2) to investigate possible deficiencies or excesses in blood of TT1 patients, PKU patients treated with BH4 (PKU-BH4), and PKU patients not treated with BH4 (PKU-nBH4); and (3) to assess possible correlations between dietary intake and metabolic control and these regular follow-up outcomes. 2. Materials and Methods {#sec2-nutrients-11-02011} ======================== 2.1. Participants {#sec2dot1-nutrients-11-02011} ----------------- In total, 12 TT1 patients (mean age 13.5 ± 9.9, 75% male) and 92 PKU patients (mean age 24.5 ± 13.9, 45% male) were included. All patients were treated at the University Medical Center Groningen, The Netherlands. Of the PKU patients, 33 patients (36%) received BH4 at the time of assessment. BH4 dosages ranged from 100 mg to 1400 mg a day. The TT1 patients were all treated with NTBC and dietary restriction of phenylalanine and tyrosine. NTBC dosages ranged from 0.51 mg/kg/day to 1.17 mg/kg/day. Both the TT1 and PKU patient groups were treated with several different amino acid mixtures (see [Supplementary Material S1](#app1-nutrients-11-02011){ref-type="app"} for information regarding micronutrient and mineral content of amino acid supplements used by our patients). The need for formal ethical review and informed consent was waived by the local medical ethical committee. 2.2. Study Parameters {#sec2dot2-nutrients-11-02011} --------------------- Data were collected retrospectively from patient files. From each patient the most recent blood measurements at the outpatient clinic for regular follow-up were included. All data came from blood samples collected between 14 August 2017 and 29 April 2019. Patients who had no blood samples taken at the hospital in that period were excluded from analyses. Concentrations were considered abnormal if they were found to be outside the reference range of the hospital laboratory information system. All blood measurements during regular outpatient clinic visits were collected, which encompassed: leucocytes, hemoglobin (Hb), hematocrit (Ht), mean corpuscular volume (MCV), thrombocytes, plasma sodium, potassium, chloride, creatinine, urea, calcium, phosphate, magnesium, albumin, total protein, alkaline phosphatase, ferritin, vitamin B12, methylmalonic acid (MMA), total homocysteine, 25-OH-vitamin D3, pre albumin, and thyroid stimulating hormone (TSH). Furthermore, plasma phenylalanine and tyrosine concentrations were collected. Plasma folic acid, alanine aminotransferase and aspartate aminotransferase concentrations were excluded from analyses, as folic acid concentrations were in the majority of cases derived from hemolytic blood samples, which can give falsely elevated results, and alanine aminotransferase and aspartate aminotransferase were only occasionally collected. For the remaining blood parameters, in total, 17% of our blood value data were missing. From the day of blood collection, the standard deviation scores (sds) of anthropometric measurements (height sds, weight sds, and body mass index (BMI) sds) were collected, as well as dietary information, which included total protein intake, natural protein intake, protein intake from amino acid supplements and total protein intake per kg body weight. 2.3. Statistical Analyses {#sec2dot3-nutrients-11-02011} ------------------------- Differences in anthropometric measurements and dietary intake between the groups were investigated using Kruskal-Wallis tests, since data were not normally distributed. The Mann--Whitney U test was used as a post-hoc test, and its *p*-value was corrected for multiple comparisons according to Bonferroni. Since some reference values from the blood measurements differed between age and gender, categorical variables were created (below normal, normal, and above normal). Because of the low expected cell frequencies for these measurements, Fisher's exact tests were performed to determine differences between TT1, PKU-nBH4 and PKU-BH4 patients and 2-sided *p*-values were adhered. Additionally, percentages of these abnormal values were calculated. To investigate possible correlations between outcomes and dietary intake, Spearman correlation tests were performed. Since dietary intake differs between age groups, the percentage of natural protein intake from total protein intake was calculated and used for these analyses. Lastly, Spearman tests were used for investigating possible correlations between metabolic control (phenylalanine and tyrosine concentrations) and observed deficiencies and/or excesses. All statistical analyses were performed using IBM SPSS Statistics 23. *p*-values \< 0.05 were considered statistically significant. 3. Results {#sec3-nutrients-11-02011} ========== 3.1. Tyrosinemia Type 1 (TT1) versus Phenylketonuria (PKU)-nBH4 versus PKU-BH4 {#sec3dot1-nutrients-11-02011} ------------------------------------------------------------------------------ Descriptives are summarized in [Table 1](#nutrients-11-02011-t001){ref-type="table"}. No significant differences were observed between the groups regarding the anthropometric measurements. When comparing percentage of natural protein intake between the three patient groups, Kruskal-Wallis tests showed a significant difference (*p* \< 0.001). Pairwise comparisons then showed that differences existed between PKU-BH4 and PKU-nBH4 patients (*p* \< 0.001) and between TT1 and PKU-BH4 patients (*p* = 0.033), with higher natural protein intake for PKU-BH4 patients, as expected. After Bonferroni correction for multiple comparisons only the difference between PKU-BH4 and PKU-nBH4 remained statistically significant (*p* \< 0.001). To investigate differences in blood measurements between the patient groups (TT1, PKU-nBH4, or PKU-nBH4) Fisher's exact tests were performed using the categorical data. A significant difference between the groups was observed regarding MCV levels (*p* = 0.038), suggesting more often higher MCV levels in the PKU-BH4 group compared to the TT1 and PKU-nBH4 groups. Other than this, no significant differences between the three groups were observed. 3.2. Deficiencies or Excesses {#sec3dot2-nutrients-11-02011} ----------------------------- [Table 2](#nutrients-11-02011-t002){ref-type="table"} shows percentages of patients with values below or above the reference ranges in the different patient groups (TT1, PKU-BH4, and PKU-nBH4). High vitamin B12 concentrations were often observed ([Figure 1](#nutrients-11-02011-f001){ref-type="fig"}). All patient groups appeared to be prone to having an excess of vitamin B12, with respectively 36%, 33% and 20% of the patients having a concentration above the reference limit. Elevated levels of MMA and total homocysteine were occasionally observed (in 5 and 2 patients respectively). Elevation was always in only one of these markers, never in both. Next to this, low vitamin D concentrations were present in our sample ([Figure 1](#nutrients-11-02011-f001){ref-type="fig"}). This was the case for all patient groups, and especially for both groups of PKU patients (8%, 29%, and 24% respectively). Since vitamin D levels are known to differ depending on time of the year \[[@B17-nutrients-11-02011]\], [Figure 2](#nutrients-11-02011-f002){ref-type="fig"} shows the vitamin D concentrations observed per month. 3.3. Correlations with Dietary Intake {#sec3dot3-nutrients-11-02011} ------------------------------------- When correlating the blood concentrations to the percentage of natural protein intake, correlations were observed for vitamin B12 (r = −0.277, *p* = 0.013), total homocysteine (r = 0.346, *p* = 0.002), and vitamin D (r = −0.232, *p* = 0.026). These correlations suggest that vitamin B12 and D concentrations are lower in patients with a higher percentage of natural protein intake, and that total homocysteine concentrations are higher with more natural protein intake. No other correlations were observed between protein intake and blood concentrations. 3.4. Correlations with Phenylalanine and Tyrosine Concentrations {#sec3dot4-nutrients-11-02011} ---------------------------------------------------------------- Spearman correlation tests were performed to investigate possible correlations between the vitamin B12 and vitamin D concentrations and phenylalanine and tyrosine concentrations. Separate analyses were performed for TT1 and PKU patients because of the opposite biochemical profile. For TT1 a significant correlation was observed between tyrosine and vitamin B12 (r = −0.627, *p* = 0.039) and a trend was observed for tyrosine and vitamin D (r = −0.566, *p* = 0.055). For PKU patients significant positive correlations were observed between tyrosine concentrations and both vitamin B12 (r = 0.279, *p* = 0.013) and vitamin D (r = 0.319, *p* = 0.002). Phenylalanine concentrations showed no significant correlations in neither the TT1 or the PKU group. When investigating PKU-nBH4 and PKU-BH4 patients separately, PKU-BH4 patients showed no significant correlations. PKU-nBH4 patients showed a significant correlation between tyrosine and vitamin D (r = 0.378, *p* = 0.003). Furthermore, trends were observed between tyrosine concentrations and vitamin B12 (r = 0.272, *p* = 0.061) and between phenylalanine and vitamin D (r = −0.255, *p* = 0.054). 4. Discussion {#sec4-nutrients-11-02011} ============= This study describes micronutrients collected for regular follow-up in TT1 and PKU patients in our hospital. The biochemical and micronutrient status of TT1 patients was largely comparable to that of PKU patients, as might be expected for the dietary treatment of PKU-nBH4 and TT1 is comparable. Especially in PKU patients, and to a lesser extent in TT1 patients, vitamin B12 concentrations were elevated, while vitamin D generally appeared to be lower than normal. In both diseases, both vitamin concentrations could to some extent be related to the natural protein intake and the tyrosine rather than the phenylalanine concentrations. Before discussing our findings in more detail, some methodological issues need to be addressed. The group of TT1 patients in this study was relatively small compared to the group of PKU patients, which is due to the difference in incidence. This smaller sample size for the TT1 patients may have been insufficient to generate significant results. Since this is a retrospective study, we could only use data that had been requested by the clinician at the time of the outpatient visit. As a consequence, 17% of the blood values were missing. Dietary information was collected from patient files. However, these may not completely reflect the actual situation since patients may not always count their protein intake or have low treatment compliance \[[@B18-nutrients-11-02011],[@B19-nutrients-11-02011]\]. It has been shown that PKU patients with high adherence to diet may have significant differences in biochemical markers compared to patients with low adherence to diet \[[@B5-nutrients-11-02011]\]. In our study, adherence could not be taken into account, and these possible differences could not be investigated. Furthermore, differences in blood concentrations for different amino acid mixtures could not be investigated because of the low number of patients per product. Lastly, some possibly relevant markers, such as blood concentrations of zinc and selenium \[[@B20-nutrients-11-02011]\], were not assessed, since these measurements are generally not included in regular follow-up in our center. With regard to the findings, several studies have shown that PKU patients, both with and without BH4 may be at risk for developing vitamin B12 deficiency especially when adherence to diet is suboptimal \[[@B4-nutrients-11-02011],[@B6-nutrients-11-02011],[@B9-nutrients-11-02011]\]. For TT1 patients, evidence on possible B12 deficiencies is currently lacking. Remarkably, in our sample, vitamin B12 concentrations in TT1 and PKU patients were elevated rather than lowered. This could possibly be caused by a high dietary adherence and thus adequate intake, as also observed previously \[[@B5-nutrients-11-02011],[@B20-nutrients-11-02011]\]. It should be noted, however, that MMA and total homocysteine are better markers of vitamin B12 status than B12 concentrations themselves, as a vitamin B12 deficiency can exist even when B12 concentrations are normal or high \[[@B4-nutrients-11-02011],[@B21-nutrients-11-02011]\]. Since increased MMA and/or total homocysteine concentrations, suggesting a B12 deficiency, were found in a small number of patients (all having normal/high B12 concentrations), this may point toward a suboptimal B12 status in these patients. Vugteveen et al. suggested that vitamin B12 intake by amino acid mixture may not result in optimal absorption of vitamin B12 \[[@B21-nutrients-11-02011]\], which may in part explain our observed results. Furthermore, our study suggests that a biochemical vitamin D deficiency may arise in approximately 25%--30% of the PKU patients, both PKU-nBH4 and PKU-BH4 patients (29% and 24%, respectively). A recent study by Kose and Arslan showed an even larger percentage of vitamin D deficiency in PKU patients in Turkey (53.6%) \[[@B5-nutrients-11-02011]\]. However, they also showed a similar percentage of healthy controls (47.2%) with this deficiency. In addition to this, a Dutch study investigating the prevalence of biochemical vitamin D deficiency in the normal population reported deficiencies in almost 60% of healthy individuals in the winter and roughly 30% in the summer \[[@B17-nutrients-11-02011]\]. These numbers considered, our PKU patients are doing relatively well. However, reference values in our hospital adhere to a lower limit of 50 nmol/L, while it has been suggested that, optimally, values should be above 75 nmol/L \[[@B17-nutrients-11-02011]\]. The use of this favorable reference range would lead to the finding that vitamin D levels were suboptimal in 62.5% of our patients (including 7/12 TT1 patients), even in some patients who were reported to use vitamin D supplements. PKU patients have also been shown to suffer from bone problems, such as reduced mineral bone density \[[@B4-nutrients-11-02011],[@B22-nutrients-11-02011]\]. Although the pathogenesis of this has not been fully elucidated yet, we recommend, as a start, to optimize vitamin D concentrations in these patients. TT1 patients on the other hand, are known to develop vitamin D-resistant rickets, if untreated \[[@B23-nutrients-11-02011]\]. For TT1 patients the effect of vitamin D supplementation might, therefore, be different than the effect on PKU patients, but this needs further investigation. Both vitamin D and B12 concentrations were correlated to percentage of natural protein intake. The results suggest that higher intake of proteins from supplements result in higher vitamin concentrations. This may be due to the high vitamin content of amino acid supplements as also suggested previously \[[@B24-nutrients-11-02011]\]. Extra attention could, therefore, be paid to patients on a higher natural protein diet or patients who discontinued their amino acid supplements. With tyrosine being the precursor of thyroxin, it can be hypothesized that altered tyrosine concentrations, as observed in PKU and TT1, may have an influence on thyroxin availability which can be assessed by measuring TSH levels. Our results did not show clear aberrations in TSH levels, which is in contrast with Sumanszki et al. who did observe lower TSH levels in adult PKU patients compared to controls \[[@B25-nutrients-11-02011]\]. Therefore, the possible effect of tyrosine (and phenylalanine) concentrations on thyroid function should be investigated in more detail. Lastly, tyrosine concentrations appeared to be correlated to the vitamin B12 and vitamin D concentrations. For TT1, higher tyrosine levels (suggestive of less treatment adherence) indicated lower vitamin concentrations. Similarly, in PKU patients, higher tyrosine concentrations (suggestive of better treatment adherence) correlated with higher vitamin B12 and vitamin D. These findings underline the conclusion of Vugteveen et al. that the vitamin B12 concentrations may be related to the intake of amino acid supplements \[[@B21-nutrients-11-02011]\]. Literature describes negative correlations between vitamin B12 levels in PKU and phenylalanine levels, however, correlations with tyrosine have not been investigated previously \[[@B5-nutrients-11-02011],[@B6-nutrients-11-02011]\]. Thus, although correlations with phenylalanine levels were not observed in our sample, both our results and the results from literature suggest a positive effect of treatment adherence on micronutrient status. 5. Conclusions {#sec5-nutrients-11-02011} ============== Diet therapy is important in the treatment of both PKU and TT1 patients. For many years, there have been concerns about nutritional deficiencies, however, the results of this study show that this is often not the case. The biomarkers of micronutrients of TT1 patients were largely comparable to those of PKU patients, and generally within the reference ranges. Vitamin B12 concentrations were high rather than low, while vitamin D concentrations were often below the reference range for which attention should be paid to the intake of protein supplements while specific supplementation can also be considered. The following are available online at <https://www.mdpi.com/2072-6643/11/9/2011/s1>: Supplementary material S1: Micronutrients in the different amino acid mixtures used in our patient group. ###### Click here for additional data file. Conceptualization, K.v.V. and F.J.v.S.; Data curation, K.v.V. and I.L.R.; Formal analysis, K.v.V.; Investigation, K.v.V. and I.L.R.; Methodology, K.v.V., I.L.R., W.G.v.G., M.R.H.-F. and F.J.v.S.; Project administration, K.v.V.; Resources, K.v.V., C.M.A.L., B.H.R.W., M.M.v.d.K., M.R.H.-F. and F.J.v.S.; Supervision, F.J.v.S.; Validation, K.v.V.; Visualization, K.v.V., I.L.R., M.R.H.-F. and F.J.v.S.; Writing---original draft, K.v.V., I.L.R. and F.J.v.S.; Writing---review and editing, K.v.V., I.L.R., W.G.v.G., C.M.A.L., B.H.R.W., M.M.v.d.K., M.R.H.-F. and F.J.v.S. This research received no external funding. F.J. van Spronsen has received advisory board fees from SOBI, Merck Serono, Biomarin, Nutricia, APR, Arla Food int., and Promethera, research grants from SOBI, Merck Serono, Biomarin, Nutricia, Alexion, Codexis, NPKUA, ESPKU and the Tyrosinemia Foundation, honoraria as consultant for Merck Serono, Biomarin, Nutricia, and APR, and honoraria as speaker for Merck Serono, Biomarin, Nutricia, Vitaflo and MendeliKABS. C.M.A. Lubout has received a speaker fee from the Recordati Rare Disease Foundation. M.M. van der Klauw received a fee from Biomarin for preparation of a study. All other authors had no conflicts of interest to declare. {#nutrients-11-02011-f001} {#nutrients-11-02011-f002} nutrients-11-02011-t001_Table 1 ###### Descriptive data. For each patient group the minimum and maximum observed values were included, as well as the number of patients for which that value was collected. Measurement TT1 PKU-nBH4 PKU-BH4 --------------------------------------- --------- -------------- ---------- --------- ------------- ------- ---- ------------- ------- ---- Age years 0.79--28.54 11.85 12 1.49--65.77 25.73 59 1.08--43.18 19.15 33 Phenylalanine µmol/L 18-- 61 43 12 63--1757 667 58 84--676 313 33 Tyrosine µmol/L 237--742 489 12 24--149 60 58 29--107 58 33 Anthropometrics Height sds −1.03--0.88 -0.18 11 −2.64--2.48 -0.46 50 −2.42--1.00 −0.17 32 Weight sds −0.61--1.91 0.70 12 −1.61--4.72 0.97 36 −1.59--3.84 0.25 26 BMI sds 0.24--2.48 1.20 11 −1.21--4.03 1.08 51 −2.10--3.60 0.61 31 Hematology Leucocytes 10^9^/L 4.2--10.3 6.9 12 3.6--13.9 6.8 54 4.4--11.4 6.9 32 Hemoglobin mmol/L 6.4--10.9 8.3 12 6.7--10.8 8.5 55 6.8--10.5 8.5 33 Hematocrit L/L 0.31--0.49 0.38 12 0.31--0.49 0.40 52 0.33--0.47 0.41 30 MCV \* fL 74.1--93.8 84.6 12 74.2--95.8 88.1 55 73.8--97.7 89.8 33 Thrombocytes 10^9^/L 114 - 464 281 12 138 - 470 258 54 151--412 266 32 Micronutrients and related parameters Sodium mmol/L 137--142 139 11 135--144 140 54 138--142 140 30 Potassium mmol/L 3.6--5.0 3.9 11 3.4--4.5 4.0 54 3.6--4.6 4.0 29 Chloride mmol/L 100--105 104 7 101--107 104 24 102--106 104 12 Creatinine µmol/L 14--84 44 12 25--87 61 52 32--90 63 29 Urea mmol/L 2.1--7.2 3.7 10 1.2--6.5 4.0 53 1.8--5.8 4.0 30 Calcium mmol/L 2.37--2.52 2.43 4 2.28--2.56 2.43 43 2.20--2.61 2.42 24 Phosphate mmol/L 0.67--1.95 1.37 11 0.56--1.65 0.98 51 0.70--1.63 1.06 28 Magnesium mmol/L 0.74--1.00 0.79 9 0.76--0.97 0.84 43 0.77--0.98 0.85 22 Albumin g/L 48--48 48 1 43--53 48 18 44--53 49 13 Total protein g/L 70--78 73 11 58--84 73 50 66--81 74 29 Alkaline phosphatase U/L 49--434 214 11 46--370 85 42 50--283 83 21 Ferritin µg/L 16--188 54 11 15--331 52 47 15--351 66 25 Vitamin B~12~ pmol/L 252--1150 630 11 174--1000 423 49 214--682 377 30 MMA nmol/L 114.8--304.7 168.7 11 47.4--527.8 158.9 56 94.3--562.9 196.5 32 Total homocysteine µmol/L 4.1--9.6 5.4 11 3.3--17.7 6.3 44 3.6--21.0 7.6 28 25-OH-Vitamin D3 nmol/L 9.1--138.6 61.0 12 10.9--138.7 67.5 59 22.7--99.5 67.8 33 Pre albumin g/L 0.15--0.48 0.32 11 0.15--0.51 0.30 54 0.17--0.45 0.30 27 TSH mU/L 0.58--3.88 3.31 10 0.73--6.00 1.71 49 0.46--7.60 1.76 23 Abbreviations: TT1 = Tyrosinemia type 1; PKU = Phenylketonuria; Sds = standard deviation scores; BMI = body mass index; MCV = mean corpuscular volume; MMA = methylmalonic acid; TSH = thyroid stimulating hormone. \* Significant difference between all groups with p \< 0.05 (Fisher's exact test). nutrients-11-02011-t002_Table 2 ###### Percentage of patients within the different patient groups with too low/deficient blood concentrations, and too high/excessive concentrations. Percentages are included when the N \> 0. Patients with a Deficiency Patients with an Excess --------------------------------------- ---------------------------- ------------------------- --------- --------- ---------- --------- Blood count Leucocytes 1 (8%) 2 (4%) 0 0 4 (8%) 3 (9%) Hemoglobin 0 2 (4%) 1 (3%) 0 0 0 Hematocrit 0 1 (2%) 0 1 (8%) 3 (6%) 5 (16%) MCV 0 0 0 0 1 (2%) 5 (15%) Thrombocytes 1 (8%) 4 (8%) 0 2 (17%) 4 (7%) 2 (6%) Micronutrients and related parameters Sodium 0 0 0 0 0 0 Potassium 0 2 (4%) 0 0 0 0 Chloride 0 0 0 0 0 0 Creatinine 1 (8%) 2 (4%) 1 (3%) 0 0 0 Urea 0 5 (10%) 1 (3%) 0 0 0 Calcium 0 0 0 0 0 1 (4%) Phosphate 1 (9%) 2 (4%) 1 (3%) 0 0 0 Magnesium 0 0 0 0 0 0 Albumin 0 0 0 0 4 (24%) 3 (23%) Total protein 0 1 (2%) 0 0 1 (2%) 3 (10%) Alkaline phosphatase 0 0 0 1 (9%) 2 (5%) 1 (5%) Ferritin 0 2 (4%) 2 (8%) 0 3 (7%) 0 Vitamin B~12~ 0 0 0 4 (36%) 16 (33%) 6 (20%) MMA 0 4 (7%) 0 0 2 (4%) 3 (9%) Total homocysteine 0 0 0 0 1 (2%) 1 (3%) 25-OH-Vitamin D3 1 (8%) 17 (29%) 8 (24%) 0 0 0 Pre albumin 1 (9%) 6 (11%) 1 (4%) 3 (27%) 4 (8%) 3 (11%) TSH 0 0 1 (4%) 0 3 (6%) 1 (4%) Abbreviations: TT1 = Tyrosinemia type 1; PKU = Phenylketonuria; MCV = mean corpuscular volume; MMA = methylmalonic acid; TSH = thyroid stimulating hormone.

1. Introduction {#sec1-ijms-19-03434} =============== Among all types of women's cancers, breast cancer has the most new cases of diagnosed cancer type and is the second cause of cancer-related mortality worldwide \[[@B1-ijms-19-03434]\]. Currently, dysregulation of metabolic pathways, including fatty acid metabolic pathways, is considered as a risk factor for promoting breast cancer progression \[[@B2-ijms-19-03434]\]. Fatty acid metabolism comprises multiple pathways including fatty acid transport, de novo synthesis, fatty acid oxidation, etc., and emerging evidence has indicated that some of the fatty acid metabolic enzymes are related to different subtypes of breast cancer \[[@B3-ijms-19-03434]\]. Fatty acid binding proteins (FABP) are a family of proteins that bind long-chain fatty acids and are involved in facilitating transport and uptake of lipids. Overexpression of FABP5 and FABP7 is associated with triple-negative breast cancer and basal-like breast cancer \[[@B4-ijms-19-03434],[@B5-ijms-19-03434],[@B6-ijms-19-03434]\]. Acyl-CoA synthetase long-chain family member 4 (ACSL4) activity leads to long-chain fatty acid transport and long-chain fatty-acid-coenzyme A ligase. High ACSL4 expression is inversely associated with estrogen receptor expression and high ACSL4 expression is a biomarker for an aggressive breast cancer phenotype \[[@B7-ijms-19-03434],[@B8-ijms-19-03434],[@B9-ijms-19-03434]\]. Thus, these enzymes might serve as therapeutic targets and diagnostic markers in different subtypes of breast cancer. CD36 is a transmembrane protein and mediates fatty acid uptake. Recent studies indicate that high CD36 expression is detected in breast cancer and CD36 function is important for cell growth in breast cancer cells and metastasis in metastasis-initiating breast cancer cells \[[@B10-ijms-19-03434],[@B11-ijms-19-03434]\]. Therefore, the fatty acid transport pathway is important for breast cancer progression. Except for CD36, ACSL, and FABP, solute carrier family 27 (SLC27) is also involved in the process of long-chain fatty acid uptake. SLC27, also named fatty acid transport proteins (FATP) or very long-chain acyl-CoA synthetases (ACSVL), is a family of six members (SLC27A1 through SLC27A6) for uptake of long-chain fatty acids \[[@B12-ijms-19-03434]\]. Each protein has different specific preferred substrates and tissue distribution \[[@B13-ijms-19-03434]\]. In addition, SLC27 family proteins, especially SLC27A1 (FATP1) and SLC27A4 (FATP4), display acyl-CoA synthetase (ACS) activity, which links to fatty acid synthesis, β-oxidation, and phospholipid synthesis \[[@B12-ijms-19-03434],[@B14-ijms-19-03434]\]. This suggests that SLC27 family proteins are involved in regulation of fatty acid uptake and down-streaming of lipid metabolic processes. Currently, the role of SLC27 family proteins is not fully understood in breast cancer although the role of ACSL, FABP, and CD36 has been investigated. The aim of this study is to investigate whether SLC27 family proteins are associated with progression of breast cancer, including cell growth, migration, invasion, and potential regulatory mechanism in breast cancer cells. 2. Results {#sec2-ijms-19-03434} ========== 2.1. Relatively High SLC27A4 Expression Was Detected in Breast Cancer Tissue {#sec2dot1-ijms-19-03434} ---------------------------------------------------------------------------- The expression levels of SLC27 mRNA in normal and cancer tissues were evaluated in the Oncomine database. Datasets selection was based on the threshold, *p*-value of 0.001, fold change of 2, and being in the top 10% gene ranking. In [Figure 1](#ijms-19-03434-f001){ref-type="fig"}a,b, the results show that only SLC27A4 expression was higher in breast cancer tissues than that in nontumor tissues. The other SLC27 family proteins showed opposite expression pattern. The data implies that SLC27A4 might be associated with malignancy of breast cancer. In addition, there is a trend toward shorter overall survival and distant metastasis-free survival in breast cancer patients with higher SLC27A4 expression ([Figure 1](#ijms-19-03434-f001){ref-type="fig"}c,d, *p* = 0.0725 and 0.033 respectively). By contrast, the high expression SLC27A1 and SLC27A6 was associated better overall survival rate ([Supplementary Figure S1](#app1-ijms-19-03434){ref-type="app"}). The SLC27A4 protein expression in normal breast and breast cancer tissues were also evaluated by the Human Protein Atlas database ([Figure 1](#ijms-19-03434-f001){ref-type="fig"}e). Compared to normal breast tissues, most breast cancer tissues revealed median to high SLC27A4 expression ([Figure 1](#ijms-19-03434-f001){ref-type="fig"}f). To further investigate whether SLC27A4 expression was associated with different subtypes of breast cancer, different stages, and races in clinical patients, the UALCAN database was used. Our results showed that significantly higher SLC27A4 expression was observed in all subtypes, stages, and races in breast cancer tissues when compared to normal breast tissue ([Figure 1](#ijms-19-03434-f001){ref-type="fig"}g--i). No significantly different levels of SLC27A4 were shown among all cancer stages; however, significant differences between luminal vs. triple negative (*p* \< 0.0001) and HER2 positive vs. triple negative (*p =* 0.0180) in different subtype analysis, and Caucasian vs. African American (*p =* 0.0013) and Caucasian vs. Asian (*p =* 0.0174) in different race analysis were observed. In general, SLC27A4 mRNA expression in breast tumor tissues was higher than that in normal breast tissues in clinical samples. 2.2. Silencing SLC27A4 in Breast Cancer Cell LINES Results in Decreasing Fatty Acids Uptake Capacity {#sec2dot2-ijms-19-03434} ---------------------------------------------------------------------------------------------------- The SLC27A4 expression was evaluated by Western blot assay in luminal A breast cancer cell lines T47D and MCF-7, and triple negative breast cell lines Hs578T and MDA-MB-231 ([Figure 2](#ijms-19-03434-f002){ref-type="fig"}a) \[[@B15-ijms-19-03434]\]. Except for MCF7, the other three cell lines express high levels of SLC27A4 protein. Hs578T and MDA-MB-231 were chosen for the following experiments. Two different targeted sequences of short hairpin RNA (shRNA), shSLC27A4\#98 and shSLC27A4\#02, were used for silencing SLC27A4 expression in Hs578T and MDA-MB-231. Because inhibition of fatty acid synthase mediates epithelial-mesenchymal transition (EMT) in the breast through FABP1 and other proteins \[[@B16-ijms-19-03434]\], the cell morphology of SLC27A4-silencing cells was also evaluated. [Figure 2](#ijms-19-03434-f002){ref-type="fig"}b--d reveal that shSLC27A4\#98 and shSLC27A4\#02 effectively suppressed SLC27A4 in Hs578T, and [Figure 2](#ijms-19-03434-f002){ref-type="fig"}e shows the morphology of shSLC27A4-knockdowned Hs578T. Furthermore, the effect of shSLC27A4\#98 and shSLC27A4\#02 in MDA-MB-231 is shown in [Figure 2](#ijms-19-03434-f002){ref-type="fig"}g--i. Because the enzyme function of SLC27 family links to fatty acids transport \[[@B12-ijms-19-03434]\], the fatty acids uptake capacity was evaluated in both cell lines. In Hs578T, only the shSLC27A4\#02-transfected group revealed lower fatty acids uptake capacity when compared to the vector control group ([Figure 2](#ijms-19-03434-f002){ref-type="fig"}j). By contrast, relatively low fatty acids uptake was detected in shSLC27A4\#98- and shSLC27A4\#02-transfected MDA-MB-231 ([Figure 2](#ijms-19-03434-f002){ref-type="fig"}k). Our results suggest that the fatty acids uptake capacity was associated with silencing efficiency in two breast cancer cell lines. 2.3. Silencing SLC27A4 in Breast Cancer Cell Lines Inhibited Cell Growth {#sec2dot3-ijms-19-03434} ------------------------------------------------------------------------ The cell proliferation in SLC27A4-knockdowned Hs578T and MDA-MB-231 was evaluated. In Hs578T, cell growth was inhibited after transfection with shSLC27A4\#02 but not shSLC27A4\#98 when compared to the vector control group ([Figure 3](#ijms-19-03434-f003){ref-type="fig"}a,b). In MDA-MB-231, relatively slower cells growth rate was observed in both shSLC27A4\#02 and shSLC27A4\#98-transfected groups ([Figure 3](#ijms-19-03434-f003){ref-type="fig"}c,d). It is interesting to note that the inhibitory effect on cell growth in both cells was associated with the inhibitory efficiency of SLC27A4 shRNAs ([Figure 1](#ijms-19-03434-f001){ref-type="fig"}c,g). This suggested that SLC27A4 is associated with enhancement of cell growth in breast cancer cells. 2.4. Silencing SLC27A4 in Breast Cancer Cell Lines Affected G0/G1 and G2/M Cell Cycle {#sec2dot4-ijms-19-03434} ------------------------------------------------------------------------------------- The cell cycle status in SLC27A4-silencing Hs578T and MDA-MB-231 was analyzed. The propidium iodide staining assay showed that decreasing cell population in G0/G1 phase and increasing cell population in G2/M phase in the shSLC27A4\#02 group but not the shSLC27A4\#98 group of Hs578T ([Figure 4](#ijms-19-03434-f004){ref-type="fig"}a). However, SLC27A4 silencing did not significantly affect the cell cycle in MDA-MB-231 ([Figure 4](#ijms-19-03434-f004){ref-type="fig"}b). The quantitative results are shown in [Figure 4](#ijms-19-03434-f004){ref-type="fig"}c,d. In addition, the regulator proteins of cell cycles were investigated in Hs578T. [Figure 4](#ijms-19-03434-f004){ref-type="fig"}e--g show that the protein expression levels of cyclin B1 and cyclin A2 significantly increased in the shSLC27A4\#02 group. 2.5. Silencing SLC27A4 in Breast Cancer Cell Lines Inhibited Capacity of Migration and Invasion {#sec2dot5-ijms-19-03434} ----------------------------------------------------------------------------------------------- The cell migration in SLC27A4-knockdowned Hs578T and MDA-MB-231 was evaluated via wound-healing assay. The results revealed that cell migration capacity was significantly inhibited in both SLC27A4-silencing groups in Hs578T and in shSLC27A4\#98 groups in MDA-MB-231 ([Figure 5](#ijms-19-03434-f005){ref-type="fig"}a,b). Furthermore, similar results were observed in the transwell migration assay ([Figure 5](#ijms-19-03434-f005){ref-type="fig"}c,d). Because MDA-MB-231 is a highly metastatic cell line, the invasion capacity was further determined. Our result indicated that the shSLC27A4\#98 group had lower invasion capacity than that in the vector control group ([Figure 5](#ijms-19-03434-f005){ref-type="fig"}e). It suggests that the SLC27A4 expression level was also associated with migration and invasion capacity of breast cancer. 2.6. Silencing SLC27A4 in Breast Cancer Cell Lines Affected Regulatory Molecules of Epithelial Mesenchymal Transition Signaling Pathways {#sec2dot6-ijms-19-03434} ---------------------------------------------------------------------------------------------------------------------------------------- The migration and invasion capacity of Hs578T and MDA-MB-231 was inhibited after silencing SLC27A4. EMT phenotype is associated with metastasis and invasion in cancer cells \[[@B17-ijms-19-03434]\]. Increasing N-cadherin, vimentin, Slug, and α-smooth muscle actin (α-SMA) and decreasing E-cadherin are biomarkers of EMT signaling \[[@B17-ijms-19-03434]\]. Therefore, the regulatory molecules of EMT introduction was investigated. In Hs578T, the protein expression levels of vimentin and Slug decreased in SLC27A4 silencing groups. The expression of α-SMA was not significantly changed ([Figure 6](#ijms-19-03434-f006){ref-type="fig"}a--d). In MDA-MB-231, the expression of N-cadherin, E-cadherin, Slug, and α-SMA was significantly affected in SLC27A4 silencing groups; however, the expression of vimentin was not changed. These results suggest that knockdown of SLC27A4 affected the regulatory molecules of EMT signaling pathways. 2.7. Potential Interacting Networks in SLC27A4-Silencing Breast Cancer Cells {#sec2dot7-ijms-19-03434} ---------------------------------------------------------------------------- To the best of our knowledge, the detailed regulatory mechanism of SLC27A4 is still unknown in breast cancer. Therefore, the biological networks of SLC27A4 were evaluated by TCSBN database, which provided biological networks in various types of cancer and non-cancer tissues \[[@B18-ijms-19-03434]\]. In this study, the interacting networks were drawn according to the top-25 genes with high co-expression correlation among normal and cancer tissues ([Figure 7](#ijms-19-03434-f007){ref-type="fig"}a,b). The results showed that most genes within interacting networks in breast cancer tissues were different from that in normal breast mammary tissues. Only three genes including *Dolichol Kinase* (*DOLK*), *TruB Pseudouridine Synthase Family Member 2* (*TRUB2*), and *Ubiquitin Related Modifier 1* (*URM1*) were identical between normal and cancer tissues. In order to determine whether the function of the these genes, these 25 genes in normal breast and breast cancer tissues were performed the functional annotation bioinformatics analysis through DAVID Bioinformatics Resources \[[@B19-ijms-19-03434],[@B20-ijms-19-03434]\]. The results were shown in [Supplementary Tables S1 and S2](#app1-ijms-19-03434){ref-type="app"}, respectively. The analysis showed that SLC27A4-correlated genes mainly involved in metabolic processes in normal breast tissues and in transport processes in breast cancer tissues. In top-25 SLC27A4 co-expression genes in breast cancer tissues, six genes including *Solute carrier family 26 member 11* (*SLC26A11*), *calcium activated nucleotidase 1* (*CANT1*), *leucine rich repeat and sterile alpha motif containing 1* (*LRSAM1*), *tubulin folding cofactor D* (*TBCD*), *LLGL2 scribble cell polarity complex component* (*LLGL2*), and *GDP dissociation inhibitor 1* (*GDI1*) were associated with poor DMFS ([Figure 7](#ijms-19-03434-f007){ref-type="fig"}c--h). These evidences might provide a possible interacting network of SLC27A4 in clinical breast cancer tissues. The summarized graph of the present study is presented in [Figure 7](#ijms-19-03434-f007){ref-type="fig"}i. 3. Discussion {#sec3-ijms-19-03434} ============= The bioinformatic analysis revealed that high SLC27A4 was associated with breast cancer tissue and poor prognosis in breast cancer patients. In addition, our results suggest that silencing SLC27A4 expression inhibited cell growth, migration and invasion capacity in breast cancer cell lines. Bioinformatic analyses of SLC27A4-interacting network are linked to several types of metabolic pathways and regulation of cell size. Phospholipids are essential components for all membranes. During growth and cell cycle progression, the regulation of DNA synthesis and phospholipids synthesis/turnover must be integrated. The intracellular fatty acids pool is contributed from de novo fatty acid synthesis and extracellular fatty acid transport in breast cancer cells \[[@B21-ijms-19-03434]\]. Therefore, we supposed that SLC27A4-silencing should affect cell growth and cell cycle in both cell lines. The present results showed that cell growth was inhibited after SLC27A4-silencing. G2/M cell cycle arrest was observed in in shSLC27A4\#02 Hs578T and increased protein expression of cyclin A2 and cyclin B1 was detected. Cyclin A2 and cyclin B1 activated cyclin-dependent kinase 1 (CDK1), which regulates mitotic entry and progression \[[@B22-ijms-19-03434]\]. Cyclin A2 regulates nuclear-envelope breakdown and then the cyclin B1-CDK1 complex is activated \[[@B23-ijms-19-03434]\]. A previous study demonstrated that inhibition of fatty acid synthase activity arrested the cancer cells at G2/M \[[@B24-ijms-19-03434]\]. When cancer cells are treated with the inhibitor of fatty acid synthase, the increased protein expression of cyclin B1 was observed. The cyclin A2 expression was significantly affected by the inhibitor \[[@B24-ijms-19-03434]\]. Fatty acid synthase and acetyl-CoA carboxylase 1 are critical enzymes involved in de novo fatty acid synthesis \[[@B25-ijms-19-03434]\]. It suggests that interference of intracellular fatty acids pool arrests cells at late G2/mitosis before anaphase/telophase. The degradation pathways of cyclin A2 and cyclin B1 might be attenuated by SLC27A4-silencing, and subsequently contribute to G2/M arrest. Perhaps CDK1 is involved in this cell cycle regulation in Hs578T. Interestingly, the cell cycle of SLC27A4-silencing MDA-MB-231 was not significantly changed. Knockdown of acetyl-CoA carboxylase 1 or fatty acid synthase disrupts fatty acids synthesis, acetyl-CoA and CoA production, and then induces apoptosis in breast cancer cells \[[@B26-ijms-19-03434],[@B27-ijms-19-03434]\]. Therefore, SLC27A4-silencing might also lead to slight cell death but not affect cell cycle progression. It will be further investigated in the future. The cell cycle results were inconsistent between MDA-MB-231 and Hs578T. Hs578T is a cell line derived from primary tumor and its pathology is distinguishing infiltrating ductal carcinoma \[[@B28-ijms-19-03434]\]. By contrast, MDA-MB-231 is derived from pleural effusion and its pathology is distinguishing adenocarcinoma \[[@B28-ijms-19-03434]\]. Both cells have mutant p53, and wild type BRCA1. Hs578T has a *HRAS* mutation, and MDA-MB-231 has *B-Raf Proto-Oncogene* (*BRAF*), *Cyclin Dependent Kinase Inhibitor 2A* (*CDKN2A*, *p16*), and *KRAS* mutation \[[@B28-ijms-19-03434],[@B29-ijms-19-03434]\]. In mammary cells, p53 is a key regulator of cell cycle \[[@B30-ijms-19-03434],[@B31-ijms-19-03434]\]. Because Hs578T has a mutant p53, p53 should not play a role in SLC27A4-mediated cell cycle regulation in Hs578T. The other tumor suppressor, such as wild type p16, might be important for regulating cell cycle in SLC27A4-silencing Hs578T. In this study, the capacity of migration and invasion was suppressed after silencing SLC27A4 ([Figure 5](#ijms-19-03434-f005){ref-type="fig"}). Generally, mesenchymal phenotype is usually associated with tumor migration, invasion, and poor clinical outcomes \[[@B32-ijms-19-03434]\]. A previous study decreasing FASN and FABP1 cause inhibition of EMT in breast cancer cells \[[@B16-ijms-19-03434]\]. The cell morphology of SLC27A4-silencing Hs578T and MDA-MB-231 was not significantly affected when compared to control groups. The Western blot analyses showed that the expression of transcription factors that promoted EMT was suppressed in SLC27A4-silencing cells. Moreover, bioinformatic analyses suggest that SLC27A4 is involved in regulation of cell size in breast cancer cells. Transforming growth factor-β (TGF-β) induced EMT and increased cell size through mammalian target of rapamycin (mTOR) signaling pathways \[[@B33-ijms-19-03434]\]. This data might suggest interaction between SLC27A4-mediated cell size regulation and TGF-β signaling pathways. BRAF is involved in the processes of EMT, stemness or metastasis in breast \[[@B34-ijms-19-03434]\]; thus, different BRAF status in two breast cancer cell lines might affect the EMT signaling pathways after SLC27A4 silencing. Palmitic acid (a common saturated fatty acid, C16:0) or a high-fat diet enhances the metastatic potential of CD36^+^ metastasis-initiating breast cancer cells \[[@B11-ijms-19-03434]\]. Interestingly, a previous study indicated that CD36 enhances fatty acid uptake but does not transport fatty acid across the plasma membrane in a mammalian cell line \[[@B35-ijms-19-03434]\]. It implies that uptake of palmitic acid or other types of fatty acid is not fully dependent on CD36 in breast cancer. The other families of transporters are necessary for utilization of fatty acid outside cells. ACSL4 has a fatty acid transporter activity and its preferred substrate is arachidonic acid (a unsaturated fatty acid, C20:4) \[[@B36-ijms-19-03434]\]. Silencing ACSL4 in breast cancer cells affects the components of cell membranes, especially arachidonic acid \[[@B37-ijms-19-03434]\]. Arachidonic acid is also known as a fatty acid that links to cancer metastasis \[[@B38-ijms-19-03434]\]; on the other hand, palmitic acid and lignoceric acid (C24:0) are known substrates of SLC27A4 \[[@B13-ijms-19-03434]\]. In [Figure 2](#ijms-19-03434-f002){ref-type="fig"}j--k, low capacity of fatty acid uptake was detected in SLC27A4-silencing Hs578T and MDA-MB-231. We speculate that knockdown of SLC27A4 might alter uptake of specific fatty acids and then change the composition of intracellular fatty acids pool. When compared to the substrates among SLC27A4, SLC27A1 and SLC27A6, oleic acid (C18:1) is a preferred substrate of SLC27A1 and SLC27A6, but not SLC27A4 \[[@B13-ijms-19-03434]\]. Oleic acid has revealed antitumor effects in several types of cancers \[[@B39-ijms-19-03434]\]. Perhaps this is why SLC27A4 expression is opposite to other SLC27 family proteins expression. Because Hs578T and MDA-MB-231 were maintained at media with normal fetal bovine serum that contained various types of fatty acids in the present study, we could not evaluate the effect of each suspicious fatty acid in SLC27A4-silencing cells. Culture medium fatty-acid withdrawal via Bio-Beads methods might be a strategy to investigate whether fatty acids are important factors to regulate cell growth, migration, and invasion. These issues will be further investigated in the future. Currently, the interaction of SLC27A4 is not well-known in cancer cells. In lung cancer cell lines, SLC27A4 directly interacts with autophagy-related 4B cysteine peptidase (ATG4B) \[[@B40-ijms-19-03434]\]. There are no related studies indicating the interacting networks of SLC27A4 in breast cancer. Because modulation of a metabolic enzyme expression might affect the entire metabolic flux, investigating the interacting networks is essential for further studies. Thus, the TCSBN database was used for predicting the possible interacting networks and DAVID Bioinformatics Resources was used for understanding the functions of these genes. In high SLC27A4-expressing breast cancer tissues and low SLC27A4-expressing normal breast tissues, different biological processes were observed. In addition, our analysis revealed six SLC27A4-correlated genes including CANT1, GDI1, LLGL2, LRSAM1, SLC26A11, and TCBD were also associated poor prognosis in clinical samples. An emerging study demonstrates that LLGL2 involves in Hippo-YAP pathway which regulates bone metastasis in breast cancer \[[@B41-ijms-19-03434]\]. The function of other genes has not been investigated in breast cancer. It is worth investigating the interactions among SLC27A4 and these genes in breast tumor cells and tumor environment in future studies. We believe that SLC27A4 is a potential diagnostic marker for breast cancer. Because SLC27A4 is a transmembrane protein, blockage of extracellular SLC27A4 via a SLC27A4 antibody might be a novel therapeutic strategy against breast cancer due to disruption of the SLC27A4/CD36-mediated fatty acids transportation pathway. This issue should be further evaluated in animal tumor models and clinical specimens in the future. 4. Materials and Methods {#sec4-ijms-19-03434} ======================== 4.1. Cell Culture {#sec4dot1-ijms-19-03434} ----------------- Human mammary cancer cell lines MDA-MB-231 (HTB-26™), Hs578T (HTB-126™), T47D (HTB-133™), and MCF-7 (HTB-22™) were purchased from American Type Culture Collection (Manassas, VA, USA). MCF-7, Hs578T, and T47D was respectively cultured in Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), and RPMI1640 supplied with 10% fetal bovine serum (Life Technologies, Grand Island, NY, USA), 100 units/mL penicillin G, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B in 5% CO~2~ air atmosphere at 37 °C. In addition, MDA-MB-231 was cultured in Leibovitz's L-15 Medium with 10% FBS, 100 units/mL penicillin G, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B in a CO~2~-free air atmosphere at 37 °C. All culture media and supplements were purchased from Lonza (Walkersville, MD, USA). 4.2. Bioinformatic Analysis {#sec4dot2-ijms-19-03434} --------------------------- The expression in SLC27 gene family (SLC27A1-6) in breast cancer samples and nontumor breast samples across available datasets was evaluated by Oncomine Research Edition (<http://www.oncomine.org>, v4.5; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The expression of SLC27A4 was further evaluated in the TCGA breast dataset in Oncomine Research Edition, either. Moreover, the expression of SLC27A4 in different subtypes and races of breast cancer samples was evaluated by the UALCAN (<http://ualcan.path.uab.edu>) \[[@B42-ijms-19-03434]\]. The association between gene expression and overall survival rate of breast cancer patients was obtained from the Human Protein Atlas (<https://www.proteinatlas.org>) \[[@B43-ijms-19-03434],[@B44-ijms-19-03434]\]. In addition, the images of SLC27A4 protein expression in normal breast and breast cancer tissues, and results staining intensity of SLC27A4 were obtained from the Human Protein Atlas database (Antibody: HPA007293). The high and low expression groups were separated by "best separation" on the website of the Human Protein Atlas. Distant metastasis-free survival (DMFS) was evaluated by Kaplan--Meier (KM) plotter (<http://kmplot.com>) \[[@B45-ijms-19-03434]\] and high- and low-expression groups were divided according to the "auto select best cutoff" in the website. The interacting networks were determined according to the TCSBN database (<http://inetmodels.com>) \[[@B18-ijms-19-03434]\]. "Maximum number of nodes" was set at 25 and "Edge Pruning Parameter (-log10 P)" was set at 3 in the TCSBN database. The interacting networks were drawn by Cytoscape version 3.6.1 \[[@B46-ijms-19-03434]\]. Functional annotation (biological process) was determined by DAVID Bioinformatics Resources (<https://david.ncifcrf.gov>) \[[@B19-ijms-19-03434],[@B20-ijms-19-03434]\]. 4.3. Western Blot Assay {#sec4dot3-ijms-19-03434} ----------------------- To collect protein, cells were cultured in a 6-cm dish for 48 h and then were lysed in radioimmunoprecipitation lysis buffer (Millipore, Billerica, MA, USA) with protease inhibitor cocktail (Millipore) at a 1000:1 ratio. Protein concentration was determined by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Billerica, MA, USA), then separated on 10--15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Polyvinylidene difluoride (PVDF) membranes (Millipore). After 1 h blocking with 5% dried skimmed milk in tris-buffered saline with Tween-20 (TBST) buffer, the membrane was hybridized with the primary antibodies including anti-E-cadherin (1:1000, Cat. No. \#610182), anti-N-cadherin (1:1000, Cat. No. \#610921), anti-Vimentin (1:3000, Cat. No. \#550513) were purchased from BD Transduction Laboratories™; anti-GAPDH (1:5000, Cat. No. \#MAB374) was purchased from Millipore (USA); anti-α-SMA (1:1000, Cat. No. \#A5228) was purchased from Sigma-Aldrich (St. Louis, MO, USA); anti-Slug (1:1000, Cat. No. \#9585S), anti-cyclin A2 (1:1000, Cat. No. \#4656) and anti-cyclin B1 (1:1000, Cat. No. \#4135) were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-SLC27A4 (1:2000, Cat. No. \#ab199719) was purchased from Abcam (UK) at 4 °C overnight. After TBST washing three times, the membrane was then hybridized with anti-rabbit IgG or anti-mouse IgG HRP-linked antibody (Cell Signaling Technology, USA). The results were acquired on Alpha Innotech FluorChem FC2 imaging system (ProteinSimple; Bio-Techne, Minneapolis, MN, USA). 4.4. Knockdown of SLC27A4 {#sec4dot4-ijms-19-03434} ------------------------- Lentivirus shRNAs were purchased from RNAi Core Facility (Taipei, Taiwan). The lentivurus-shRNA clones included: Lenti-emptyT (clone ID, TRCN0000089107; a vector control); Lenti-shSLC27A4 \#98 (clone ID, TRCN0000043398; targeting sequence: 5′-CTTCACAGATAAACTGTTCTA-3′); Lenti-shSLC27A4 \#02 (clone ID, TRCN0000043402; targeting sequence: 5′-CCGGGTCTTCATCAAGACCAT-3′). To silencing the gene expression, the Hs578T and MDA-MB-231 cells lines were complete culture media containing 8 μg/mL polybrene (EMD Millipore, Billerica, MA, USA) in a 6-cm dish at 37 °C for 30 min. Lentiviruses for Hs578T and MDA-MB-231 were added for infection at multiplicity of infection (MOI) = 5 and MOI = 3, respectively. After 24 h of incubation, the culture medium was refreshed with fresh culture media, with 2 μg/mL puromycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), for 48 h. The infected cells were maintained in medium with 2 μg/mL puromycin, and subsequently used in assays. 4.5. Fatty Acid Uptake Assay {#sec4dot5-ijms-19-03434} ---------------------------- The capacity of fatty acid uptake was determined by using the Free Fatty Acid Uptake Assay Kit (Fluorometric) according to the manufacturer's instructions (cat. no. ab176768; Abcam, Cambridge, UK). Before fatty acid uptake assay, 1 × 10^4^ Hs578T and MDA-MB-231 were seeded on a 96-well plate overnight. The cells were preincubated in serum-free media for 1 h after phosphate-buffered saline washing. Subsequently, cells were incubated in a fluorescent fatty acid mixture for 30 min. The results were evaluated by using a microplate fluorescence reader at 485/528 nm (FL × 800; BioTek Instruments Inc., Winooski, VT, USA). The fluorescence signals from wells containing assay mix without cells were used as the background and fluorescence quantification in vector control groups was set to 100% for relative quantification. 4.6. Real-Time Quantitative PCR {#sec4dot6-ijms-19-03434} ------------------------------- Total RNA was isolated via TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcription of cDNA was performed via the PrimeScript RT reagent kit (Clontech Laboratories, Inc., Kusatsu, Japan). The relative PCR levels were determined by Fast SYBR-Green Master Mix (Applied Biosystems, Foster City, CA, USA) with the specific primer targeting human Solute Carrier Family 27 Member 4 (SLC27A4), 5′-TCCTGTGGGCTTTTGGTTGT-3′ and 5′-TGGCACCCAACTCAACACAT-3′, and human Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-GAGTCAACGGATTTGGT CGT-3′ and 5′-TTGATTTTGGAGGGATCTCG-3′, on a Real-Time PCR system (StepOnePlus Real-Time PCT system; Applied Biosystems, Foster City, CA, USA). The relative mRNA expression was normalized to the GAPDH expression and calculated using the 2^−ΔΔ*C*t^ method \[[@B47-ijms-19-03434]\]. 4.7. WST-1 Assay {#sec4dot7-ijms-19-03434} ---------------- The cell proliferation of Hs578T and MDA-MB-231 was evaluated by WST-1 (4-\[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio\]-1,3-benzene disulfonate) (Clontech, Mountain View, CA, USA). Briefly, 3 × 10^3^ cells were respectively seeded in 96-well plates overnight. The culture media were replaced with 100 μL mixture containing 95 μL of fresh culture media and 5 μL of WST-1 reagent. For 24- or 48-h incubation, the absorbance at 450 nm was determined on a microplate spectrophotometer (PowerWave X340; BioTek, Winooski, VT, USA). 4.8. Colony Formation Assay {#sec4dot8-ijms-19-03434} --------------------------- To determine the long-term effect, 500 cells were seeded in a six-well plate. Cell culture media were replaced each 3 days until 14 days after seeding. Colonies were stained with crystal violet (0.4 g/L; Sigma-Aldrich, St. Louis, MO, USA) and the number of colonies was counted. 4.9. Cell Cycle Analysis {#sec4dot9-ijms-19-03434} ------------------------ Hs578T and MDA-MB-231 cells were harvested at 48 h incubation after subculture. Harvested cells were fixed with 70% ethanol overnight at 4 °C and then were washed by phosphate-buffered saline. Subsequently, cells were incubated with 1 U/mL of DNase-free RNase A and 5 μg/mL of propidium iodide for 30 min at 4 °C in the dark (Sigma-Aldrich, St. Louis, MO, USA). The cell cycle distribution was determined on a flow cytometry (BD Accuri C6 flow cytometer, BD Biosciences San Jose, CA, USA). Amount of G0/G1, S and G2/M phase cells were determined as a percentage of the total number of cells. 4.10. Wound Healing Assay {#sec4dot10-ijms-19-03434} ------------------------- 1.5 × 10^5^ breast cancer cells were seeded into 24-well plates. A scratch was made by a 200 μL pipette tip when cells reached a complete confluent monolayer. After scratching, the suspended debris was removed by phosphate-buffered saline (PBS) washing. Subsequently, the cells were cultured in serum-free culture media (MDA-MB-231) or culture media with 1% FBS (Hs578T) for 24 h. The images were captured via a Leica inverted microscope and quantification was performed by TScratch software (version 1.0. Available at <http://www.cse-lab.ethz.ch>). 4.11. Transwell Migration and Invasion Assay {#sec4dot11-ijms-19-03434} -------------------------------------------- Before performing the transwell migration assay, 3 × 10^4^ breast cancer cells were seeded into a 24-well insert (Millicell Cell Culture Inserts 24-well Hanging Inserts, 8-μm PET, Millipore, St. Charles, MO, USA) in 300-μL serum-free medium, while 500 μL medium with 10% FBS was placed in the lower chamber. After culturing for 24 h, the transwell membrane on the 24-well insert was fixed with 500 μL 4% formaldehyde solution followed by 1% crystal violet staining. After removal of the cells on the upper surface, four images of each bottom membrane were captured using a Leica inverted microscope at ×100 magnification via Leica Applications Suite version 4.5.0™ (LAS v4.5) software (Leica Microsystems, Wetzlar, Switzerland). Invasion assay was performed by QCM ECMatrix Cell Invasion Assay, 24-well (8 μm), fluorimetric (Millipore, Billerica, MA, USA) according to the manufacturer's instruction. Briefly, 1.2 × 10^5^ cells in 300 μL serum-free culture media was added in the insert and 500 μL media with 10% FBS was placed in the lower chamber for 48 h. The results were evaluated on a Bio-tek FLX-800 Fluorescence & Luminescence Reader at the excitation (Ex) and emission (Em) wavelengths = 485/528 nm. 4.12. Statistics {#sec4dot12-ijms-19-03434} ---------------- All graphs and statistics were made by the GraphPad Prism 7 software (GraphPad Software, Inc., La Jolla, CA, USA). To examine statistical differences among all groups, a one-way analysis of variance (ANOVA) with Tukey's multiple comparison test was used. *p* \< 0.05 was considered to indicate a statistically significant difference. 5. Conclusions {#sec5-ijms-19-03434} ============== High expression of SLC27A4 was associated with breast cancer tissues and poor prognosis in breast cancer patients. In addition, knockdown of SLC27A4 decreased not only fatty acid uptake capacity in Hs578T and MDA-MB-231 but cell growth in Hs578T also, as well as capacity of migration and invasion in Hs578T and MDA-MB-231. Although detailed regulatory signaling pathways of SLC27A4 was not completely investigated in this study, our results firstly demonstrated that SLC27A4 was involved in progression of breast cancer. It is worth investigating whether SLC27A4 could serve as a diagnostic marker and treatment target in further studies. Supplementary materials can be found at <http://www.mdpi.com/1422-0067/19/11/3434/s1>. ###### Click here for additional data file. M.-C.Y., and Y.-L.H. conceived and designed the experiments; M.-C.Y. and S.-K.C. performed the experiments; M.-C.Y., S.-K.C., J.-Y.K. analyzed the data; J.-Y.K., P.-L.K., M.-F.H. and Y.-L.H. contributed reagents/materials/analysis tools; M.-C.Y. wrote the first draft of the paper, and all authors contributed to the editing and final approval of the paper. This study was supported by grants from the Ministry of Science and Technology (MOST 104-2314-B-037-053-MY4; MOST 105-2314-B-037-037-MY3; MOST 106-2314-B-037-046; MOST 106-2320-B-037-029-MY3), the Kaohsiung Medical University Hospital (KMUHS10701; KMUHS10712; KMUH106-6R34; KMUH106-6R77), and the Kaohsiung Medical University (KMU-DK108008). The authors thank the Center for Research Resources and Development of Kaohsiung Medical University. The authors declare no conflicts of interest. {#ijms-19-03434-f001} {#ijms-19-03434-f002} {#ijms-19-03434-f003} {#ijms-19-03434-f004} {#ijms-19-03434-f005} {#ijms-19-03434-f006} {#ijms-19-03434-f007}

Introduction ============ Renal fibrosis is a hallmark of most forms of progressive renal disease [@B1]. The unilateral ureteral obstruction (UUO) method, involving ligation of the ureter, most commonly of the left one, has been widely adopted to establish animal models with interstitial fibrosis. The kidney of the ligated ureter is conventionally termed as the obstructed kidney, which exhibits several important events such as mechanical stretching, activation of renin-angiotensin-aldosterone system (RAS), loss of renal epithelial cells, inflammation, oxidative stress, macrophage infiltration and fibroblast activation, together leading to the final fibrosis [@B2]. Histologically, the obstructed kidney is characterized by tubular dilation, interstitial expansion, loss of renal parenchyma, infiltration of inflammatory cells, and accumulation of extracellular matrix (ECM) [@B2]. Some molecular mechanisms have been identified involved in renal interstitial fibrosis. Signaling pathways such as Notch, Wnt and Hedgehog, which are crucial for kidney development, were shown overactivated and to play positive roles in UUO-induced fibrosis [@B3], [@B4], [@B5], [@B6]. Moreover, Erk and Akt signaling pathways were revealed to have a pro-proliferative effect on myofibroblasts by affecting molecules involved in cell cycle such as c-Myc, cyclin D1 and p21 [@B7], [@B8]. Recently, the mammalian target of rapamycin (mTOR) was found implicated in fibroblast proliferation and activation [@B9], [@B10], [@B11], [@B12]. Activation of Stat3 signaling may be another mechanism underlying renal interstitial fibrosis. Expression of phosphorylated Stat3 was enhanced in both tubular epithelial cells and interstitial fibroblasts after UUO [@B13]. Furthermore, blockade of Stat3 pathway was shown to inhibit fibroblast activation in obstructive nephropathy [@B14], [@B15]. Ruxolitinib is a potent and selective inhibitor of Jak1/2. It has been approved for the treatment of myelofibrosis, a neoplasm characterized by bone marrow fibrosis, and in which Jak/Stat pathway is over-activated. Ruxolitinib ameliorates disease-related symptoms, improves health-related quality of life, and increases patient survival [@B16], [@B17], [@B18]. Notably, a phase 3 study demonstrated that 15.8% patients with Ruxolitinib treatment had improved fibrosis [@B19]. Based on the above findings, in the present study we explored the effect of Ruxolitinib on UUO-induced renal interstitial fibrosis. We found that Ruxolitinib treatment suppressed fibroblast activation and reduced ECM deposition. Mechanistically, Ruxolitinib treatment attenuated Stat3, Akt, and mTOR signaling pathways in both obstructed kidneys and*in vitro* activated fibroblasts. Results ======= Ruxolitinib alleviates renal damage ----------------------------------- UUO was used to establish mouse models of obstructive nephropathy. After two weeks, PAS and Masson′s trichrome staining were used to evaluate renal damage and fibrosis. The obstructed kidneys from UUO mice without Ruxolitinib treatment (later called UUO kidneys) exhibited severe structural disorders, characterized by tubular dilation and atrophy, intratubular cast formation, inflammatory cell infiltration, and ECM deposition (Figure [1](#F1){ref-type="fig"}A-D). However, kidneys from UUO mice with Ruxolitinib treatment displayed remarkably less tubular injuries and ECM deposition, indicating Ruxolitinib treatment alleviated UUO-induced renal damage (Figure [1](#F1){ref-type="fig"}A-D). Ruxolitinib ameliorates ECM deposition -------------------------------------- To evaluate the effect of Ruxolitinib on UUO-induced fibrosis, we analyzed the deposition of several ECM including Collagen I, Collagen III and Fibronectin. Immunohistochemistry staining showed that UUO kidneys expressed much higher levels of Collagen I (Figure [2](#F2){ref-type="fig"}A-B), Collagen III (Figure [2](#F2){ref-type="fig"}C-D) and Fibronectin (Figure [2](#F2){ref-type="fig"}E-F) compared with normal control (Sham group), and indicating UUO successively induced renal interstitial fibrosis. Ruxolitinib treatment attenuated the deposition of all these ECM (Figure [2](#F2){ref-type="fig"}A-F). Western blot detection similarly revealed Ruxolitinib treatment reduced Collagen I, Collagen III and Fibronectin expression in UUO kidneys (Figure [2](#F2){ref-type="fig"}G-H). Furthermore, Western blot detection indicated that Ruxolitinib treatment suppressed UUO-induced metallopeptidase inhibitor 1 (Timp-1) upregulation (Figure [2](#F2){ref-type="fig"}G-H), suggesting that Ruxolitinib treatment may not only reduce ECM production, but also promote ECM degradation. Ruxolitinib blocks renal fibroblast activation ---------------------------------------------- Fibroblast activation is crucial for renal interstitial fibrosis. Activated fibroblasts or myofibroblasts are sources of ECM. We assessed the effect of Ruxolitinib on fibroblast activation using UUO models and TGF-β1 -treated NRK-49F cells. Staining of α-SMA by Immunohistochemistry revealed much more activated fibroblasts in UUO kidneys compared with normal control. Ruxolitinib treatment notably blocked UUO-induced fibroblast activation, which was indicated as a decrease in α-SMA expression (Figure [3](#F3){ref-type="fig"}A-B). This finding was further confirmed by Western blot detection (Figure [3](#F3){ref-type="fig"}C-D). TGF-β1 was used to activate NRK-49F cells. MTT measurement showed that Ruxolitinib treatment suppressed TGF-β1 -induced NRK-49F cell proliferation (Figure [3](#F3){ref-type="fig"}E). Furthermore, Ruxolitinib treatment downregulated TGF-β1 -induced α-SMA expression (Figure [3](#F3){ref-type="fig"}F-G). Consistently, Ruxolitinib treatment counteracted TGF-β1 -induced Collagen I and Fibronectin expression in NRK-49F cells (Figure [3](#F3){ref-type="fig"}F-G). Ruxolitinib suppresses renal tubular cell EMT --------------------------------------------- Epithelial-mesenchymal transition (EMT) endows tubular epithelial cells a mesenchymal phenotype, with increased migratory capacity and ECM production [@B2]. We next investigated the effect of Ruxolitinib on renal tubular cell EMT using UUO models and TGF-β1 -treated NRK-52E cells. Both Immunohistochemistry and Western blot detection showed that E-cadherin, an epithelial marker, was remarkable downregulated in UUO kidneys, while Ruxolitinib treatment effectively restored its expression (Figure [4](#F4){ref-type="fig"}A-D). Western blot detection also revealed that Ruxolitinib treatment inhibited UUO-induced upregulation of Snail and Twist, two EMT transcription factors (Figure [4](#F4){ref-type="fig"}C-D). TGF-β1 was used to induce NRK-52E cell EMT. Ruxolitinib treatment compensated for TGF-β1 -induced loss of E-cadherin, and blocked TGF-β1 -induced upregulation of α-SMA, Snail and Twist (Figure [4](#F4){ref-type="fig"}E-F). Ruxolitinib inhibits inflammatory responses ------------------------------------------- Inflammation is a hallmark of obstructed kidney characterized by inflammatory cell infiltration and inflammatory cytokine production. We examined F4/80 expression to assess macrophage infiltration. As shown by Immunohistochemistry, F4/80 expression level is higher in UUO kidneys compared with normal control. Ruxolitinib treatment reduced F4/80 expression in UUO kidneys significantly, indicating macrophage infiltration was effectively inhibited (Figure [5](#F5){ref-type="fig"}A-B). NFκB signaling was activated in UUO kidneys, whereas Ruxolitinib treatment suppressed p-65 phosphorylation (Figure [5](#F5){ref-type="fig"}C-D). We further evaluated the effect of Ruxolitinib on the expression of several inflammatory cytokines. Real-time PCR detection revealed that Ruxolitinib treatment reduced levels of TNF-α, IL-1β, IL-6 and MCP-1/CCL2 mRNA (Figure [5](#F5){ref-type="fig"}E-H). Ruxolitinib reduces renal tubular epithelial cell apoptosis ----------------------------------------------------------- Renal tubular epithelial cell apoptosis is an important event in obstructed kidney. TUNEL staining was used to determine cell apoptosis. It was shown that apoptotic cells increased in UUO kidneys. Ruxolitinib treatment ameliorated the situation (Figure [6](#F6){ref-type="fig"}A-B). Cleaved caspase-3 was subsequently examined. As shown by both Immunohistochemistry and Western blot, cleaved caspase-3 expression increased in UUO kidneys, while decreased after Ruxolitinib treatment (Figure [6](#F6){ref-type="fig"}C-F). As oxidative stress is one mechanism underlying cell apoptosis, we thereafter investigated levels of Malondialdehyde (MDA) and total Superoxide dismutase (T-SOD). As expected, UUO resulted in an increase in MDA, and a reduction in T-SOD; Ruxolitinib treatment restored T-SOD and MDA towards normal level (Figure [6](#F6){ref-type="fig"}G-H). Ruxolitinib attenuates Akt/mTOR/Yap pathway ------------------------------------------- We finally explored signaling pathways targeted by Ruxolitinib. UUO kidneys exhibited increased expression of phosphorylated Stat3 and Erk. Ruxolitinib treatment significantly suppressed Stat3 and Erk phosphorylation (Figure [7](#F7){ref-type="fig"}A-B). Similarly, Ruxolitinib treatment inhibited Stat3 and Erk phosphorylation in TGF-β1 -treated NRK-49F cells (Figure [7](#F7){ref-type="fig"}C-D). We further analyzed downstream molecules of Jak signaling such as Akt and mTOR which have been shown involved in obstructed kidney [@B9], [@B10]. Consistent with these reports, UUO kidneys overexpressed Akt, p-Akt, mTOR and p-mTOR. Ruxolitinib treatment reduced Akt and mTOR phosphorylation (Figure [7](#F7){ref-type="fig"}E-F). Recently, yes-associated protein (Yap) was shown as a downstream of mTOR and involved in UUO kidney [@B12], [@B20]. Indeed, Ruxolitinib treatment inhibited Yap expression (Figure [7](#F7){ref-type="fig"}E-F). Moreover, Ruxolitinib treatment suppressed Akt and mTOR phosphorylation and Yap expression in TGF-β1 -treated NRK-49F cells (Figure [7](#F7){ref-type="fig"}G-H). Discussion ========== Activated fibroblasts expressing α-SMA are conventionally called myofibroblasts [@B1]. Myofibroblasts produce ECM; therefore they are crucial for organ fibrosis. Study by LeBleu and colleagues demonstrated that in UUO kidneys, 50% of myofibroblasts originate from local resident fibroblasts through proliferation, and 35%, 10% and 5% of myofibroblasts arise from bone marrow differentiation, endothelial-to-mesenchymal transition and EMT, respectively [@B21]. Our study revealed that Ruxolitinib treatment reduced α-SMA expression in both UUO kidneys and TGF-β1 -treated NRK-49F cells, and consistently, Ruxolitinib treatment resulted in a reduced ECM production; Moreover, Ruxolitinib treatment interfered with TGF-β1 -induced NRK-49F cell proliferation. Together, these findings indicate that Ruxolitinib has a potentiality to suppress fibroblast activation or myofibroblast generation. As renal tubular epithelial cell EMT contributes to UUO-induced renal interstitial fibrosis, we observed the effect of Ruxolitinib on E-cadherin expression. Just as expected, UUO resulted in a decreased E-cadherin expression, which was partially recovered by Ruxolitinib treatment. Furthermore, Ruxolitinib treatment blocked TGF-β1 -induced E-cadherin downregulation in NRK-52E cells. Transcription factors snail and twist have been shown involved in renal tubular epithelial cell EMT [@B22], [@B23]. Our study demonstrated that Ruxolitinib treatment suppressed snail and twist upregulation in both UUO kidneys and TGF-β1 -treated NRK-52E cells. However, study by Pang and colleagues indicated that a specific Stat3 inhibitor, S3I-201, has no effect on UUO-induced snail overexpression [@B15]. These findings suggest that Ruxolitinib inhibits snail upregulation independent on Stat3. UUO kidneys overproduce chemokines and their receptors [@B24], [@B25], [@B26]. These chemokines are responsible for the recruitment of inflammatory cells, which, when activated, produce more cytokines to sustain and enhance inflammation. MCP-1 recruits macrophages into renal interstitium [@B27], [@B28], [@B29]. Our study showed that Ruxolitinib treatment reduced MCP-1 expression as well as macrophage infiltration in the obstructed kidneys. In contrast, blockade of Stat3 by S3I-201 failed to suppress UUO-induced MCP-1 upregulation, but still reduced macrophage infiltration, suggesting that other cytokines were involved in macrophage recruitment [@B15]. Besides growth factor such as TGF-β1, cytokine such as IL-6 also contributes to fibroblast activation and fibrosis [@B30]. A recent study showed that blockade of IL-6 improved UUO-induced fibrosis [@B31]. Several factors have been identified to induce tubular epithelial cell apoptosis, including mechanical stretch, Angiotensin II, TGF-β1, Fas/FasL and oxidative stress [@B32], [@B33]. In our study, Ruxolitinib treatment reduced expression of TNF-α, which was shown to stimulate apoptosis in UUO kidneys [@B34], [@B35]. Moreover, Ruxolitinib treatment attenuated the oxidative stress response. It has been shown that UUO impairs renal antioxidant enzyme activation [@B36]. Ruxolitinib treatment recovered T-SOD level in the obstructed kidneys, and consistently, alleviated UUO-induced apoptosis. Our study finally identified that Ruxolitinib treatment attenuated Akt/mTOR pathway. Both mTOR complex 1 (mTORC1) and mTORC2 were activated in UUO kidneys [@B9], [@B10]. Furthermore, Yap was revealed to mediate mTORC2-induced renal interstitial fibrosis [@B12]. Increasing evidence indicated that Yap functioned as a pro-fibrotic factor in kidneys, and targeting Yap improved renal interstitial fibrosis [@B20], [@B37], [@B38], [@B39]. Ruxolitinib treatment inhibited Yap expression in UUO kidneys, further suggesting that Akt/mTOR/Yap is a potential target signaling by Ruxolitinib. In summary, Ruxolitinib treatment alleviated inflammation and oxidative stress in UUO kidneys, and suppressed fibroblast activation, tubular cell EMT and ECM production in both UUO kidneys and *in vitro* cultured cells. Mechanistically, Ruxolitinib treatment blocked UUO or TGF-β1 -induced activation of both Stat3 and Akt/mTOR/Yap pathways. These findings indicate that Ruxolitinib treatment can ameliorate UUO-induced renal interstitial fibrosis, and suggest that Ruxolitinib could be potentially used to treat fibrotic kidney disease. Materials and Methods ===================== Chemicals and antibodies ------------------------ Ruxolitinib phosphate (Jakavi, Novartis) and Ruxolitinib (INCB018424; Selleck chemicals) were used for *in vivo* and *in vitro* experiment, respectively. Antibodies to collagen I (ab34719), collagen III (ab7778), Fibronectin (ab2413), Timp-1 (ab86482), and α-SMA (ab124964) were purchased from Abcam. Antibodies to E-cadherin (\#3195), Snail (\#3879), Twist (\#46702), F4/80 (\#70076), mTOR (\#2972), p-mTOR (\#2971), Akt (\#9272), p-Akt (Ser473, \#9271), Stat3 (\#12640), p-Stat3 (Tyr705, \#9145), Erk 1/2 (\#4695), p-Erk 1/2 (\#4370), and Yap (\#14074) were purchased from Cell Signaling Technology. Antibodies to p-NFκB p65(sc-33020) and NFκB p65(sc-109) was purchased from Santa Cruz Biotechnology. TUNEL assay kit (KGA7061) for apoptosis was purchased from KeyGEN BioTECH (Nanjing, China). UUO models and Ruxolitinib treatment ------------------------------------ Male C57BL/6 mice (Beijing Huafukang Biotechnology, China) that weighed 22-24g were randomly assigned to three groups with 5 mice in each group as follows: (1) Sham-operated mice with vehicle (Sham); (2) UUO mice with vehicle (UUO); (3) UUO mice treated with Ruxolitinib (UUO+RUX). To establish UUO model, mice were given general anesthesia by intraperitoneal injection of pentobarbital (50mg/kg body weight). The left ureter was exposed via a left flank incision, ligated with 4-0 silk at two points, and cut between the 2 ligation points. The Sham-operated group had no ligation. For *in vivo* experiments, Ruxolitinib was dissolved in PEG300/dextrose 5% in a ratio of 1:3 (PEG/dex) and administered to mice by oral gavage at a dosage of 30 mg/kg twice daily for 14 days immediately after UUO or Sham-operation. The Sham and UUO group received PEG/dex alone as vehicle. The mice were sacrificed, and the left kidneys were collected at days 14 after surgery. All procedures were performed in accordance with guidelines approved by the Institutional Animal Care and Use Committee of China Medical University. PAS and Masson′s trichrome staining ----------------------------------- The paraffin-embedded sections were stained with PAS (Solarbio, China, G1281) and Masson\'s trichrome (Solarbio, China, G1340) to evaluate histological change and fibrosis. Ten non-repeating fields were randomly selected. Tubular lesions were scored from 0 to 4 [@B31]. 0: normal; 1: mild (\<25% of the cortex); 2: moderate (25\~50%); 3: severe (50\~75%); 4: extensive damage (\>75%). The positive area of Masson\'s trichrome staining (blue) was calculated with the Image-Pro Plus. Cell culture and treatment -------------------------- Rat fibroblast NRK-49F and rat renal tubular epithelial cell NRK-52E were cultured in Dulbecco\'s modified Eagle\'s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C with 5% CO~2~. 2ng/ml TGF-β1 was used to activate NRK-49F cells or induce NRK-52E cell EMT. These cells were starved for 12 h and then exposed to TGF-β1 with or without 5μM Ruxolitinib for 24 hours. Immunohistochemistry -------------------- Tissue sections were deparaffinized, hydrated, and incubated with 3% H~2~O~2~ to remove endogenous peroxidase. Then the sections were incubated with primary antibody overnight at 4 ℃, and next with biotinylated secondary antibody for 30 min at 37 °C. Subsequently the sections were stained with DAB, re-stained in hematoxylin, dehydrated, and sealed with cover slides. Ten non-repeating images of each sample were acquired using microscope image system. Positive signals and numbers of positive cells were quantified using Image-Pro Plus software. Western blot ------------ Equal amount of protein was isolated by SDS-PAGE, and transferred to PVDF membranes which was blocked by 5% bovine serum albumin or skimmed milk for 2 h at room temperature, and incubated with primary antibody overnight at 4°C. The membrane was subsequently incubated with peroxidase-conjugated goat secondary antibody (1:5000, ZSGB Bio) for 2 h at room temperature. Protein content was determined by SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific). Real-time PCR ------------- Total RNAs were extracted using RNAiso Plus (Takara, Cat\#9108), and reversely transcribed into cDNA using PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, Cat\#RR047a) according to the instructions. Real-time PCR was carried out using TB Green™ *Premix Ex Taq*™ II (Tli RNaseH Plus) (RR820A, TaKaRa). The primers for TNF-α are, forward: 5′-gcgacgtggaactggcagaag-3′ and reverse: 5′-gccacaagcaggaatgagaagagg-3′; for IL-6, forward: 5′-acttccatccagttgccttcttgg-3′ and reverse: 5′-ttaagcctccgacttgtgaagag-3′; for IL-1β, forward: 5′-tcgcagcagcacatcaacaagag-3′ and reverse: 5′-tgctcatgtcctcatcctggaagg-3′. for MCP-1, forward: 5′-ccactcacctgctgctactcattc-3′ and reverse: 5′-ctgctgctggtgatcctcttgtag-3′. GAPDH was used as internal control. Expression difference was assessed using 2^-ΔΔCT^ method. TUNEL ----- The apoptotic cells in kidney tissue sections were detected using a TUNEL apoptosis detection kit (KeyGEN BioTECH, China, KGA7061) according to the instructions provided by the manufacture. Briefly, paraffin-embedded sections were treated with fresh diluted proteinase K for 20 min at 37 ℃ after deparaffinage and rehydration. TdT enzyme reaction mixture was applied to each slide at 37 ℃ for 60 min. Then, Streptavidin-TRITC was added into the slides at 37 ℃ for 30 min. At last, the slides were reacted with DAPI solution for 10 min at room temperature. The number of TUNEL-positive nuclei per field was counted in 10 non-repeating micrographs for each sample. MTT --- Cell viability was measured at 48 h using an MTT assay kit (Beyotime, China, C0009). 2,000 Cells in 96-well plate were washed and incubated with MTT staining solution for 4 h. Then Formazan solving solution was added. The cell viability was indicated by the absolution value at 570nm. T-SOD and MDA measurement ------------------------- T-SOD (A001-1) and MDA (A003-1) kits were purchased from Jiancheng Bioengineering Institute (Nanjing, China). Briefly, kidney tissues were prepared into 10% homogenates which were centrifuged at 3,000 rpm for 10 min. The supernatants were collected to measure levels of T-SOD and MDA according to the instructions. Statistical analysis -------------------- All the experiments were conducted at least three times. GraphPad Prism 7.0 was used to analyze the data, and all data were expressed as the Mean ± SEM. Inter-group comparisons were performed using One-way analysis of variance (ANOVA). Multiple means were compared by Tukey′s test. *P*\<0.05 is considered as significant. This work was supported by a grant from Department of Science and Technology Liaoning Province (2017225028). Contributions ============= YB and CZ designed the study. YB and PY performed animal experiments. YB, JG, LN, ZW and ZZ performed protein detection. YB and WW performed mRNA detection. YB, PY and YS performed cell experiments. YB and WW analyzed data. YB, WW and CZ wrote the manuscript. UUO : unilateral ureteral obstruction ECM : extracellular matrix EMT : epithelial-mesenchymal transition RAS : renin-angiotensin-aldosterone system mTOR : mammalian target of rapamycin Timp-1 : metallopeptidase inhibitor 1 MDA : malondialdehyde T-SOD : total superoxide dismutase Yap : yes-associated protein mTORC1 : mTOR complex 1 {#F1} {#F2} {#F3} {#F4} {#F5} {#F6} {#F7} [^1]: ✉ Corresponding author: Chenghai Zhao (E-mail: chzhao\@cmu.edu.cn), Department of Pathophysiology, College of Basic Medical Science, China Medical University, Shenyang, China. Tel 86+24+31939318. [^2]: Competing Interests: The authors have declared that no competing interest exists.

Background {#Sec1} ========== Thrombocytopenia is a frequent hematologic disorder in patients infected with the human immunodeficiency virus (HIV) \[[@CR1]\]. It can occur independently of other cytopenias and at all stages of HIV infection \[[@CR2]\]. Although often asymptomatic, the thrombocytopenia in these patients may be associated with a serious complication including major bleeding and death \[[@CR3]\]**.** It is a frequent disorder occurring in about 30--40% of individuals with HIV infection \[[@CR4]\]. Thrombocytopenia may indicate the initial manifestation of HIV diseases and it may enhance the progress of the disease into AIDS or advanced immunological deterioration \[[@CR5]\]. The underlying mechanisms for the development of thrombocytopenia has not yet been well described \[[@CR6]\]. The suggested mechanisms that may account for the development thrombocytopenia includes; increased destruction of platelets due to the presence of anti-platelet antibodies, and direct infection of megakaryocytes by HIV leading to low production of platelets from those precursor cells \[[@CR6]\]. Multicenter AIDS cohort study was conducted in Asia, America and Africa \[[@CR7]\]. The result showed that the average frequencies of thrombocytopenia at initiation of antiretroviral therapy were 7% and varied by country \[[@CR7]\]. For instance, a study conducted in India showed that the prevalence of thrombocytopenia before initiation of Zidovudine was 16.6% which rises to 30% after initiation of Zidovidine. It was suggested mechanistically that immune mediated destruction of both platelets and megakaryocytes occurs in Zidovudine therapy \[[@CR8]\]. The prevalence of thrombocytopenia showed an increasing trend with decreasing CD4 count \[[@CR6], [@CR9]\] but, the prevalence of thrombocytopenia did not differ by sex, ethnicity or age \[[@CR9]\]. A study conducted in Uganda also reported that the prevalence of thrombocytopenia was 17.8% among HAART naive and was 13.0% for clients who were on ART for up to 6 months. The study found a significant association between thrombocytopenia and other cytopenias, CD4^+^ T cell counts, antiretroviral treatment(ART), and deteriorating HIV stage \[[@CR10]\]. A comparative cross sectional study carried out at Gondar University hospital, Ethiopia showed that the prevalence of thrombocytopenia was 9% in HAART naïve patients and 4.1% in patients on HAART \[[@CR11]\]. Although cytopenias have been widely reported in HIV infection, there is little data regarding prevalence and associated factors of thrombocytopenia among HIV infected patients before and after initiation of HAART in Ethiopia. This study will provide further information and it can serve as a reference material for further researches with regards to HIV related thrombocytopenia. The aim of this study was therefore to determine the prevalence of thrombocytopenia before and after initiation of antiretroviral therapy among HIV patients who attended at ART clinic of Black Lion Specialized Hospital, Addis Ababa, Ethiopia. Methods {#Sec2} ======= The methodological approach of this study is summarized based on previous study \[[@CR12]\]. Institution based cross sectional study design was conducted in Black Lion Specialized Hospital, Addis Ababa, Ethiopia from February to April 2017. During the data collection period, a total 2675 HIV infected adults were on ART, of which 176 HIV infected patients taking HAART for at least six months were selected randomly. Sample size was determined using a statistical formula for single population proportion (*n* = Z^2^ p(1-p) / d^2^), taking *p* = 12.7% (prevalence rate of thrombocytopenia from previous study) \[[@CR4]\], 5% level of precision (d) with 95% confidence interval. Pregnant women, patients transferred from other health institutions, diagnosed as having hematological diseases, severely sick due to other medical conditions and those who took other medication were excluded from the study. The structured questionnaire was adapted (see additional file [1](#MOESM1){ref-type="media"}) after the review of different literatures and the data was collected by trained ART nurses. Data concerning socio-demographic, clinical characteristics and pre ART information of the study participants were collected by interviewer administered questionnaire and review of medical records. Then, blood sample was collected and sent to the hematology laboratory. Based on the standard procedures, platelet counts and CD4+ T cell counts were determined using Sysmex XT 2000i hematology analyzer and BD FACS Count System respectively. To maintain good quality of the data; standard procedures were followed during all laboratory procedures and the quality of CD4 and hematology analyzer were checked by running quality control samples along the patients sample. Additionally, there was training of data collectors, pre testing of questionnaires and the data collection process were supervised in daily fashion. Thrombocytopenia was defined as platelet counts less than 150,000cells/μl. It was further classified into mild (100,000--150,000/mm^3^), moderate (50,000/mm^3^--100,000/mm^3^) and severe thrombocytopenia (platelet counts \< 50,000/mm^3^). The data were coded, checked and entered into SPSS version 20 for analysis. Descriptive statistics (mean and standard deviation) were used for continuous variables in the course of analysis. To assess the association between dependent variables and independent variables, logistic regression was done. A *p*- value of \< 0.05 was considered to be statistically significant. Result {#Sec3} ====== General characteristics of study participants {#Sec4} --------------------------------------------- A total of 176 HIV positive patients, of which 107(60.8%) women and 69(39.2%) men were involved in this study. The mean age of the patients were 40.08 ± 9.38 years, ranging from 20 to 62 years. The majority of study participants were within the WHO stage III category at the baseline. The most widely used HAART regimen in this study was 1e (TDF-3TC-EFV) (Table [1](#Tab1){ref-type="table"}).Table 1Socio-demographic and clinical characteristics of HIV positive patients taking HAART at Black Lion Specialized Hospital, Addis Ababa, Ethiopia, 2017VariablesFrequency (*n* = 176)Percentage (%)Age (in years) 20--292815.9 30--395833 40--496134.7 50--592614.8 60--6931.7Sex Male6939.2 Female10760.8Marital Status Single5028.4 Divorced2413.6 Married7542.6 Widowed2715.3Educational status illiterate2111.9 Primary school7442 High school6235.2 Certificate and above1910.8WHO clinical stages at the baseline Stage I3218.2 Stage II3922.2 Stage III5933.5 Stage IV4626.1Types of ART regimens 1c3017 1d4022.7 1e9453.4 1f126.8Note: 1c = AZT-3TC-NVP, 1d = AZT-3TC-EFV, 1e = TDF-3TC-EFV, 1f = TDF-3TC-NVP Platelets and CD4^+^ T cell counts of study participants {#Sec5} -------------------------------------------------------- The mean platelet count of the study participants were 218.44 ± 106.6 × 10^3^/μl at the baseline and 273.65 ± 83.8 × 10^3^/μl after HAART initiation (*p* \< 0.001). Similarly, the mean CD4+ T cell counts showed an increment from 162.35 ± 113.2 cells/μl at the baseline to 360.76 ± 196.2 cells/μl after HAART initiation (*p* \< 0.001). Prevalence of thrombocytopenia and associated factors before HAART initiation {#Sec6} ----------------------------------------------------------------------------- The prevalence of thrombocytopenia was 25% before HAART initiation. From the total thrombocytopenic subjects at the baseline, 54.6, 31.8 and 13.6% had mild, moderate and severe thrombocytopenia, respectively (Fig. [1](#Fig1){ref-type="fig"}). The overall prevalence of thrombocytopenia was 26.1% among males and 24.3% among females. However, the difference was not statistically significant. In this study, the majority of thrombocytopenia cases (31%) were observed in the aged subjects (≥ 50 years). HIV patients whose CD4 counts less than 200 cells /mm^3^ were 4.4 times more likely to have thrombocytopenia than HIV patients whose CD4 counts greater than or equals to 350cells/mm^3^ (Table [2](#Tab2){ref-type="table"}).Fig. 1Degree of thrombocytopenia among HIV positive adult patients at baseline and after six months of HAART initiation at Black Lion Specialized Hospital, Addis Ababa, Ethiopia, 2017Table 2Thrombocytopenia and its associated factors before HAART initiation in HIV positive patients attending Black Lion Specialized Hospital, Addis Ababa, Ethiopia, 2017VariablesThrombocytopeniaAdjusted OR(95%CI)*P* valueYes (%)No (%)Age(in years) 20--293(10.7%)25 (89.3%)0.3(0.07--1.24) 30--3914(24.1%)44(75.9%)0.7(0.26--1.97)0.45 40--4918(29.5%)43(70.5%)0.9(0.34--2.40)  ≥ 509(31%)20 (69%)1Sex Female26(24.3%)81(75.7%)0.9(0.47--1.99)0.93 Male18(26.1%)51(73.9%)1WHO clinical stage Stage III/IV27(25.7%)78(74.3%)1.1(0.54--2.34)0.76 Stage I/II17(23.9%)54(76.1%)1CD4 count (cells/mm^3^)  \< 20034(28.9%)88(72.1%)4.4(0.52--36.28)0.35 200--3499(22.5%)31(77.5%)3.3(0.37--29.84)  ≥ 3501(7.1%)13(92.9%)1 Prevalence of thrombocytopenia and associated factors after HAART initiation {#Sec7} ---------------------------------------------------------------------------- The prevalence of thrombocytopenia was 5.7% after HAART initiation, of which 90% had mild and 10% had moderate thrombocytopenia (fig. [1](#Fig1){ref-type="fig"}). The prevalence of thrombocytopenia after HAART initiation was significantly decreased by 19.3% (*P* \< 0.001). From thrombocytopenic patients after HAART initiation, about 10.1% were males and 2.8% were females. The prevalence of thrombocytopenia was higher (10.3%) among patients whose age group was ≥50 years. HIV patients on AZT based therapy were more likely to have thrombocytopenia than HIV patients on TDF based therapy. Increased percentage of thrombocytopenia were observed in HIV patients whose CD4 count was \< 200 cells/μl (*P* \< 0.05), but there was no significant association in the thrombocytopenia between patients who were categorized in to different CD4 count categories (*P* \> 0.05) (Table [3](#Tab3){ref-type="table"}).Table 3Thrombocytopenia and its associated factors after HAART initiation in HIV positive patients attending Black Lion Specialized Hospital, Addis Ababa, Ethiopia, 2017VariablesThrombocytopeniaAdjusted OR(95%CI)*P* valueYes (%)No (%)Age(in years) 20--292(7.1%)26 (92.6%)0.95(0.13--6.98) 30--391(1.7%)57(98.3%)0.18(0.02--1.94)0.53 40--494(6.6%)57(93.4%)0.66(0.13--3.41)  ≥ 503(10.3%)26 (89.7%)1Sex Male7(10.1%)62(89.8%)3.45(0.79--15.05)0.09 Female3(2.8%)104(97.2%)1Types of ART regimenTDF based6(4.8%)119(95.2%)0.86(0.22--3.38)0.83AZT based4(7.8%)47(92.2%)1CD4 count (cells/mm^3^)  \< 2005(12.2%)36(87.8%)3.39(0.74--15.63)0.19 200--3492(3.8%)51(96.2%)0.96(0.15--6.29)  ≥ 3503(3.6%)79(96.3%)1 Discussion {#Sec8} ========== It's well documented that hematological abnormalities are common in HIV infected patients \[[@CR2]\]. Thrombocytopenia, for instance, is a condition frequently seen in HIV infected individuals regardless of HIV status, gender, or age. Consequently, it's tempting to deduce that the presence of thrombocytopenia is associated with rapid disease progression, and by complicating the management of AIDS patients, thrombocytopenia has become a medical challenge \[[@CR13]\]. This study revealed that the prevalence of thrombocytopenia was 25% at baseline and 5.7% after six months of HAART initiation. A study conducted in Uganda reported that the prevalence of thrombocytopenia was 17.8% among antiretroviral HAART-naive and was 13.0% for clients who were on ART for up to 6 months \[[@CR10]\]. Another study conducted in Ethiopia reported that the prevalence of thrombocytopenia was 4.1% in patients on HAART and 9% in HAART naive patients \[[@CR11]\]. The difference in results seen from the present study might be due to the difference in the definition of thrombocytopenia, study design and size of the study population. The decrease in the prevalence of thrombocytopenia after HAART initiation might be due to; disorders of hematopoiesis, opportunistic infections and immune causes related to HIV leading to low platelets count could be reverted after HAART initiation \[[@CR14]\]. Additionally the presence of intervention protocol for HIV subjects will decrease the incidence of thrombocytopenia \[[@CR13]\]**.** The study found that older age (age \> 50 years) was a risk factor for thrombocytopenia, In Lai et al.'s study, for every 1-year increase in age, the prevalence of thrombocytopenia increased by 1.04 fold \[[@CR15]\]. The increase in the prevalence of thrombocytopenia with age might be due to a higher incidence of myelodysplasia in older patients \[[@CR15]\]. However, thrombocytopenia had not showed statistical significance difference with sex and age. This was in agreement with previous studies \[[@CR9], [@CR16]\]. According to the present study, the prevalence of thrombocytopenia was increased with decreasing CD4 count both before and after HARRT initiation. Thrombocytopenia was more prevalent among HIV positive patients who had a CD4 + T cell count of \< 200 cells/μl. This finding was consistent with several studies, which reported that thrombocytopenia was more prevalent among patients with CD4 count \< 200 cells/mm^3^ \[[@CR11], [@CR17], [@CR18]\]. However, the increase in prevalence of thrombocytopenia with decreased CD4 cell count was not statistically significant. This might be due to increase in the frequency of bone marrow abnormalities as the disease progresses \[[@CR19]\] and thrombocytopenia is greater in advanced HIV infection \[[@CR20]\]. The present study showed that patients on AZT based HAART regimen had a higher prevalence of thrombocytopenia compared to TDF based HAART regimen. Similar to the current finding study done by Suma et al. showed an increment in the prevalence of thrombocytopenia after initiation of Zidovudine \[[@CR8]\]. However, thrombocytopenia had not showed statistical significance difference with the type of HAART regimen. The high prevalence of thrombocytopenia might be due to immune mediated destruction of both platelets and megakaryocytes occurs in Zidovudine therapy \[[@CR8]\]. Conclusions {#Sec9} =========== In conclusion, this study has shown that the prevalence of thrombocytopenia after HAART initiation was decreased significantly. HIV patients with old age (age greater than 50 years), lower CD4 + T cell count and AZT based HAART regimen had an increased risk of developing thrombocytopenia. Based on our results, a number of study participants still had thrombocytopenia after initiation of HAART. Therefore continuous screening for thrombocytopenia among HIV infected patients should be performed to decrease the risk of morbidity and mortality. Additional file =============== {#Sec10} Additional file 1:Questionnaires The data within additional file [1](#MOESM1){ref-type="media"} contains questionnaires, which were used to collect information from the study participants for this study. The questionnaires had two parts; the first part is for collecting data about socio-demographic characteristics of the study subjects. The second part is for collecting data concerning clinical characteristics and immunohematological profiles of the study participants before and after HAART initiation. (DOCX 19 kb) 3TC : Lamivudin AIDS : Acquired immunodeficiency syndrome ART : Antiretroviral treatment AZT/ZDV : Azidothymidine/ Zidovudine CD4 : Cluster of differentiation 4 EVF : Efavirenz HAART : Highly active antiretroviral therapy HIV : Human immunodeficiency virus NVP : Nevirapine TDF : Tenofovir **Electronic supplementary material** The online version of this article (10.1186/s12878-018-0103-6) contains supplementary material, which is available to authorized users. The authors would like to extend their deepest appreciation to staff member of ART clinic of Black Lion Specialized Hospital for their cooperation, who providing the necessary information for this study. We also would like to express our thanks for all of the study participants for their cooperation. We are grateful to thank Addis Ababa University for sponsoring this research project. Funding {#FPar1} ======= This study was funded by Addis Ababa University with a reference number phy/402/2017. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Availability of data and materials {#FPar2} ================================== The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. GGW: Develop proposal and data collection sheet, collected data, analyzed it and wrote the draft of the manuscript. DHW: Conceived the study, supervised the data collection and reviewed the draft of the manuscript. Both authors read and approved the final draft of the manuscript. Ethics approval and consent to participate {#FPar3} ========================================== The study was conducted after ethical letters obtained from Research and Ethics Review Committee of the Department of Medical Physiology, Addis Ababa University, Ethiopia. The research proposal has been submitted to the department of Medical Physiology for review. The departmental research committee (DRC) had reviewed and looked into the originality, feasibility, laboratory setting and ethical aspects of the study. Following through discussion, the committee approved the research proposal developed by the authors with ethical approval reference number phy/219/2016. Then permission was taken from hospital higher management and data was collected after obtaining written informed consent from the study subjects. To keep confidentiality codes were used and unauthorized person didn't have access to the data. Competing interests {#FPar4} =================== The authors declare that they have no competing interests. Publisher's Note {#FPar5} ================ Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

All relevant data are within the paper and its Supporting Information files. Introduction {#sec001} ============ The major histocompatibility complex (MHC) is an essential component of the adaptive immune system for all vertebrates. One of the most remarkable characteristics of the *MHC* genes is the presence of extreme polymorphism within loci \[[@pone.0135922.ref001],[@pone.0135922.ref002]\]. However, among the genes within the porcine *MHC* class I region, the detailed characteristics and functions of the *MHC* class I chain-related sequences (*MIC*) are not well understood. The *MIC* gene was first described in primates and other mammals \[[@pone.0135922.ref003]\]. More than one functional *MIC* gene has been identified in several species; in addition, a number of pseudogenes have been reported \[[@pone.0135922.ref004],[@pone.0135922.ref005]\]. Seven *MIC* genes were identified in the human genome, including *MICA* and *B*, which produce functional transcripts, and *MICC*-*G*, which is nonfunctional \[[@pone.0135922.ref006]\]. Analysis of the cattle genome \[[@pone.0135922.ref007]\] has led to the identification of three complete *MIC* genes within the *MHC* class I region, temporarily referred to as *BoLA MIC1-3*. In pigs, two *MIC* genes, *SLA-MIC1* and *2*, have been reported \[[@pone.0135922.ref008]\]; *SLA-MIC2* is functional, whereas *SLA-MIC1* is a truncated pseudogene \[[@pone.0135922.ref005],[@pone.0135922.ref009]--[@pone.0135922.ref011]\]. As a member of the MHC class I system, MIC has a similar molecular structure to classical MHC class I molecules. The organization of MIC proteins consists of one transmembrane, and one cytoplasmic, and three external (α1--3) domains, which are encoded by six exons \[[@pone.0135922.ref003],[@pone.0135922.ref008],[@pone.0135922.ref012]\]. Distinguished from their classical MHC class I counterparts, the MIC protein binds neither β~2~-microglobulin (β~2~m) nor present class I peptides \[[@pone.0135922.ref013],[@pone.0135922.ref014]\]. In addition, *SLA-MIC2* expression is not affected by interferon, which is the main regulatory factor for classical *MHC* I and II \[[@pone.0135922.ref015]\]. On the other hand, the MIC protein acts as ligand of NKG2D, a transmembrane receptor, activating the cytolytic response, which is found in many cells within the immune system, including the natural killer cells γδ Τ and αβ CD8+ T \[[@pone.0135922.ref016],[@pone.0135922.ref017]\]. In humans, *MIC* is transcribed in several immune cells and most epithelial tissues. However, cell surface expressions of *MIC* were reported only from freshly isolated endothelial cells, fibroblasts \[[@pone.0135922.ref018]\], and gastric epithelium \[[@pone.0135922.ref013]\]. On the other hand, there were reports showing the up-regulation of transcripts and cell surface protein expression of MIC in many cell lines, including immune cells when stimulated with cellular stress inducers \[[@pone.0135922.ref014],[@pone.0135922.ref019]\]. Consistently, heat-shocked, viral-infected, and cellular-transformed upregulation of *MIC* has led to the impression that it is probably a 'marker of stress', especially in epithelial cells \[[@pone.0135922.ref013],[@pone.0135922.ref020],[@pone.0135922.ref021]\]. Several studies have demonstrated the possible associations between *MIC* genes and diseases \[[@pone.0135922.ref022]--[@pone.0135922.ref024]\]. For example, a strong association has been shown between specific *MICA* alleles and autoimmune disorders such as Behˎçet's disease \[[@pone.0135922.ref024],[@pone.0135922.ref025]\]. Other studies have also demonstrated an association between *MICA* alleles and human brucellosis resistance or susceptibility \[[@pone.0135922.ref022]--[@pone.0135922.ref024]\]. However, the linkage disequilibrium to classical *MHC I* or other genes in the *MHC* region may complicate disease association studies \[[@pone.0135922.ref026]\]. Therefore, high-resolution typing of candidate genes may be beneficial to the reduction of possible bias. In this study, we experimentally confirmed the molecular organization and expression pattern of *SLA-MIC* transcripts, characterized the polymorphism using a genomic DNA-based high resolution typing method, and performed a comparative analysis of *MIC* genes for seven mammalian species. Our results contribute to a more complete understanding of the molecular complexity and genetic variation of *SLA-MIC2* and provide novel tools for genotyping. Materials and Methods {#sec002} ===================== Animals and preparation of DNA {#sec003} ------------------------------ The Institutional Animal Care and Use Committee (IACUC) of Konkuk University approved the ear tissue and peripheral blood sampling methods. The IACUC approval number of this study is KU13101. Initially, 28 samples were selected on the basis of *SLA1* genotypes (22 different alleles, data not shown) as reference samples for the development of the *SLA-MIC2* typing method. To estimate *SLA-MIC2* diversity, we further typed 117 randomly selected pigs from seven different pig breeds and resulted in typing a total of 145 animals: 22 Seoul National University (SNU) miniature pigs, 25 Korean native pigs (KNPs), 13 National Institutes of Health (NIH) miniature pigs, 22 Duroc pigs, 20 Landrace pigs, 19 Yorkshire pigs, and 24 Berkshire pigs. Genomic DNA was extracted from 0.5 g of ear tissue obtained by ear punching, or 1 mL peripheral blood containing 6% ethylene diamine tetra acetic acid (EDTA), according to a previously described protocol \[[@pone.0135922.ref027]\]. Polymerase chain reaction (PCR) primer design {#sec004} --------------------------------------------- We aligned available genomic sequences of *SLA-MIC2* (accession numbers CT737281, AJ251914, and NM_001114274) using ClustalW software (<http://www.genome.jp/tools/clustalw/>), and analyzed the exon-intron organization. After we determined the correct exon-intron sequences, primers for the amplification of *SLA-MIC2* genomic DNA (gDNA) and complementary DNA (cDNA) were designed against a reference sequence (CT737281) using Primer Designer software (Version 2.0; Scientific and Educational Software, State Line, PA, USA). Primer sequences, annealing temperatures, and the size of the PCR products are summarized in [Table 1](#pone.0135922.t001){ref-type="table"}. 10.1371/journal.pone.0135922.t001 ###### Primer sequences and amplification conditions used for *MIC2* analysis. {#pone.0135922.t001g} Target regions Primer ID Primer sequences (5'-3') Annealing temperature (°C) Product size (bp) --------------------------------------------------- ---------------------------------------- ----------------------------------------- ---------------------------- ------------------- **gDNA-PCR** *MIC-2* (Exons 2 to 4) MIC2-gDNA-F1 TGTCCTCTGCTTGCCGATCTC 66 2512 MIC2-gDNA-R1 ATCCAGAACCACCTAGATCC **Sequencing primers** *MIC2*: Exon 2 MIC2-E2sF TTCTGGCCCCTTGTACACAT 55 MIC2-E2sR TCCATGCTCAGCTCACAGAC *MIC2*: Exon 3 MIC2-E3sF CCTTGACTCAGCAGCACAGG 55 MIC2-E3sR GGACTGACCAGAAGAGCAAG *MIC2*: Exon 4 MIC2-E4sF TGCATGAAGGCTCAGCCAG 55 MIC2-E4sR AGCCTGGCCTCTGGATCTC *MIC2*-cDNA **cDNA PCR 5' UTR to 3' UTR** MIC2-cDNA-F GAGCGAGTGTCCCATTTGGGA 48 1262 MIC2-cDNA-R GGCCAGAACAGGGAGTTGAATTC **cDNA PCR partial, exon 2 to 3' UTR** MIC2-cDNA-F1 GGTACAACTTCACGGTGATG 48 1080 MIC2-cDNA-R GGCCAGAACAGGGAGTTGAATTC **cDNA PCR partial, exon 3 to 3' UTR** MIC2-cDNA-sF[\*](#t001fn001){ref-type="table-fn"} GGAGAAGACGTGCGACATGG 48 670 Oligo dT(17) TTTTTTTTTTTTTTTTT **Full-length cDNA sequencing primers** MIC2-cDNA MIC2-cDNA-sF GGAGAAGACGTGCGACATGG 55 MIC2-cDNA-sR CTCTGTGAAGCTGGTCCAGG **GAPDH PCR primers** *GAPDH* GAPDH-F ACTCACGGCAAATTCAACGGC 48 294 GAPDH-R ATCACAAACATGGGGGCATCG \*MIC2-cDNA-sF primer was used for both PCR and cDNA sequencing. Amplification of *SLA-MIC2* and direct sequencing {#sec005} ------------------------------------------------- PCR reactions were performed in a 20 μL volume containing 50 ng DNA, 0.5 μM of each primer, 200 μM dNTPs, PCR buffer \[10 mM Tris (pH = 8.3), 50 mM KCl, and 1.5 mM MgCl~2~\], and 0.5 U LA-Taq polymerase (Takara Biotechnology Inc., Otsu, Shiga, Japan). PCR thermal cycling was performed using a T-3000 thermal cycler (Biometra, Goettingen, Germany), and consisted of an initial denaturation of 95°C for 5 minutes, followed by 35 cycles of 35-second denaturation at 94°C, 45-second annealing at 66°C, and 2-minute extension at 72°C; a final extension at 72°C for 10 min was then performed. PCR products were confirmed by electrophoretic separation on a 1.5% agarose gel in 1X Tris-acetate-EDTA (TAE) buffer for approximately 25 minutes at 100 V. The gel was stained with ethidium bromide and visualized under ultraviolet light. For the direct sequencing of PCR products, 5 μL of the product was incubated with 4 U exonuclease I (Fermentas, St. Leon-Rot, Germany) and 0.8 U shrimp alkaline phosphatase (USB Corporation, Cleveland, OH, USA) for 30 min at 37°C in 2.5× reaction buffer to degrade primers and dephosphorylate dNTPs that were not consumed in the amplification reaction. The purification reaction was stopped by a 15-minute incubation at 80°C. Sequencing reactions were performed using the ABI PRISM BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) using specific forward and reverse primers for each exon according to the manufacturer's instructions. The products were analyzed using an automated DNA analyzer (Applied Biosystems, Foster City, CA, USA). All sequences were checked for ambiguous bases and manually edited using BioEdit V7.0 software \[[@pone.0135922.ref028]\]. Confirmation of new *SLA-MIC2* alleles {#sec006} -------------------------------------- New *SLA-MIC2* alleles or any alleles that appeared for the first time in our analysis were confirmed by cloning PCR products and bidirectional sequencing. PCR products were gel purified using a QIAquick gel extraction kit (QIAGEN, Venlo, the Netherlands) and ligated into the pGEM-T Easy Vector System (Promega Corporation, Madison, WI, USA). Ligation products were transformed into *Escherichia coli* DH-5α competent cells. These cells were grown overnight on agar containing 50 μg/mL ampicillin, 40 mg/mL X-gal (Norgen Biotek Corp. Thorold, ON, Canada), and 100 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Thermo Scientific, Waltham, MA, USA) at 37°C. Five white colonies were picked from each ligation to amplify inserts using T7 and SP6 universal primers. Sequencing reactions were performed using exon-specific primers for each exon, as indicated in [Table 1](#pone.0135922.t001){ref-type="table"}. After confirming the accuracy of the sequencing results by manual inspection of the chromatograms, a sequence similarity search (BLAST) against the NCBI (National Center for Biotechnology Information) database, as well as local *SLA-MIC2* databases, was performed. The complete sequences of *SLA-MIC2* exons 2, 3, and 4 were aligned using CLUSTALW \[[@pone.0135922.ref029]\], as implemented in MEGA 6.0 \[[@pone.0135922.ref030]\]. RNA isolation, reverse transcription PCR (RT-PCR), and *SLA-MIC2* cDNA typing {#sec007} ----------------------------------------------------------------------------- Total RNA was isolated from fifteen different tissues (liver, stomach, lung, small intestine, heart, skin, tongue, spleen, muscle, large intestine, testis, kidney, ovary, neocortex, and olfactory epithelium) of a nine-week-old female pig using the R&A-BLUE total RNA extraction kit (iNtRON Biotechnology, Seoul, Korea), following the manufacturer's protocol. Reverse transcription was carried out in a 25 μL reaction using oligo-(dT)~17~ and Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) for 50 min at 50°C, and incubated for 15 min at 72°C to stop the reaction. RT-PCR was performed using a T-3000 thermal cycler. The PCR mixture consisted of 50 ng cDNA, 0.5 U Super-Therm DNA polymerase (JMR Holdings, Kent, UK), 0.5 μM primers ([Table 1](#pone.0135922.t001){ref-type="table"}), 1X PCR reaction buffer (1.5 mM MgCl~2~), and 0.1 mM dNTPs. The amplifications were performed in a 20 μL reaction volume. The thermal cycling profile included a 5-minute denaturation step at 95°C, followed by 35 cycles of denaturation for 1 minute at 95°C, annealing for 1 minute at 48°C, and extension for 1 minute at 72°C, followed by a final extension of 5 minutes at 72°C. Direct sequencing was performed on the products using cDNA sequencing primers ([Table 1](#pone.0135922.t001){ref-type="table"}). Analysis of *SLA-MIC2* expression by semi-quantitative RT-PCR {#sec008} ------------------------------------------------------------- To evaluate the levels of *SLA-MIC2* mRNA in tissues, primers for amplifying full-length (5′ to 3′ UTRs) and partial sequences (exon 2 to 3′ UTR) of *SLA-MIC2* cDNA were designed, and semi-quantitative RT-PCR was performed. PCR mixtures and RT-PCR conditions were identical to the experimental conditions described above, except that the number of cycles was decreased to 32. Glyceraldehyde-3-phosphate dehydrogenase (*GAPDH*) was used as a control for experimental variation. The photodensity ratio was calculated by comparing the density of the *SLA-MIC2* amplicon relative to that of *GAPDH* using Image Studio Analysis Software Version 4.0 (LI-COR Biosciences, USA). Statistical and phylogenetic analysis {#sec009} ------------------------------------- Population statistics, including allele frequencies, number and effective number of alleles, observed and expected heterozygosity, and the Hardy-Weinberg equilibrium, were estimated by using POPGENE 1.32 \[[@pone.0135922.ref031]\]. Phylogenetic analysis of *SLA-MIC2* alleles was performed using the neighbor-joining method \[[@pone.0135922.ref032]\] with bootstrap analysis of 1000 replicates, and evolutionary distances among alleles were calculated using the Kimura 2-parameter model \[[@pone.0135922.ref033]\] using MEGA 6 software \[[@pone.0135922.ref030]\]. Results and Discussion {#sec010} ====================== Characterization of *SLA-MIC2* gene structure {#sec011} --------------------------------------------- We designed sets of PCR primers against the *SLA-MIC2* sequence contained within a bacterial artificial chromosome (BAC) sequence, AJ251914, and attempted to amplify the region between introns 1 and 4, or the full-length *SLA-MIC2* cDNA, according to the exon-intron information of the pig *SLA-MIC2* gene from a previous report \[[@pone.0135922.ref005]\]. Following the results of *MICA* and *MICB* expression from humans \[[@pone.0135922.ref034]\], we selected the lung and small intestine as initial RNA sources \[[@pone.0135922.ref013],[@pone.0135922.ref021],[@pone.0135922.ref034]\]. However, we were unable to obtain amplicons from either genomic DNA PCR or RT-PCR. Through additional database searches, we identified another BAC sequence (CT737281) that contained the *SLA-MIC2* gene, but with a sequence discrepancy from the end of exon 4 to exon 6 of *SLA-MIC2*, as compared to AJ251914. We also realized that the exon-intron organization from AJ251914 was different from the current *in silico* annotation (NM_001114274) of the *SLA-MIC2* gene according to the Ensemble genome browser (<http://asia.ensembl.org/index.html>). To resolve the discrepancy, we performed RT-PCR with *SLA-MIC2*-specific forward primers and a poly A specific oligo (dT)~17~ reverse primer. Among the several primer combinations used, a *SLA-MIC2* exon 3-specific forward primer (MIC2-cDNA-sF) produced a 670-bp cDNA product. From this result, we were able to identify the differences in both the nucleotide sequence and the position of exon-intron boundaries for *SLA-MIC2* exons 5 and 6 between the reported information and our findings, resulting in the precise characterization of the exon-intron structures of full-length *SLA-MIC2* cDNA ([Fig 1](#pone.0135922.g001){ref-type="fig"} and [S1 Fig](#pone.0135922.s001){ref-type="supplementary-material"}). The porcine *MIC2* gene encodes a polypeptide of 374 amino acids consisting of a leader sequence (exon 1), three extracellular domains α1--3 (exons 2, 3 and 4), a transmembrane domain (exon 5), and a cytoplasmic domain (exon 6) ([Fig 2](#pone.0135922.g002){ref-type="fig"} and [S1 Fig](#pone.0135922.s001){ref-type="supplementary-material"}), which were defined by comparative analysis with human *MICA*. {#pone.0135922.g001} {#pone.0135922.g002} Analysis of *SLA-MIC2* expression in 15 different pig tissues {#sec012} ------------------------------------------------------------- We examined the expression of *SLA-MIC2* in 15 different pig tissues using semi-quantitative RT-PCR. The comparison of band intensity and semi-quantitative measures of *SLA-MIC2* expression (photodensity ratio) showed that *SLA-MIC2* was expressed in only the small intestine, lung, and heart, with the most abundant expression in the lung ([Fig 3](#pone.0135922.g003){ref-type="fig"}). These findings suggested that the expression patterns of *MIC* genes among different species are not identical, and that the expression of *MIC* genes could vary even within a species. The difference in the expression pattern between pigs and humans suggests the presence of possible differences between the *MIC*-related immune systems of the two species. Further studies are necessary to understand the consequences of these differences. {#pone.0135922.g003} Development of a genomic DNA-based high-resolution typing method for *SLA-MIC2* {#sec013} ------------------------------------------------------------------------------- The availability of an efficient typing method for highly polymorphic loci is fundamental to understanding the underlying biology associated with their genetic polymorphisms, especially for MHC or related molecules. In contrast to a considerable amount of data from human *MIC* gene \[[@pone.0135922.ref003],[@pone.0135922.ref013],[@pone.0135922.ref022]--[@pone.0135922.ref024],[@pone.0135922.ref035],[@pone.0135922.ref036]\] studies, detailed information on the genetic diversity of *SLA-MIC2* has not been available. Therefore, we carried out the development of a high-resolution typing method of the *SLA-MIC2* gene covering exons 2, 3, and 4, which is the region of functional importance for *MICA* and *B*, required for the typing of *MICs* in humans. After we confirmed the gene structure of *SLA-MIC2* ([S1 Fig](#pone.0135922.s001){ref-type="supplementary-material"}), systematic trials of different primer sets to amplify all of the *SLA-MIC2* alleles regardless of the existing sequence variations were performed. PCR amplification, cloning of PCR products, and subsequent sequence analyses were performed using genomic DNA from 28 animals that were selected based on the diversity of *SLA-1* corresponding to 22 different *SLA-1* alleles (data not shown). The possible linkage between *SLA1* and *SLA-MIC2*, which are \~0.7 Mb apart \[[@pone.0135922.ref011]\], should allow maximum diversity of the *SLA-MIC2* gene by choosing animals with different *SLA-1* alleles. Among the pairs of primers tested, MIC2-gDNA-F1 and MIC2-gDNA-R1 ([Table 1](#pone.0135922.t001){ref-type="table"}) showed successful amplification of *SLA-MIC2* for all samples ([S2 Fig](#pone.0135922.s002){ref-type="supplementary-material"}). The general strategy of our *SLA-MIC2* high-resolution typing method is described in [Fig 1](#pone.0135922.g001){ref-type="fig"}. PCR amplification using the gDNA PCR primer set resulted in a 2512-bp PCR fragment, covering partial intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, and partial intron 5. For sequence-based discrimination of *SLA-MIC2* alleles, several sequencing primers were designed for each of exons 2--4. Among them, MIC2-E2sF and MIC2-E2sR for exon 2, MIC2-E3sF and MIC2-E3sR for exon 3, and MIC2-E4sF and MIC2-E4sR for exon 4 showed consistent results when used for direct sequencing ([Table 1](#pone.0135922.t001){ref-type="table"}), leading to the establishment of the sequence analysis method. To assemble the sequence information of each exon into a single sequence contig, the eight possible combinations of assembled typing results were compared to the available sequence information of existing alleles in the public database, or to previously confirmed alleles in our local *SLA-MIC2* database. Complete matches to the known alleles in public or local databases were determined to be valid alleles. In cases of heterozygotes, alleles were separated according to the complete sequence identity to the existing alleles in our *SLA-MIC2* database. However, due to the possible appearance of unknown alleles, we originally carried out the cloning, sequencing, and subsequent allelic characterization in all genotyped samples to increase the amount of information in our *SLA-MIC2* allele database. Since the probability of a hybrid exon occurring between different alleles in the population by genetic recombination should be extremely low, we believed that the best-fit prediction approach for allele interpretation should result in accurate typing results. Because we were able to amplify the target region in all samples using the initial set of primers, we did not analyze the sequence variations of intronic regions of different *SLA-MIC2* alleles, which was necessary for the development of typing methods for *MHC* class II genes in pigs \[[@pone.0135922.ref037]\]. Verification of *SLA-MIC2* typing accuracy {#sec014} ------------------------------------------ The reliability of our *SLA-MIC2* genomic sequence-based typing (GSBT) method was evaluated by cloning PCR products and subsequent sequencing, followed by comparison to the typing results for cDNA and artificial heterozygote samples. First, the typing results for all of the newly identified *SLA-MIC2* alleles, or genotyping results that appeared for the first time in our analysis, were subjected to cloning-based analysis with at least five clones in both the forward and reverse direction. Samples with typing results that contained even a single base mismatch to known alleles were subjected to cloning-based analysis to eliminate any possible typing errors. This process strictly followed the requirements of the SLA Nomenclature Committee of the International Society for Animal Genetics (ISAG) for acceptance of new alleles. At least two PCR reactions were performed for one sample: one for direct sequencing and the other for cloning; the result was a collation of the two reactions. The *SLA-MIC2* GSBT and cloning-based typing resulted in identical outcomes for all comparisons (n = 15 alleles). Second, we performed cDNA typing using the primer set MIC2-cDNA-F and MIC2-cDNA-R for samples with available RNA, and compared the results to those obtained using the *SLA-MIC2* GSBT method. The results were identical for all samples (n = 10). The alleles verified by cDNA typing are shown in [Table 2](#pone.0135922.t002){ref-type="table"}. Third, allelic dropout or preferential amplification is one of the most common genotyping errors, leading to the preferential amplification of a single allele from heterozygotes. The occurrence of allelic dropout was examined by mixing equal concentrations of DNA (50 ng/μL) from five different *SLA-MIC2* homozygote samples containing the alleles *MIC2*\*01, \*05, \*04, \*07, and \*kn15. This mixture was used to prepare six different simulated heterozygotes, including *SLA-MIC2*\*01/05, 04/05, 05/07, 01/kn15, 04/kn15, 05/kn15, which were not available from the animals in the study. We found that the genotyping results from the artificial heterozygotes were identical to the predicted genotypes for all samples (data not shown). Combined, these results suggest that the amplification of each allele from at least the confirmed combinations in this study did not appear to be affected by allelic biases in PCR amplification, indicating that our genotyping primers are located in intronic regions of high sequence conservation across breeds of pigs. 10.1371/journal.pone.0135922.t002 ###### Comparison of the allele frequency of porcine *MIC2* among seven pig breeds using high-resolution genomic sequence-based typing. {#pone.0135922.t002g} Allele BER KNP NIH SNU YOR DUR LAN All (n = 145) -------------------------------------------------- ------- ------- ------- ------- ------- ------- ------- --------------- MIC2\*01[^a^](#t002fn002){ref-type="table-fn"} 0.208 0.308 0.109 0.167 0.046 0.075 0.117 MIC2\*03[^a^](#t002fn002){ref-type="table-fn"} 0.021 0.120 0.024 MIC2\*04[^a^](#t002fn002){ref-type="table-fn"} 0.280 0.022 0.417 0.103 MIC2\*05[^a^](#t002fn002){ref-type="table-fn"} 0.042 0.020 0.692 0.869 0.056 0.546 0.025 0.300 MIC2\*07[^a^](#t002fn002){ref-type="table-fn"} 0.479 0.139 0.227 0.131 MIC2\*kn08 0.100 0.023 0.021 MIC2\*kn09 0.056 0.050 0.014 MIC2\*kn10 0.125 0.280 0.083 0.100 0.097 MIC2\*kn11 0.040 0.007 MIC2\*kn12 0.021 0.003 MIC2\*kn13 0.023 0.275 0.041 MIC2\*kn14 0.050 0.007 MIC2\*kn15[^a^](#t002fn002){ref-type="table-fn"} 0.021 0.114 0.350 0.069 MIC2\*kn16[^a^](#t002fn002){ref-type="table-fn"} 0.083 0.083 0.050 0.031 MIC2\*kn17 0.160 0.046 0.025 0.038 Note: SNU, Seoul National University miniature pigs; KNP, Korean native pig; NIH, National institute of Health miniature pig; DUR, Duroc; LAN, Landrace; YOR, Yorkshire; BER, Berkshire. "^a^" Alleles were verified by cDNA typing. *SLA-MIC2*\**01*, \**03*, \**04*, \**05* and \**07* are existing alleles in the GenBank database. Ten new alleles were submitted to GenBank under the accession numbers KM514686, KM514687, KM514688, KM514689, KM514690, KM514691, KM514692, KM514693, KM514694 and KM514695, which were provisionally named as *SLA-MIC2*\**kn08*, \**kn09*, \**kn10*, \**kn11*, \**kn12*, \**kn13*, \**kn14*, \**kn15*, \**kn16* and \**kn17*, respectively. The assignment of tentative names for new *SLA-MIC2* alleles followed SLA Nomenclature Committee guidelines. Combining genomic PCR and subsequent direct sequencing of PCR products can serve as a reliable technique for the detection of genetic polymorphisms in population studies, due to the presence of a larger number of correct template copies, compared to a smaller number of mutated templates generated during PCR and cloning processes, ultimately leading to more accurate results. This approach was proven successful for the typing of *MHC* class II loci \[[@pone.0135922.ref038]\]. Considering the high fidelity of typing results from genomic PCR and subsequent direct sequencing analyses, we propose that it may not be necessary to perform bidirectional sequencing when the typing results perfectly match previously confirmed alleles. Structural comparison of *MIC* genes among pigs, humans, and cattle {#sec015} ------------------------------------------------------------------- *MICA* in humans encodes a full-length polypeptide of 383 amino acid residues, with the relative molecular mass of 43 kDa \[[@pone.0135922.ref003]\]; *BoLA MIC1* and *MIC2* consist of 384 amino acids \[[@pone.0135922.ref007]\]. In contrast, pig *SLA-MIC2* encodes a 374-amino acid polypeptide ([Fig 2](#pone.0135922.g002){ref-type="fig"}). When we compared the exon-intron organization of the *SLA-MIC2* gene to that of the reported functional *MIC* genes of cattle and humans, the organization was almost identical except for slight differences in the length of certain exons ([Fig 2](#pone.0135922.g002){ref-type="fig"}). The similarities between the amino acid sequence of *SLA-MIC2* and the orthologs in cattle and humans are shown in [Fig 4](#pone.0135922.g004){ref-type="fig"}. Depending on the domain, *SLA-MIC2* shared approximately 11--70% amino acid sequence similarity with these orthologs. When we compared *MIC* sequences between humans and cattle, *SLA-MIC2* extracellular domains α1 and α2 showed 2 and 6 amino acid inserts or deletions (indel), respectively, while no length variation was observed for the α3 domain ([Fig 2](#pone.0135922.g002){ref-type="fig"}). The transmembrane and cytoplasmic domains displayed less sequence similarity than extracellular domains with 14 and 8 indel, respectively. For the leader peptide region, the sequence similarity was weak among the different species. The high sequence similarity of the extracellular domains α1--3 suggests that structural conservation of the extracellular domains is important for protein function. The amino acid sequence identity between pigs and cattle was higher than that between pigs and humans. {#pone.0135922.g004} Glycosylation is important for protein stability and biological function \[[@pone.0135922.ref039]--[@pone.0135922.ref041]\]. The effects of glycosylation often depend on the position and number of N-linked oligosaccharides added to a protein chain. We identified seven putative N-linked glycosylation sites (3 sites in α1 and 4 sites in α3) from *SLA-MIC2*, indicating that all the predicted glycosylated residues reside in the extracellular domains ([Fig 2](#pone.0135922.g002){ref-type="fig"}). It has been reported that cattle *MICs* have six potential sites for N-linked glycosylation \[[@pone.0135922.ref007]\], while human *MICA* has eight \[[@pone.0135922.ref003]\]. Comparing the patterns of amino acid conservation of *MIC* genes in pigs, cattle, and humans, we found three conserved N-linked glycosylation sites (Asn-8, -255, and -283) among the species. Cysteine is one of the least abundant amino acids in an organism. It is often present in functionally important protein sites. We have detected 10 cysteine residues concentrated in the extracellular domains of *SLA-MIC2*, one in α1 (Cys-76), five in α2 (Cys-99, -123, -130, -158, and -176) and four in α3 (Cys-207, -219, -276, and -291; [Fig 2](#pone.0135922.g002){ref-type="fig"}). Human *MICA* contains seven cysteine residues that are also located in the extracellular domains \[[@pone.0135922.ref003]\]. Pig, cattle, and human genes were shown to share conserved cysteine in the α2 (Cys-99) and α3 (Cys-276) domains ([Fig 2](#pone.0135922.g002){ref-type="fig"}). In addition, pigs and cattle were found to share three conserved cysteines in the α2 domain. The differences in positions and the number of cysteines in *MIC* proteins from different species may result in changes in the protein structure and stability \[[@pone.0135922.ref042]\]. Comparisons of the *MIC* protein structures, which are currently unavailable, are necessary to verify the influence of these variations on the structure and function of *MIC*s in different species. Genetic diversity of *SLA-MIC2* {#sec016} ------------------------------- Seven different sequences corresponding to pig *SLA-MIC2* exons 2--4 are currently identifiable using the NCBI database. In this study, we identified 15 different *SLA-MIC2* alleles ([Table 2](#pone.0135922.t002){ref-type="table"}) from typing 145 animals from seven pig breeds which are consisted of 22 non-randomly selected reference individuals (19.3%) of high *SLA-1* allele diversity and 117 randomly selected individuals from seven pig breeds. Therefore, the allele frequencies of *SLA-MIC2* in [Table 2](#pone.0135922.t002){ref-type="table"} were not estimated entirely from randomly chosen animals for each breed. However, they still should not significantly deviate from the patterns of allele distribution specific to each breed. At the amino acid level, nine *SLA-MIC2* alleles can be discriminated. There are considerably fewer *SLA-MIC2* alleles compared to the number of alleles from *SLA* class I (116 alleles) and class II (167 alleles). This is similar to what is observed in humans, where there are 100 *MICA* alleles in contrast to 3,105 human leukocyte antigen (*HLA*) class II alleles or 9,308 *HLA* class I alleles (Immuno Polymorphism Database \[IPD\], <https://www.ebi.ac.uk/ipd/>). Among the alleles, five were previously reported with tentative names, including *MIC2*\**01*, *MIC2*\**05*, *MIC2*\**03*, *MIC*\**04*, and *MIC2*\**07*; ten were new alleles. The common alleles, *MIC2*\**05*, *MIC2*\**07*, and *MIC2*\**01*, accounted for approximately 30.3%, 13.1%, and 12% of the *SLA-MIC2* gene pool, respectively. A low frequency was observed for the remainder of the alleles, and the frequency varied among different breeds. The frequency of *MIC2*\**05* was the highest in the NIH and SNU miniature pigs, as well as the Duroc breed. Besides, the three alleles including *MIC2-*\**kn11*, *MIC2-*\**kn12*, and *MIC2-*\**kn14* were unique to KNPs, Berkshire and Landrace breeds, respectively. Of the new alleles, *MIC2*\**kn12* and *MIC2*\**kn11* were observed from only one and two heterozygote individuals, and *MIC2*\**kn09* and *MIC2*\**kn16* occurred in several heterozygote animals. The remaining new alleles occurred in at least one homozygote and several heterozygote individuals. We compared 15 *SLA-MIC2* alleles that were identified in this study with the *SLA-MIC2* sequence from BAC CT737281 ([S3 Fig](#pone.0135922.s003){ref-type="supplementary-material"}). All of the allelic variations that were detected were nucleotide substitutions. We examined the patterns of amino acid changes for each nucleotide substitution in *SLA-MIC2* sequences. Seven non-synonymous and five synonymous coding mutations were detected ([S3 Fig](#pone.0135922.s003){ref-type="supplementary-material"}). Three polymorphic positions were identified in *SLA-MIC2* exon 2, resulting in three distinguishable *SLA-MIC2* exon 2 sequences. For exon 3, five polymorphic positions were observed, resulting in eight distinguishable *SLA-MIC2* exon 3 sequences. For exon 4, four polymorphic sites defined seven different sequences in exon 4. Heterozygosity may contribute to increased resistance to infectious diseases. In particular, high heterozygosity at the *MHC* locus has been shown to be beneficial to species by conferring a selective advantage through enhancing resistance to infectious diseases \[[@pone.0135922.ref043]\]. The average level of observed heterozygosity from 145 typed animals in seven different breeds was 52.1%, ranging from 26% for inbred SNU miniature pigs, to 72.8% for outbred Duroc animals. The average level of expected heterozygosity for the same data set was 63.4%, ranging from 23.6% in SNU miniature pigs to 79.7% in Landrace pigs ([Table 3](#pone.0135922.t003){ref-type="table"}). The levels of *MIC2* heterozygosity among different breeds were diverse and even higher than those of the *SLA* class II genes that were reported in previous studies (0.1--0.69 in *SLA-DQB1* and 0.28--0.77 in *SLA-DRB1*) \[[@pone.0135922.ref037],[@pone.0135922.ref038]\]. A potential explanation for this finding is that some animals (19.3%) employed for S*LA-MIC2* typing in this study had non-random distribution of *SLA1* alleles according to our typing strategy. Inbred pigs, including NIH and SNU miniature breeds, showed lower heterozygosity than outbred breeds, as expected. There was no excess or deficiency of heterozygotes in six breeds, which is consistent with previous reports on *MHC* genes \[[@pone.0135922.ref037],[@pone.0135922.ref044]\]. Only the Landrace breed (P \< 0.001) deviated from Hardy-Weinberg equilibrium in our study, which may be attributed to the limited sample size or use of animals with selected haplotypes according to *SLA-1*. 10.1371/journal.pone.0135922.t003 ###### Differences in porcine *MIC2* heterozygosity among seven breeds of pigs. {#pone.0135922.t003g} Breed N Number of alleles ne Het-O Het-E HWE (P values) ------- ----- ------------------- ------- --------------------------------------------- --------------------------------------------- ---------------------------------------------- BER 24 8 3.348 0.625 0.716 0.52 KNP 25 7 4.562 0.56 0.796 0.06 NIH 13 2 1.742 0.307 0.443 0.244 SNU 22 3 1.301 0.26 0.236 0.935 YOR 19 7 4.153 0.666 0.781 0.552 DUR 22 7 2.916 0.728 0.672 0.288 LAN 20 9 4.494 0.5 0.797 0.001[\*\*](#t003fn003){ref-type="table-fn"} Total 145 15 6.646 0.521[^a^](#t003fn004){ref-type="table-fn"} 0.634[^b^](#t003fn005){ref-type="table-fn"} Note: Het-O: observed heterozygosity; Het-E: Nei's expected heterozygosity; ne: effective number of alleles; HWE shows P-value for heterozygous protein S deficiency from the Hardy--Weinberg equilibrium likelihood ratio test \*\*P \< 0.00 ^a^average observed heterozygosity ^b^average expected heterozygosity Phylogenetic analysis {#sec017} --------------------- A phylogenetic tree incorporating all of the identified pig *SLA-MIC2* sequences corresponding to exons 2, 3, and 4, as well as those from humans (*MICA* and *B*), chimpanzees (*Patr-MICA/B*), rhesus macaques (*Mamu-MIC1* and *2*), cattle (*BoLA-MIC1-3*), mice and rats (*Mr1*), are shown in [Fig 5](#pone.0135922.g005){ref-type="fig"}. The tree depicts a low phylogenetic resolution for all sequences within a locus, particularly in the case of *SLA-MIC2*. This may suggest that the current genetic variations in *MIC* genes, including *SLA-MIC2*, have recently emerged, or that the genes have been under selective pressure to limit sequence changes. The result of phylogenetic analysis of *MIC* genes from the mammals selected for this study was consistent with reported evolutionary relationships. For example, rodent species (mice and rats) are distantly related to primates (humans, chimpanzees, and rhesus macaques) and ungulates (pigs and bovines). Interestingly, in primate branch, human *MICA* alleles are closer to *MICA/B* from chimpanzee than human *MICB*. This possibly indicates a vertical evolution of *MIC* genes in primate species. *SLA-MIC2* alleles are clustered tightly with *BoLA-MIC* genes, indicative of the close evolutionary relationship between them. {#pone.0135922.g005} Applications {#sec018} ------------ Investigations into the diversity and evolution of *MHC* related genes is essential to understanding relationships between genetic differences, as well as the resistance and susceptibility to infectious or immune-related diseases. Bacterial infection is considered the most important cause of neonatal and post-weaning diarrhea in pigs. For example, *E*. *coli*-related diseases were ranked as the largest threat to economic loss in suckling pigs, and third largest threat to economic loss in weaned pigs \[[@pone.0135922.ref045]\]. In the swine industry, loss of productivity, as well as the morbidity and mortality from *E*. *coli*, costs producers enormous economic losses annually \[[@pone.0135922.ref046]\]. There is evidence that pathogenic strains of *E*. *coli* trigger a rapid *MICA* expression at the surface of the intestinal epithelium \[[@pone.0135922.ref047]\]. Understanding *SLA-MIC2* polymorphisms in pig populations and their expression may be important from the viewpoint of porcine immunogenetics. Moreover, understanding *SLA-MIC2* polymorphisms could benefit biomedical research using porcine models, considering *MIC* molecules are involved in the rejection of grafted tissues in humans \[[@pone.0135922.ref048],[@pone.0135922.ref049]\]. The importance of the *MIC* genes has been highlighted by their implication in different human diseases \[[@pone.0135922.ref022],[@pone.0135922.ref023]\]. Furthermore, the genetic improvement of livestock resistance to pathogens holds promise in the animal breeding arena; e.g., by the selection of pigs with an increased resistance to infectious diseases. Therefore, the typing method that we developed to analyze the *SLA-MIC2* polymorphisms, and the information that we obtained about the genetic diversity of the *SLA-MIC2* gene in pigs, could be used as novel tools, and could serve to promote other advancements in understanding porcine immune responses. Our results can also be used to establish *MIC* polymorphism for the *SLA* database in the IPD (<http://www.ebi.ac.uk/ipd/mhc>), which is currently only available for *HLA*. Supporting Information {#sec019} ====================== ###### The nucleotide and amino acid sequences of the *SLA-MIC2* coding region and corresponding exons. The boundaries for each exon are indicated by vertical lines. (TIF) ###### Click here for additional data file. ###### PCR amplification results of fifteen different alleles using a set of porcine *SLA-MIC2*-specific primers. A 2512-bp segment of the genomic *SLA-MIC2* locus was amplified consistently from all alleles. The number on the top of the lane corresponds to the respective allele: 1. *MIC2*\*01, 2. *MIC2*\*03, 3. *MIC*\*kn08, 4. *MIC2*\*04, 5. *MIC2*\*kn09, 6. *MIC2*\*kn10, 7. *MIC2*\*kn11, 8. *MIC2*\*kn12, 9. *MIC2*\*kn13, 10. *MIC2*\*05, 11. *MIC2*\*07, 12. *MIC2*\*kn14, 13. *MIC2*\*kn15, 14. *MIC2*\*kn16, 15. *MIC2*\*kn17, and N, negative control. The plus signs above the bands indicate the detected heterozygous PCR products. (TIF) ###### Click here for additional data file. ###### Analysis of nucleotide polymorphisms of *SLA-MIC2* exons 2, 3, and 4 for fifteen detected alleles. Allele names are indicated on the left. Identical nucleotides are shown as a dot. The sequences were compared to a *MIC2* corresponding region (exon2: 169421--169675, exon3: 169957--170241 and exon4: 170818--171095) of a BAC sequence (accession number CT737281) from NCBI as a reference sequence. All non-synonymous mutations are outlined in grey. (DOCX) ###### Click here for additional data file. This work was supported by grants from the Next-Generation BioGreen 21 Program (No. PJ011130) and the "Cooperative Research Program for Agriculture Science & Technology Development (PJ009103)" of the Rural Development Administration, Republic of Korea. Soundrarajan was supported by the 2013 KU Brain Pool of Konkuk University. [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: CP. Performed the experiments: H. Dadi ML H. Dinka H. Cho MC NS. Analyzed the data: H. Dadi DN. Contributed reagents/materials/analysis tools: H. Choi. Wrote the paper: H. Dadi CP JK JP. Revised the manuscript: CP ML.

Introduction {#s1} ============ Haemagioma arising in the knee is a rare cause of knee swelling. The diagnosis frequently is delayed for long. We are presenting the case report of a 12- year boy who had a swelling on the anteromedial aspect of the left knee which remained undiagnosed for more than a year. Patient had all baseline blood investigations and plain radiograph of knee which were normal. He Aspiration of the knee had yielded only blood. The aim of presenting this case report is to create awareness about the possibility of a haemangioma arising from a joint which although rare should be considered as a differential diagnosis. Case Report {#s2} =========== Haemangiomas arising in a joint are rare. Amongst all the joints involved, the knee is the most common followed much less commonly by elbow, wrist and ankle^1^. Haemangioma of the knee can present with a visible swelling, associated with pain, and can be a cause of spontaneous haemarthrosis in children and young adults. Two different forms have been observed synovial haemangioma and arteriovenous malformation (Hemangio-hamartoma)^2^. Both these types can cause a chronic haemorrhagic synovitis with ultimate joint degeneration as they tend to remain undiagnosed for long periods. We report a case of a 12- year old boy who presented with a localized swelling over the antero-medial aspect of left knee for a year. The patient had no history of trauma or complaints in any other joint. There were no constitutional symptoms. Pain was moderate and bearable. Predominantly it was the swelling for which patient came to the hospital. On examination patient had a well-defined swelling on the superior antero-medial -aspect of right knee, measuring 10 x 7 cm x 4 cm. It was mildly tender, not attached to the deeper structures or skin, and soft in consistency. The knee had a full range of movements without any - demonstrable instability. There was no knee effusion. The swelling was more prominent with the knee flexed and less so with the knee extended. [(Figure 1)](#F1){ref-type="fig"} Antero-posterior and lateral views of the left knee were normal apart from the soft tissue shadow of the lesion seen on AP view. Blood counts and coagulation parameters were normal. Patient gave a history of aspiration on one occasion in another hospital which according to the records had yielded only blood. An MRI of the right knee showed a large lobulated altered intensity mass in the superomedial region of knee extending into the suprapatellar space. Multiple hypointense septae were seen on T2W images [(Figure 2)](#F2){ref-type="fig"}. The appearances were suggestive of a benign lesion, possibly a haemangioma. A decision was made for an excisional biopsy of the lesion under spinal anaesthesia. A thigh tourniquet was applied but not inflated. Through a n antero-medial arthrotomy of the knee the lesion was excised in toto carefully using electrocautery and meticulous haemostasis at all stages. The macroscopic appearance of the lesion was a reddish brown lobulated mass [(Figure 3)](#F3){ref-type="fig"}. Contrary to the expectation, there was not much bleeding even though the tourniquet was not inflated. A compression dressing was applied after skin closure. Histopathological examination of the excised specimen confirmed the diagnosis of a cavernous synovial haemangioma Post operative recovery was uneventful. The knee was kept in a compression dressing for two weeks. Knee mobilization exercises were started within 48 hours of the surgery. At one month post-operative follow up the patient was doing extremely well, with a full range of left knee movement. There was no evidence of recurrence of the swelling. Discussion {#s3} ========== Synovial haemangiomata are a rare cause of knee swelling and pain. Up to the present time, about 200 cases have been reported in the world literature3 As early as 1949 Julian E Jacobs et al ^4^ had stated "Articular hemangiomata can be diagnosed prior to surgery in practically all cases, provided the correlation between the clinical picture and the pathological process was fully appreciated. These signs are significant: ^1^ the presence of a circumscribed mass, which is covered by normal skin and which increases in size when the extremity is in the dependent position; ^2^ the presence of blood after puncture of the mass; and ^3^ the disappearance of the contrast substance roentgenographically after injection into the vascular area. Surgical excision offers excellent end results." In our patient localized swelling over the supero-medial aspect of the knee was the only presenting symptom. History of recurrent non-traumatic haaemarthrosis in a patient with a normal coagulation profile should raise a suspicion of synovial haemangioma. The X-rays are essentially normal in most of these cases and may show just a soft tissue shadow. At times calcification or phleboliths may be seen on the X-rays. Less than 5% of patients show periosteal reaction, cortical destruction, osteoporosis, advanced maturation of the epiphyses and a discrepancy in leg length or even arthropathy simulating haemophilia . Angiography as an investigation also provides the opportunity for therapeutic embolisation of a major feeder vessel in the same sitting. Angiography may fail to show the hamangioma if the vessels are thrombosed. MRI is the investigation of choice for diagnosis of synovial haemangiomata. Contrast MRI may be used to differentiate haemangioma from joint fluid in cases where there is a knee effusion. Several treatment methods have been advocated for synovial haemangioma in the past, like radiotherapy, surgical excision (open or arthroscopic), and arthroscopic laser ablation. Arthroscopic excision is possible for pedunculated or focal lesions of small size. As the results of open excision are good it is the treatment of choice for large lesions. Synovial hemangiomata should be treated early as they can cause arthropathy, due to recurrent episodes of intra-articular bleeding, and they can even infiltrate muscles, fat and cortical bone 5. {#F1} {#F2} {#F3} Conclusion {#s4} ========== Synovial hemangioma arising in a joint is rare. Knee joint is most commonly affected. Recurrent episodes of nontraumatic haemarthrosis along with normal coagulation parameters should raise the possibility of synovial haemangioma. Plain radiographs are of limited help and MRI of the knee is the investigation of choice for confirming the diagnosis. Angiography is of value but is invasive and not available at all centers. Once the diagnosis is confirmed, early excision should be instituted to reduce the risk of arthropathy.

INTRODUCTION ============ The gene regulatory information encoded in the primary DNA sequence is interpreted and transmitted to the transcription machinery, that it is assembled at the promoter, by the binding of transcription factors to the appropriate sequence elements ([@B1],[@B2]). Among these, enhancers are described as DNA elements that increase the level of transcription of the associated gene in a position- and distance-independent manner relative to the transcription start site ([@B3]). As a consequence, in complex genetic loci with multiple promoters, mechanisms ought to be put in place to make an enhancer prefer one specific promoter. Indeed, some transcriptional enhancers can discriminate between core promoters that contain either a *TATA* box or a DPE element ([@B4]). For instance, in *Drosophila*, the *AE1* enhancer preferentially interacts with the *ftz* promoter rather than with the *Scr* promoter, in spite of its intergenic position and comparable distance from both promoters; in transgenic embryo, promoter competition dictates the *IAB5* enhancer, placed between two divergently transcribed transgenes, to preferentially activate transcription from the *TATA*-containing promoter ([@B5],[@B6]). In addition, in the sea urchin *Hemicentrotus pulcherrimus*, a combination of the upstream and either *TATA*-containing or *TATA*-less core promoter sequences seems to contribute to the establishment of the specific spatial and temporal expression profiles of developmentally regulated genes ([@B7],[@B8]). Besides promoter competition, chromatin insulators may also be involved in promoter selectivity by a given enhancer. Most of these regulatory elements may have two activities: (i) a boundary function blocks the spread of the heterochromatin into the euchromatic region and protects the transgenes from the negative influence of chromatin at the site of insertion; and (ii) an enhancer-blocking activity that restricts enhancer function in one direction and only when interposed between the enhancer and promoter ([@B8; @B9; @B10; @B11; @B12; @B13]). The DNA replication-dependent sea urchin early or α-histone gene cluster represents an interesting model system to investigate the specificity of function of an enhancer element in close proximity of different gene promoters. These genes are development-regulated and organized in a single large cluster made up of ∼2000 tandem repeat units, each containing one copy of each of the five histone genes, in the order 5′*-H2B-H3-H2A-H1-H4-*3′ ([@B14],[@B15]). As embryogenesis occurs, transcription of these genes is limited to the rapid early cleavage and reaches its maximum at the morula/early blastula stage. After hatching, they become repressed and are maintained in such a transcriptional state for the whole life cycle of the animal ([@B16]). We have previously described the *cis*-regulatory sequences and the necessary transcription factor for the timing of transcription of the α-*H2A* gene during the embryogenesis of the sea urchin *P. lividus*. In the 5′ region, a 30-nucleotide-long regulatory sequence termed modulator or simply *M30* ([@B17],[@B18]) specifically binds the MBF-1 activator ([@B19]). The *H2A* modulator has a bidirectional enhancer activity, both in the homologous and heterologous (*Xenopus laevis* oocytes) system ([@B20],[@B21]). Worth mentioning is the capability of tandem copies of the MBF-1-binding sites to activate transcription from a viral promoter independent of distance and orientation ([@B21],[@B22]). As shown in this article ([Figure 1](#F1){ref-type="fig"}), this enhancer function is maintained also by a single MBF-1 recognition sequence. Remarkably, the MBF-1 regulator, although essential for *H2A* transcriptional activation, is constitutively bound to the *H2A* enhancer, which in a transgene construct can elicit transcription from a viral promoter also after silencing of the endogenous α-histone genes at the gastrula stage ([@B23],[@B24]). Figure 1.A single copy of the MBF-1 activator binding site enhances transgene expression. The *M30-tk*-CAT transgenes, bearing one, two or three copies of the 30 bp *H2A* modulator sequence in different location and orientation, were microinjected into sea urchin zygotes. Total RNA from 30 to 50 gastrula stage embryos, microinjected with the indicated transgenes, were hybridized with a ^32^P-labelled CAT antisense probe and processed for the RNase protection assay described in 'Materials and Methods' section. Asterisk indicates the protected RNA band for the CAT transcript. Down-regulation at the gastrula stage relies on the functional interaction between the 5′ dispersed *GA* repeats, located upstream of the enhancer, and the *sns5* insulator placed at the 3′-end of the *H2A* transcription unit ([@B24]). The repressed *H2A* gene is characterized by the specific positioning of two nucleosomes in the promoter/enhancer region, histone de-acetylase recruitment, and histone H3K9 dimethylation in the insulator and 5′ regulatory sequences ([@B25]). Interestingly, the *sns5* element contains the enhancer blocker *sns* that, in an enhancer-blocking assay, restricts enhancer function in a directional and polar manner in both sea urchin and mammalian cells ([@B22],[@B26],[@B27]). In addition, *sns5* exhibits the other property of insulators, the ability to block repressive chromatin effects on the flanking regions of transgenes ([@B28]). The five histone genes are coordinately expressed during early development. Despite this need for nucleosome assembly in the newly replicated chromatin, the number of *H1* linker mRNA molecules is less than the value for histone nucleosomal mRNAs, being about half, as determined by kinetic and hybridization studies ([@B29],[@B30]) or even less (this article). In this article, we have investigated the specificity of the *H2A* enhancer and the molecular mechanism that allows differential transcription of linker versus core histones genes during sea urchin development. In principle, the *H2A* enhancer-bound MBF-1 activator could interact, at least, with the promoters of the neighboring *H3* and *H1* genes, unless promoter competition and the 3′ located *sns5* insulator restrain the activity of the *H2A* enhancer specifically to the associated *H2A* gene. Our results exclude competition between core promoter elements and indicate that the MBF-1 transcription factor participates also in the expression of the *H3* gene in the resident chromatin. In addition, we show that the enhancer-blocking activity of the *sns5* insulator buffers the downstream *H1* promoter from the *H2A* enhancer. These results provide evidence for insulator action in a normal genomic context and suggest that both the *H2A* enhancer and the *sns5* insulator are involved in the different regulation of nucleosomal core and linker histone transcription during early development of the sea urchin embryo. MATERIALS AND METHODS ===================== DNA constructs -------------- The Δp*H3-H2A-*Δp*H1* DNA plasmid, containing the core promoters of *H3* and *H1*, and a wild-type *H2A* transcription unit, was constructed by polymerase chain reaction (PCR) amplification of histone DNA cluster Ph70 and cloning in the pBS vector. The *M30-CAT* plasmids were constructed by shotgun cloning of ligated double-stranded oligonucleotides bearing the *H2A* modulator sequence either upstream or downstream the chloramphenicol acetyltransferase (CAT) coding sequences of the *tk*-70 pBL2 vector under the control of the *thymidine kinase* gene (*tk*) promoter ([@B31]). A dominant negative construct was obtained by fusing the MBF-1 DNA-binding domain encoding sequences ([@B19]) to those of the Engrailed repressor domain cloned in the CS2+nls expression vector. All DNA clones were checked by sequencing. Microinjection of DNA constructs, double-strand oligonucleotides and synthetic RNA ---------------------------------------------------------------------------------- Microinjection in *P. lividus* and *Sphaerechinus granularis* was conducted as previously described ([@B32]). Approximately 5000 molecules of the desired plasmid DNA were injected into the zygote, together with Texas Red-conjugated dextran added at a concentration of 5% in a 2 pl volume of 30% glycerol. In the *in vivo* competition experiments, double-stranded *M30*, *M30mut*, or *BoxA* oligonucleotides were ligated with T4 DNA ligase and fractionated onto polyacrylamide gel. DNA fragments containing four to six tandem copies were eluted from the gel and mixed with the plasmid solution to be microinjected at a molar ratio of the specific genetic element to construct of 50:1. In the competition experiments for the endogenous histone genes, the purified double-strand oligonucletides were mixed with glycerol and Texas Red-conjugated dextran, and injected at a final concentration ranging from 1 to 15 ng/µl. The oligonucleotide sequences used in the competition assays are listed in [Supplementary Table S1A](http://nar.oxfordjournals.org/cgi/content/full/gkp859/DC1). For mRNA injection, dn-MBF-1 and control CS2+nlsEn ([@B33]) constructs were linearized and transcribed *in vitro* using the Sp6 mMessage mMachine kit (Ambion). Capped mRNAs were resuspended in ultrapure RNase-free water (Gibco) and 2 pl, corresponding to the amounts of 0.1, 0.5 and 1.0 pg, respectively, were injected. Injected embryos at the desired stage were harvested and total RNA extracted. RNase protection assay ---------------------- Total RNA samples, extracted from microinjected embryos, were hybridized with antisense ^32^P-labelled RNA probes. The *H3*, *H2A* and *H1* antisense RNA probes did not protect the endogenous *S. granularis* RNA bands. Hybridization conditions, RNase digestion and gel fractionation of the RNase resistant hybrids were as described ([@B22]). Real-time quantitative PCR -------------------------- The amounts of histone gene transcription in control and injected embryos at morula stage were evaluated as described ([@B34]). Briefly, total RNA from batches of 150 microinjected embryos was extracted by using the High Pure RNA Isolation kit (Roche). RNA samples were treated with reagents provided by the Turbo DNA-free kit (Ambion) and resuspended in a final volume of 30 μl. Reverse transcription into cDNA was performed in an 80 μl reaction using random hexamers and the TaqMan Reverse Transcription Reagents kit (Applied Biosystems). The resulting cDNA sample was further diluted and the equivalent amount corresponding to one embryo was used as template for Q-PCR analysis. Q-PCR experiments were performed from two different batches and all reactions were run in triplicate on the 7300 Real-Time PCR system (Applied Biosystems) using SYBR Green detection chemistry (Applied Biosystems). ROX was used as a measure of background fluorescence and MBF-1 mRNA ([@B19]) was used to normalize all data, in order to account for fluctuations among different preparations. At the end of the amplification reactions, a 'melting-curve analysis' was run to confirm the homogeneity of all Q-PCR products. Calculations from QPCR raw data were performed by the RQ Study software version 1.2.3 (Applied Biosystems), using the comparative *C*~t~ method (ΔΔ*C*~t~). The oligonucleotide sequences, length and predicted amplicon size are described in [Supplementary Table S1B](http://nar.oxfordjournals.org/cgi/content/full/gkp859/DC1). RESULTS ======= A single copy of the MBF-1 activator-binding site enhances transgene expression ------------------------------------------------------------------------------- As reported previously, the MBF-1 activator has been identified as being capable of specifically binding a 30-bp sequence of the modulator, and tandem copies of this sequence, denoted as *M30*, enhanced transcription from a viral promoter in a position- and orientation-independent manner in transgenic sea urchin embryos ([@B22]). To gain more details on the promoter specificity of the MBF-1 activator, we assessed, in the first place, whether a single copy of the *M30* sequence was capable of activating the basal *tk* promoter. The constructs schematically drawn in [Figure 1](#F1){ref-type="fig"} were microinjected into sea urchin zygotes, embryos were raised, and expression of the reporter *CAT* gene was determined at the gastrula stage by RNase protection assays with RNA samples from the same number of injected embryos. The results shown in [Figure 1](#F1){ref-type="fig"} demonstrate that the MBF-1-binding sequence enhanced transcription from the viral promoter to a similar extent as did multiple copies. Furthermore, this enhancer activity was displayed independently of location and orientation and occurred also at 2.7-kb distance from the *tk* promoter. Promoter specificity of the MBF-1 activator ------------------------------------------- To elucidate the role of the MBF-1 activator in the expression of the early histone genes, we inhibited its binding to the *H2A* enhancer by performing an *in vivo* competition assay. This assay involves the titration of a given DNA-binding factor by molar excess of tandem copies of a *cis-*regulatory sequence. We have routinely used this approach to knockdown the function of regulatory sequences of the *sns5* insulator and of histone and *Hbox12* gene promoters. As demonstrated in several instances, the effect on transgene expression is identical to that obtained by the mutation of the same sites ([@B24],[@B27],[@B34]). Concatameric ligation products containing on average six tandem copies of the *M30* oligonucleotide were co-injected with the *P. lividus H3-H2A-H1* histone gene constructs, depicted in [Figure 2](#F2){ref-type="fig"}A, into the sea urchin *S. granularis* zygotes*,* to distinguish between endogenous and transgene histone transcripts. Embryos were raised and the expression of the injected genes was detected by RNAse protection assays. All three injected genes followed the embryonic temporal expression profile of the endogenous histone genes, i.e. they are highly transcribed at morula stage and silenced at gastrula stage (lanes 1, 2, 13 and 14). As a control, we used the mutant *M30* sequence (*M30* mut), which as reported cannot bind the MBF-1 protein translated *in vitro* ([@B19],[@B21]). Injection of *M30* mut at the same doses as *M30* had no effect on histone gene transcription ([Supplementary Figure S1](http://nar.oxfordjournals.org/cgi/content/full/gkp859/DC1)). Conversely, the injected *M30* sequence selectively inhibited transcription of the *H2A* gene, while it did not affect the expression of both *H3* and *H1* genes at the morula stage (lanes 3 and 15). These results would suggest that the *H2A* enhancer prefers to interact with the cognate promoter. Figure 2.*In vivo* competition assay to knock-down the *H2A* enhancer function in transgenic embryos. (**A**) Annotated map of the *P. lividus* wild type and deletion mutants early *H3*, *H2A* and *H1* histone genes, highlighting the *cis*-regulatory sequence elements. The horizontal black line and arrow-shaped boxes represent, respectively, the genomic DNA and coding sequences, while the bent arrows denote the putative transcription start site. (**B**) The *P. lividus* histone gene constructs, orientated as in the endogenous histone gene repeat, were co-injected with excess of the modulator binding site (*M30*) into *S. granularis* zygotes. RNase protection was carried out by hybridizing antisense ^32^P-labelled RNA, transcribed *in vitro* from *H3*, *H2A* and *H1* subclones, with total RNA exctracted from 25 injected embryos at morula (Mor) and gastrula (Gas) stages. The two *H2A* and *H3* probes were hybridized together. The protected 409, 357 and 209 nt RNA bands, respectively, for the *H2A*, *H3* and *H1* transcripts are indicated. To assess whether competition between core promoter elements is one of the molecular mechanisms that lead the MBF-1 activator to selectively interact with the *H2A* promoter, we performed the *in vivo* competition assay for the *H2A* enhancer function on the deletion mutant Δp*H3*-*H2A*-Δp*H1* ([Figure 2](#F2){ref-type="fig"}A). In such a construct, the *H2A* transcription unit is a wild type. All the regulatory sequence elements upstream the *TATA* box were deleted from the *H3* promoter ([@B31],[@B35]), while the 5′ deletion of the linker *H1* promoter occurred up to half of the essential regulatory sequence *USE* ([@B36]). We have already described that the expression of *H3* driven by only the *TATA* box and other core promoter elements is up-regulated in a *H3*-*H2A* transgene ([@B24]). The result displayed in [Figure 2](#F2){ref-type="fig"}B extends this observation, by showing that the *H2A* enhancer-bound MBF-1 transcription factor is most probably responsible for the up-regulation. In fact, while the *H2A* gene followed the time of expression of the endogenous gene, the core promoter of the *H3* gene, which by itself displays a barely detectable transcriptional activity (not shown), gave rise to comparable levels of transcripts. Significantly, co-injection of an excess of the *M30* enhancer abolishes transcription of both genes ([Figure 2](#F2){ref-type="fig"}B, lanes 5--8), suggesting that the *TATA* boxes and other core promoter elements of *H3* and *H2A* genes do not compete for the interaction with the MBF-1 activator. A different result was obtained with the *H1* gene driven by the mutated *H1* promoter. The core promoter elements displayed a very low transcriptional activity and did not respond to the *trans*-activating signal emanated by the MBF-1 transcription factor ([Figure 2](#F2){ref-type="fig"}B, lanes 17--20), suggesting that *H1* gene expression is autonomously regulated. This independence from the *H2A* enhancer can be explained either with a core promoter competition mechanism or by the action of the *sns5* insulator located at 3′ of the *H2A* gene, between the *H2A* enhancer and *H1* promoter. The MBF-1 activator is involved also in the transcription of the early H3 gene ------------------------------------------------------------------------------ The results displayed in [Figure 2](#F2){ref-type="fig"} seem to be in apparent contrast. In one case, we have observed a lack of influence of the *M30* competitor on the function of the wild type *H3* promoter. In the other case, we showed the interaction of the MBF-1 activator with the *H3* core promoter. To gain insights on this issue, we have determined by quantitative PCR (Q-PCR) whether competition of the *H2A* enhancer activity affected, and to what magnitude, the expression of the endogenous *H2A*, *H3* and *H1* genes. The histone mRNAs prevalence in the un-competed embryos, shown in [Figure 3](#F3){ref-type="fig"}A, indicates that, in the sea urchin *P. lividus* at morula stage, the *H1* linker histone peaks at about one-third of the value for the nucleosomal *H3* and *H2A*. As expected, the mRNA levels for *H2A* decreased with the microinjection of increasing amounts of the wild type MBF-1-binding site. Once again, the mutant *M30* sequence had no effect. Conversely, the number of mRNA molecules for the linker histone did not change with the rise of either *M30* or *M30* mut competitors, confirming the independence of *H1* transcription from the *H2A* enhancer. Very interestingly, we found that inhibition of MBF-1 binding reduced also the *H3* gene transcripts, although to a lesser extent than the *H2A* mRNA. Figure 3.*In vivo* competition assay for endogenous *H3*, *H2A* and *H1* histone gene expression. (**A**) Relative abundance of histone mRNAs in the *P. lividus* embryo at morula stage. A similar prevalence is detected for the two nucleosomal *H3* and *H2A* mRNAs, while the *H1* linker histone mRNA peaks at about one-third of the value for the formers. (**B**) Endogenous histone gene expression analysis carried out in embryos at morula stage microinjected with excess of the *M30* sequence element or the mutated *M30* mut oligonucleotide as a control. Graphs show *n*-fold changes in mRNA expression level of histone genes based on the threshold cycle number (*C*~t~) of injected embryos compared to that of the uncompeted control embryos. *C*~t~ numbers were normalized for the endogenous MBF-1 in the same sample. Data were derived from two independent microinjection experiments and each bar represents the average of triplicate samples from the two batches of embryos. In order to obtain clear-cut evidence on the involvement of the MBF-1 activator in the expression of the *H3* gene, we performed a more direct experiment to knock down its function. To this end, we made a dominant negative construct, termed dn-MBF-1, in which the DNA-binding domain of MBF-1 ([@B19]) was joined to the repression domain of *Drosophila* engrailed ([@B33]). An *in vitro* transcribed mRNA was injected into the sea urchin zygote and the expression of *H3*, *H2A* and *H1* genes analyzed by Q-PCR at morula stage. The results obtained ([Figure 4](#F4){ref-type="fig"}) are in accordance with the *in vivo* competition analysis described above. In fact, we found a dose-dependent negative effect of the MBF-1 forced repressor on the expression of the nucleosomal *H2A* and *H3*, but not *H1* genes. Once again, transcription of *H3* was less affected compared with *H2A*. Figure 4.Knock-down MBF-1 function by microinjection of a synthetic mRNA encoding for as dominant repressor (dnMBF-1). Increasing amounts (0.1--1 pg) of the chimeric RNA were injected in *P. lividus* zygotes and RNA extracted from embryos at morula stage. Graphs show *n*-fold changes in mRNA expression level of histone genes based on the threshold cycle number (*C*~t~) of dnMBF-1 injected embryos compared to that of the uninjected control embryos. *C*~t~ numbers were normalized for the endogenous MBF-1 in the same sample, by amplifying a fragment of the coding region external to the DNA binding domain. Data were derived from two independent microinjection experiments and each bar represents the average of triplicate samples from the two batches of embryos. In summary, these results confirmed that the MBF-1 transcription factor is absolutely necessary for the activation of the *H2A* gene. In addition, they suggest that the MBF-1 activator is involved also in the transcription of the upstream *H3* gene and has no role on the expression of the linker *H1* gene. The sns5 insulator confers transcriptional independence to the linker H1 histone gene promoter ---------------------------------------------------------------------------------------------- The location of the *sns5* insulator between the *H2A* enhancer and *H1* promoter ([Figure 1](#F1){ref-type="fig"}) prompted us to examine its possible involvement in the mechanism of enhancer specificity. Wild type and mutated *H3-H2A-H1* three gene constructs were microinjected, respectively, in the absence and in the presence of excess *BoxA*-ligated oligonucleotides. As reported, *BoxA* is one of the *cis-*acting sequences absolutely required for the enhancer blocking and silencing function of the *sns5* insulator at gastrula stage ([@B24],[@B27]). This is further shown in [Figure 5](#F5){ref-type="fig"}. The competitor *BoxA* up-regulated the *H2A*, while the wild type *H3* gene followed the temporal regulatory program of the endogenous gene (lanes 1--4). In addition, the inhibition of the insulator activity did not influence the constitutive *trans*-activation of the *H3* core promoter (lanes 5--8). What is most important, however, is the effect of the competition of the insulator activity on the mutant *H1* promoter. In this case, although it appeared that the expression of the wild type *H1* was not affected, we did find a strong *trans*-activation of the mutated *H1* transgene, at both early and late developmental stages, caused most probably by the interaction of MBF-1 with the *H1* core promoter (lanes 13--20). Figure 5.*In vivo* competition assay to inhibit the *sns5* insulator function in transgenic embryos. Wild-type histone gene construct *H3*-*H2A*-*H1* and the deletion mutant *ΔpH3-H2A-ΔpH1* (showed in [Figure 2](#F2){ref-type="fig"}A), were microinjected with excess of the *BoxA* sequence element into *S. granularis* zygotes. RNase protection was carried out as for [Figure 2](#F2){ref-type="fig"}. The protected 409, 357 and 209 nt RNA bands, respectively, for the *H2A*, *H3* and *H1* transcripts are indicated. It should be noted that, as for the wild-type *H3* transgene, the RNase protection assay did not reveal possible subtle differences in the transcription of the wild type *H1* transgene between the un-competed and competed samples. For this reason, to eventually validate the regulatory role of the *sns5* insulator, we looked at the expression by Q-PCR of the endogenous histone genes upon microinjection of the competitor *BoxA* sequence. As can be seen in [Figure 6](#F6){ref-type="fig"}, both nucleosomal histone *H2A* and *H3* mRNAs did not vary their prevalence at the different doses of competitor. By contrast, the number of molecules of the linker *H1* mRNA increased monotonically with the augmentation of the *BoxA* oligonucleotide. Altogether, these results represent a strong indication that the *sns5* insulator blocks the *H2A* enhancer in the interaction with the downstream *H1* promoter. Figure 6.Endogenous gene expression analysis upon impairment of the *sns5* insulator function by *in vivo* competition assay with excess of the *BoxA* sequence element. Graphs show *n*-fold changes in mRNA expression level of histone genes based on the threshold cycle number (*C*~t~) of injected embryos compared to that of the uncompeted control embryos. *C*~t~ numbers were normalized for the endogenous MBF-1 in the same sample. Data were derived from two independent microinjection experiments and each bar represents the average of triplicate samples from the two batches of embryos. DISCUSSION ========== The experiments described in this article highlight the regulatory function of the modulator/enhancer and insulator flanking the *H2A* transcription unit in the expression of S-phase-dependent histone genes during sea urchin development. These genes are organized in a large cluster and are coordinately regulated during embryogenesis. Notwithstanding this need, the transcripts of linker and core histones accumulate at the morula stage at different levels, with the *H1* mRNA being, in *P. lividus*, ∼30% of the abundance of the two nucleosomal *H3* and *H2A* mRNAs. This differential regulation occurs despite the presence of a strong enhancer in the 5′-flanking region of the *H2A* gene. Although some evidence suggests that the enhancer has a bipartite organization ([@B16]), our results indicate that a single binding site for the MBF-1 activator suffices the *trans*-activation of a distant promoter and in both orientations ([Figure 1](#F1){ref-type="fig"}). Because of this property, we raised the issue of whether competition between promoter *cis-*regulatory sequences specifically directs the MBF-1 transcription factor toward the cognate *H2A* promoter. Although the activity of the wild type promoter of the *H3* transgene seemed refractory to the inhibition of MBF-1 binding, several lines of evidence substantiate the involvement of the MBF-1 transcription factor in the expression also of the α-*H3* gene, as previously suggested ([@B31]). First, we found a robust activation of the *H3* minimal core promoter that is inhibited by the titration of the MBF-1 factor. Second, we found a reduction of the number of endogenous *H3* and *H2A* mRNA molecules with microinjection of increasing amounts of *M30*, but not the *M30* mutant, competitor ([Figure 3](#F3){ref-type="fig"}). The third evidence is even stronger, in that, the expression of a forced MBF-1 repressor had a severe impact on the transcription level of the two nucleosomal core histone genes ([Figure 4](#F4){ref-type="fig"}). Clearly, the accumulation of the *H2A* transcripts is more affected than that of *H3*, indicating a different responsiveness of their gene promoters to MBF-1 knockdown. A straightforward interpretation of this observation is that the MBF-1 activator is the essential transcription factor for *H2A* gene, as suggested by previous experiments ([@B19]), but it only participates, together with other factor(s), in the transcription of the nucleosomal *H3* histone gene. We speculate that one of the factors with which the enhancer-bound MBF-1 activator interacts is the protein complex containing the homeodomain CDP/cut that, as described ([@B37]), binds to the *CCAAT* sequence element for the maximum expression of the *H3* gene. The results described in this article demonstrate that the linker *H1* histone gene is differentially regulated relative to the patterns of core histone gene transcription. In *Drosophila*, the cell cycle-dependent histone genes are tandemly arrayed and coordinately regulated, and the ratio of linker and core histones varies during embryonic development ([@B38]). However, the regulatory mechanism involved in the lower accumulation of *H1* gene transcripts, with respect to the nucleosomal histones, are profoundly different between fly and sea urchin. In *Drosophila*, two distinct sets of core promoter recognition factors, the TBP and TBP-related factors TRF2 ([@B39]), are responsible for directing transcription, respectively, of the *TATA*-containing nucleosome core histone genes and the *TATA*-less linker histone *H1* ([@B40]). Although the molecular mechanism is not clear, upstream promoter-bound regulatory factors are probably involved in the interaction with the proper core promoter transcription complex. In the sea urchin *P. lividus*, the *H1* core promoter lacks a canonical *TATA* box and yet, as shown here, there is no competition between core promoter elements for the *H2A* enhancer. Instead, we obtained compelling evidence for the involvement of a chromatin insulator in making the linker *H1* promoter independent from the action of the *H2A* enhancer. Chromatin insulators are genetic regulatory elements that may possess both a boundary function and a directional enhancer-blocking activity ([@B41]). Thus, insulators, by restricting enhancer function, may impart promoter selectivity to a given enhancer in the eukaryotic genome. This role has been demonstrated in several cases. The *Drosophila SF1* insulator, for instance, of the Antennapedia complex, placed in the *scr-ftz* intergenic region, restricts promoter selection by the *ftz*-distal enhancer in transgenic embryos ([@B42]) and separates the fushi tarazu from the neighboring *Hox* gene ([@B43]). As an additional example, the human and mouse imprinting controlling region (ICR) contain an insulator activity that depends on the binding of the CTCF regulator. In the maternal allele, the CTCF factor binds to the unmethylated ICR and prevents the downstream enhancer from interacting with the upstream *IGF2* promoter. The blocked enhancer can activate transcription of the *H19* gene ([@B44]). Intriguingly, the *P. lividus sns5* DNA fragment, located at the 3′ of the *H2A* transcription unit, has been identified as an essential element for the silencing of the *α-H2A* gene at the gastrula stage ([@B27]). In addition, as for the most known vertebrate insulator, the *HS4* insulator of the chicken β-globin locus ([@B13]), the *sns5* sequence element displays, on transgene constructs, both enhancer-blocking and boundary activities ([@B22],[@B28]). The directional enhancer blocking activity is achieved by the cooperative interactions between all three different protein factors bound to their specific *cis-*regulatory sequences, in that, titration of any of them abolishes the function ([@B24],[@B27]). Here, we have shown that microinjection of molar excess of the *cis-*acting *BoxA* element allowed *trans*-activation of the *H1* transgene, driven by the core promoter elements. Most importantly, the *BoxA* competitor injected at the maximum dose (higher concentration is toxic for the embryo) specifically increased the expression of only the endogenous *H1* gene. The most obvious interpretation of this result is that the chromatin insulator *sns5* buffers the downstream *H1* promoter from the activity of *H2A* enhancer-bound MBF-1 transcription factor. The *sns5* insulator should leave also the expression of *H4* and *H2B* genes of the repeating unit not linked to the action of MBF-1 input from the *H2A* promoter. In fact, the high level of expression of these two genes depends on strong promoter upstream elements ([@B45],[@B46]). Altogether, the results presented in this article suggest that the *sns5* insulator is most probably responsible for the different level of accumulation of nucleosomal and linker transcripts during sea urchin embryogenesis*.* Finally, our findings provide a clear demonstration of the enhancer-blocking function of a chromatin insulator in a natural gene context. An additional important issue to be clarified concerns the mechanism that prevents the activation of the *H1* gene by the *H2A* enhancer in the adjoining histone gene repeat. As a possibility, the enhancer might not elicit a perceptible effect at the resulting genomic distance of more than 5 kb. Alternatively, it could be speculated that the *H1* gene is flanked, at the 3′, by a supplementary insulator element. Experiments have been planned to distinguish between these two possibilities. SUPPLEMENTARY DATA ================== [Supplementary Data](http://nar.oxfordjournals.org/cgi/content/full/gkp859/DC1) are available at NAR Online. FUNDING ======= The University of Palermo (ex 60%). Funding for open access charge: University of Palermo (ex 60%). *Conflict of interest statement*. None declared. Supplementary Material ====================== ###### \[Supplementary Data\] The participation of F. Palla and D. Di Caro in the experiment described in [Figure 1](#F1){ref-type="fig"} is acknowledged.

**Core tip:** Based on the esophageal cancer-associated RNA-seq in The Cancer Genome Atlas, we studied differentially expressed genes of esophageal cancer at various stages, constructed a protein-protein interaction network, obtained 14 dysfunctional modules, and screened Hub genes. We performed enrichment analysis to predict non-coding RNA and transcription factors as well as methylation analysis of the genes in the module. A series of regulatory factors was predicted to regulate to a certain degree the potential dysfunction mechanism of esophageal cancer, which provides new insight for future studies of esophageal cancer. INTRODUCTION ============ Esophageal cancer is one of the world\'s most common cancers with poor diagnosis and high mortality because of invasiveness and a fast growth rate\[[@B1]\]. From a therapeutic point of view, esophageal cancer can be divided into early esophageal cancer, locally advanced resectable esophageal cancer, locally advanced unresectable esophageal cancer, and metastatic esophageal cancer. Because of the anatomical features of esophageal cancer, esophageal cancer is usually detected in the late stage, which vitally affects the treatment options and prognosis of patients\[[@B2]\]. During the development of esophageal cancer, the rs11473 polymorphism of the miR-483-5p binding site plays a vital role in the 3\'-untranslated region of the basigin gene\[[@B3]\]. Single nucleotide polymorphisms in telomerase reverse transcriptase may be associated with susceptibility to esophageal cancer and contribute to the development of esophageal cancer\[[@B4]\]. MiR-20b may play an essential role in the tumorigenesis of esophageal cancer by regulating phosphatase and tensin homolog expression, which may be a potential therapeutic target for the treatment of esophageal cancer\[[@B5]\]. Growing evidence has revealed molecular targets for diagnosis and prognosis using bioinformatic analysis in the field of oncology\[[@B6]-[@B16]\]. These findings have deepened our understanding of the pathogenesis of esophageal cancer and have guided the direction of our research. However, the molecular pathogenesis of the disease is still elusive. To explore comprehensively the molecular processes and potential therapeutic targets of esophageal cancer progression, we conducted a systematic module analysis. Overall, our work details the role of multifactorial mediated dysfunction modules in the growth of esophageal cancer and identifies essential genes and related biological processes, finding potential molecular mechanisms and therapeutic targets \[G protein subunit gamma transducin 2 (GNGT2)\] for esophageal cancer. MATERIALS AND METHODS ===================== Patient samples and cell lines ------------------------------ All esophageal cancer analyses in this study involving human participants were in accordance with the ethical standards of the Second Hospital of Jilin University and with the Declaration of Helsinki. A total of 40 esophageal cancer patients and healthy control volunteers, who matched for age and sex, were involved in this study. Informed consent was obtained from all participants. The esophageal cancer cell lines EC109 and KYSE70 were kindly provided by Laboratory in The Second Hospital of Jilin University. The cells were maintained in RPMI-1640 medium containing 10% fetal bovine serum. Quantitative real-time PCR and cell proliferation experiment ------------------------------------------------------------ Total RNA was extracted from case/control group using TRIzol. The quantitative real time-polymerase chain reaction (PCR) experiment was conducted in a real-time PCR detection system using SYBR Green qPCR Master Mix. Primers were designed and synthesized by Novogene (Beijing, China). Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control. Cell counting kit-8 assay was used to measure cell proliferation. Transfected cells were cultured for 0-96 h and incubated at 37 °C for 2 h. A spectrophotometer (450 nm) was used to quantitate samples. Data resource ------------- The Cancer Genome Atlas (TCGA) is a joint project of the National Cancer Institute and the American Human Genome Research Institute. High-throughput genomic analysis technology is a useful tool for people to understand better cancer, and it improves their abilities to prevent, diagnose, and treat disease. We downloaded esophageal cancer RNA-Seq data from the TCGA database and screened non-coding RNA (ncRNA)-mRNA interaction pairs with a score ≥ 0.5 from RNA Associated Interaction Database v2.0\[[@B17]\], including 431937 interacting pairs involving 5431 ncRNAs. All human transcription factor target data were downloaded and used in the general database-Transcriptional Regulatory Relationships Unraveled by Sentence-based Text mining v2 database for transcriptional studies, including 2492 transcription factors and 9396 interaction pairs. Differential expression analysis -------------------------------- In order to explore the molecular process of esophageal cancer staging, we selected four stages of esophageal cancer and normal samples for differential expression analysis, including healthy tissue samples *vs* stage 1 disease samples, stage 1 disease samples *vs* stage 2 disease samples, stage 2 disease samples *vs* stage 3 disease samples, and stage 3 disease samples *vs* stage 4 disease samples. We used the limma package for analysis\[[@B18]-[@B20]\]. Using the Correct background function, we performed background correction and normalization on the data. The normalize Between Arrays function quantile normalization method can filter out the control probe and the low expression probe. The differentially expressed genes of the data set were identified based on the lmFit and eBayes functions (*P* \< 0.01) using default parameters. Establishing a protein interaction network to identify esophageal cancer related functional modules --------------------------------------------------------------------------------------------------- A protein-protein interaction system was constructed based on Search Tool for the Retrieval of Interacting Genes/Proteins database data (score \> 500). The gene module of more than 30 nodes was screened throughout the network using the ClusterONE plug-in\[[@B18]\] of the Cytoscape software\[[@B19]\]. We use the Cytoscape plugin CytoHubba\[[@B20]\] to identify hub genes in the module subnet, while CytoHubba contains 12 methods for identifying hub genes. We obtained the top 10 genes and then screened the repeat genes in the 12 sets of genes for survival analysis. Enrichment analysis ------------------- The study of the functions and signal transduction pathways involved in genes contributes to our understanding of the molecular mechanisms of disease. Gene ontology function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using the R language Cluster profiler package\[[@B21]\]. The Cluster profiler is a bioconductor software package that provides statistical analysis of functional clustering of gene sets. Predictive transcriptional factors and ncRNAs for significant regulatory modules -------------------------------------------------------------------------------- The transcription and post-transcriptional regulation of genes are often dominated by the regulation of transcription factors (TF) and ncRNA. Therefore, we have scientifically predicted its role in the esophageal cancer dysfunction module. If the regulatory effect between the regulator and the module exceeds 2, and the number of organizational relationships between the regulator and the module is essential (hypergeometric test, *P* value \< 0.01), it can be confirmed that it is a regulator of the critical regulatory module. RESULTS ======= Identifying the expression of dysregulated molecules in esophageal cancer ------------------------------------------------------------------------- Biologists have conducted many experiments and studies on the pathogenesis of esophageal cancer and have thus identified potential pathogenic genes for the deterioration of esophageal cancer. To observe molecular changes in the progression of esophageal cancer, we performed differential expression analysis based on RNA-Seq data from four stages of esophageal cancer in the TCGA database. Based on analysis of phase 1 disease samples of normal tissue samples and esophageal cancer, analysis of phase 1 disease samples and stage 2 disease samples, analysis of stage 2 disease samples and phase 3 disease samples, and analysis of stage 3 disease samples and stage 4 disease samples, we obtained differential expression genes (DEGs) associated with each stage of esophageal cancer. A total of 7457 differentially expressed genes were received (Figure [1B](#F1){ref-type="fig"}). We believe that the presence of these differentially expressed genes is closely related to the development of various stages of esophageal cancer. Of the 7457 DEGs, there were 13 common genes (Table [1](#T1){ref-type="table"}). The genes that were continuously down-regulated are *CPLX2, DPEP1, EPHA5, SCGB1A1, ST18*. The genes that were continuously up-regulated are *FGF14, KCNH6, LOC100506136, RGS7, SH3GL2, THBS4* (Figure [1A](#F1){ref-type="fig"}). ###### Differential expression of 13 common genes in four different stages of esophageal cancer **Common genes** **DEG_S1** **DEG_S2** **DEG_S3** **DEG_S4** ------------------ ------------ ------------ ------------ ------------ *CPLX2* -4.85047 2.288678 1.290368 -4.17468 *DPEP1* -1.33466 2.340936 1.604402 -4.41133 *EPHA5* -6.3307 1.829821 0.991225 -3.72048 *FGF14* -2.70845 1.254105 1.339303 -2.01758 *INSM1* -2.80099 1.759985 2.787167 -3.27386 *KCNH6* -3.87431 1.636848 1.717225 -2.87864 *LOC100506136* 0.757741 0.789295 -0.58427 0.955725 *RGS7* -3.27316 2.095696 1.45118 -3.13742 *SCGB1A1* -3.02471 3.325037 -1.81283 -2.46682 *SH3GL2* -6.28822 2.761197 1.269058 -2.63088 *SLITRK1* -4.12073 2.309863 1.635813 -3.57252 *ST18* -2.02067 1.550856 1.289301 -2.51987 *THBS4* -2.15432 2.753388 1.06135 -2.2027 DEG_S: Differential analysis of four different stages *vs* control. {#F1} Identify functional esophageal cancer staging related modules ------------------------------------------------------------- Gene module analysis helps us to study the complex collaborative relationships between multiple genes. Based on the protein interaction data of the STING database, the interaction network of differentially expressed genes was constructed, and 14 functional barrier modules were explored. Using the 12 methods in Cyto-hubba, a total of 758 hub genes were identified in the interaction sub-network of the module genes, including the gene *SH3GL2*, which is continuously up-regulated in Module 8. Further, 23 hub genes shared by the top10 gene set in 12 methods were screened for survival analysis. The results show that *GNGT2* in module 6 is the related gene (*P* = 0.014) (Figure [2A](#F2){ref-type="fig"}). A decrease in survival rate accompanied the high expression of *GNGT2* gene, and the expression level of *GNGT2* gene was negatively correlated with survival rate. Function and pathway are essential mediators of the physiological response of the disease. We performed GC enrichment analysis on 14 module genes (Figure [2B](#F2){ref-type="fig"}) and KEGG (Figure [2C](#F2){ref-type="fig"}). The main biological processes include positive regulation of protein transport, gastric acid secretion, and insulin-like growth factor receptor binding. The main signal transduction pathways involved are the p53 signal transduction, the epidermal growth factor signal transduction, and the epidermal growth factor receptor signal transduction pathways. These pathways play crucial roles in the dysfunctional module for the functions and pathways involved in multiple genes. {#F2} TFs and ncRNAs that drive esophageal cancer progression ------------------------------------------------------- From the perspective of systems biology and systems genetics, transcription and post-transcriptional regulation of genes have long been recognized as crucial regulators of disease development, while transcription factors and ncRNAs are universal regulators of expression and function. Although the role of TFs and ncRNA regulation of esophageal cancer progression has been evaluated by many biologists, few studies have focused on their overall global effect on dysfunctional mechanisms and the role they play in development. Therefore, in this study, based on the targeted regulation relationship between TF and ncRNA on the module gene, we performed a pivotal analysis of the conventional module to explore the crucial regulator that regulates the progression of esophageal cancer. The results showed that a total of 54 transcription factors involved 54 TF-module target pairs and 853 ncRNAs involved 944 ncRNA-module regulatory pairs. Statistical analysis revealed that TF HIF1A and ncRNA CRNDE regulate the most dysfunctional modules. These crucial transcription factors and ncRNAs may influence the development and progression of esophageal cancer by mediating dysfunctional modules. Thus, we identified these potential factors as regulators of dysfunction in esophageal cancer. Notably, hsa-miR-330-3p up-regulates the differentially expressed gene *SH3GL2* throughout the esophageal cancer process, suggesting that hsa-miR-330-3p plays a crucial role in four stages of esophageal cancer. GNGT2 expression is upregulated in the esophageal epithelial cells of esophageal cancer and cell lines ------------------------------------------------------------------------------------------------------ To investigate changes in *GNGT2* expression during esophageal cancer development, samples from esophageal cancer patients (*n* = 20) and esophagus controls (*n* = 20) were subjected to quantitative real time-PCR analysis. As shown in Figure [3A](#F3){ref-type="fig"}, expression of *GNGT2* gene was significantly upregulated in the esophageal epithelial cells of esophageal cancer patients. The experiment in esophageal cancer cell lines (EC109 and KYSE70) showed consistent results (Figure [3B](#F3){ref-type="fig"}) (*P* \< 0.05). {#F3} GNGT2 could promote the proliferation of esophageal cancer cell lines --------------------------------------------------------------------- To explore further the role of GNGT2 in the proliferation of EC109 and KYSE70 cells, cells were transfected with *GNGT2* siRNA. As shown in Figure [3C](#F3){ref-type="fig"}, *GNGT2* mRNA expression level was significantly decreased in EC109 and KYSE70 cells. Moreover, the proliferation of *GNGT2* transfected group was significantly lower than that of the control group (Figure [3D](#F3){ref-type="fig"}). Taken together, the above results demonstrated that GNGT2 could promote the proliferation of esophageal cancer cell lines. DISCUSSION ========== Esophageal cancer is one of the most deadly cancers, mainly because it is extremely aggressive and has a poor survival rate. Its 5-year survival rate is about 15%-25%\[[@B1]\]. The underlying cause of this disappointing low survival rate is that most patients have reached the late stage of disease at the time of detection. For patients with metastatic and unresectable disease, their chances of survival are limited\[[@B22]\]. In the present study, we collected RNA-Seq data from TCGA esophageal cancer and selected four stages of esophageal cancer disease samples and normal samples for differential analysis, and obtained four sets of time series differentially expressed genes. After screening, we found 13 common genes in four groups of DEGs. Komatsu et al\[[@B23]\] studied clinical biomarkers of pulmonary neuroendocrine tumors (LNET) and found that CPLX2 was strongly positive in 16.3% of the examination groups. Importantly, positive CPLX2 expression is associated with lymphatic invasion, pathological staging, and adverse disease-specific survival in LNET patients. It was concluded that CPLX2 is a novel clinical biomarker for LNET\[[@B23]\]. In the study of breast cancer diagnostic markers, Fu et al\[[@B24]\] found that changes in gene expression, such as DPEP1, may lead to cancer progression. DPEP1 has been identified as a prognostic gene for colorectal cancer (CRC). We found that *DPEP1* is overexpressed in CRC. After knocking out the *DPEP1* gene, cells (SW480 and HCT116) exhibited increased apoptosis and attenuated cell proliferation and cell invasion\[[@B25]\]. In the study of CRC, Eisenach\[[@B26]\] found that the expression of DPEP1 was increased in CRC tissues compared with normal mucosa. Zhang et al\[[@B27]\] also noted the DPEP1 gene in the study of pancreatic ductal adenocarcinoma and found that its gene expression was negatively correlated with histological grade and that lower expression of *DPEP1* in tumors was associated with poor survival. Chen et al\[[@B28]\] analyzed the gastric cancer-associated Gene Expression Omnibus data and found that thrombospondin 4 (THBS4) was up-regulated in patients with recurrent gastric cancer and was positively correlated with the pathological stage and poor prognosis of gastric cancer. THBS4 stimulates the proliferation of gastric cancer cells. The breast-related gene explored by Huang et al\[[@B29]\] contains the gene *THBS4*, which is up-regulated in breast cancer. In the study of hepatocellular carcinoma, Su et al\[[@B30]\] found that knockdown of THBS4 inhibited migration and invasion of hepatocellular carcinoma cells as well as hemangiocarcinoma-induced angiogenesis. THBS4 as a target is very promising for the treatment of advanced liver cancer. Both of the above genes were present in the differential genes of the four stages of esophageal cancer in this study and were continuously down-regulated. Moreover, THBS4 was identified as a clinical biomarker gene and a therapeutic target gene in various cancers. Therefore, we can reasonably speculate that this gene plays an important role in the occurrence and development of esophageal cancer, providing a reasonable direction for further study of esophageal cancer. The results of the methylation test showed that the *SST* gene was up-regulated extensively, which may be a key gene involved in methylation modification to regulate the progression of esophageal cancer. Jin et al\[[@B31]\] found that hypermethylation of the *SST* promoter is common and is associated with early tumor progression in Barrett\'s esophagus. The *SH3GL2* gene is up-regulated. The gene is not only the common DEGs of the four-stage time series but also the Hub gene in module 6. It also may play an important role in the regulation of esophageal cancer by methylation modification. Ghosh et al\[[@B32]\] studied the effect of SH3GL2 methylation on the pathogenesis of head and neck squamous cell carcinoma, and abnormal *SH3GL2* is an independent pathway for early developmental abnormalities of the head and neck. ARTICLE HIGHLIGHTS ================== Research background ------------------- Esophageal cancer is one of the most lethal malignant tumors in the world. In the past decades, although the treatment methods for esophageal cancer have improved, the overall efficacy is still poor. Research motivation ------------------- In-depth analysis of molecular mechanisms related to esophageal cancer. Research objectives ------------------- Exploring the molecular process of esophageal cancer comprehensively and deeply. Research methods ---------------- This study used differential expression analysis, enrichment analysis, methylation analysis, survival analysis, and statistical analyses. Research results ---------------- A total of 7457 differentially expressed genes and 14 gene interaction modules were identified. These module genes were significantly involved in the positive regulation of protein transport, gastric acid secretion, insulin-like growth factor receptor binding and other biological processes as well as p53 signaling pathway, epidermal growth factor signaling pathway and epidermal growth factor receptor signaling pathway. In addition, transcription factors (including HIF1A) and ncRNAs (including CRNDE and hsa-mir-330-3p) that significantly regulate dysfunction modules were identified. Further, survival analysis showed that GNGT2 was closely related to survival of esophageal cancer. Differentially expressed genes with strong methylation regulation ability were identified, including *SST* and *SH3GL2*. Research conclusions -------------------- Overall, our work describes in detail the role of multifactor-mediated dysfunction module in the whole process of esophageal cancer, identifying key genes for staging and related biological processes, which may help to identify potential molecular mechanisms and therapeutic targets for the deterioration of esophageal cancer. Research perspectives --------------------- This work not only helps us to reveal the potential regulatory factors involved in the development of disease but also deepen our understanding of its deterioration mechanism. Manuscript source: Unsolicited manuscript Specialty type: Gastroenterology and hepatology Country of origin: China Peer-review report classification Grade A (Excellent): A Grade B (Very good): B Grade C (Good): C Grade D (Fair): 0 Grade E (Poor): 0 Institutional review board statement: This study was reviewed and approved by the Ethics Committee of the Second Hospital of Jilin University. Conflict-of-interest statement: The authors declare no conflict of interest. Peer-review started: September 29, 2019 First decision: November 27, 2019 Article in press: December 14, 2019 P-Reviewer: Gazouli M, Rodrigo L, Sterpetti AV S-Editor: Gong ZM L-Editor: Filipodia E-Editor: Zhang YL Data sharing statement ====================== No additional data are available. [^1]: Author contributions: Liu GM and Ji X designed the research; Lu TC and Duan LW performed the research; Jia WY and Liu Y analyzed the data; Sun ML and Luo YG wrote the paper. Supported by Construction of Engineering Laboratory of Jilin Development and Reform Commission (grant no. 3J115AK93429) and Jilin Provincial Science and Technology Department Medical Health Project (grant no. 3D5195001429). Corresponding author: Yun-Gang Luo, MD, Attending Doctor, Department of Stomatology, Jilin Provincial Medicine Anti-Tumor Engineering Center, The Second Hospital of Jilin University, No. 218, Ziqiang Street, Changchun 130041, Jilin Province, China. <[email protected]>

Introduction {#Sec1} ============ Hand is one of the most frequently injured parts of the body \[[@CR1]\]. Functional outcome in case of fractures of small bones of hand depends upon injury severity and management \[[@CR2]\]. Ultimate functional outcome is more important than just fracture healing in case of hand fracture \[[@CR3]\]. Most hand fractures can be treated by nonoperative methods with good outcome \[[@CR4], [@CR5]\]. In the small percentage of unstable hand fractures, results of closed treatment remain unsatisfactory. Closed multiple metacarpal fractures are considered highly unstable and are more prone to poor functional outcome compared with open single metacarpal fracture \[[@CR6]--[@CR10]\]. A small number of prospective studies have been published on treatment of unstable metacarpal and phalangeal fractures using miniature plate (mini plate) and screws \[[@CR11], [@CR13], [@CR14]\]. After thorough literature review we did not find any prospective studies in which ipsilateral multiple metacarpal fractures were treated with plating system. We carried out a study in which 21 patients with closed ipsilateral multiple metacarpal fractures were treated with mini fragment plates and screws. Patients and methods {#Sec2} ==================== A prospective study was conducted from January 2005 to December 2008. Thirty-one consecutive patients with closed ipsilateral multiple metacarpal fractures who were admitted to our institution were enrolled in the study. Patients with two or more metacarpal fractures were included. Two patients died due to associated head injury. Eight patients were lost to follow-up. Finally, a total of 21 patients with 55 metacarpal fractures were included in the study. The minimum age of the patients in our series was 16 years, and the maximum was 75 years, with mean age of 49.5 years. Of all 21 cases, the majority (\>50%) were in either the second or fifth decade of life, with the maximum number of patients in the 21--30-year-old age group, accounting for 28% of total patients. Nineteen patients were male, and two patients were female. Right hand was involved in 11 patients and left in 10 patients. Roadside accidents with high-energy trauma were the mode of injury in most cases (11 cases). The second most common cause of these fractures was assault (seven cases), while few patients suffered these fractures during industrial accidents (two cases) or fall (one case). Eleven patients had two metacarpal fractures. The most common pattern was ring finger with little finger (five patients), and the least common was little finger with index finger (one patient). Seven patients had three metacarpal fractures, and three patients had four metacarpal fractures. There are different sizes of plate available to fix metacarpal fractures (1.5-mm screws and titanium mini plates, 2.0-mm screws and stainless-steel AO mini plates, and 2.7-mm screws and stainless-steel AO mini plates). Ultralow-profile plates are also available. We used 2.0-mm stainless-steel AO mini plates with 2.0-mm screws. Souer et al. describe the use of "escape" screws, i.e., a 2.4-mm screw through a 2.0-mm plate, in metaphyseal bone if satisfactory purchase is not obtained with a 2.0-mm screw \[[@CR9]\]. However, in our cases we were able to get satisfactory purchase with 2.0-mm screws. The DASH score and the American Society for Surgery of the Hand (ASSH) Total Active Flexion (TAF) score (Table [1](#Tab1){ref-type="table"}) were used to grade results. The ASSH TAF score grades results as excellent (flexion ≥220), good (flexion 120--80), or poor (flexion ≤80).Table 1American Society for Surgery of the Hand (ASSH) Total Active Flexion (TAF) score systemDegree of flexionRatingTAF from MCPJ to DIPJ: digit 2--5 \>220Excellent 120--80Good \<80PoorTAF from MCPJ to IPJ: thumb \>220Excellent 120--80Good \<80Poor*Clinical Assessment Committee*. Total Active Flexion (TAF) scale, American Society for Surgery of the Hand (ASSH) report. New Orleans, 1976. TAF, total active flexion; MCPJ, metacarpophalangeal joint; DIP, distal interphalangeal joint; IPJ, interphalangeal joint Surgical technique {#Sec3} ------------------ The metacarpal fractures were exposed by dorsal incisions in the space between the involved metacarpals. Extensor tendons were retracted. Fractures were fixed with the plate best suited to the fracture configuration. Reduction and screw sizes were confirmed by image intensifier. Adequate soft tissue closure was achieved over the plate to avoid extensor tendon irritation. Wound was closed without drainage. The hand was rested in elevation for 24--48 h to control pain and swelling, and mobilized actively thereafter. Fracture union was monitored by serial radiographs during fortnightly follow-up visits. Clinical progress in terms of range of movement and complications was recorded at each outpatient visit until healing of fractures, and union was noted. The final range of motion of operated finger was noted in degrees after fracture union. Average follow-up was 1 year. The study was performed in accordance with the ethical standards of the 1964 Declaration of Helsinki and was approved by the local ethical committee. Written informed consent was obtained from all patients. Results {#Sec4} ======= Bone union was seen in all patients, with average period of 6.22 weeks (range 4.5--7.5 weeks). Final functional outcome (as assessed by ASSH TAF score) was excellent in 18 patients, good in 2 patients, and poor in 1 patient. Mean DASH score was 8.47 (range 1--26). The results were satisfactory, as shown in Figs. [1](#Fig1){ref-type="fig"} and [2](#Fig2){ref-type="fig"}.Fig. 1Case 1: **a** preoperative and **b** postoperative X-raysFig. 2Case 1: **a** full flexion, **b** full extension, and **c** pen-holding, showing pinch Deep infection was seen in two patients and was managed with daily dressings and antibiotics. Of these two patients, one had four metacarpal fractures and the other had three metacarpal fractures. Superficial infection was seen in three patients and was managed with daily dressings and antibiotics. There were no cases of angular or rotational displacement. No cases of implant breakage were noted. None of the patients in our study had tendon irritation. This may because we were extra cautious during soft tissue suturing over plate. In none of the patients was implant removal required. Discussion {#Sec5} ========== Most hand fractures can be treated by nonoperative methods with good outcome \[[@CR4], [@CR5]\]. In the small percentage of unstable hand fractures, results of closed treatment are usually unsatisfactory. Indications for accurate open reduction and internal fixation in hand fractures are few, probably accounting for less than 5% of all hand fractures \[[@CR15]--[@CR17]\]. James reported loss of function in 77% of fingers with unstable phalangeal fracture treated by closed methods \[[@CR18]\]. Open reduction and internal fixation of metacarpal fractures with K-wires produces a less rigid fixation with little rotational stability. Protruding ends of the K-wires cause other problems. Interosseous wiring along with K-wire provides more rigid stabilization; however, this method is useful in transverse diaphyseal fractures only. Metacarpal fracture fixation with external fixator has been described in literature \[[@CR19]--[@CR27]\]. Return of total range of motion was achieved in up to 100% of metacarpal fractures fixed with external fixator by Shehadi et al. \[[@CR20]\]. Tun et al. compared the biomechanical properties, clinical versatility, ease of application, and financial cost of seven mini external fixation systems used to treat unstable metacarpal shaft fractures with segmental bone loss \[[@CR25]\]. Those authors discouraged routine use of such fixators because of unacceptable loosening at the pin--cement interface during testing and because of difficulties encountered during construction and application. Transverse and short oblique metacarpal fractures may be splinted with intramedullary wires \[[@CR28]--[@CR37]\]. Flexible bent intramedullary fascicular wires may be used to support oblique fractures. In a study of 21 metacarpal fractures, a J-shaped nail formed from a curved 2.0-mm-diameter Kirschner wire bent sharply at the proximal end was found to be useful in neck or transverse shaft fractures of the metacarpals without concomitant injuries such as severe soft tissue damage \[[@CR31]\]. A recent uncontrolled retrospective consecutive study of 22 metacarpal fractures suggested that transcutaneous intramedullary wire fixation of oblique extra-articular metacarpal shaft fracture wires achieves good results and has few complications \[[@CR36]\]. In a study of 52 consecutive closed, displaced, extra-articular metacarpal fractures, results of intramedullary nail (IMN) fixation were compared with those of plate--screw (PS) fixation. No significant differences in clinical outcomes were found, but the incidences of loss of reduction, penetration to the metacarpal--phalangeal joint, and secondary surgery for hardware removal in the operating room were much higher in the IMN group \[[@CR37]\]. In the literature, several studies have reported satisfactory results for unstable metacarpal and phalangeal fractures fixed with AO mini plates and screws \[[@CR11], [@CR12], [@CR27], [@CR38]--[@CR47]\]. In literature, we found only one study, by Souer et al., in which results of plate fixation in closed ipsilateral multiple metacarpal fractures were evaluated \[[@CR9]\]. The study was retrospective, unlike our study. They found total active motion (TAM) \>230° in 18 of 19 patients. They had two patients with plate-related complications and one delayed union. Their functional results as evaluated by TAM were quite similar to our results. Gupta et al., in their prospective study, divided patients with fractured metacarpals into four groups. They found TAM \>230 in all patients in the group where fracture was fixed with plates \[[@CR27]\]. Dabezies and Schutte reported no complications in 27 unstable metacarpal fractures fixed with plates \[[@CR40]\]. Our low complication rate is similar to their results. Other authors have reported that patients with open fractures and severe soft tissue injury have high rate of complications \[[@CR45], [@CR48]--[@CR50]\]. Nonunion and delayed union are infrequent findings in metacarpal fractures. Souer et al. reported 1 of 19 patients having delayed union \[[@CR9]\]; the patient was a smoker. Page and Stern \[[@CR49]\] found nonunion in 1 of 66 patients, and Stern et al. \[[@CR45]\] found nonunion in 3 of 17 patients. Their low rate of nonunion and delayed union was similar to our results. Infection was seen in 5 of 21 patients. Two patients had deep and three had superficial infection. In all three patients who had superficial infection, there was discharge from the wound from postoperative day 1, which was settled within postoperative day 3 with daily dressings and antibiotics. In two cases with deep infection, the discharge persisted up to postoperative day 7. Though the rate of infection was quite high, all patients were managed with dressings and antibiotics, and the final outcome was not affected. In closed multiple metacarpal fractures, plate fixation is a good option for several reasons. These fractures are highly unstable, and stable fixation is required in these fractures \[[@CR9]\]. Metacarpal length is very likely to be shortened in multiple metacarpal fractures, causing instability \[[@CR6], [@CR7]\]. This effect is greater in internal metacarpals (third and fourth metacarpals) than in border metacarpals (second and fifth metacarpals), because the latter are anchored on both sides of the metacarpal head \[[@CR8]\]. Closed ipsilateral multiple metacarpal fractures are frequently associated with more soft tissue injury as compared with single fracture, making them more susceptible to stiffness and poor functional results. Osteosynthesis using miniature plates and screws in these unstable fractures produces anatomical reduction of fractures with stabilization that is rigid enough to allow early mobilization of adjacent joints without allowing loss of reduction, thereby preventing stiffness and hence good functional results. In our study, we found a 100% union rate, with 85.71% (18 of 21) excellent and 9% (2 of 21) good results according to the American Society for Surgery of the Hand (ASSH) Total Active Flexion (TAF) score. Mean DASH score was 8.47 (range 1--26). Rigid and stable fixation with mini plates allowed early mobilization, which prevented stiffness and achieved good functional result. Though the infection rate was high, it was managed with dressings and antibiotics in all patients. In conclusion, plate fixation is a good option for treating closed multiple metacarpal fractures, providing rigid fixation for early mobilization and good functional outcome. None. Open Access {#d30e564} =========== This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited.

Background {#Sec1} ========== Ventriculo-atrial defects are rare defects, first described in 1958 by Gerbode \[[@CR1]\]. These defects until now were subdivided into two types Type-1 Gerbode defect is an acquired defect through the ventriculo-atrial membranous septum, resulting in a direct left ventricle to right atrium shunt. Type-2 Gerbode defect is an indirect congenital defect, there are two defects present, a ventricle septum defect and a defect in the tricuspid septal leaflet, thus creating an indirect left to right shunt. In this paper we present a case of a third type Gerbode defect, a peri-prosthetic defect with a left-to right shunt due to prosthesis dehiscence, creating a direct shunt from the left ventricle and aortic annulus into the right atrium. Acquired ventriculo-atrial defects can occur due to infectious endocarditis, ischemic heart disease or following tricuspid or mitral valve surgery \[[@CR2]\]-\[[@CR4]\]. On echocardiographic examination a systolic high-pressure jet can usually be detected, caused by the defect. This jet is sometimes misinterpreted as severe asymmetrical tricuspid regurgitation, wrongly suggesting increased pulmonary pressure \[[@CR5]\],\[[@CR6]\]. In this report we present a successful surgical treatment of an acquired ventriculo-atrial defect, tricuspid insufficiency, anterior mitral leaflet and bioprosthetic aortic valve endocarditis. Case presentation {#Sec2} ================= A 71-year-old Caucasian male, with a history of aortic valve replacement in 2010 (Carpentier Edwards Perimount Magna Ease® stented bioprothesis, Ø 27 mm), was one year later referred to our academic center, with symptoms of fever (38.5°C) and malaise after two weeks of unsuccessful treatment (Amoxicillin) for suspected urinary-tract infection. A trans-thoracic echocardiographic examination was performed it revealed mild tricuspid regurgitation and an mobile echo density with clear delineated borders, adjacent to the tricuspid valve, raising the suspicion for infective endocarditis. Nine days after admission patient suddenly developed a 3rd degree atrioventricular block and signs of forward cardiac failure. Transesophageal echocardiography (TEE) revealed a mobile structure near the septal leaflet of the tricuspid valve, grade III Tricuspid Regurgitation (TR) and a suspected abscess of the aortic annulus at the level of the Non Coronary Cusp (NCC) (Figures [1](#Fig1){ref-type="fig"} and [2](#Fig2){ref-type="fig"}).Figure 1**Vegetation can be seen at the tricuspid annulus near the septal leaflet.** Echocardigraphy suggests tricuspid leaflet involvement, however the intra-operative image shows the vegetation on the atrial septal wall.Figure 2**Transesophageal echocardiograpy views.** Panel A: Mid-esophageal RV inflow/outflow view shows a paravalvular abcess near the atrial septum and involvment of the tricuspid valve insertion (arrow). Panel **B**: Mide-Esophageal long axis view showing vegetation on the aortic valve prosthesis. Panel **C**: zoomed in on the atrial septum, showing the Gerbode like defect with turblent flow (arrow). Panel **D**: severe tricuspid insufficiency (arrow). The need for external pacing with increasing systemic malperfusion (oliguria), combined with a positive response to antibiotics, signified by dropping infectious parameters led to the decision for surgical treatment at this stage. Surgical procedure {#Sec3} ------------------ After induction of anesthesia, sternotomy and careful dissection of the severe adhesions it was noted that the aortic ascending aortic arch was dilated (Ø 5 cm). After initiation of cardio pulmonary bypass the aorta was clamped and combined antegrade & retrograde cold blood cardioplegia was given. After aortotomy the previously implanted aortic valve was noted to have vegetations most prominently on the non-coronary cusp. A paravalvular abscess cavity was found, which extended subvalvular into the left ventricle and continued to the right atrium, Gerbode-like defect (Figure [3](#Fig3){ref-type="fig"}). After atriotomy inspection of the Tricuspid valve revealed vegetations of the septal leaflet and a severe annular dilation (\>40 mm) causing severe Tricuspid regurgitation. After explantation of the aortic prosthetic valve, vegetations were seen on the Anterior Mitral Leaflet (AML). A partial resection of the base of the AML was also performed. Reconstruction of the aorto-mitral continuity was done with a pericardial patch using single stitch technique.Figure 3**Intraoperative image showing a clamp going into the right atrium (arrows) and exiting in the left ventricle (the defect runs from subvalvular to epi-annular).** The aortic root and dilated ascending aorta were reconstructed with a Vascutec® prosthesis (Ø 26 mm Vascutec Vascular prosthesis, Glasgow, United Kingdom). The bioprosthetic aortic valve was replaced by a new stentless aortic bioprosthesis. Tricuspid annuloplasty was performed using the DeVega technique. Finally the Gerbode defect was closed with felt pledges and sutures (Figure [4](#Fig4){ref-type="fig"}).Figure 4**Schematic images of the situation before and after surgery, top right, shows the Gerbode-like defect and initial prosthetic valve.** Bottom right shows the situation after surgery with the Vascutek prosthesis, new biological valve, reconstruction of the Aorto-Mitral continuity and the DeVega annuloplasty. TEE examination showed adequate pump function at the end of surgery. It also showed normal function of the mitral and aortic valves and only grade I Tricuspid regurgitation. After achieving adequate hemostasis, closure was performed in the standard fashion and the patient was transported to the ICU in a hemodynamically stable condition. Recovery was uneventful, patient was discharged in a good clinical condition. Conclusions {#Sec4} =========== Our patient had a ventriculo-atrial defect due to infective endocarditis, valve cultures were positive for streptococcus agalactiae, originating from the aortic root adjacent to his bioprosthetic aortic valve, implanted 1 year earlier. The diagnosis of infective endocarditis is usually made according to the Duke criteria \[[@CR7]\]. Based on a combination of minor and major criteria to confirm endocarditis, as a possible diagnosis or reject it. In our case positive blood cultures, fever and predisposition (artificial valve), together with findings on TEE, confirmed the diagnosis. Infective endocarditis is a serious illness with mortality rates ranging from 9.6-26% \[[@CR8]\]-\[[@CR10]\]. Mainstay of treatment for infective endocarditis is antibiotic treatment, patient was treated with penicillin. Surgical treatment is reserved for treatment of complications due to degenerative disease process or resistant micro-organisms \[[@CR11]\]. The indications for surgery can be divided into heart failure, uncontrolled infection and prevention of embolism \[[@CR11]\]. This case represents a severe endocarditis; not only did our patient have heart failure (as evidenced by the oliguria and need for pacing) in addition there was uncontrolled infection (paravalvular abscess) as well as fistula formation leading to hemodynamic compromise. Atrioventricular conduction runs through fibers in the atrioventricular septum our patient developed a 3rd degree AV block indicating inter-atrial septum conductive pathway involvement. Cause of which was likely the formation of the paravalvular abscess. During the surgical procedure the extent of the damage was greater than initially anticipated with involvement of not only the aortic, but also the tricuspid and mitral valve. The Gerbode defect was not seen initially on echocardiography due to the existence of severe tricuspid regurgitation hiding the jet from the left ventricle entering the right atrium. Decision to proceed to surgery was based on the suspicion of infective endocarditis with clinical deterioration despite antibiotic treatment. Combination of forward cardiac failure together with arrhythmia ultimately dictated surgical management. In retrospect the peculiar object seen in the right atrium (Figure [1](#Fig1){ref-type="fig"}) and vegetations on the bioprosthesis (Figure [5](#Fig5){ref-type="fig"}) could have been seen as evidence of septal involvement.Figure 5**Aortic valve prosthesis after explantation with severe vegetations.** Ventriculo-atrial defects are complex entities, which require a thorough understanding of pathophysiology. Echocardiographic images should be looked at with great care since inter-septal jets can be mistaken for valve insufficiencies, and the true extent of valvular involvement can be underestimated. Patients with endocarditis are continuously at risk for severe complications, heart failure, sepsis and abscess formation. Rapid decision-making should involve a multi-disciplinary approach. These complex cases need to be addressed with utmost care in experienced clinical centers. Consent {#Sec5} ======= Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Authors' original submitted files for images {#Sec7} ============================================ Below are the links to the authors' original submitted files for images.Authors' original file for figure 1Authors' original file for figure 2Authors' original file for figure 3Authors' original file for figure 4Authors' original file for figure 5 TEE : Transesophageal echocardiography TR : Tricuspid regurgitation (TR) NCC : Non coronary cusp (NCC) ICU : Intensive care unit **Competing interest** The authors declare that they have no competing interests. **Authors\' contribution** JJ performed literature search and wrote the draft, RB participated in drafting the article JW provided the necessary photo\'s and critically revised the text, AW contributed to the design of the drawings, GM critically revised the date, EN performed the surgery, designed the paper and gave final approval for submission of this manuscript. All authors read and approved the final manuscript.

All nucleotide sequence files are available from the NCBI GenBank database (accession numbers KT279554 to KT279561). Introduction {#sec001} ============ *Staphylococcus aureus* is one of the leading human pathogens worldwide. It causes a broad range of diseases from superficial infections to life-threatening invasive diseases. Antimicrobial therapy is sometimes ineffective, owing to the development of antimicrobial-resistant strains, such as methicillin-resistant *S*. *aureus* (MRSA) \[[@pone.0149112.ref001]\]. *S*. *aureus* expresses various virulence factors, including a broad range of exotoxins. Some of these toxins, such as most of the phenol-soluble modulins and α-hemolysin (also known as α-toxin), are encoded in the core-genome; while others, such as Panton-Valentine leucocidin, are encoded by acquired mobile genetic elements \[[@pone.0149112.ref002], [@pone.0149112.ref003]\]. The α-hemolysin, which belongs to a class of small β-barrel pore-forming cytotoxins, is a major virulence factor in *S*. *aureus* infections \[[@pone.0149112.ref002], [@pone.0149112.ref004]\]. It is encoded by the 960-bp *hla* gene, which is initially produced as a 319-residue precursor, then processed to a 293-residue (approximately 33 kDa) mature toxin \[[@pone.0149112.ref004], [@pone.0149112.ref005]\]. Previous studies have proven that changes in key amino acid residues of α-hemolysin, such as a histidine substitution at amino acid 35, can result in reduction or even loss of virulence \[[@pone.0149112.ref006], [@pone.0149112.ref007]\]. In this study, we determined the genotypes of *hla* in 47 *S*. *aureus* isolates collected in China, and compared this with the clonal background of the isolates. For comparison, *hla* genotype and clonal background were also determined for well-characterized and published whole genome sequences of 318 global strains by *in silico* analysis. Methods {#sec002} ======= Ethics {#sec003} ------ The study was approved by the Human Research Ethics Committee of Peking Union Medical College Hospital (No. S-263). Written consent was obtained from patients, and the study was carried out in accordance with approved guidelines. *Staphylococcus aureus* isolates {#sec004} -------------------------------- Isolates were collected from patients with *S*. *aureus* infections in Beijing Electric Power Hospital during January 2011 to June 2011. A total of 47 *S*. *aureus* isolates were collected consecutively, each from different individuals. The most common type of infection was pneumonia (n = 31, 66.0%), followed by soft tissue infection (n = 6, 12.8%), urinary tract infection (n = 6, 12.8%), bloodstream infection (n = 2, 4.3%), joint infection (n = 1, 2.1%), and gallbladder infection (n = 1, 2.1%). Molecular identification of isolates and *mecA* gene detection {#sec005} -------------------------------------------------------------- DNA extraction of *S*. *aureus* isolates was performed as previously described \[[@pone.0149112.ref008]\]. A multiplex PCR was used for simultaneous amplification of 16S rRNA, *femA* and *mecA* genes for identification and differentiation of methicillin-susceptible *S*. *aureus* (MSSA) and MRSA isolates \[[@pone.0149112.ref009]\]. Genotyping of the 960-bp *hla* gene {#sec006} ----------------------------------- Primer pair of hlaF1 (`5’- TTAGCCGAAAAACATCATTTC-3’`) and hlaR2 (`5’- TTATTCCCGACGAAATTCCAA-3’`) was designed for amplification of the complete 960-bp *hla* gene encoding the α-hemolysin precursor, which was made up of a 78-bp nucleotide sequence encoding the 26 aa signal peptide, followed by the 882-bp nucleotide sequence encoding the 293 aa mature α-hemolysin. Each PCR mix contained 12.5 μL of 2× EasyTaq PCR SuperMix (TransGen Biotech, Beijing, China), 2 μL of DNA template, 0.5 μM of each forward and reverse primer, and molecular biology grade water (TransGen Biotech) added to make a total volume of 25 μL. PCR was performed as follows: initial denaturation at 95°C for 15 min, followed by 30 cycles of 94°C for 2 min, 55°C for 2 min, 72°C for 2 min, with a final extension at 72°C for 10 min. The products were sequenced in both directions using the inner primer pair hlaF2 (`5’- GAAGTTATCGGCTAAAGTTATAA-3’`) and hlaR1 (`5’- CATAATTAATACCCTTTTTCTC-3’`) on the DNA analyzer ABI 3730XL system (Applied Biosystems, Foster City, CA). The obtained 960-bp *hla* gene sequences were compared to a wild-type reference sequence from *S*. *aureus* strain WOOD 46 (GenBank accession no. X01645) \[[@pone.0149112.ref010]\], aligned using CLC sequence viewer (version 7, QIAGEN Aarhus, Denmark) to detect single nucleotide polymerases (SNPs), and designate genotypes (the *hla* DNA sequence was identical for all isolates belonging to a given genotype). Further, the α-hemolysin peptide sequences of isolates were deduced and aligned to determine the presence of amino acid substitutions. Assignment of clonal background {#sec007} ------------------------------- All isolates were analyzed by multilocus sequence typing (MLST) and *spa* typing using previously established methods \[[@pone.0149112.ref011], [@pone.0149112.ref012]\]. Assignment of related sequence types (STs) into clonal complexes (CCs) was conducted using eBURST \[[@pone.0149112.ref013]\]. In addition, all MRSA isolates were characterized by staphylococcal cassette chromosome *mec* (SCC*mec*) typing as described by Chen et al. \[[@pone.0149112.ref014]\]. *S*. *aureus* clones were named in the format of ST-*spa* type or ST-SCC*mec* type-*spa* type (e.g. ST5-t002 for a MSSA clone, or ST239-III-t030 for a MRSA clone). *In silico* analysis of published whole genome sequences {#sec008} -------------------------------------------------------- Because only limited number of *S*. *aureus* isolates was involved in the present study, to better define the *hla* gene diversity and association with clonal background amongst *S*. *aureus* with more different genetic background, 318 selected well-characterized published genomes derived from other geographic regions were further studied. The 318 selected published genomes included i) genetic information of all *S*. *aureus* isolates with complete assembled whole genome sequences as at July 8 2015 were obtained from the NCBI Genome database (70 isolates, [S1 Table](#pone.0149112.s001){ref-type="supplementary-material"}), and ii) *S*. *aureus* genome sequences from four previous publications (248 isolates, [S2 Table](#pone.0149112.s002){ref-type="supplementary-material"}) \[[@pone.0149112.ref015]--[@pone.0149112.ref018]\]. These genomes comprised examples from the major worldwide lineages of MRSA, e.g. CC8 (including USA-300), CC1 (including USA 400), CC22 (including EMRSA15), CC93 (including Queensland CA-MRSA), CC30 (including EMRSA16) and CC121 isolates. The MLST STs and *hla* genotypes of the above isolates were determined by *in silico* mapping the paired-end reads of 318 isolates to seven gene loci sequences of *S*. *aureus* ST1 and wild-type *hla* reference sequence (GenBank accession no. X01645), respectively, using Burrows-Wheeler Alignment \[[@pone.0149112.ref019]\]. Base coverage of each position of genes was assessed using SAMtools mpileup packages (<http://samtools.sourceforge.net/mpileup.shtml>). The support number of reference base (ref) and alternative base (alt) are examined at each position in each strain. High quality SNPs were defined when SNPs satisfied the criteria of alt/(alt+ref)\>0.8. According to these SNPs, the ST and *hla* genotype for each isolate was identified. MLST STs and *hla* genotypes were entered into BioNumerics software v7.5 (Applied Maths, Austin, TX) for minimum-spanning-tree analysis. The diversity of the sequences of the *hla* gene and seven genes utilized in the MLST scheme for *S*. *aureus* was analyzed by DnaSP (version 5.1, University of Barcelona, Spain). Results {#sec009} ======= Molecular identification and detection of the *mecA* gene {#sec010} --------------------------------------------------------- All 47 isolates were confirmed as *S*. *aureus* by molecular methods. Thirty-three isolates (70.2%) were determined to be MRSA by detection of the *mecA* gene. Genotypes of the 960-bp *hla* gene {#sec011} ---------------------------------- Amongst the 47 isolates from China, a total of eight *hla* genotypes (genotype 1 to 8) and six peptide sequence types were identified (Tables [1](#pone.0149112.t001){ref-type="table"} and [2](#pone.0149112.t002){ref-type="table"}). Of the eight *hla* genotypes, genotype 1 was predominant (n = 27, 57% of 47 isolates), followed by genotype 3 (n = 7, 15%) and genotype 5 (n = 3, 6%). The remaining four genotypes were rare, with one or two isolates belonging to each ([Table 1](#pone.0149112.t001){ref-type="table"}). 10.1371/journal.pone.0149112.t001 ###### Nucleotide mutations and corresponding amino acid substitutions of the eight *S*. *aureus hla* genotypes identified from 47 isolates from China. {#pone.0149112.t001g} Nucleotide mutation position/corresponding amino acid substitution[^a^](#t001fn002){ref-type="table-fn"} ------------------------------------------------- ---------------------------------------------------------------------------------------------------------- ----- -------- ----- ----- ----- ----- ----- ------------------------------------------- ------------------------------------------- ------------------------------------------- ------------------------------------------- ------------------------------------------- ------------------------------------------- ------------------------------------------- --------------------------------------------- ------------------------------------------- ------------ Wild type[^a^](#t001fn002){ref-type="table-fn"} G G G G C T C G T C T T G A T T C A X01645.1 Genotype 1 A/ A/ AA/ T/ --- --- --- --- --- C/ --- --- --- C/ C/ T/ --- KT279554.1 R(-23)H Syn G(-2)N Syn Syn Syn Syn T275I Genotype 2 A/ A/ AA/ T/ --- --- --- --- --- C/ --- --- G/ C/ C/ T/ --- KT279555.1 R(-23)H Syn G(-2)N Syn Syn Syn Syn Syn T275I Genotype 3 --- A/ AA/ --- --- --- --- --- --- --- G/ --- --- C/ --- --- --- KT279558.1 Syn G(-2)N D208E Syn Genotype 4 --- A/ AA/ --- A/ T/ C/ G/ --- C/ --- T/ --- C/ --- --- --- KT279556.1 Syn G(-2)N Syn Syn Syn Syn Syn Syn Syn Genotype 5 --- A/ AA/ --- --- --- --- --- --- --- --- --- --- --- --- --- --- KT279559.1 Syn G(-2)N Genotype 6 --- A/ AA/ --- --- --- --- --- --- --- G/ --- --- C/ --- --- T/ KT279557.1 Syn G(-2)N D208E Syn Syn Genotype 7 --- A/ AA/ T/ --- --- --- --- --- C/ --- --- --- C/ C/ T/ --- KT279561.1 Syn G(-2)N Syn Syn Syn Syn T275I Genotype 8 --- A/ AA/ T/ --- --- --- --- Del[^b^](#t001fn003){ref-type="table-fn"} C/ ---[^b^](#t001fn003){ref-type="table-fn"} ---[^b^](#t001fn003){ref-type="table-fn"} ---[^b^](#t001fn003){ref-type="table-fn"} C/ C/ T/ ---[^b^](#t001fn003){ref-type="table-fn"} KT279560.1 Syn G(-2)N Syn Syn[^b^](#t001fn003){ref-type="table-fn"} Syn[^b^](#t001fn003){ref-type="table-fn"} Syn[^b^](#t001fn003){ref-type="table-fn"} T275I[^b^](#t001fn003){ref-type="table-fn"} Abbreviations: Syn, synonymous mutation; Del, deletion mutation; N/A, not applicable; "---", no mutation. ^a^Nucleotide/peptide positions were designated relative to the first nucleotide/amino acid of the mature α-hemolysin (*S*. *aureus* strain WOOD 46, nucleotide sequence GenBank accession no. X01645). ^b^The deletion at nucleotide position 479 of genotype 8 resulted in peptide termination at residue position 164. 10.1371/journal.pone.0149112.t002 ###### Relationship between *hla* genotypes and molecular clone backgrounds of 47 *S*. *aureus* isolates from China. {#pone.0149112.t002g} *hla* genotype Molecular clone Clonal Complex (CC) No. of isolates ----------------------------- ---------------------- --------------------- ----------------- **Peptide sequence type 1** Genotype 1 ST239-MRSA-III-t030 CC8 13 Genotype 1 ST239-MRSA-III-t037 CC8 8 Genotype 1 ST239-MRSA-III-t2270 CC8 5 Genotype 1 ST239-MRSA-III-t459 CC8 1 Genotype 2 ST239-MRSA-III-t030 CC8 2 **Peptide sequence type 2** Genotype 3 ST5-MRSA-II-t002 CC5 3 Genotype 3 ST5-MSSA-t002 CC5 3 Genotype 3 ST25-MSSA-t078 CC25 1 **Peptide sequence type 3** Genotype 4 ST59-MRSA-IV-t437 CC59 1 Genotype 4 ST59-MSSA-t163 CC59 1 Genotype 5 ST1-MSSA-t127 CC1 3 **Peptide sequence type 4** Genotype 6 ST6-MSSA-t2467 CC6 1 Genotype 6 ST6-MSSA-t701 CC6 1 Genotype 6 ST7-MSSA-t091 CC7 1 Genotype 6 ST7-MSSA-t796 CC7 1 **Peptide sequence type 5** Genotype 7 ST188-MSSA-t189 CC1 1 **Peptide sequence type 6** Genotype 8 ST188-MSSA-t189 CC1 1 Eighteen SNPs were found, including four on the signal peptide encoding portion (nucleotide positions -68, -22, -6 and -5 relative to the first nucleotide of mature α-hemolysin portion), and 14 on the mature α-hemolysin encoding sequence (nucleotide positions 144, 177, 399, 438, 453, 479, 606, 624, 669, 708, 765, 777, 824 and 864) ([Table 1](#pone.0149112.t001){ref-type="table"}). The majority of the SNPs (12/18, 67%) were synonymous---only five (28%, nucleotide positions -68, -6, -5, 624 and 824) were nonsynonymous. Lastly, a deletion mutation was detected in nucleotide position 479 of genotype 8, presumably resulting in a prematurely terminating transcript ([Table 1](#pone.0149112.t001){ref-type="table"}). Of the 18 SNPs identified, nucleotide position -68 was ST239-specific, positions 177, 399, 438,453 and 669 were ST59-specific, position 765 was ST1-specific, whilst the other 11 were not lineage specific. Clonal background of *S*. *aureus* isolates {#sec012} ------------------------------------------- A total of six ST-SCC*mec*-*spa* types and nine ST-*spa* types were identified amongst 32 MRSA and 15 MSSA isolates from China, respectively ([Table 2](#pone.0149112.t002){ref-type="table"}). 88% (29/33) of the MRSA isolates belonged to CC8, 9% (3/33) belonged to CC5, and 3% (1/33) belonged to CC59. The ST239-III-t030 clone comprised over half of the CC8 MRSA isolates (15/29, 52%), followed by ST239-III-t037 (8/29, 28%), ST239-III-t2270 (5/29, 17%) and ST239-MRSA-III-t459 (1/29, 4%). All three CC5 MRSA isolates were ST5-II-t002 ([Table 2](#pone.0149112.t002){ref-type="table"}). In comparison, the distribution of MSSA clones was more diverse, with no clone comprised of more than three isolates. ST5-t002 was represented by three MRSA and three MSSA isolates in this study, and ST59 was represented by one MRSA and one MSSA isolates with differing *spa* types. No other ST or CC was common to both MRSA and MSSA isolates ([Table 2](#pone.0149112.t002){ref-type="table"}). Relationship between *hla* genotypes and clonal background {#sec013} ---------------------------------------------------------- A strong correlation was observed between both *hla* genotypes and α-hemolysin peptide sequence types, and the clonal background of isolates from China. Of the eight *hla* genotypes, six were restricted to either MRSA (genotypes 1 and 2) or MSSA (genotypes 5, 6, 7 and 8) strains. The predominant MRSA clone, ST239-MRSA-III-t030, was represented by *hla* genotype 1 (13/15 isolates, 86.7%) or genotype 2 (2/15 isolates, 13.3%). All of the remaining ST239-MRSA-III isolates possessed *hla* genotype 1. Both genotype 1 and 2 *hla* genes encoded α-hemolysin of peptide sequence type 1 ([Table 2](#pone.0149112.t002){ref-type="table"}). *hla* genotypes 5 and 6 were found in one and four MSSA clones, respectively. Of the two ST188-MSSA-t189 isolates, one possessed *hla* genotype 7 and the other genotype 8, which differed by one deletion mutation at nucleotide position 479 ([Table 1](#pone.0149112.t001){ref-type="table"}). The remaining two *hla* genotypes, genotypes 2 and 3, were represented by both MRSA and MSSA isolates. Genotype 2 was found in six isolates of ST5-t002 (including three isolates each of ST5-MRSA-II-t002 and ST5-MSSA-t002) and one isolate of ST25-MSSA-t078. Genotype 3 was found in two ST59 isolates (one isolate each of ST59-MRSA-IV-t437 and ST59-MSSA-t163), and one isolate of ST1-MSSA-t127. The *in silico* analysis of 318 well-characterized genomes {#sec014} ---------------------------------------------------------- Amongst 318 well-characterized *S*. *aureus* genomes, 25 *hla* genotypes were identified, including 20 genotypes not found in the 47 isolates from China ([S1](#pone.0149112.s001){ref-type="supplementary-material"} and [S2](#pone.0149112.s002){ref-type="supplementary-material"} Tables), for a total of 28 *hla* genotypes identified in this study. The SNPs identified for all clinical isolates and published genomes are summarized in [S3 Table](#pone.0149112.s003){ref-type="supplementary-material"}. Substantial diversity amongst *S*. *aureus hla* gene was found. Compared to the seven loci utilized in the MLST, the *hla* gene had higher nucleotide diversity (0.0256 vs. 0.0042--0.0119), more haplotypes identified (28 vs. 10--19), greater haplotype diversity (0.899 vs. 0.517--0.804) and higher non-synonymous polymorphisms/ synonymous sites ratio (3.598 vs. 3.059--3.569) ([Table 3](#pone.0149112.t003){ref-type="table"}). Of note, 79 of 107 ST22 *S*. *aureus* isolates and one ST188 isolate (*hla* genotype 8) had one to 16 deletion mutations in their *hla* sequences ([S3 Table](#pone.0149112.s003){ref-type="supplementary-material"}). In addition, all 20 ST36 isolates had a SNP C→T at sequence position 259, which resulted in a premature stop codon ([S3 Table](#pone.0149112.s003){ref-type="supplementary-material"}). These mutations would presumably inhibit production of the toxin protein. 10.1371/journal.pone.0149112.t003 ###### Comparison of genetic diversity between the *hla* gene and seven gene loci utilized in the multilocus sequence typing scheme. {#pone.0149112.t003g} Multilocus sequence typing loci ------------------------------------------------------ -------- --------------------------------- -------- -------- -------- -------- -------- ---------- Nucleotide diversity 0.0256 0.0083 0.0093 0.0042 0.0083 0.0079 0.0119 0.0073 No. of haplotypes 28 13 15 12 10 18 19 19 Haplotype diversity 0.899 0.760 0.796 0.517 0.726 0.804 0.800 0.799 No. of non-synonymous sites 730.09 350.45 356.18 350.45 327.30 362.89 312.13 400.19 No. of synonymous sites 202.91 105.55 99.82 114.55 91.70 111.11 89.87 115.81 No. of non-synonymous sites/ no. of synonymous sites 3.598 3.320 3.568 3.059 3.569 3.266 3.473 3.455574 In combining the 47 isolates from China and the 318 globally distributed genomes for composite analysis, it was again noted that *hla* genotype was closely related to the clonal background of the isolate, in particular the ST, with little association with the geographic origin, host source or methicillin-resistance phenotype ([Table 2](#pone.0149112.t002){ref-type="table"}, [S1](#pone.0149112.s001){ref-type="supplementary-material"} and [S2](#pone.0149112.s002){ref-type="supplementary-material"} Tables and [Fig 1](#pone.0149112.g001){ref-type="fig"}). The minimum-spanning tree analysis of MLST data shown that for 30 of 33 STs identified in the present study, isolates belonging to the same ST shared a unique *hla* genotype, and ST22 was the only sequence type that comprised more than three (seven in all) *hla* genotypes ([Fig 1](#pone.0149112.g001){ref-type="fig"}). In addition, STs belonging to the same CC frequently shared the same or close-related *hla* genotype, e.g. ST5, ST105, ST225 and ST228 of CC5 was comprised of 24 isolates belonging to *hla* genotype 3, ST8 and ST250 of CC8 was comprised of 18 isolates belonging to *hla* genotype 7, and ST95, ST121 and ST123 of CC121 was comprised of seven isolates belonging to *hla* genotype 26 ([Fig 1](#pone.0149112.g001){ref-type="fig"}). {#pone.0149112.g001} Although the same *hla* genotype may be shared by unrelated STs, this was observed uncommonly. For instance, one isolate each that belonged to ST72 and ST25 isolates were *hla* genotype 3, which was mostly associated with CC5 *S*. *aureus* isolates. Likewise, one ST188 isolate was identified as *hla* genotype 25, which was mostly associated with non-ST239 CC8 isolates. Discussion {#sec015} ========== Infections due to antimicrobial-resistant pathogens are a growing problem all over the world. In developing countries like India and China, antimicrobial resistance is particularly prevalent, owing to previous unregulated overuse of antimicrobials \[[@pone.0149112.ref020], [@pone.0149112.ref021]\]. *S*. *aureus* is one of the commonest Gram-positive bacterial pathogens, and in many places, the majority of *S*. *aureus* infections are now caused by multidrug-resistant strains, including MRSA and vancomycin-resistant *S*. *aureus* (VRSA). Immunotherapies are now being investigated as an alternative therapeutic options for staphylococcal infections in the hope that these may avoid the selection pressure associated with the use of antimicrobials \[[@pone.0149112.ref003], [@pone.0149112.ref022]\]. The *S*. *aureus* α-hemolysin was the first described of a family of bacterial pore-forming β-barrel toxins, which play an important role in the pathogenesis of staphylococcal disease \[[@pone.0149112.ref004], [@pone.0149112.ref023]\]. As such, it was chosen as a potential target for the development of vaccines to combat *S*. *aureus* infections, and positive results have been obtained in some preclinical trials targeting pneumonia and skin and soft tissue infections \[[@pone.0149112.ref023]--[@pone.0149112.ref026]\]. It has been noted that substitutions in amino acid residues may reduce the activity of α-hemolysin. For instance, a α-hemolysin mutant with a H35L substitution was found to have no hemolytic or lethal activity, despite retaining the ability to bind to target cells \[[@pone.0149112.ref006], [@pone.0149112.ref007]\]. The EMRSA-16 CC30 *S*. *aureus* isolates were another example. As observed in the present study, and as reported elsewhere, CC30-ST36 isolates had a SNP C→T at nucleotide sequence position 259, which resulted in a premature stop codon \[[@pone.0149112.ref027], [@pone.0149112.ref028]\]. It has been proven that CC30 isolates possessed this SNP had significantly reduced toxin production and decreased lethality in a mouse model \[[@pone.0149112.ref027]\]. These α-hemolysin mutants could be considered as candidate immunogens in prototypic *S*. *aureus* vaccines \[[@pone.0149112.ref023]--[@pone.0149112.ref026], [@pone.0149112.ref029]\]. Despite this work, little has been described regarding the genetic polymorphism of the *hla* gene in *S*. *aureus*. This is an important consideration, since variation in α-hemolysin peptide sequences could potentially lead to failure in antigen-antibody binding and thus compromise vaccine efficacy. In this study, we have illustrated the diversity of the *hla* gene in *S*. *aureus* and the relationship of *hla* sequence with clonal background, using 47 *S*. *aureus* clinical isolates from China supplemented with 318 well-characterized and globally distributed isolates with published whole genome sequences. All ST239 from China were MRSA, and carried either genotype 1 or genotype 2 *hla*. These two genotypes differed by just one synonymous nucleotide mutation, (peptide sequence type 1). Amongst the 70 global *S*. *aureus* isolates with published genomes, seven isolates were ST239-MRSA-III, all of which also carried genotype 1 *hla*, regardless of the isolates' geographic origins. The ST239-MRSA-III clone has been reported largely to be hospital-acquired and widely disseminated in Brazil, Australia, New Zealand and many Asian countries in the past decade, although the prevalence different *spa* types within this clone (e.g. *spa* type t030 and t037) vary in different regions \[[@pone.0149112.ref008], [@pone.0149112.ref030], [@pone.0149112.ref031]\]. In a previous genome-based phylogeographic analysis, it was determined that human movement played an important role in the global dissemination of ST239-MRSA-III \[[@pone.0149112.ref032]\]. Therefore, the consistent *hla* genotype of this clone across different regions is not surprising. Interestingly, all of the 14 ST5 *S*. *aureus* isolates (six clinical isolates from China and eight global strains), including nine ST5-MRSA-II and five ST5-MSSA strains, possessed genotype 3 *hla*. Genotype 3 *hla* was also found in other CC5 *S*. *aureus* clones, including ST228-MRSA-I-t041 (n = 8), ST105-MRSA-II-t002 (n = 1) and ST225-MRSA-II-t003 (n = 1). Likewise all six ST59 isolates analyzed in this study, despite diverse methicillin-resistance phenotypes, SCC*mec* and *spa* types, carried genotype 4 *hla*. The ST59 lineage is primarily a community-acquired MRSA clone predominant in China and several other Asian countries \[[@pone.0149112.ref033]\]. These results again indicate that the *hla* genotype correlated closely with the ST. Meanwhile, ST22 isolates shown significantly higher *hla* genotype diversity (comprised seven *hla* genotypes in all) than other *S*. *aureus* clones. Only occasional discrepancies between *hla* genotype and ST were observed. Future vaccine development will need to account for the influence of this diversity on vaccine effect. Conclusion {#sec016} ========== We have found substantial diversity amongst *S*. *aureus hla* gene and amino acid sequences. Strong correlations between *hla* genotypes and clonal background were found in *S*. *aureus*, regardless of the isolates' geographic origins and methicillin-resistance phenotype. Although the relative virulence of different *hla* genotypes remain undetermined, our investigation has provided some preliminary epidemiologic data which will be essential for future vaccine development. Supporting Information {#sec017} ====================== ###### *In silico* analysis of *hla* genotype and clonal background of 70 complete assembled whole genome sequences of *S*. *aureus*. (DOCX) ###### Click here for additional data file. ###### *In silico* analysis of *hla* genotype and clonal background of and 248 *S*. *aureus* genomes from four previous publications. (DOCX) ###### Click here for additional data file. ###### Alignment of 365 *hla* sequences obtained from 47 China clinical isolates and 318 published genomes, and SNPs identified. (XLSX) ###### Click here for additional data file. [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: MX RZ YCX. Performed the experiments: MX QZ XF. Analyzed the data: MX MOS DFL XYW HLW. Contributed reagents/materials/analysis tools: MX FK. Wrote the paper: MX MOS FK.

Dara are available at: <https://datadryad.org/review?doi=doi:10.5061/dryad.jh655f3>. Introduction {#sec001} ============ Human beings are constantly confronted with choices that have explicit moral dimensions. There has been much philosophical research about how to properly guide and judge these choices from an ethical point of view, leading to two popular traditions: *consequentialism* and *deontological ethics*. Consequentialism states that choices are to be assessed solely by their expected consequences and the states of affairs they bring about. Positions of this kind are often seen to have their intellectual predecessor in Utilitarianism as promoted by Jeremy Bentham and the work of John Stuart Mill, exemplified in Mill's statement that "actions are right in proportion as they tend to promote happiness, wrong as they tend to produce the reverse of happiness" \[[@pone.0205066.ref001]\]. Along the lines of the consequentialist tradition, the consequences of an action (and thereby their moral value) have often been classified in terms of happiness or use--thus generally stating that an action is morally *good* if it brings about favorable consequences. Consequentialist views are often contrasted with deontological ethics. Here, the moral value of actions and choices is not to be evaluated solely on grounds of their consequences. An action is to be evaluated as morally good or bad if it is instantiating or violating certain ethical norms, respectively. Deontological ethics often claim to be of Kantian origin as Immanuel Kant prominently advocated an ethical framework relying on categorical norms and duties \[[@pone.0205066.ref002]\]. One example of fundamental importance is his *practical imperative* that demands respect for human beings as such: "So act that you use humanity, whether in your own person or in the person of any other, always at the same time as an end, never merely as a means" \[[@pone.0205066.ref002]\]. In Kantian and much of deontological ethics in general, it is thus prohibited to use human beings solely to achieve one's goals--thereby demanding a certain respect for human life as such. Norms and duties of this kind have to be followed even if by that unfavorable overall consequences are to be expected. This philosophical discourse thus offers a possible background against which one can evaluate moral decision making and ethical behavior. While the co-existence of the deontological and the consequentialist positions is defendable from a philosophical viewpoint, the persistence of deontological choices is puzzling from an evolutionary perspective, as deontological decisions may have suboptimal consequences for both decision makers and the society as a whole. As a particularly emblematic example, consider a hypothetical deontological world in which nobody lies, regardless of consequences, compared to a corresponding consequentialist world in which all people lie if the consequences of lying are good for themselves and for the society as a whole (lies which benefit all parties involved are usually called *Pareto white lies* \[[@pone.0205066.ref003]--[@pone.0205066.ref005]\]). By definition, both the individual and the society as a whole would be worse off in the deontological world than in the competing consequentialist world. Of course, this argument is not restricted to the case of lying but applies to any moral rule X: a deontological world in which people follow X regardless of consequences would soon be invaded by people who follow X whenever following X benefits all parties involved, and do otherwise if not. In light of these and similar considerations, why do deontological choices persist in human societies? Several explanations have been put forward. One stems from the remark that consequentialist judgments require the ability to evaluate all possible alternatives in a window of time that is often prohibitive for the limited cognitive capabilities of humans. In the hypothetical consequentialist world introduced above, its inhabitants would have great difficulty to assess whether lying or telling the truth is more advantageous--simply because it is beyond their cognitive capacities to see and evaluate all the consequences of their actions. This implies that the moral value of an action may in practice be inaccessible if one adopts a consequentialist view. This practical inaccessibility makes the development of heuristics, simple short-cuts and rules of thumb, that generally work well on common circumstances, plausible \[[@pone.0205066.ref006]\]. Following this line of argument, it has been proposed that "deontological philosophy, rather than being grounded in moral *reasoning*, is to a large extent an exercise in moral rationalization" \[[@pone.0205066.ref007]\]. (See also \[[@pone.0205066.ref008]--[@pone.0205066.ref009]\]). From this point of view, deontological ethics is not inconsistent with an evolutionary framework, as it may have developed as a set of simple rules that allows us to make reasonably good decisions with little effort, in situations in which it is practically impossible to assess all the consequences of one's actions \[[@pone.0205066.ref010]\]. Even more radically, it has been argued that "outside the very narrow domain in which consequences can be unambiguously anticipated, it is not clear at all that calculation processes optimize the outcomes" \[[@pone.0205066.ref011]\]. See \[[@pone.0205066.ref012]\] for a criticism of this radical position. In any case, consistent with this general viewpoint, empirical studies have repeatedly shown that intuitive judgments tend to be characteristically deontological, whereas characteristically consequentialist judgments tend to result from deliberative cognitive processes \[[@pone.0205066.ref013]--[@pone.0205066.ref015]\]; that emotional engagement drive deontological choices in personal dilemmas \[[@pone.0205066.ref016]\]; that enhanced accessibility of consequentialist outcomes boosts utility maximization \[[@pone.0205066.ref017]\] and can account for the recent finding that people believe that autonomous vehicles should be utilitarian, while reporting they would buy a (non-utilitarian) car that protects the passenger over other people \[[@pone.0205066.ref018]--[@pone.0205066.ref019]\]. Another view maintains that deontological rules allow decision makers to avoid moral condemnation and punishment. In a world in which third parties judge, condemn, and punish particular actions, avoiding these actions may be an optimal strategy for avoiding condemnation \[[@pone.0205066.ref020]\]. But then, why do people condemn and punish certain behaviors, rather than others? It has been proposed that moral judgment is primarily designed as a dynamic coordination device to take sides during conflicts. Specifically, according to this view, each individual comes with a set of moral wrongs, each of which equipped with a magnitude, representing the wrongness of the respective wrong. When a conflict starts, third parties choose sides against the individual who has chosen the action with the greatest wrongness magnitude. Morally motivated punishment serves to signal which side the punisher is on. This mechanism gives rise to a secondary strategic game in which individuals try to influence the set of moral rules to serve their interests. Rawlsian moral rules (i.e., moral rules that people would choose if they did not know their own identity in society, e.g., do not kill) are favored by most people and thus are the most common. However, other moral rules may spread due to the competitive advantages they provide to groups, and they may even change over time within and between cultures due to competition within and between groups. This may explain why the sets of moral wrongs appear not to be universal \[[@pone.0205066.ref021]\]. However, the main focus of this work is a somewhat different account that has recently originated from the observation that people making deontological judgments may receive indirect evolutionary benefits, as they display (at least) two features that might signal commitment to prosociality \[[@pone.0205066.ref022]\]. This implies that people making deontological choices may have better chances to be selected as social partners, which, in turn, brings obvious long-term benefits, for example, due to direct and indirect reciprocity \[[@pone.0205066.ref023]--[@pone.0205066.ref024]\]. The first of these features is that deontological judgments are driven by the explicit prohibition of certain actions, regardless of their consequences. For example, typically deontological rules are: Don't lie, Don't steal, Don't harm--regardless of consequences. The path through which commitment to follow these rules regardless of consequences may favor social interactions is particularly well described by the following example from \[[@pone.0205066.ref022]\]: an individual who claims that stealing is always wrong will be less likely to steal from me than an individual who believes that stealing may be morally acceptable, depending on its consequences. Thus, people making deontological judgments may be particularly attractive, since potential partners know that they are unlikely to be damaged by them. Direct support for this interpretation comes from recent empirical studies, which uncovered that people making deontological judgments are perceived as being guided less by their self-interest, as being more trustworthy, and as expressing morally stronger views \[[@pone.0205066.ref022], [@pone.0205066.ref025]--[@pone.0205066.ref026]\]. Additional indirect support comes from the work showing that deciding not to tell a Pareto white lie (a characteristically deontological behavior) is positively correlated with altruism and cooperation in economic games \[[@pone.0205066.ref005]\]. Second, making consequentialist judgments often requires the suppression of strong emotional responses driven by socially desirable values. For example, sacrificing one life to save a greater number of lives requires overriding an emotional response guided by harm aversion--and pushing towards the deontological decision. Being incapable of overriding such emotional responses may favor partner selection along a path similar to the aforementioned one: people displaying strong emotional aversion to harm others will be less likely to harm me, which makes them attractive social partners. Symmetrically, the typically consequentialist emotion of compassion towards all humankind may not be perceived as socially desirable: people helping everyone in the world will be less likely to help me, which makes them little attractive as social partners. In line with this interpretation, recent experimental studies have demonstrated that deontological judgments are positively correlated with harm aversion, and negatively correlated with anti-social personality traits \[[@pone.0205066.ref027]--[@pone.0205066.ref030]\], and that people making deontological judgments are rated as being more empathic and having a superior moral character \[[@pone.0205066.ref031]\], as well as warmer \[[@pone.0205066.ref032]--[@pone.0205066.ref033]\], compared to those who make consequentialist decisions. In sum, deontological decisions may be favored if they work as a mechanism to signal social desirability. Of course, this mechanism would be evolutionarily favorable only if people preferring the deontological course of action in a given dilemma are actually more socially desirable than people preferring the competing consequentialist course of action. Otherwise, potential partners would ultimately learn that people making deontological choices in that dilemma are not socially more desirable than those making consequentialist choices, which would eventually lead to the loss of deontologists' evolutionary advantage. However, although previous research has reported that people making deontological decisions in some dilemmas are *perceived* to be more attractive than people making consequentialist decisions along a number of measures of social desirability, little is known about the direct question: are people making deontological judgments in these dilemmas *actually* more desirable social partners? In particular, to the best of our knowledge, no one has explored this question using the methodology of economic games. (A very recent paper makes use of similar methodology: they compare actual behavior of people making deontological decisions with actual behavior of people making consequentialist decisions, but only in *hypothetical* games \[[@pone.0205066.ref034]\]. We will discuss their work and its implications in the discussion). Here we wish to move a first step in this direction. Study overview {#sec002} -------------- We investigate whether behavior in moral dilemmas, i.e. having to choose between a characteristically deontological and a characteristically consequentialist option in a fictitious situation, can significantly predict prosocial behavior, which we see as one of the main ingredients of actual social desirability. Prosociality has its manifestation in various forms--with trustworthiness and altruism as some of its primary instantiations. We will test for these using economic games: We examine trustworthiness and altruism with one-shot trust games (henceforth TG) and dictator games (henceforth DG), respectively. We will shortly introduce the TG and the DG, and then move on to a brief discussion of our moral dilemma--a version of the well-known trolley problem--as it was presented to the participants. ### Trust game (TG) {#sec003} Two players, Player A and Player B, are paired anonymously. Player A is given \$0.20 and has to decide whether or not to transfer it to Player B. If Player A decides to transfer their money to Player B, then Player B receives \$0.60. If this happens, Player B is then asked how much of this \$0.60, if any, she wants to transfer back to Player A. Then the game ends. (We chose to use small stakes for two reasons. First of all, these stakes are essentially the same as the ones used in \[[@pone.0205066.ref022]\], which represents the starting point of our analysis. Moreover, previous research has found that DG-altruism \[[@pone.0205066.ref035]--[@pone.0205066.ref036]\] and TG-trustworthiness \[[@pone.0205066.ref037]\] are stake-independent, at least as long as stakes are not *too high*--some studies indeed suggest that these prosocial motivations may decrease at very high stakes \[[@pone.0205066.ref036],[@pone.0205066.ref038]\]). It is clear that Player A's best strategy depends on her beliefs about the amount that Player B is going to return: If Player A believes that Player B is going to return more than \$0.20, then she is better off by transferring the money, otherwise, she is better off by keeping the money. For this reason, Player A's behavior in the Trust Game is considered as an individual measure of trust, and Player B's behavior is taken as an individual measure of trustworthiness \[[@pone.0205066.ref022],[@pone.0205066.ref039]--[@pone.0205066.ref040]\]. ### Dictator game (DG) {#sec004} Two players, Player A and Player B, are paired anonymously and Player A (the dictator) gets \$0.20. Player A can then decide to transfer an amount of their \$0.20 to Player B (available options: \$0.00, \$0.02, \$0.04, ..., \$0.20). Player B has no active role and the money is distributed as proposed by Player A. Since Player A has no incentive to transfer their money, and since Player B has no possibility to reciprocate Player A's action, Player A's donation is usually taken as an individual measure of altruism \[[@pone.0205066.ref041]--[@pone.0205066.ref043]\]. ### Trapdoor dilemma (TD) {#sec005} In the TD, participants read the following scenario: "A runaway trolley is heading down the tracks toward five workers who will all be killed if the trolley proceeds on its present course. Adam is on a footbridge over the tracks, in between the approaching trolley and the five workers. Next to him on this footbridge is a stranger who happens to be very large. The only way to save the lives of the five workers is to flip a switch to release a Trapdoor that will drop the stranger off the bridge and onto the tracks below where his large body will stop this trolley. The stranger will die if Adam does this but the five workers will be saved. Participants are asked to report what they think Adam should do in this situation." There are theoretical motivations for choosing this dilemma over more classical ones, such as the Trolley problem, or the Footbridge dilemma. On the one hand, the TD allows us to discriminate among people who violate Kant's practical imperative that humans should never be used solely as a means from those who do not violate this imperative, by, at the same time, avoiding the confounding of emotional salience that is present for example in the footbridge dilemma \[[@pone.0205066.ref022],[@pone.0205066.ref044]\]. This is crucial, because, as observed by Everett and colleagues \[[@pone.0205066.ref022]\], the footbridge dilemma "highlights the possibility that deontologists are simply more averse to physical harm, and not necessarily that they are more reliable cooperators". On the other hand, the rationale for employing the TD instead of the classical Trolley problem is that the consequentialist choice in the TD requires a more blatant violation of Kant's imperative than it is the case in the Trolley problem. In line with this intuition, it has been shown that, while deontological judgments in the TD work as a signal of trustworthiness, deontological judgments in the Trolley problem do not \[[@pone.0205066.ref022]\]. In agreement with these results, in a Pilot Study, we also found that deontological decisions in the Trolley problem do not have a significant effect on neither trustworthiness, nor expectation of other's trustworthiness (N = 246, all p's \> 0.7). Study 1: Trapdoor-deontologists are perceived to be more trustworthy than Trapdoor-consequentialists, but they are actually not {#sec006} =============================================================================================================================== We start by exploring whether people making deontological decisions in the Trapdoor dilemma (Trapdoor-deontologists) are more trustworthy than those making consequentialist decisions in the same dilemma (Trapdoor-consequentialists). We thus have to test for deontological judgment and for trustworthiness. Concerning the former, we present subjects with the Trapdoor dilemma. Concerning the latter, we measure a person's perception of others' trustworthiness by having her play a TG in the role of Player A, and we test a person's trustworthiness by having her play a TG in the role of Player B. The aim of our first study is to replicate the finding that Trapdoor-deontologists are perceived to be more trustworthy than Trapdoor-consequentialists \[[@pone.0205066.ref022],[@pone.0205066.ref034]\] *and* to explore whether Trapdoor-consequentialists are actually less trustworthy than Trapdoor-deontologists. Hypotheses {#sec007} ---------- *H1*.*1*. Trapdoor-deontologists are perceived to be more trustworthy than Trapdoor consequentialists. *H1*.*2*. Trapdoor-deontologists are actually more trustworthy than Trapdoor-deontologists. Method {#sec008} ------ ### Participants {#sec009} We recruited 300 American participants using Amazon Mechanical Turk (AMT) \[[@pone.0205066.ref045]--[@pone.0205066.ref048]\], to participate on an incentivized online survey that we prepared using Qualtrics. The experiments reported in this paper were conducted in 2016, when all authors were based at the University of Amsterdam. According to the Dutch legislation, this is a non-WMO study, that is (i) it does not involve medical research and (ii) participants are not asked to follow rules of behavior. See <http://www.ccmo.nl/attachments/files/wmo-engelse-vertaling-29-7-2013-afkomstig-van-vws.pdf>, §1, Article 1b, for an English translation of the Medical Research Act. Thus (see <http://www.ccmo.nl/en/non-wmo-research>) the only legislations which apply are the Agreement on Medical Treatment Act, from the Dutch Civil Code (Book 7, title 7, §5), and the Personal Data Protection Act (a link to which can be found in the previous webpage). The current study conforms to both. Implied consent via survey was obtained by all subjects prior to participating and data were fully anonymized before analysis. The participation fee was \$0.50. Participant could also earn additional money depending on the choice they make during the experiment, as detailed in the *Design* subsection. After the survey was completed, we downloaded the datafile from Qualtrics, which contained 370 observations. The number of observations resulting on Qualtrics is usually higher than the number of submissions on AMT as Qualtrics includes observations of subjects that get excluded along the survey because they fail to correctly answer the comprehension questions. A total of 130 subjects were excluded because they either failed the comprehension questions or took the survey more than once, leaving us with a final sample of 240 participants and valid surveys. The number of participants who pass the comprehension questions is never equal to the number of actual submissions on AMT, because some subjects submit their survey on AMT even if they are eliminated along the survey. The proportion of participants who were excluded is in line with previous experiments using economic games on AMT \[[@pone.0205066.ref046]\]. For this and the subsequent studies we did not conduct an a priori power analysis, but sample sizes were based on earlier studies testing behavioral changes in economic games involving prosociality. In Study 1, we recruited 100 participants per condition. In the subsequent studies, to increase power, we recruited 150 participants per condition. Data of each study were collected all together and analyzed after the experiment. Data collection was not continued after data analysis. All measures, manipulations, and exclusions in this and the following studies are disclosed. ### Design {#sec010} Participants were randomly assigned to one of three conditions. In the *PlayerB-Trapdoor-Consequentialist* condition, participants played a Trust Game in the role of Player A with a Trapdoor-consequentialist in the role of Player B. More precisely, participants were shown the instructions of the Trapdoor dilemma and were informed that a participant who had already completed the survey, named Player B, opted for the consequentialist course of action. A comprehension question (regarding what happens if Adam flips the switch) was asked to make this point as clear as possible. Participants failing this comprehension question were automatically excluded from the survey. Participants in this condition were not asked to make a choice in the Trapdoor dilemma. They were only informed about Player B's action. Subsequently, participants were shown the instructions of the Trust game. After reading these instructions and before making a choice, participants were asked four comprehension questions (regarding the actions that maximize players' payoffs in four different scenarios). Participants failing any comprehension question were automatically excluded from the survey. The *PlayerB-Trapdoor-Deontologist* condition was similar to the previous condition, but participants played a Trust Game in the role of Player A with a Trapdoor-deontologist in the role of Player B. In the *Trapdoor-PlayerA* condition, participants first made a choice in the Trapdoor dilemma, and then played a Trust Game in the role of Player B with one Player A who had decided to transfer their \$0.20. After the survey was completed, we matched participants according to their choices and according to the conditions they participated in, we computed the bonuses and we paid them on top of their participation fee. ### Results and discussion {#sec011} The N = 240 subjects who passed the comprehension questions were distributed across conditions as follows: N = 83 in the *PlayerB-Trapdoor-Consequentialist* condition; N = 85 in the *PlayerB-Trapdoor-Deontologist* condition; and N = 72 in the *Trapdoor-PlayerA* (34 of whom chose the consequentialist option, while the remaining 38 chose the deontological option). [Fig 1](#pone.0205066.g001){ref-type="fig"} provides visual evidence that Trapdoor-deontologists were perceived to be more trustworthy than Trapdoor-consequentialists, but they were actually not. To show this, we define a variable "prosocial" which: for those who participated as Player A, it takes value 1 if they transferred their \$0.20 to Player B, and 0 otherwise; for those who participated in the role of Player B, it measures the amount returned to Player A, normalized such that the maximum return, which is \$0.60, corresponds to 1. Linear regression predicting Prosocial as a function of three dummy variables, Player A (1 if a subject participated as Player A, and 0 otherwise), Consequentialist (1 if a subject was/was-paired-with a Trapdoor-consequentialist, and 0 otherwise), and their interaction, reveals indeed a significant interaction (F(3,236) = 11.50, coeff = -0.24, t = -2.05, p = 0.041). We now look at main effects. We find that the participants playing the TG in the role of Player A transferred significantly more to Trapdoor-deontologists than to Trapdoor-consequentialists playing as Player B (69.2% vs 57.7%, F(1,166) = 8.40, coeff = 0.21, t = 2.90 p = 0.004). This suggests that Trapdoor-deontologists were expected to be more trustworthy than Trapdoor-consequentialists. However, the amount returned by Trapdoor-deontologists is not significantly different than the amount returned by Trapdoor-consequentialists (33.2% vs 34.4%, F(1,70) = 0.27, coeff = -0.03, t = -0.52, p = 0.602). This suggests that Trapdoor-deontologists were not actually more trustworthy than Trapdoor-consequentialists. We also conducted a Bayesian hypothesis test \[[@pone.0205066.ref049]--[@pone.0205066.ref050]\] as follows: we conducted linear regression predicting Pro-sociality with and without the Consequentialist variable to compute the corresponding BICs and we used these values to compute the posterior probability of the null hypothesis (no differences between trapdoor-consequentialists and trapdoor-deontologists) given the prior that the null hypothesis and the alternative hypothesis are equally likely. In doing so, we found a posterior probability of 92.23%, which provides indeed strong support for the null hypothesis that there is no difference in trustworthiness between Trapdoor-consequentialists and Trapdoor-deontologists. {#pone.0205066.g001} ### Conclusion {#sec012} Trapdoor-deontologists are perceived to be more trustworthy in the Trust Game than Trapdoor-consequentialists, but actually they are not. Study 2: Trapdoor-deontologists are perceived to be more altruist than Trapdoor-consequentialists, but they are actually not {#sec013} ============================================================================================================================ Study 1 uncovered a perception gap according to which Trapdoor-deontologists are not significantly more trustworthy than Trapdoor-consequentialists, although they are perceived to be so. However, trustworthiness is only one particular dimension of prosociality. Study 2 aims at exploring what happens if we adopt altruistic behavior instead of trustworthiness as a measure of prosociality. (As in Study 1, also in this case we conducted a Pilot Study with the standard Trolley problem, instead of the Trapdoor dilemma. And, also in this case, we found no statistically significant effect: Trolley-deontologists were neither perceived to be significantly more altruistic than Trolley-consequentialists, nor they were significantly more altruistic than Trolley-consequentialists). Hypotheses {#sec014} ---------- *H2*.*1*. Trapdoor-deontologists are perceived to be more altruistic than Trapdoor-consequentialists. *H2*.*2*. Trapdoor-deontologists are actually more altruistic than Trapdoor-consequentialists. Method {#sec015} ------ ### Participants {#sec016} 1,050 American participants (none of which had participated in the previous study) were recruited using AMT. They were paid \$0.50 as a participation fee and they could earn additional money depending on the choice they make during the experiment, as detailed below. After the survey was completed, we downloaded the datafile from Qualtrics, which contained 1,079 observations. A total of 238 subjects were excluded because they either failed the comprehension questions or took the survey more than once, leaving us with a final sample of 841 participants. ### Design {#sec017} Participants were randomly divided between three conditions. In the *Guess-Trapdoor-Consequentialist* condition, participants had to guess the DG donation of a randomly selected Trapdoor-consequentialist participant, with a \$0.20 reward in case they make the right guess. More precisely, participants were shown the Trapdoor dilemma and were informed that another participant, named Player A, who had already completed the survey, had chosen the consequentialist option. One comprehension question was asked to make this point as clear as possible. Participants failing this comprehension question were automatically excluded from the survey. Participants in this condition were not asked to make a decision in the Trapdoor dilemma. They were only informed about Player A's choice. Subsequently, participants were informed that Player A was playing a DG with a third participant, named Player B. After reading the instructions of the DG, participants were asked two comprehension questions (one regarding which action maximizes the dictator's payoff, and the other one regarding which action maximizes the recipient's payoff). Participants failing any comprehension question were automatically excluded from the survey. Participants who passed the comprehension questions were asked to guess Player A's donation to Player B. The *Guess-Trapdoor-Deontologist* condition was similar to the previous condition, with the difference that participants had to guess the DG donation of a Trapdoor-deontologist participant. In the *DG-Trapdoor* condition, participants first made a choice in the Trapdoor Dilemma and then in the DG. (In reality, participants were divided in seven conditions, because each "Guess condition" was actually made by three conditions: the one described, and two more conditions in which participants were informed about the gender of the donor. However, knowing the gender did not have any significant impact on guesses, and thus we collapse across conditions). After the survey was completed, we paired participants according to their decisions and we computed and paid their bonuses. ### Results and discussion {#sec018} The N = 841 subjects were distributed across conditions as follows: N = 326 in the *PlayerB-Trapdoor-Consequentialist* condition; N = 369 in the *PlayerB-Trapdoor-Deontologist* condition; and N = 146 in the *Trapdoor-PlayerA* (69 of whom choose the consequentialist option, while the remaining 77 choose the deontological option). [Fig 2](#pone.0205066.g002){ref-type="fig"} provides visual evidence that Trapdoor-deontologists were perceived to be more altruistic than Trapdoor-consequentialists, but actually they were not more altruistic. In line with Study 1, linear regression predicting prosocial behavior as a function of Player A, Consequentialist, and their interaction, reveals a marginally significant interaction (F(3,837) = 4.08, coeff = -.08, t = -1.74, p = 0.081). Looking at main effects, in line with Study 1, we find that the participants expected Trapdoor-deontologists to give significantly more than Trapdoor-consequentialists (30% of the total share vs 24%, F(1,166) = 8.40, coeff = -.21, t = -2.90, p = 0.004). However, the actual donation of Trapdoor-deontologists was not significantly different than the actual donation of Trapdoor-consequentialists (22% vs 24%; F(1,144) = 0.34, coeff = .024, t = 0.58, p = 0.563). We also conducted a Bayesian hypothesis following a similar procedure as in Study 1. In doing so, we found a posterior probability of 91.06%, which provides indeed strong support for the null hypothesis that there is no difference in altruistic behavior between Trapdoor-consequentialists and Trapdoor-deontologists. {#pone.0205066.g002} ### Conclusion {#sec019} Trapdoor-deontologists are perceived to be more altruistic in the Dictator Game than Trapdoor-consequentialists, but they are not actually so. General discussion {#sec020} ================== We tested the hypothesis that people making deontological judgments in the Trapdoor dilemma are more trustworthy and more altruistic towards strangers than those making consequentialist judgments. In doing so, we found a perception gap such that people perceive Trapdoor-deontologists to be more altruistic and more trustworthy towards strangers than Trapdoor-consequentialists, but they actually are not. Our results are naturally situated in a framework that stems from evolutionary considerations. As mentioned above, deontological judgments often lead to positive character evaluations which, in turn, can lead to evolutionary advantages when it comes to partner choice mechanisms \[[@pone.0205066.ref022],[@pone.0205066.ref051]--[@pone.0205066.ref054]\]. Our findings indicate that this procedure might not promote favorable outcomes as the positive character evaluation itself is not manifested in prosocial behavior--at least when prosociality is operationalized using trustworthiness in the TG and altruism in the DG. It is thus reasonable to wonder: how and why did people's perception concerning the selection of their social partners evolve in order to favor deontological decisions? This point highlights the main limitation of our study. We operationalized social desirability using prosociality in the trust game and the dictator game. (We have also conducted a pilot using the Prisoner's Dilemma (PD), as a measure of cooperative behavior towards strangers \[[@pone.0205066.ref055]--[@pone.0205066.ref057]\]. Again we found that Trapdoor-consequentialists are neither significantly more nor significantly less cooperative than Trapdoor-deontologists. However, in this pilot, we did not measure beliefs. So, we do not know whether the perception gap discussed in Study 1 and Study 2 extends to cooperative behavior in PD). This procedure is inherently limited, as it does not take into account a myriad of other, potentially different, behaviors of crucial importance in social relationships. Although previous research has shown that TG-trustworthiness and DG-altruism correlate with a number of other prosocial behaviors in the lab \[[@pone.0205066.ref040], [@pone.0205066.ref058]--[@pone.0205066.ref060]\], and in real-life \[[@pone.0205066.ref061]--[@pone.0205066.ref062]\], it is possible that using other measures of prosociality would lead to different results. For example, one may wonder whether deontological judgments in the Trapdoor dilemma, while not predicting prosociality towards anonymous strangers, work as a signal of commitment to prosociality towards people belonging to the same social group--the so-called *ingroup favoritism* \[[@pone.0205066.ref063]--[@pone.0205066.ref064]\]. This is a promising stream of research because consequentialism is radically impartial, while deontological ethics focus on notions of duties, rights, and obligations, which may be context-dependent. Thus, everything else being constant, Trapdoor-deontologists might favor ingroup members over outgroup members to a larger extent than consequentialists do. In line with this, a recent work found that people prefer those making deontological choices (in a dilemma between volunteering for cause or helping a family member) as a friend or spouse, but prefer those making consequentialist choices as political leaders \[[@pone.0205066.ref022]\]. In any case, an important direction for future research is to expand our study to include other economic games and thereby examining in more depth how expectations of prosocial actions of Trapdoor-deontologists and their actual behaviors are connected in different contexts. Additionally, we operationalized social desirability using *actual* behavior, and this led us to use economic games. This procedure has two limitations. First, actual behavior in economic games pertains to the domain of prescriptions, while judgments in moral dilemmas pertain to the domain of proscriptions \[[@pone.0205066.ref065]--[@pone.0205066.ref066]\]. Since it is possible that the psychology underlying decisions is different from the psychology underlying judgments \[[@pone.0205066.ref030]\], this might be at the origin of the observed gap between expectations and actual behavior. Second, it is possible that Trapdoor-deontologists are more desirable than Trapdoor-consequentialists along dimensions that are not easily measurable using economic games, as, for example, warmth and empathy, as some recent studies using different moral dilemmas seem to suggest \[[@pone.0205066.ref032]--[@pone.0205066.ref033]\]. Thus, another interesting strand of further research is to extend our study to include other scales of social desirability, centered around proscriptions, instead of prescriptions, also including psychometric measures \[[@pone.0205066.ref067]--[@pone.0205066.ref068]\]. Symmetrically, we measured deontological judgment using a hypothetical Trapdoor dilemma. However, previous research suggests that there might be significant disparities between judgment and actual behavior in moral dilemmas \[[@pone.0205066.ref069]--[@pone.0205066.ref070]\]. This ultimately suggests that it is possible that deontological choices become an honest signal of prosociality if less hypothetical measures of deontology are used. Exploring this possibility is an important direction for future research. A similar point regards our measure of deontological judgment. As in \[[@pone.0205066.ref022]\], we operationalized deontological judgment by using the Trapdoor dilemma, because this dilemma allows us to discriminate among people who violate Kant's practical imperative from those who do not violate this imperative, by, at the same time, avoiding the confounding of emotional salience that is present for example in the footbridge dilemma. This distinction is crucial, because it has been proposed that deontological judgments signal commitment to prosociality through signaling commitment to follow Kant's practical imperative that other people should never be used solely as a means \[[@pone.0205066.ref016]\]. The choice of the Trapdoor dilemma has, however, also two limitations. One is that this and similar dilemmas have been criticized for evoking humor, rather than serious consideration of moral concerns \[[@pone.0205066.ref071]\]. The second one is that the practical imperative is not the only dimension in which deontological ethics differs from consequentialism. Thus, in future research, it would be important to extend our study to include more dimensions of deontological judgment, in order to determine the boundary conditions of our results. We have done a first step in this direction. As mentioned earlier, in line with \[[@pone.0205066.ref022]\], in a Pilot Study, we have also found that deontological judgments in the standard Trolley problem do *not* signal trustworthiness. Understanding which deontological judgments signal pro-sociality and which give rise to the observed gap between expectations and behavior is certainly an important direction for future research, that may shed light on which moral principles are involved in prosocial behavior and in the perception of others' prosocial attitudes. Another potential source of criticism is the use of small stakes. We chose to use such small stakes for two reasons. First of all, we used essentially the same stakes as in \[[@pone.0205066.ref022]\], which represents the starting point of our analysis. Second, previous research has found no stake effect on DG-altruism \[[@pone.0205066.ref035]--[@pone.0205066.ref036]\] and TG-trustworthiness \[[@pone.0205066.ref037]\]--some studies suggest that these prosocial motivations may decrease at very high stakes \[[@pone.0205066.ref035],[@pone.0205066.ref038]\]. Thus we believe that it is unlikely that future research will reveal that Trapdoor-consequentialists becomes less trustworthy and altruistic than Trapdoor-deontologists, at least when using standard medium-size stakes. Potentially more intriguing is what could happen at very large stakes, as previous research suggests that prosocial motivations may decrease in such situations. It is possible that this decrease in prosocial motivations is driven by consequentialists and that, at very large stakes, Trapdoor-consequentialists become indeed less trustworthy and altruistic than Trapdoor-deontologists. Nonetheless, we should say that we *did* find a significant gap between expectations and actual behavior. Even though future research may uncover that people making deontological decisions are actually more prosocial than those making consequentialist decisions along other dimensions of prosociality or at higher stakes, our study shows that, as a matter of fact, in our tasks, Trapdoor-deontologists are perceived to be more trustworthy and more altruistic than Trapdoor-consequentialists, although they are actually equally trustworthy and equally altruistic. Our results are, however, silent regarding the psychological underpinnings behind this gap. We hope that future research may shed light on *why* Trapdoor-deontologists are perceived to be more prosocial than Trapdoor-consequentialists, in spite of not being so. Therefore, besides the aforementioned limitations, our work has also several positive implications. For example, the result that respecting Kant's practical imperative in the Trapdoor dilemma is perceived as a signal of altruism and trustworthiness is theoretically remarkable as it suggests that people believe that Kant's practical imperative is one of the determinants of prosociality, and thus that prosociality and morality are linked, at least in some contexts. This mirrors recent findings that morality drives prosociality in a variety of contexts \[[@pone.0205066.ref072]--[@pone.0205066.ref074]\], and suggests that exploring the links between morality and prosociality can be a fruitful avenue for future research. Designing the boundaries of our results along the dimensions of the deontology and prosociality measures are not *just-academic* questions, but can elucidate underlying theoretical relations between motivations, behaviors, and beliefs that have been left uncovered by previous research. These links can turn out to be useful to find novel descriptions of people's behavior and people' expectations in other people's behavior in terms of underlying moral principles. A very recent work is the most similar to ours that we are aware of \[[@pone.0205066.ref034]\]. They compared the actual behavior of people making deontological judgments and people making consequentialist judgments in a hypothetical Trust Game and in a hypothetical 4-player Public Goods Game (PGG, which represents a way to measure cooperative behavior in groups of four people). In line with our study, \[[@pone.0205066.ref034]\] found that people making deontological judgments are perceived to be more trustworthy than people making consequentialist judgments while, actually, they are not. However, they found that people making deontological judgments are perceived to be more cooperative in the hypothetical 4-player Public Goods game than people making consequentialist judgments *and* actually are more cooperative--there is thus no perception gap observable in the PGG. Thus, our work extends the result of \[[@pone.0205066.ref034]\] along two dimensions: first, we show that Trapdoor-deontologists are perceived to be more trustworthy than Trapdoor-consequentialists in a real, incentivized Trust Game, rather than a hypothetical one. This is an important extension, because economic motivations can be an important drive of human behavior, especially among people making consequentialist choices. Second, we showed that this perception gap between the expected level of prosociality of Trapdoor-deontologists versus Trapdoor-consequentialists and their actual levels of prosociality does not only regard the domain trustworthiness, but also that of altruistic behavior towards strangers. A somewhat related work is \[[@pone.0205066.ref075]\]. Here the authors asked subjects to self-report the motivation behind their choice in a dictator game, and then classified the responses in several classes, including to whether they were consequentialist or deontologist. The authors found no differences in dictator game giving between subjects who left a consequentialist motivation and those who left a deontological motivation. This result is thus in line with the current results of ours. Our work extends \[[@pone.0205066.ref075]\] along two directions: first, we do not only consider altruistic behavior in the dictator game, but we also consider trustworthiness in the trust game; second, we do not use self-report motivations to classify subjects into consequentialists or deontologists, but we use actual judgments in a specific moral dilemma, that we have chosen because of specific theoretical motivations. In sum, deontological judgments do not work as a universal reliable signal of prosociality. Understanding if, when, and why deontological judgments correlate with prosocial behavior is an important direction for future research with important consequences for our understanding of human sociality. [^1]: **Competing Interests:**The authors have declared that no competing interests exist.

Background ========== At the initial stages of drug discovery and design, there are often millions of candidate drug molecules under consideration. Therefore, the early prediction of activity for drug candidates using computational methods is very important to save time and resources. Due to importance of early prediction of activity of drug candidates on the target protein, a large number of computational methods were developed. QSAR (Quantitative Structure-Activity Relationship) analysis is one of the most widely used methods to relate structure to function. QSAR analysis can be described as the quantitative effort of understanding the correlation between the chemical structure of a molecule and its biological and chemical activities such as biotransformation ability, reaction ability, solubility or target activity\[[@B1]\]. QSAR assumes that structurally similar molecules should have similar activities, which draws attention to the importance of detecting the most significant chemical and structural descriptors of the drug candidates. The drug activity behavior can be predicted using a wide range of descriptors. Some of the most widely used 3D QSAR methods can be listed as follows: comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA), eigenvalue analysis (EVA). In CoMFA, molecular descriptors are calculated and selected by calculating the electrostatic and steric potential energies between a positively charged carbon atom located at each vertex of a rectangular grid and a series of molecules embedded within the grid\[[@B2]\]. The sensitivity to small changes in the alignment of compounds is reduced and hydrogen-bonding and hydrophobic fields are introduced to in CoMSIA\[[@B3]\]. In these methods aligning of the structures is essential, therefore EVA was used due to the fact that methods that are sensitive to 3D structure but do not require superposition were introduced\[[@B4]\]. The generation of descriptors in EVA is based on molecular vibrations, where a normal mode calculation is required to simulate the IR spectrum of a molecule \[[@B5]\]. In this study E-Dragon \[[@B6]-[@B8]\], which is a remote version of the DRAGON descriptor calculation program, was used to calculate the molecular descriptors for drugs. It applies the calculation of molecular descriptors developed by Todeschini et. al\[[@B9]\] and provides more than 1,600 molecular descriptors, which are divided into 20 blocks, including atom types, functional group and fragment counts, topological and geometrical descriptors, autocorrelation and information indices, 3D molecular descriptors, molecular properties \[[@B6]-[@B8]\]. DRAGON incorporates two steps; the first step eliminates low-variable descriptors, the second step optimizes the descriptor subset using a Q^2^-guided descriptor selection by means of a genetic algorithm using several data analysis methods: Unsupervised Forward Selection (UFS)\[[@B10]\], Associative Neural Network (ASNN)\[[@B11],[@B12]\], Polynomial Neural Network (PNN)\[[@B13],[@B14]\] and Partial Least Squares (PLS) \[[@B6]-[@B8]\]. In most studies, Partial Least Squares (PLS)\[[@B15]\] is used to develop QSAR models by reducing the number of attributes in the descriptor set to a small number of attributes correlated with the defined property being modeled. In our approach, to classify activities of drug compounds, we used the mixed-integer programming (MILP) based hyper-boxes method that takes the molecular descriptors as attributes of the model. The problem of QSAR analysis and the classification of drug candidates are addressed based on their published IC~50~values by introducing an algorithm that combines PLS regression with mixed-integer linear programming based hyper-boxes classification method. The strength of the algorithm not only comes from combining regression with classification but also the ability to improve the classification accuracies by its iterative approach. The algorithm that links QSAR descriptor model generation with inhibitory activity classification was applied to inhibitors of Acetylcholinesterase (ACHE), Benzodiazepine Receptor (BZR), Dihydrofolate Reductase (DHFR) and Cyclooxygenase-2 (COX-2) and finally for Cytochrome P450 C17 (CYP17). The comparison of our classification accuracies with the accuracies of the classification methods available in the WEKA data mining package \[[@B16]\] is also made. WEKA contains 63 different classification methods, but here only 16 of those, which had the best classification accuracies for the data sets considered in this paper are discussed. Brief overview of these classifiers is further presented in the Methods section. Our approach outperformed all of the classifiers available in WEKA for each model of the all of the 7 data sets, even reaching 100% accuracy in predicting the activity classification of the inhibitor sets, Ache inhibitors and Cytochrome P450 C17. A total number of 21 QSAR models were built in this study for 7 inhibitor sets, and in 18 of them the accuracy of our methodology exceeded the accuracy of the second best classifier with more than 10%. Through all of the 21 models, the smallest difference in the accuracies is 6.31% and the largest difference is 27.47%. Results ======= To determine the threshold values, which divide the low and high classes, for all datasets the IC~50~values were statistically analyzed. In this study, we consider 6 datasets, of which IC~50~values and structures were reported \[[@B16]-[@B26]\]. In addition to these datasets we introduced a new dataset for Cytochrome P450 C17 inhibitors that we collected from the literature. Cytochrome P450 C17 is a well-recognized target for prostate cancer treatment, since selective inhibition of the enzyme exerts control over androgen synthesis \[[@B27]\]. After building the descriptor models by e-Dragon \[[@B8]\], three models were constructed during the PLS analysis as: 7, 10 and 15 descriptor models. The reason that we build 3 models with different number of variables is due to the fact that we might come up with insignificant descriptors within one of these models, so that we can replace them by a more significant one from the other models. The QSAR models with the most significant descriptors, as they were concluded as a result of the initial PLS study for the 7, 10 and 15 attribute models are listed in Table [1](#T1){ref-type="table"} with their R^2^values. Table [1](#T1){ref-type="table"} shows the optimal R^2^values of our PLS models given by Minitab\[[@B28]\] with predefined number of descriptors from the descriptors calculated by e-Dragon software, and the R^2^values of the PLS models calculated by Sutherland et al\[[@B26]\] with the same data sets but different methods and models. ###### Comparison of R^2^values for PLS models. Data set CoMFA\* CoMSIAbasic\* CoMSIAextra\* EVA\* HQSAR\* 2D\* 2.5D\* e-DragonPLS-7 e-DragonPLS-10 e-DragonPLS-15 --------------------- --------- --------------- --------------- ------- --------- ------ -------- --------------- ---------------- ---------------- AchE 0.88 0.86 0.86 0.96 0.72 0.40 0.38 0.84 0.90 0.95 BZR 0.61 0.62 0.62 0.51 0.64 0.51 0.52 0.51 0.67 0.79 COX-2 0.70 0.69 0.69 0.68 0.70 0.62 0.68 0.53 0.61 0.73 DHFR_RL 0.79 0.76 0.75 0.81 0.81 0.61 0.65 0.42 0.53 0.64 DHFR_PC N/A N/A N/A N/A N/A N/A N/A 0.44 0.54 0.65 DHFR_TG N/A N/A N/A N/A N/A N/A N/A 0.40 0.51 0.66 Cytochrome P450 C17 N/A N/A N/A N/A N/A N/A N/A 0.84 0.91 0.95 \* PLS results reported by Sutherland et al. \[[@B26]\]. The R^2^values shows that, the models we developed with 10 and15 descriptors for Ache BZR and COX-2 are stronger than or at least as strong as the other models reported by Sutherland et al\[[@B26]\] in representing the IC~50~values in terms of selected descriptors, but our model for DHFR_RL is not as good as the other reported models. High R^2^values of Cytochrome P450 C17 models also suggest good prediction of the IC~50~values and a promising initial model for classification. It is worth noting that, our study is not simply a regression study, but we develop these regression models in order to use the selected descriptors from this step as attributes for accurate classification. Iterations ---------- At the end of the initial runs of the hyper-boxes classification method, classification results are obtained. The next step is the significance analysis and the improvement of the classification accuracies by iterations. The weakest and the strongest descriptors were calculated by significance analysis and, the weakest descriptor in the current model was replaced by the most significant one from other models at each iteration. The classification runs are repeated after each replacement, by MILP based hyper-boxes method. When the classification accuracy is not improved at the end of iteration, the algorithm stops and final results are reported (Table [2](#T2){ref-type="table"}). ###### Classification Accuracies of each iteration. **Iter \#0** **Iter \#1** **Iter \#2** **Iter \#3** -------------- ------------------- -------------- -------------- -------------- -------------- **ACHE** **7 Attributes** 91.89 100.00 **10 Attributes** 86.48 89.19 91.89 **15 Attributes** 86.05 89.18 **BZR** **7 Attributes** 90.90 96.36 **10 Attributes** 92.73 94.55 **15 Attributes** 90.09 92.73 **COX-2** **7 Attributes** 94.39 95.33 97.20 98.13 **10 Attributes** 91.58 97.20 **15 Attributes** 88.78 89.72 90.65 **DHFR_RL** **7 Attributes** 94.73 96.99 **10 Attributes** 93.98 97.74 **15 Attributes** 94.73 **DHFR_PC** **7 Attributes** 95.23 96.83 97.62 **10 Attributes** 94.44 95.24 98.41 **15 Attributes** 92.06 93.65 **DHFR_TG** **7 Attributes** 96.24 97.74 **10 Attributes** 93.23 93.98 96.24 **15 Attributes** 96.24 97.74 **P450 C17** **7 Attributes** 86.36 90.00 97.20 100.00 **10 Attributes** 100.00 **15 Attributes** 100.00 While choosing the weakest descriptor to leave the model, the descriptor with the maximum p-value (failed to reject H~0~with the greatest error, see methods section for our hypothesis) for one of the high or low classes was selected. The weakest descriptor was replaced by the strongest one. The strongest descriptor defined as the attribute whose maximum p-value for high and low classes is the minimum among the other descriptors. Final Classification -------------------- As shown in Table [3](#T3){ref-type="table"}, we compared the classification accuracies of our model with the results that calculated using all of the classification methods in WEKA. We report only the results of the16 best performing WEKA classifiers. Our method performed better than all of the other classifiers for every model of each dataset. Our integrated approach of regression and classification for Ache and Cytochrome P450 C17 inhibitors datasets displayed an activity prediction accuracy of 100%. The activity of BZR inhibitors was calculated with the accuracy of 96.36%. We were able to predict the activities of COX-2 inhibitors with 98.13% in a 7-descriptor model. In addition, the prediction accuracy of activity of DHFR_RL, DHFR_PC, and DHFR_TG inhibitors were 97.74%, 98.41% and 97.74% respectively. The best performing WEKA classifiers are also highlighted in Table [3](#T3){ref-type="table"}. ###### Comparison of classification accuracies of best WEKA classifiers with the MILP based hyper-boxes classification. **% accuracy** **% accuracy** ----------------------------------- ----------------- ------------------ ------------------ ----------------------------------- ----------------- ------------------ ------------------ **MILP based hyper-boxes method** **100** **91.89** **89.19** **MILP based hyper-boxes method** **96.36** **94.55** **92.73** Bayes Network 79.28 77.48 78.38 Bayes Network 77.91 77.3 73.62 Naive Bayes 80.18 80.18 81.08 Naive Bayes 80.37 77.91 66.26 Naive Bayes Simple 81.08 80.18 81.98 Naive Bayes Simple 79.14 77.3 68.71 Naive Bayes Updatable 80.18 80.18 81.08 Naive Bayes Updatable 80.37 77.91 66.26 Lojistic 79.28 ***84.68*** 80.18 Lojistic ***83.44*** 80.98 80.98 Multilayer Perceptron 82.88 81.08 81.08 Multilayer Perceptron 79.75 80.98 79.14 SimpleLogistic ***83.78*** 82.88 79.28 SimpleLogistic 80.98 ***82.82*** 79.14 SMO (WEKA SVM) 79.28 80.18 80.18 SMO (WEKA SVM) 79.14 77.91 77.91 IB1 70.27 80.18 77.48 IB1 72.39 74.85 75.46 Ibk 70.27 80.18 77.48 IBk 72.39 74.85 75.46 Logit Boost 82.88 81.08 ***82.88*** Logit Boost 78.53 77.3 77.91 Multi Class Classifier 79.28 ***84.68*** 80.18 Multi Class Classifier ***83.44*** 80.98 ***80.98*** Threshold Selector 47.75 68.47 60.36 Threshold Selector 78.53 76.69 75.46 LMT ***83.78*** 82.88 79.28 LMT 80.98 ***82.82*** 79.14 RandomForest 80.18 80.18 81.98 RandomForest 77.3 79.75 ***80.98*** OneR 81.08 72.97 72.97 OneR 74.85 74.23 79.14 **% accuracy** **% accuracy** **DHFR_TG** **7-attribute** **10-attribute** **15-attribute** **COX-2** **7-attribute** **10-attribute** **15-attribute** **MILP based hyper-boxes method** **97.74** **96.24** **97.74** **MILP based hyper-boxes method** **98.13** **97.2** **90.65** Bayes Network 77.33 78.09 73.05 Bayes Network 67.2 67.2 66.88 Naive Bayes 76.57 ***79.35*** 72.54 Naive Bayes 71.66 70.06 64.65 Naive Bayes Simple 75.57 78.84 67 Naive Bayes Simple 72.29 70.06 64.65 Naive Bayes Updatable 76.57 ***79.35*** 72.54 Naive Bayes Updatable 71.66 70.06 64.65 Lojistic 75.82 78.84 75.57 Lojistic 72.29 70.38 70.06 Multilayer Perceptron 76.32 77.08 75.06 Multilayer Perceptron ***72.61*** 72.29 ***75.16*** SimpleLogistic 74.56 77.83 75.31 SimpleLogistic 72.29 71.97 68.47 SMO (WEKA SVM) 72.54 79.09 72.54 SMO (WEKA SVM) 71.02 69.43 69.43 IB1 75.31 79.09 75.82 IB1 69.11 71.02 70.06 Ibk 75.31 79.09 75.82 IBk 69.11 71.02 70.06 Logit Boost 77.33 78.34 78.34 Logit Boost 71.66 70.06 70.7 Multi Class Classifier 75.82 78.84 75.57 Multi Class Classifier 72.29 70.38 70.06 Threshold Selector 69.77 74.81 73.55 Threshold Selector 68.47 65.29 64.65 LMT 76.07 76.57 77.83 LMT 71.34 71.02 68.15 RandomForest ***77.58*** 79.09 ***80.35*** RandomForest 71.97 ***74.2*** 70.06 OneR 69.77 69.77 70.53 OneR 70.7 70.38 70.06 **% accuracy** **% accuracy** **DHFR_RL** **7-attribute** **10-attribute** **15-attribute** **DHFR_PC** **7-attribute** **10-attribute** **15-attribute** **MILP based hyper-boxes method** **96.99** **97.74** **94.73** **MILP based hyper-boxes method** **97.62** **98.41** **93.65** Bayes Network 63.72 71.78 70.5 Bayes Network 80.42 80.42 78.04 Naive Bayes 63.97 68.76 71.7 Naive Bayes 82.54 81.48 80.95 Naive Bayes Simple 63.97 67.75 71 Naive Bayes Simple 82.8 79.89 81.22 Naive Bayes Updatable 63.98 68.77 71.78 Naive Bayes Updatable 82.54 81.48 80.95 Lojistic ***69.52*** 73.8 78.58 Lojistic 81.75 83.33 81.75 Multilayer Perceptron 62.72 76.57 77.58 Multilayer Perceptron 82.8 82.8 84.13 SimpleLogistic 66.75 73.55 78.33 SimpleLogistic 80.42 ***84.13*** 81.22 SMO (WEKA SVM) 64.99 73.05 79.59 SMO (WEKA SVM) 82.28 83.33 79.1 IB1 62.97 75.06 81.11 IB1 82.28 80.16 81.75 Ibk 62.97 75.06 ***81.11*** IBk 82.28 80.16 81.75 Logit Boost 64.99 75.06 77.33 Logit Boost 83.33 81.48 81.48 Multi Class Classifier 69.52 73.8 78.59 Multi Class Classifier 81.75 83.33 81.75 Threshold Selector 64.99 69.52 78.59 Threshold Selector 83.33 79.1 81.22 LMT 65.24 ***77.33*** 77.83 LMT ***83.6*** 83.07 ***85.19*** RandomForest 68.51 77.08 77.83 RandomForest 82.8 80.95 83.07 OneR 61.46 66 62.72 OneR 79.89 79.89 80.16 To verify the reliability of the accuracies given by 10-fold cross validation standard deviations of the classification accuracies were also calculated for each run of MILP based hyper-boxes method. The sensitivity of classification accuracy to the number of descriptors is also examined and the results are reported in Table [4](#T4){ref-type="table"}. Small number of descriptors may lead to poor models while a large number of descriptors may lead to inefficient models that incorporate non-informative descriptors for classification. In all of the datasets considered in this paper, this trend is observed from the accuracy values and standard deviation of accuracies for 10-fold cross validation. ###### Final average classification accuracies and corresponding standard deviations of classification with 10-fold cross validation with various number of descriptors. Average Accuracy Std. Dev Average Accuracy Std. Dev ----------- --------------- ------------------ ---------- ------------- --------------- ------------------ ---------- **ACHE** 4 Attributes 80.83 4.36 **DHFR_RL** 4 Attributes 82.15 2.76 6 Attributes 83.36 3.67 6 Attributes 91.67 1.86 7 Attributes 100 0 7 Attributes 96.99 2.14 8 Attributes 96.36 1.89 8 Attributes 96.64 0.72 10 Attributes 91.89 2.22 10 Attributes 97.74 0.82 12 Attributes 86.63 3.28 12 Attributes 97.37 1.33 15 Attributes 89.18 1.18 15 Attributes 94.73 1.94 20 Attributes 83.65 3.26 20 Attributes 95.25 3.28 **BZR** 4 Attributes 86.83 1.36 **DHFR_PC** 4 Attributes 81.27 4.72 6 Attributes 88.36 2.57 6 Attributes 94.48 3.97 7 Attributes 96.36 2.06 7 Attributes 97.62 2.22 8 Attributes 93.65 3.83 8 Attributes 96.15 0.82 10 Attributes 94.55 2.37 10 Attributes 98.41 1.18 12 Attributes 95.63 1.06 12 Attributes 92.18 2.83 15 Attributes 92.73 1.46 15 Attributes 93.65 0.98 20 Attributes 86.25 2.12 20 Attributes 94.25 4.02 **COX-2** 4 Attributes 91.86 3.86 **DHFR_TG** 4 Attributes 84.94 1.47 6 Attributes 94.36 1.42 6 Attributes 94.03 3.49 7 Attributes 98.13 1.73 7 Attributes 97.74 1.62 8 Attributes 97.65 1.23 8 Attributes 96.05 0.72 10 Attributes 97.2 2.29 10 Attributes 96.24 2.47 12 Attributes 96.63 2.16 12 Attributes 95.42 1.79 15 Attributes 90.65 3.06 15 Attributes 97.74 2.78 20 Attributes 88.06 1.41 20 Attributes 93.5 2.67 Detailed analysis: Cytochrome P450 C17 inhibitors ------------------------------------------------- We applied our approach to classify activities of drug molecules in a new data set (P450 C17) that is constructed from data in literature \[[@B27],[@B29]\]. The molecular structures and IC50 values for these molecules are given in Additional File [1](#S1){ref-type="supplementary-material"}. This approach may be utilized for the new molecules that inhibit activity of Cytochrome P450 C 17 before channeling them into experiments. For the 7, 10 and 15 attribute models the selected most significant descriptors as a result of the initial PLS study are listed with R^2^values (Table [1](#T1){ref-type="table"}) of 0.946, 0.907 and 0.838 respectively. When the hyper-boxes model was implemented, 10 and the 15-attribute models reached 100% accuracy, by 10-fold cross validation. The 7-attribute model, however, still needed to be improved since the classification results reached an average accuracy of 96.35%. This led us to conclude that there may be some overestimated descriptors actually having low significance in terms of classifying the drug activity. Therefore, significance tests were performed after the preliminary classification runs. Table [5](#T5){ref-type="table"} shows the *p*-values for the descriptors at each iteration. At iteration 1, C-027 was detected as insignificant since it has the largest *p*-value among the other descriptors. Then from the significance analysis of 10 descriptor model, EEig04x was chosen to replace it, since its maximum *p*-value is the minimum among the other descriptors. After each replacement, the hyper-boxes classification model was built and performed with the new attributes and, average classification accuracy was determined. The runs were stopped after iteration 3 since we reached 100% accuracy. The classification results are reported in Table [6](#T6){ref-type="table"}. ###### The descriptors leave the 7 descriptor model and the descriptors replacing them. **Iteration 1** **Iteration 2** **Iteration 3** ---------- ----------------- ----------------- ----------------- Leaving maxmax1 maxmax2 maxmax3 *C-027* *EEig01d* *Mor22m* 0.96416 0.9491 0.67855 Entering minmax1 minmax2 minmax3 *EEig04x* *nHAcc* *Mor14e* 0.5455 0.5783 0.5946 The results of the final run of hyper-boxes classification for the 7-descriptor model shows that the effect of changing the less significant descriptors with the more significant ones improved the accuracy of the classification from 96.36% to 100%. Since we have reached 100% accuracy in 7-descriptors models, the significant ones may be included in this model among 912-descriptors that are initially calculated by e-DRAGON. Brief explanations of the descriptors can be found in Table [7](#T7){ref-type="table"}. \[[@B9]\]. ###### Comparison of classification accuracies of best WEKA classifiers with MILP based hyper-boxes classification on P450 C17 inhibitors. **% accuracy** ----------------------------------- ---------------- ------------- ------------- **MILP based hyper-boxes method** **100.00** **100.00** **100.00** Bayes Network ***81.25*** ***81.25*** ***81.25*** Naive Bayes 62.50 71.88 53.13 Naive Bayes Simple 62.50 68.75 50.00 Naive Bayes Updatable 62.50 71.88 53.13 Lojistic 71.88 56.25 62.50 Multilayer Perceptron 62.50 71.88 59.38 SimpleLogistic 75.00 75.00 ***81.25*** SMO ***81.25*** ***81.25*** ***81.25*** IB1 59.38 59.38 ***81.25*** IBk 59.38 59.38 62.50 Logit Boost 71.88 62.50 62.50 Multi Class Classifier 71.88 56.25 62.50 Threshold Selector 43.75 40.63 62.50 LMT 75.00 75.00 ***81.25*** RandomForest 75.00 68.75 65.63 OneR 75.00 71.88 75.00 ###### Brief explanation of the most significant descriptors. **Descriptor** Brief explanation ---------------- ------------------------------------------------------------------------ **Mor10m** 3D-MoRSE -- signal 10/weighted by atomic masses **DISPp** d COMMA2 value/weighted by atomic polarizabilities **Mor14e** 3D-MoRSE -- signal 14/weighted by atomic Sanderson electronegativities **Mor08m** 3D-MoRSE -- signal 08/weighted by atomic masses **nHAcc** number of acceptor atoms for H-bonds (N. O. F) **EEig04x** Eigenvalue 04 from edge adj. matrix weighted by edge degrees **DISPv** d COMMA2 value/weighted by atomic van der Waals volumes Discussion ========== Early analysis and estimation of the drug activities by computational methods are widely studied in order to narrow down drug candidates for further experimental tests. The accuracy comparison of our algorithm with other QSAR algorithms suggests that drug activities can be classified with a significantly higher accuracy with the method introduced in this study. After model building by E-dragon QSAR software, the PLS runs were performed to determine the best model in representing the depended variables (IC~50~values) in terms of the independent variables (the attributes). The corresponding R^2^values were calculated to determine the reliability of the PLS models, where a model with a higher R^2^value can be regarded as a more reliable model to proceed to the classification step. The R^2^values for the 15, 10 and 7 descriptor models of P450 C17 were obtained by PLS runs and, with a considerable strength in representing the IC~50~values we accepted the initial models. While the high R^2^values of the Ache inhibitor models also were promising on its own to build the classification model, the initial models of BZR and COX-2 inhibitor sets were chosen after the comparison of PLS results with the results reported in the literature as presented in Table [3](#T3){ref-type="table"}. For DHFR inhibitors data sets such comparison was not also possible, therefore the models with the best R^2^values in PLS studies were chosen among all other possible models calculated. R^2^value directly depends on the values of attributes (the descriptors) and the number of attributes in the corresponding model. We first applied our iterative algorithm to the large and widely used QSAR data sets in order to validate our methodology. The strength of our algorithm was presented by comparing our classification accuracies with the classification accuracies of 63 WEKA classifiers, on 7 inhibitor sets. The attribute sets prepared as the input for WEKA classifiers were the same as the ones, by which we built the last iteration of our MILP based hyper-boxes classification model. In other words, those were the most significant attributes that we used to develop the final classification models and reached our best accuracies. Our approach outperformed all of the classifiers available in WEKA for each model of the all of the 7 data sets, even reaching 100% accuracy in predicting the activity classification of the inhibitor sets, Ache inhibitors and Cytochrome P450 C17. A total number of 21 QSAR models were built in this study for 7 inhibitor sets, and in 18 of them the accuracy of our methodology exceeded the accuracy of the second best classifier with more than 10%. Through all of the 21 models, the smallest difference in the accuracies is 6.31% and the largest difference is 27.47%. The higher prediction accuracy of the model not only comes from the choice of initial models by PLS analysis but also the characteristics of MILP based hyper-boxes method. The MILP based hyper-boxes approach allows using more than one hyper-box in order to define a single class \[[@B30]\]. Moreover, this approach deals with problematic and non-problematic instances separately and prevents overlapping of final hyper-boxes \[[@B31]\]. Therefore, these strengths significantly improve the accuracy and efficiency of the MILP based hyper-boxes approach compared to other data classification methods. Data on true positive and false positive rates for accuracy comparison of classifiers for all data sets are given in Additional File [2](#S2){ref-type="supplementary-material"}. Conclusion ========== Drug molecules can be classified as low active or high active based on IC~50~values. In this study an integrated approach was introduced, which combines the MILP based hyper-boxes method with partial least squares regression to effectively classify the drug candidates. As a result, the most significant molecular descriptors of the drug molecules were also reported. WEKA is used to compare the classification accuracies of the developed model with the classifiers in the WEKA data mining package. The best classification algorithm in WEKA database gave an accuracy of maximum 85% in classifying the activity of drug molecules, through the datasets used in this study. Our method was applied in order to predict the activities of widely known inhibitor datasets for Acetylcholinesterase (ACHE), Benzodiazepine Receptor (BZR), Dihydrofolate Reductase (DHFR), Cyclooxygenase-2 (COX-2) with known IC50 values. The results suggests that the approach used in this paper results in better classification accuracies compared to other classification methods reported in literature. This approach also applied to Cytochrome P450 C17 inhibitors and their activities were predicted with 100% accuracy. Methods ======= In this paper, we present an integrated approach combining statistical analysis and MILP based hyper-boxes classification method for early prediction of drug behavior targeting Ache, BZR, COX-2, DHFR_TG, DHFR_RL, DHFR_PC, and finally Cytochrome P450 C17. The approach used in this paper is composed of five main steps. In the first step, molecular structures of the drug candidates is built and optimized the by Marvin Sketch\[[@B32]\]. Then, the molecular descriptors of these drug candidates are obtained using the web server E-Dragon \[[@B6]-[@B8]\]. The second step consists of building the regression model using PLS, which will result in selecting the most significant descriptors. Then drug candidates are classified based on the most significant descriptors that are obtained by the previous step, using MILP based hyper-boxes method. This primary classification may result in relatively lower classification accuracy due to the existence of a few insignificant descriptors in the model; therefore, a significance testing analysis is conducted in order to determine the insignificant descriptors that might interfere with our classification accuracy in fourth step. If there are insignificant descriptors in the model we replace the insignificant descriptors with more significant ones; then return to the third step where we classify the drug activities again with the new model that is obtained in step five. After the significance tests if all of the descriptors are significant we build our model with the most significant ones, and report the classification results. We use an iterative algorithm such that, some of the steps can be repeated when the significance tests give unsatisfactory results for the selected descriptors of a particular model. Less significant descriptors are replaced with a more significant ones affecting the final classification of the drugs at each iteration, thus improves the success of the study. The outline of our method is given in Figure [1](#F1){ref-type="fig"}. {#F1} Data sets --------- We applied our algorithm to widely known QSAR data sets available in literature. Dihydrofolate Reductase (DHFR), Acetylcholinesterase (AchE), Benzodiazepine Receptor (BZR) and Cyclooxygenase-2 (COX-2) inhibitor sets are used for classification. We also introduce a new dataset of Cytochrome P450 C17 inhibitors, which we have derived from the literature and calculated their 3D structures. Seven data sets were used for the validation of our methodology by applying the algorithm on these large and known data sets and comparing our classification accuracy on these data sets with the other widely used classifiers available in the WEKA data mining package. Representative compounds from each data set are shown in Figure [2](#F2){ref-type="fig"}. The experimental IC~50~values for the dihydrofolate reductase (DHFR) inhibitor set were calculated and reported \[[@B16],[@B19],[@B22],[@B26]\] for the DHFR enzyme from three different species: *P. carinii*(PC), *T. gondii*(TG) and rat liver (RL), where the activity of the DHFR inhibitors to the enzymes from different species differ. Therefore, activities of the inhibitors towards the enzymes from these three species for DHFR inhibitors are studied separately in our study. A set of 397 dihydrofolate reductase inhibitors (DHFR) were used for *P. carinii*DHFR with IC~50~values from 0.31 nM to 3700 μM, a set of 378 inhibitors were used for *T. gondii*DHFR with values from 0.88 nM to 392 μM and 397 inhibitors were used for rat liver DHFR with values from 0.156 nM to 7470 μM. A set of 111 acetylcholinesterase (AchE) inhibitors were used with experimentally calculated IC~50~values, reported by within the range of 0.3 nM to 100 μM \[[@B23]-[@B26]\]. The data set of the benzodiazepine receptor (BZR) inhibitors consisted of 163 inhibitors, whose IC~50~values were calculated experimentally from 1.2 nM to 5 μM\[[@B20],[@B26]\]. The 322 molecules of cyclooxygenase-2 (COX2) inhibitor set were derived such that IC~50~values from 1 nM to 100 μM \[[@B17],[@B21],[@B26]\]. The QSAR sets used in this study were also used in a comparison study of QSAR methods by Sutherland et al\[[@B26]\]. We also compared the R^2^values of our 3D descriptor models, which were calculated by the Minitab PLS runs in the first phase of our algorithm, with the reported R^2^values by Sutherland et al \[[@B27]\] for several PLS models on the same data sets. {#F2} Structure building and obtaining the descriptor model ----------------------------------------------------- As outlined above, in our study the first step is finding molecular descriptors for the drug candidates. Therefore, Marvin Sketch \[[@B32]\] was used to calculate the molecular structures of each drug candidate should be constructed by building their structure and optimize their energy by minimization to determine their confirmation in 3-D space. Next, the optimized 3-D structures are loaded to E-Dragon and molecular descriptors are calculated by using the web server. E-Dragon suggests many descriptor blocks, each of which contains parameters that describe the characterization of molecules, and the ones that are used in this study can be listed as follows: constitutional descriptors (48), topological descriptors (119), connectivity indices (33), information indices (47), edge adjacency indices (107), topological charge indices (21), geometrical descriptors (74), 3D-MoRSE descriptors (160), functional group counts (154), atom-centered fragments (120), molecular properties (29)\[[@B9]\]. Therefore, the total number of descriptors considered is 912 while building our QSAR descriptor model. PLS\[[@B15]\] is selected for regression analysis because the number of instances is much smaller than the number of attributes (descriptors) by using MINITAB\[[@B28]\]. As we mentioned before, PLS is widely used to develop QSAR models by reducing the number of attributes in the descriptor set to a small number of attributes correlated with the defined property being modeled, which is experimental IC~50~values in our study. Model building with PLS for the selection of the most informative descriptors ----------------------------------------------------------------------------- The main purpose of the regression analysis is to determine the model that predicts the activity (IC~50~) of the drug candidates in terms of the descriptors. PLS can be referred as an MLR method closely related to principal component regression. Basically, by conducting a PLS study we can predict a set of dependent variables *Y*based on a set of independent variables *X*by MINITAB\[[@B28]\], which gave us the PLS runs automatically based on the upper limit we determined on the number of most significant descriptors. Each PLS run provides a linear model of the dependent variable (IC~50~values) with respect to the independent variables (most significant descriptors). At this point, the relevant model is built and the most significant descriptors are determined. The next step would be the initial classification of the drugs based on the descriptors. The choice of the significant descriptors by the first PLS runs may not be the most effective ones in classification. Therefore, we perform significance tests on the selected descriptors by the regression analysis to increase the classification accuracies. Classification of drug candidates with MILP based hyper-boxes method -------------------------------------------------------------------- The third step is devoted to the classification of drugs; we apply the MILP based hyper-boxes method \[[@B30],[@B31]\] by using the selected descriptors from the previous step. The objective in data classification problems is to assign data points, which are described with certain number of attributes, into predefined classes. The strength of hyper-boxes classification method is from its ability to use more than one hyper-box when defining a class as shown in Figure [3](#F3){ref-type="fig"}, and this ability prevents overlapping in the classes, which would not be prevented if the classes were defined with a single hyper-box only\[[@B30]\]. {#F3} The data classification problem is solved in two steps: training step and testing step. In the training step, the boundaries of the classes are formed by the construction of hyper-boxes, where as the effectiveness of the constructed classes are tested in the testing step\[[@B30]\]. The MILP problem for the classification is constructed such that the objective function is the minimization of the misclassifications in the data set with the minimum number of hyper-boxes in the training step. The minimization of the number of hyper-boxes, i.e. the elimination of unnecessary use of hyper-boxes, is enforced by penalizing the existence of a box with a small scalar in the objective function. In the training part the upper and lower bound of each hyper-box also calculated by the data points enclosed in that hyper-box\[[@B30]\]. In the testing step, the data points are assigned to classes by calculating the distance between the data point to the each box, and determining the box that is closest to the data point. Finally, the original and assigned classes of test data points are compared and the effectiveness of the classification is obtained by means of correctly classified instances\[[@B30]\]. Solving the proposed MILP problem to optimality is computationally challenging for large datasets due to the large number of binary variables. Hence, a three-stage decomposition method for obtaining optimal solutions of large data classification problems is developed\[[@B31]\]. Instances that are difficult to classify are identified in the first stage that we refer to as preprocessing. Moreover, seeds are determined for each class to improve the computational efficiency. With greater emphasis given to these observations, a solution to the problem is obtained in the second stage with the modified model. Last, final assignments and intersection eliminations are carried out in the third step\[[@B31]\]. In this paper, we apply this method described above in classifying the activities of drug molecules for the data sets considered. We perform 10-fold cross validation while choosing the training and test sets, where we partition the datasets randomly into 10 subsamples with equal number of members. From these 10 subsamples 9 of them are combined and used as the training set, and the remaining 1 sub sample is used as the test set. Then the classification is performed 10 times with each of the 10 subsamples used exactly once as the test set. Finally, the accuracy of the classification is reported as the average of these 10 classifications. We classify each of the drug candidates in the test set as having a low or high IC~50~value. In this iterative study, this classification step is performed several times: first with the initial set of descriptors then using the enhanced set of descriptors derived from significance analysis. Significance analysis --------------------- In the fourth step, significance tests are performed. After the PLS runs it is possible to conclude a descriptor as significant while it is not in reality and this problem is resolved by conducting significance tests after primary classification. The main idea behind the significance test is as follows: If *Z*is the whole set of drug candidates, assume after the classification it is divided into two classes, A and B. For a successful classification, the variances of descriptor values should be smaller within classes *A*and *B*than it is for the whole population, *Z*. The equation given below in Eq. 2.1 exhibits the *F*distribution. $$\frac{S_{ij}^{2}/\sigma_{i}^{2}}{S_{k}^{2}/\sigma_{i}^{2}} = S_{ij}^{2}/S_{ik}^{2} = f_{\nu\eta}$$ where, $S_{ij}^{2}$ is the sample variance of values for descriptor *i*for drug set *j*, ν = *n*-1 and η = *m*-1 are degrees of freedom, and *n*is the number of values of descriptor *i*for the drug set *j*, and *m*is the number of values of descriptor *i*for the drug set *k*. Then the hypothesis testing is performed by the null hypothesis $S_{ij}^{2} = S_{ik}^{2}$, which suggests that the variance of the whole set of drug candidates is equal to the variance of the drugs within the same class. Since the variance of the whole set of drugs should be larger than the variance within the class, we define our alternative hypothesis as: $H_{a} = S_{ij}^{2} \succ S_{ik}^{2}$, where j is a member of a whole data set and k is a member of the class. Note that the *p*-value of *f*~vη~in the current should be smaller than the *p*-value of *f*~vη~in the previous model to accept the alternative hypothesis. Building the new classification model ------------------------------------- This last step is performed when we conclude that there are overestimated descriptors in the model during step four. Therefore, a total number of 3 models are constructed through regression analysis by selecting 7, 10 and 15 descriptors respectively as representative variables of each model, and the significance analysis is applied to all of the descriptors in these 3 models. If we conclude existence of an insignificant variable in one of these models, we replace them with the ones that are significant in the other models. This adjustment is proved to improve our classification accuracy. When we are replacing the less significant ones, the remaining 880 descriptors that are eliminated during the PLS analysis are ignored, since these 7, 10, and 15 attributes were chosen by the PLS regression analysis and have a proven strength in describing the IC~50~values. The main purpose of the PLS regression study in fact is eliminating the statistically meaningless features, and provide us with the most meaningful sample space to further work with. The results obtained by our method are compared with all of the 63 classification methods available in WEKA, and 16 best WEKA classifiers reported with the results obtained by our algorithm in Table [3](#T3){ref-type="table"}, with the corresponding classification accuracy. The attributes used in WEKA classifiers are the same descriptors that are found after the significance tests, and 10-fold cross validation was applied to each classifier including our classification method. WEKA is a powerful data mining tool to use for comparison purposes, since it includes all widely known machine-learning algorithms among its 63 classifiers. The success of these existing machine learning algorithms in binary classification of active and inactive compounds based on their descriptor values were also previously reported\[[@B33]\]. Following is a brief overview of the best performing data classification methods available in WEKA. A Bayesian network\[[@B34]\]*B*= \<*N*, *A*, Φ \> is a directed acyclic graph \<N, A\> with a conditional probability distribution attached to each node, collectively represented by Φ. Each node *n*∈ *N*represents a dataset attribute, and each arc *a*∈ *A*between nodes represents a probabilistic dependency. The Naive Bayes classifier assumes that all of the variables are independent of each other, where the classification node is represented as the parent node of all other nodes\[[@B35]\]. Naive Bayes Simple uses the normal distribution for the modelling of the attributes and handle numeric attributes using supervise discretization, where as Naive Bayes Updateable is an incremental version, which processes one instance at a time, and uses a kernel estimator instead of discretization. The Logistic classifier\[[@B35]\] builds a two-class logistic regression model. It is a statistical regression model, where logistic regression assumes that the log likelihood ratio of class distributions is linear in the observations. The Simple Logistic classifier builds linear logistic regression models based on a single attribute\[[@B35]\]. The model is a generalized model of the ordinary least squares regression model. Multilayer perceptron\[[@B35]\] is a neural network that uses back propagation. The perceptron, which is a processing element, computes a single output, a nonlinear activation function of linear combination of multiple inputs, whose parameters are learned through the training phase. SMO (sequential minimal optimization)\[[@B36]\], also called the WEKA SVM (support vector machine), is a method to train a support vector classifier using polynomial kernels by breaking a large quadratic programming optimization problem into smaller QP optimization problems. IB1\[[@B35]\] is listed as a lazy classifier, in a sense that it stores the training instances and it does not really do any work until the classification time. IB1 is an instance based learner. It finds the training instance closest in Euclidian distance to the given test instance. IBk is a k-nearest-neighbor classifier that uses the same idea. Logit Boost\[[@B37]\] uses additive logistic regression. The algorithm can be accelerated by assigning a specific threshold for weights. Multi Class Classifier\[[@B38]\] uses four distinct two-class classification methods for multiclass problems. The Threshold Selector\[[@B35]\], which is a meta learner optimizes the F-measure by selecting a probability threshold on the classifiers output. Random forest and LMT are decision tree methods. Random Forest generates random trees by collecting ensembles of random trees, where as LMT builds logistic model trees, and uses cross validation to determine the number of iterations while fitting the logistic regression functions at each node. OneR (one rule)\[[@B35]\] builds a one-level decision tree and learns a rule from each attribute and selects the rule having the smallest error rate as the one rule. Authors\' contributions ======================= PA gathered all data used in this study, made all computational runs, analysis of the results and drafted the paper. MEO performed the annotation of the drug data and helped in the biological analysis of the results. FUY worked on the development of the mixed-integer programming based hyper-boxes method and helped in the use of computer programs for classification. IHK supervised the biological analysis of the input data, interpretation of the results and helped draft the manuscript. MT instigated and guided the research project, supervised the development of the methods used in this project and helped draft the manuscript. All authors read and approved the manuscript. Supplementary Material ====================== ###### Additional file 1 Cytochrome P450 C17 inhibitors, their IC50 values and reference IC50 values with ketoconazole. The molecular structures and IC50 values for Cytochrome P450 C17 inhibitors. ###### Click here for file ###### Additional file 2 True positive and false positive rates for accuracy comparison of classifiers. Data on true positive and false positive rates for accuracy comparison of classifiers for all data sets. ###### Click here for file Acknowledgements ================ The computing hardware support from IBM SUR Award is gratefully acknowledged. IHK thanks for the support of Turkish National Academy of Science of Turkey for young investigator program (TUBA-GEBIP).

Introduction ============ Human norovirus (HuNV) is currently a leading cause of acute gastroenteritis in adults and predicted to become the predominant cause of diarrhea in all age groups worldwide ([@B31]; [@B5]). HuNV can establish chronic infection and becomes life-threatening in immunocompromised patients ([@B5]). However, no effective antiviral or vaccine against HuNV are available yet, mainly due to the lack of an efficient *in vitro* and *in vivo* infection system of HuNV. The recent developments of immortalized B cell lines, the stem-cell-derived organoid system, and BALB/c Rag-γc-deficient mice for HuNV infection provide new models to study HuNV ([@B40]; [@B24]; [@B12]). Since its isolation from immunocompromised mice ([@B43]), murine NoV (MNV) has been serving as an effective surrogate model ([@B25]; [@B44]) to elucidate the molecular mechanism of NoV replication and pathogenesis. Norovirus (NoV) is a positive-sense single-stranded RNA (+RNA) virus belonging to the *Caliciviridae* family. The ∼7.6 kb +RNA genome of NoV contains three open reading frames (ORFs) ([@B19]), with an additional ORF4 for MNV ([@B28]). ORF1 encodes a polyprotein that is cleaved into non-structural proteins, including virion protein genome-linked (VPg) and RNA-dependent RNA polymerase (RdRp) ([@B35]). RdRp amplifies the viral genome using ribonucleotide triphosphates (rNTPs) as substrates in low-fidelity due to the lack of an effective proofreading mechanism. The initiation of RNA synthesis by RdRp on an RNA template can be either primer-dependent or -independent in *Caliciviridae*, *Picornaviridae*, and *Potyviridae* ([@B15]; [@B14]; [@B23]). In the former case, VPg serves as a primer for these viruses, providing free hydroxyl groups from a tyrosine or serine residue. VPg covalently attached to the 5′ end of viral RNAs plays crucial roles in viral protein translation and genome replication ([@B7]; [@B20]; [@B14]; [@B23]). VPg is an intrinsically disordered molten globule-like protein with multiple functions. It is highly diverse in sequence and size, ranging from 2 to 90 kDa, of which the largest belongs to *Birnaviridae* possessing bisegmented dsRNAs with its RdRp linked as VPg ([@B8]). The genomic RNA of feline calicivirus, a member of *Caliciviridae*, is not infectious after proteolytic cleavage of the VPg ([@B20]). VPg was shown to bind to its cognate RdRp in enterovirus71 (EV71), foot and mouth disease virus (FMDV), and coxsackievirus (CV) ([@B13]; [@B16]; [@B9]), with which the VPg of caliviruses shares no sequence homology. No caliciviral RdRp-VPg complex structure is yet available and the role of the RdRp--VPg interaction in NoV replication remains unknown. In this study, we characterized some biochemical and biophysical properties the MNV RdRp-VPg(1-73) complex. The MNV VPg induced the formation of higher-order multimers or tubular fibrils of RdRp and enhanced the RdRp activity. The replication of MNV mutants with VPg-binding defective RdRps was completely blocked in a cell culture system. Moreover, the crystal structure of the complex provided the evidence that the interaction between VPg and RdRp plays a crucial role in NoV replication through the higher-order multimer formation of RdRp molecules. Materials and Methods {#s1} ===================== Subcloning, Protein Expression, and Purification ------------------------------------------------ MNV RdRp and different lengths of VPg including VPg(1-73) were cloned into pET14b or pET22b vectors and introduced into *Escherichia coli* ER2566 or BL21 (DE3), as described previously ([@B18]; [@B26]). VPg(1-124) and eight truncates with different lengths, VPg(1-73, 1-86, 1-96, 1-104, 1-119, 20-124, 40-124, and 64-124), were used to construct clones. Single-amino-acid-changed mutants of R239A, D331A, and L354D of RdRp were constructed via site-directed mutagenesis by PCR amplification (**Table [1](#T1){ref-type="table"}**). The purification of MNV RdRp protein used for the structural and biochemical studies has been described previously ([@B18]; [@B26]). Briefly, protein expression was induced with isopropyl β-[D]{.smallcaps}-1-thiogalactopyranoside at 37°C for 4 h or at 15°C overnight. The cells were disrupted by sonication after treatment with DNase and RNase. The recombinant proteins were purified using a nickel-nitrilotriacetic acid (Ni-NTA) affinity and gel filtration chromatography. For the biochemical assay and electron microscopy studies, recombinant RdRp, VPg(1-124) and VPg(1-73) were prepared at 1--1.5 mg/mL. The proteins were quantified by using Nanodrop 1000 (Thermo Scientific) and SDS-PAGE. ###### Primers for mutants involved in the RdRp-VPg complex. Name Sequence Purpose ---------------- ------------------------------------------ ----------------------------- RdRp-F^1^ GGAATTCCATATGCTTCCCCGCCCCTCAGGCACCTAT RdRp amplification RdRp-R^1^ ATAAGAATGCGGCCGCATCCTCATTCACγGACTGCTGA RdRp amplification RdRp-F^2^ CATATGGGACCCCCCATGCTTCCCC RdRp amplification RdRp-R^2^ GGATCCTCACTCATCCTCATTCACγGA RdRp amplification R239A-F CACGCCAATTTC[GCA]{.ul}TACCACATGGATGCTGAC Arg^239^ to Ala mutant RdRp R239A-R GTCAGCATCCATGTGGTA[TGC]{.ul}GγTTGGCGTG Arg^239^ to Ala mutant RdRp D331A-F GTAACCCGAGTT[GCA]{.ul}CCTGACATTGTG Asp^331^ to Ala mutant RdRp D331A-R CACAATGTCAGG[TGC]{.ul}AACTCGGGTTAC Asp^331^ to Ala mutant RdRp L354D-F GTTTCGACCAAC[GAU]{.ul}GAGTTGGATATG Lys^354^ to Asp mutant RdRp L354D-R CATATCCAACTC[ATC]{.ul}GTTGGTCGγC Lys^354^ to Asp mutant RdRp VPg (1-124)-F GGAATTCCATATGGGγGAAGGGCAAGAACAAGAAG VPg (1-124)-R CCGCTCGAGCTCγGTTGATCTTCTCGCCGTA VPg(20-124)-F GGAATTCCATATGCTCACGGATGAGGAGTACGATGAA VPg(20-124)-R CCGCTCGAGCTCγGTTGATCTTCTCGCCGTA VPg (40-124)-F GGAATTCCATATGTCCATTGATGATTACCTCGCTGAC VPg (40-124)-R CCGCTCGAG CTCγGTTGATCTTCTCGCCGTA VPg (64-124)-F GGAATTCCATATGTTCGGGGATGGCTTCGGGTTGAAG VPg (64-124)-R CCGCTCGAGGCCCAGTTTGGCTCTCTCTGCCTT VPg (1-73)-F GGAATTCCATATGGGγGAAGGGCAAGAACAAGAAG VPg (1-73)-R CCGCTCGAGCCCGAAGCCATCCCCGAAGATAGCCTC VPg (1-86)-F GGAATTCCATATGCTCACGGATGAGGAGTACGATGAAG VPg (1-86)-R CCGCTCGAGGCCCAGTTTGGCTCTCTCTGCCTT VPg (1-96)-F GGAATTCCATATGTCCATTGATGATTACCTCGCTGAC VPg (1-96)-R CCGCTCGAGGCGGGCGCGGATGTCGCCACCAGA VPg (1-104)-F GGAATTCCATATGTTCGGGGATGGCTTCGGGTTGAAG VPg (1-104)-R CCGCTCGAGGGGGCCAACCACATTCCAGTCGAT VPg (1-119)-F GGAATTCCATATGTTCGGGGATGGCTTCGGGTTGAAG VPg (1-119)-R CCGCTCGAGCTCGCCGTAGTCGACCTGGCGGTC 1,2 RdRp constructs were cloned into the pET22b vector with C-terminal His-taq 1 or the pET14b vector with N-terminal His-taq 2 . Cross-Linking Assays -------------------- Inter- and intramolecular interactions between RdRp native or mutants and VPg were tested by glutaraldehyde cross-linking assay. The reaction mixture containing 50 mM HEPES (pH 7.4), 5 mM MgCl~2~, 5 mM DTT, 1 μM oligo(A)~15~, 2 μM RdRp, and 6 μM VPg, was incubated with or without 0.5% SDS. The reaction mixture was treated with 0.001% glutaraldehyde and analyzed by SDS-PAGE followed by western blotting with anti-RdRp or anti-VPg antisera (from immunizing rabbits, Cosmo Genetech, Seoul, South Korea). Electron Microscopy ------------------- The reaction mixture was placed on carbon-coated copper grids, followed by staining with 0.75% uranyl formate. Images were collected on a 4K × 4K Eagle HS CCD camera (2.1 A/pixel) on a Tecnai T120 microscope (FEI) operating at 120 kV. The defocus and nominal magnification for all images were 1.5 μm and ×52,000, respectively (pixel size: 2.10 Å). In order to identify the RdRp hexamer, a total of 5,149 particles were semi-automatically selected from 150 micrographs using EMAN2 boxer and bad particles were manually excluded ([@B39]), and it was subjected to two-dimensional (2D) reference-free alignment, multivariate statistical analysis (MSA), and MSA classification, which were iterated by using the IMAGIC software ([@B42]). Five representative class average images were compared with the corresponding forward projection images of the hexamer model. To observe the multimerization, samples were prepared for electron microscopic studies by mixing (a) 2 μM RdRp, 6 μM VPg(1-73) or 1 μM VPg(1-124) and 16 μM oligo(A)~8~, (b) 2 μM RdRp, 6 μM VPg(1-73) or 1 μM VPg(1-124), (c) 2 μM RdRp or 6 μM VPg(1-73) or 1 μM VPg(1-124), and (d) 2 μM RdRp or 6 μM VPg(1-73) or 1 μM VPg(1-124) and 16 μM oligo(A)~8~, in the presence of 2 mM UTP, 2 mM MnCl~2~, and 2 mM MgCl~2~. For mutants, 2 μM of R239A, D331A, or L354D protein was used instead of the native protein. The reaction mixture was incubated for 1 h at room temperature, and then 3 μL of the reaction mixture was placed on carbon-coated copper grids. *In Vitro* Binding Affinity Using SPR and RdRp Assay ---------------------------------------------------- In order to confirm the binding of VPg to RdRp native or mutants, SPR experiments were carried out to determine the affinity (*K*~D~) of the protein--protein interactions for MNV RdRp-RdRp or RdRp-VPg by using the Biacore2000 system (GE Healthcare, Korea Basic Science Institute, Seoul Branch). RdRp or mutant proteins in sodium acetate buffer (pH 5.0) were immobilized onto a CM5 chip after pH scouting, regeneration scouting, and chip activation with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/*N*-hydroxysuccinimide. Proteins were injected onto the chip by the kinetics method after being serially diluted in 20 mM Tris--HCl, pH 8.0, 100 mM NaCl, and 5 mM MgCl~2~. The affinity constant *K*~D~ was determined from the association (*k*~a~) and dissociation (*k*~d~) rates, by evaluation of the 1:1 Langmuir binding model kinetics in the sensorgrams. The RdRp assay was performed as described previously ([@B18]), with minor modifications. *In Vitro* RNA-Dependent RNA Polymerase Assay --------------------------------------------- Template DNAs for the *in vitro* transcription of sgRNA for RNA polymerase assay were amplified by PCR and the RdRp assay was performed as described previously ([@B18], [@B17]), with minor modifications. For RdRp assay with (+)sgRNA as a template, 50 μL of reaction mixture containing 50 mM HEPES, pH 7.4, 5 mM MgCl~2~, 10 mM dithiothreitol (DTT), 1 μg of *in vitro* transcribed (+)sgRNA, 250 μM of each NTP, 2.5 μCi \[α-^32^P\]UTP (3000 Ci mmol^-1^, 10 mCi/mL), and 1 μg RdRp (0.33 μM) was incubated with 0.6--2.4 μg (0.7--2.8 μM) of VPg, or with BSA as a control, for 1 h at 37°C. The reaction was stopped by adding an equal volume of 200 mM EDTA (pH 8.0), and the RNA was extracted by using the RNeasy Mini Kit and analyzed by agarose gel electrophoresis in Tris--borate--EDTA buffer. After electrophoresis, the gel was dried and the \[α-^32^P\]UTP labeled RNA was detected with a BAS-1500 imaging system (Fujifilm, Tokyo, Japan). Crystallization and Structure Determination ------------------------------------------- MNV RdRp protein was incubated with VPg at a molar ratio of 1:2 in the presence of 2 mM uridine triphosphate (UTP) or guanidine triphosphate (GPT), 10 nM oligo(A) (A~8∼10~) or oligo C (C~6∼10~), 2 mM MnCl~2~, and 2 mM MgCl~2~ at 4°C overnight. The hanging drop vapor-diffusion method was used for the crystallization by mixing complex protein and 0.1 M cacodylate (pH 6.5) buffer containing 1.0 M sodium citrate. Triangular-shaped crystals in the P2~1~ space group appeared within a week, with six monomers in the asymmetric unit. Diffraction data were collected using synchrotron radiation sources at beamline 17A of Photon Factory (Tsukuba) and 5C of PAL (Pohang, South Korea), with the crystals flash-cooled at 100 K in a stream of liquid N~2~. The data were integrated, scaled, and processed using the HKL2000 program ([@B30]). The initial model of the complex structures was built using molecular replacement and AutoBuild from the PHENIX suite ([@B1]), employing a previously solved native structure (PDB 3QID) as a search model. Initial difference Fourier maps of the RdRp-VPg(1-73)-RNA complex, with coefficients 2\| F~o~\| -\| F~c~\| and \| F~o~\| -\| F~c~\|, showed an elongated extra density at the interface of the RdRp hexamers that could be interpreted as VPg, mainly in α-helical conformation. VPg was built manually in the Coot program ([@B11]). Using a high-resolution structure as a template, exploiting geometric redundancies (NCS) and B-sharpening ([@B6]) did not improve the electron density maps sufficiently to build a complete VPg model. The VPg structure was verified by examining a composite simulated annealing omit map at the 1.2σ level. The *R* and *R*~free~ values for RdRp-VPg(1-73)-RNA were 20.3 and 24.1%, respectively, after rigid body, NCS, TLS refinements coupled with Ramachandran restraints using the PHENIX program ([@B6]; [@B1]). Ramachandran analysis revealed 97.9, 1.9, and 0.2% in the favored, allowed, and outlier regions, respectively. The structural figures were generated with PyMOL.^[1](#fn01){ref-type="fn"}^ The data quality and refinement statistics are presented in **Table [2](#T2){ref-type="table"}**. Production of Plasmid-Derived Recombinant MNV --------------------------------------------- The expression cassette of MNV CW3 strain was subcloned from pCW3 ([@B37]) to pcDNA3 via restriction enzyme cloning. This pcDNA3-MNV system was used to produce recombinant MNV with mutated RdRp. Specific RdRp mutations were generated using QuikChange II Site-Directed Mutagenesis kit (Agilent Technologies) according to the manufacturer's instructions; the sequence of wild type RdRp were modified to R239A (from TTC[AGG]{.ul}TAC to TTC[gca]{.ul}TAC), D331A (from GTT[GAC]{.ul}CCT to GTT[Gca]{.ul}CCT), L354D (from AAC[CTC]{.ul}GAG to AAC[gat]{.ul}GAG), and DDAA (from GGT[GATGAC]{.ul}GAG to GGT[gcggcg]{.ul}GAG). Five hundred nanograms of the recombinant plasmids were transfected into 293T-CD300LF cells (1 × 10^5^ cells/well in 24-well plate) using Lipofectamine 2000 (ThermoFisher Scientific), and the transfected cells were harvested by freezing at 24, 48, and 72 h post-transfection (hpt) to titrate produced infectious viruses. Four micrograms of the recombinant plasmids were transfected into 293T-CD300LF cells (5 × 10^5^ cells/well in 6-well plate) using standard Calcium/Phosphate transfection method, and the transfected cells were lysed to analyze expressed viral proteins; were lysed in TRI reagent (Sigma-Aldrich, T9424) at 24, 48, and 72 hpt to measure viral genome replication and transcription; and fixed on coverslips with PBS containing 2% formaldehyde (Ted Pella) at 24, 48, and 72 hpt to examine the localization of viral proteins and dsRNAs. Cell Culture ------------ 293T cells transduced with the MNV receptor CD300LF (293T-CD300LF) ([@B29]) were used to analyze MNV replication and viral gene expression upon transfection of plasmids expressing recombinant MNVs. BV-2 cells were used to titer infectious virus for TCID~50~ analysis. Both cell lines were cultured as previously described ([@B22]). Western Blot Analysis --------------------- The transfected cells were lysed with sample buffer (0.1 M Tris, pH 6.8, 4% SDS, 4 mM EDTA, 286 mM 2-mercaptoethanol, 3.2 M glycerol, and 0.05% bromophenol blue). The resultant cell lysates were resolved by SDS-PAGE and proteins were specifically detected using the following antibodies: rabbit anti-ProPol \[detecting both protease and polymerase (RdRp) of MNV\] ([@B22]) and anti-Actin. Tissue Culture Infectious Dose 50 (TCID~50~) -------------------------------------------- The transfected cells with media were harvested by freezing and lysed by one cycle of freeze-and-thaw. The infectious viruses were tittered via TCID~50~ analysis as previously described ([@B4]). Quantitative PCR ---------------- The transfected cells were lysed in TRI reagent and RNAs were extracted and column purified (Zymo Research) after DNAse treatment according to the manufacturer's instruction. cDNAs were reverse transcribed from 1 μg of the DNAse-treated RNAs using IMPROM-II reverse transcriptase (Promega) according to the manufacturer's instruction. Quantitative PCR analysis was conducted as previously described ([@B4]) using SYBR-green reagents with the following sets of primers: 5′-AGCTCAGGATGGTCTCGGAT-3′ and 5′-TCAAGAGCAAGGTCGAAGGG-3′ for both positive and negative-strand of MNV, 5′-GAGTCAACGGATTTGGTCGT-3′ and 5′-TTGATTTTGGAGGGATCTCG-3′ for GAPDH. Immunofluorescence Analysis --------------------------- The transfected cells were fixed on coverslips and permeabilized with PBS containing 0.05% Saponin and blocked and probed in PBS containing 0.05% Saponin and 5% normal donkey serum, which were mounted on glass slides with ProLong Diamond Antifade Mountant (Life Technologies). Nuclei were stained with Hoechst 33342 and the following antibodies were used: guinea pig anti-ProPol ([@B22]), rabbit anti-VPg, rabbit anti-VP1 ([@B22]), mouse anti-dsRNA, Alexa Fluor 647 Donkey anti-Guinea Pig, Alexa Fluor 488 Donkey anti-Rabbit, and Alexa Fluor 555 Goat polyclonal anti-Mouse. Images were acquired using the EVOS FL Cell Imaging System. Results ======= Chracterization of Higher-Order Structures of RdRp Mediated by VPg ------------------------------------------------------------------ Our initial attempt to prepare the MNV proteins of RdRp and VPg was not successful due to the instability of the full-length VPg(1-124) and RdRp. The N- and C-terminal regions of VPg were predicted to be disordered according to the disorder prediction program ([@B33]), similar to the VPg of rabbit hemorrhagic disease virus and Sapporo virus in the *Caliciviridae* family ([@B27]). Nine constructs with various deletions at the N- or C-termini of the full length VPg(1-124) were prepared (**Table [1](#T1){ref-type="table"}**). Among them, VPg(1-73) was found to be stable (Supplementary Figure [S1](#SM1){ref-type="supplementary-material"}). RdRp tends to readily form aggregates at high concentrations and the experiments were performed at the concentration where it is readily soluble. We initially examined the RdRp--VPg interaction using chemical crosslinking with glutaraldehyde, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. RdRp was found to form dimers (119.4 kDa), trimers (179.1 kDa), and hexamers (358.2 kDa) (**Figure [1A](#F1){ref-type="fig"}**, lane 5), whereas VPg existed as a monomer (lane 4). Protein bands of approximately 76 kDa were detected by both anti-RdRp and anti-VPg antisera when VPg was incubated with RdRp (lanes 6--8), matching the sum of the RdRp (59.7 kDa) and VPg (16.6 kDa) masses. This 76 kDa band was not detected when RdRp or VPg was incubated separately (lanes 4 and 5) or when the protein interaction was interrupted by the addition of 0.5% SDS (lanes 1--3). Protein bands with molecular masses higher than the RdRp hexamer were observed upon addition of VPg in a dose-dependent manner (lanes 6--8). {ref-type="fig"}**. **(D)** Electron microscopic images of the RdRp-VPg(1-73)-oligo(A)~8~ or RdRp-VPg(1-124)-oligo(A)~8~ complexes are shown (1D,  and ). An RdRp tubular fibril (approximately 18 nm in diameter) formed after incubation with VPg(1-124) and oligo(A)~8~ is shown (1D, ). A comparison between VPg(1-73) and VPg(1-124) in complex with RdRp is shown in the absence of oligo(A)~8~ (1D,  and ).](fmicb-09-01466-g001){#F1} To quantitatively examine the interaction between RdRp and VPg, surface plasmon resonance (SPR) assays were carried out to measure binding affinities using immobilized RdRp. The affinity constants (*K*~D~) were 2.2 ± 1.1 and 8.7 ± 0.6 nM for the RdRp--RdRp and RdRp--VPg(1-124) interactions, respectively (data not shown and **Figure [1B](#F1){ref-type="fig"}**), indicating a comparable binding affinity between RdRp and VPg(1-124) to that between RdRp molecules. The *K*~D~ for RdRp-VPg(1-73) complex was 17 ± 0.4 nM (data not shown), which is slightly lower affinity than that of the RdRp-VPg(1-124) complex, suggesting a role of the disordered C-termini of VPg in its interaction with RdRp. The interaction of RdRp with VPg was further examined using transmission electron microscopy. The RdRp molecules by themselves were found to form densely packed individual hexamers or fibrous hexameric oligomers like hexamers in tandem stacks (**Figure [1C](#F1){ref-type="fig"}**, left panel, arrows). The formation of hexamers was supported by 2D averaged images (**Figure [1C](#F1){ref-type="fig"}**, right panel). Interestingly, addition of VPg(1-73) or VPg(1-124) induced the formation of aggregates or higher-ordered structures of RdRp in the presence of oligo(A)~8~ RNA, although less with VPg(1-73) than with VPg(1-124) (**Figure [1D](#F1){ref-type="fig"}**). No aggregate was observed with either VPg or RdRp alone (data not shown), although in the presence of oligo(A)~8~ the aggregated forms of RdRp or VPg were occasionally observed (**Figure [1D](#F1){ref-type="fig"}**, lower panel). Notably, RdRp often formed a fibril-like structure in the presence of VPg(1-124) and oligo(A)~8~ (**Figure [1D](#F1){ref-type="fig"}**, upper right). Such fibril formation of RdRp was also observed with the RdRp of FMDV and poliovirus ([@B36]; [@B26]; [@B3]). Crystal Structure of the RdRp-VPg(1-73)-RNA Complex --------------------------------------------------- Next, we determined the complex structure of RdRp-VPg(1-73) at 3.1 Å resolution (**Table [2](#T2){ref-type="table"}**). Continuous electron density was observed at the base of the palm domain of RdRp in the initial map. Two helix backbones (α1 and α2) modeled for VPg were fitted to the electron density with lower B factor (**Figure [2A](#F2){ref-type="fig"}**), which was verified by examining a simulated annealing composite omit map at the 1.2σ level (Supplementary Figure [S2](#SM2){ref-type="supplementary-material"}). However, the electron densities for the side chains of VPg were not well defined, whereas those of RdRp were clearly interpretable. Superposition of the VPg backbone in the complex with previously determined nuclear magnetic resonance (NMR) structure of MNV VPg(11-85) (PDB 2M4G) gave the root-mean-square deviation (RMSD) of 1.5 Å based on secondary structure matching ([@B27]), indicating little change in VPg structure upon the complex formation. MNV VPg had a distinct helix-loop-helix conformation and bound to the palm domain of RdRp, and the binding site was relatively similar to that of EV71 RdRp ([@B9]) but far from that of VPg at the active site of FMDV or the thumb domain of CV RdRps (**Figure [2B](#F2){ref-type="fig"}**) ([@B13]; [@B16]). Viral VPgs show diverse conformations among the members of the same family ([@B13]; [@B16]; [@B9]); the VPg proteins of EV71, FMDV, and CV in the *Picornaviridae* family exhibit V-shaped, loop-rich, and extended conformations, respectively. In contrast, viral RdRps have very similar structure; superposition of the RdRp structures of MNV, EV71, FMDV, and CV gave RMSD values of 2.5--2.7 Å. At the interface between VPg and the palm domain of RdRp, the side chains of MNV RdRp were well-resolved (**Figure [2C](#F2){ref-type="fig"}**). Although the sequences of RdRp residues at the interface with VPg were not highly conserved in the members of the *Caliciviridae* and *Picornaviridae* (Supplementary Figure [S3](#SM3){ref-type="supplementary-material"}), Asp331 and Leu354 were selected for a mutational analysis to examine their roles in RdRp--VPg interactions. The electron density for the oligo(A)~8~ RNA was not visible in the complex structure, despite the presence of the oligonucleotide in the crystallization solution. Superposition of the complex with the HuNV RdRp-RNA complex (3BSO) suggested a potential location of RNA at the active site and within the closed conformation of fingers and thumb domains ([@B45]; [@B17]). ###### Data collection and refinement statistics. RdRp-VPg(1-73)-RNA complex ----------------------------- ---------------------------- **Data statistics** Space group P2~1~ Cell dimensions  *a*, *b*, *c* (Å) 109.3, 159.8, 121.7  *α*, *β*, *γ* (°) 90.0, 97.2, 90.0 Resolution (Å) 50-3.1 *R*~sym~ or *R*~merge~ 19.8 (51.9)^a^ *I*/σ 5.2 (1.4)^a^ Completeness (%) 96.4 (90)^a^ Redundancy 3.6 (2.4)^a^ **Refinement statistics** Resolution (Å) 3.1 No. of reflections 69,633 *R*~work~/*R*~free~ 20.3/24.1 No. of atoms 23,161  RdRp 22,819  VPg 324  Water 18 *B*-factors^b^  RdRp 54.5  VPg 65.8  Water 29 Root-mean-square deviations  Bond lengths (Å) 0.004  Bond angles (°) 0.545 a Values in parentheses are for the highest-resolution shell. RdRp-VPg(1-73)-RNA: RdRp complexed to VPg(1-73) in the presence of RNA. b Average B value for whole chain atoms . {ref-type="fig"}**. Scale bars are noted.](fmicb-09-01466-g002){#F2} Spatial Arrangement of the RdRp-VPg(1-73) Complex ------------------------------------------------- The overall structure of MNV RdRp in the RdRp-VPg(1-73) complex was very similar to that in the native RdRp and RdRp-ligand complex structures as previously reported ([@B26]; [@B2]). A large ball-like hexameric arrangement of RdRp was observed in the crystal structures (**Figure [2D](#F2){ref-type="fig"}**). A face-to-face contact of the palm domains was capable of forming a dimeric structure, and the trimer interface was largely made up of the thumb and fingers domains in the hexameric complex. Previous size-exclusion chromatography and cross-linking assays showed that MNV RdRp exists primarily as a monomer in room temperature solution but oligomerize at 37°C, resulting in heavy precipitation ([@B26]; [@B2]). HuNV RdRp showed dimeric arrangements only in its crystal structure ([@B21]). The RdRp-VPg(1-73) complex structure showed that the RdRp hexamers were more closely packed in the presence of the VPg(1-73). When viewed from the top along the threefold axis, the hexamers in the native were aligned to form a triangular shape via interactions at the triangle vertices (**Figure [2E](#F2){ref-type="fig"}**, left). In contrast, those in the RdRp-VPg(1-73) complex were packed more tightly, face to face, by six neighboring hexamers, which could drastically alter the RdRp hexamer clustering (**Figure [2E](#F2){ref-type="fig"}**, right). Further, the interactions between RdRp molecules in the RdRp-VPg(1-73) complex were distinct from that of the native; interactions between Arg329 and Gln389 and between Asp91 and Arg411 of adjacent RdRp molecules were observed in the RdRp-VPg(1-73) complex structure but absent in the native (Supplementary Figure [S4](#SM4){ref-type="supplementary-material"}). D331A and L354D mutants did not promote the formation of higher-order structures of RdRp in the presence of VPg and oligo(A)~8~, suggesting that the point mutations inhibited the interaction of RdRp with VPg and consequently the multimerization process (**Figure [2F](#F2){ref-type="fig"}**). An R239A mutant of RdRp was prepared as a control for D331A and L354D mutants, which is located near the interface but not involved in the interaction between RdRp and VPg in the complex model (**Figure [2C](#F2){ref-type="fig"}**), and it induced multimerization as significantly as the native. When we compared the binding of D331A and L354D mutants to VPg(1-124) with that of the WT RdRp by a Langmuir 1:1 binding kinetic analysis, the binding affinity of the mutants was significantly decreased to 23 ± 0.8 and 210 ± 1.6 nM, respectively, but not completely abolished (Supplementary Figure [S5](#SM5){ref-type="supplementary-material"}). Therefore, these results strongly suggested that the higher-order multimerization or tubular fibril formation of RdRp molecules was mediated by VPg and that Asp331 and Leu354 of RdRp were involved in the interaction of RdRp with VPg for the multimer formation in the presence of RNA. Since the exact residues of VPg at the interface were not discernible in the electron density maps due to low resolution, the side chains from the known VPg structure (PDB 2M4G) were used to deduce the residues of VPg at the interface. Accordingly, L55D of VPg was prepared as a potential non-interactor of RdRp and examined for its effect on the multimerization of RdRp. However, we did not observe any difference between the native VPg and VPg/L55D mutant via cross-linking assays (data not shown), indicating the necessity of a better structural resolution of VPg for its functional study in RdRp multimerization. RdRp--VPg Interaction in RNA Synthesis and Viral Replication ------------------------------------------------------------ We investigated the functional consequence of the VPg-mediated multimerization of RdRp. In an *in vitro* RNA synthesis assay, MNV RdRp synthesized negative-stranded RNA *de novo* from poly(A) tailed subgenomic RNA (sgRNA), consequently forming double-stranded RNA (**Figure [3A](#F3){ref-type="fig"}**). Upon addition of purified VPg into the reaction mixture, the formation of double-stranded RNA was substantially increased in a dose-dependent manner. In contrast, the addition of an unrelated protein (bovine serum albumin, BSA) into the reaction mixture did not affect the RNA synthesis. To determine the role of VPg priming in this VPg-mediated enhancement of RdRp activity, priming-defect mutants of VPg were subjected to the same RNA synthesis assay. Both Y26F and Y117F mutants blocking the canonical and non-canonical guanylylation ([@B18]; [@B38]), respectively, enhanced the *in vitro* RNA synthesis like the wild type VPg, indicating that the VPg-mediated enhancement of RNA synthesis was independent of VPg-priming (**Figure [3A](#F3){ref-type="fig"}**, lower panel). ![RdRp--VPg interaction for RNA synthesis and viral replication. **(A)** Representative autoradiograph of *in vitro* RNA synthesis assay. The RNA synthesis was monitored by the generation of double strand RNA (dsRNA) from poly(A) tailed sgRNA template in the presence of \[α-^32^P\]UTP. T7 indicates T7 RNA polymerase as a non-polymerizing control (upper panel). The experiments were repeated in duplicates. The RNA polymerase activity of recombinant RdRp (1 μg) in 50 mM HEPES, pH 7.4, 5 mM MgCl~2~, 10 mM dithiothreitol (DTT), 1 μg of *in vitro* transcribed (+)sgRNA, 250 μM of each NTP, 2.5 μCi \[α-^32^P\]UTP was determined in the presence of VPg native, Y26F or Y117F, analyzed by agarose gel electrophoresis in Tris--borate--EDTA buffer (lower panel). BSA was used as a control. **(B)** Representative western blot data showing the expression of RdRp at 24 hpt of plasmids producing the indicated recombinant MNVs into 293T-CD300LF cells. As a positive control, the cells were infected with MNV at the multiplicity of infection (MOI) of five for 24 h (MNV inf.). Actin as loading controls. *N* = 2 replicates. **(C)** Growth analysis of recombinant MNVs upon transfection of 293T-CD300LF cells with plasmids producing MNVs with the indicated mutations. Data as mean ± SEM. Dashed line indicates the limit of detection. *N* = 3 replicates. **(D)** Quantitative PCR analysis for the positive and negative strands of MNV generated at 24, 48, and 72 hpt as described in **(C)**. \# in negative strand indicates inclusion of false-positive signals due to remaining plasmid DNAs even after DNAse treatment. Gel pictures under bar graph show PCR products after 40 cycles of amplification. As a positive control, the cells were infected with MNV at MOI = 0.05 for 72 h (MNV inf.). *N* = 3 replicates.](fmicb-09-01466-g003){#F3} To examine the role of the RdRp--VPg interaction in the lifecycle of MNV, we utilized a plasmid-based reverse-genetic system of MNV ([@B37]); we mutated the relevant sequences in the plasmid to generate recombinant MNVs harboring R239A, D331A, or L354D mutants of RdRp. We also made an MNV plasmid with D346A/D347A (henceforth DDAA) mutations in RdRp as a non-replicating control; DDAA is a polymerase-activity-dead mutant of RdRp ([@B26]). We transfected these recombinant plasmids into 293T cells stably expressing the MNV receptor CD300LF (293T-CD300LF) ([@B29]) for efficient transfection and amplification of produced virus. In the transfected cells, all RdRp mutants were properly processed and expressed like native RdRp in the MNV-infected cells, although there was less RdRp/L354D compared to the others (**Figure [3B](#F3){ref-type="fig"}**). Strikingly, we were not able to recover any infectious MNV from the plasmids containing D331A or L354D mutants of RdRp (**Figure [3C](#F3){ref-type="fig"}**). In contrast, the recombinant MNV with the RdRp/R239A mutation replicated similarly to the control plasmid-derived MNV with native RdRp. We further checked the synthesis of positive and negative strand MNV RNA from the transfected plasmids (**Figure [3D](#F3){ref-type="fig"}**). Positive strand MNV RNA was detected from all constructs including the polymerase-dead mutant (DDAA), indicating a basal-level signal of positive strand MNV RNA from remaining plasmid DNAs and the active CMV promoter of all constructs. However, the plasmids with the native or R239A RdRp produced about 1,000-fold more positive strand RNA at 72 hpt. The data suggested that there was an approximately 1,000-fold amplification of the MNV genome by RdRp only from the plasmid-derived MNV with the native or R239A mutant RdRp. Consistently, we detected the specific synthesis of negative strand MNV RNA, the intermediate of the MNV genome replication, only in the cells transfected with MNV plasmids with the native or R239A mutant of RdRp (**Figure [3D](#F3){ref-type="fig"}**). Taken together, these data suggested that the RdRp--VPg interaction was required for MNV replication in the host cell. To understand the essential function of the RdRp--VPg interaction, we examined the steps of MNV replication in the cell. MNV non-structural proteins generated from the proteolytic processing of ORF1 ([@B35]) reorganize the cellular membranes to form replication compartment (RC), where RdRp replicates the MNV genome ([@B43]); at 24 hpt, RdRps from all the plasmid-derived MNVs showed perinuclear localization, a typical pattern of MNV RC ([@B22]) (**Figure [4A](#F4){ref-type="fig"}**). RdRp/L354D showed substantial cytoplasmic localization compared to the other RdRps, suggesting that the L354D mutation of RdRp might interfere with forming and/or maintaining the RC structure. VPgs from the plasmid-derived MNVs showed localization patterns similar to those of corresponding RdRps. Collectively, these data suggested that the RdRp--VPg interaction was not required to form and/or maintain the MNV RC *per se*, although the L354 residue of RdRp might play an additional role in the RC structure. In all the transfected cells, RdRps were still detectable and showed the 24 hpt-like localization pattern at 72 hpt (**Figure [4B](#F4){ref-type="fig"}**). However, double-strand RNA (dsRNA), the intermediate of +RNA virus replication, was detected only at the RCs of plasmid-derived MNV with the native or R239A mutant RdRp, even though only a few cells survived the cytopathic effect of MNV replication; VP1, the structural protein expressed from the sgRNA of MNV after genome replication ([@B41]), showed the same expression pattern and was detectable only from MNV with the native or the R239A mutant RdRp. Taken together, our data demonstrated that the RdRp--VPg interaction enhanced the activity of MNV RdRp and was required for the efficient replication of MNV in cells. These data further suggest that the VPg-mediated multimerization of RdRp might be essential for efficient MNV infection in the host. {#F4} Discussion ========== +RNA viruses can synthesize viral RNAs *de novo* and/or with primers. Caliciviruses utilize both *de novo* and primer-dependent RNA synthesis, in which VPg functions as the proteinaceous primer ([@B32]; [@B34]). In fact, VPg is a multifunctional protein involved in protein synthesis and viral genome packaging into virions; degradation or mutation of VPg abolishes the infectivity of viral genomic RNA ([@B7]; [@B38]). To understand the interaction between this multifunctional VPg and viral RNA polymerase in the lifecycle of MNV, we characterized the interaction of RdRp with VPg(1-73) in the presence of RNA. Higher-order multimers or tubular fibrils of RdRp molecules emerged in the presence of VPg. Moreover, VPg enhanced the polymerizing activity of RdRp and the RdRp--VPg interaction was required for efficient replication of MNV in a cell culture system. MNV RdRp is predominantly a monomer in solution and tends to aggregate slowly when the concentration is higher than 5--6 mg/mL at room temperature. A temperature jump to 37°C results in heavy precipitation of RdRp. On the other hand, when RdRp and VPg are mixed together in the presence of oligo A, rapid aggregation is observed. Like the native RdRp, the D331A and L354D mutants can oligomerize as a hexamer by themselves in the absence of VPg. However, they do not promote the formation of higher-order multimers in the presence of VPg and oligo A. Aggregation of RdRp may follow a slow process of precipitation when the condition is changed, e.g., temperature jump, whereas VPg interaction with RdRp induces the rapid formation of higher-ordered multimers in the presence of RNA. Collectively, our data suggest that the multifunctional VPg might play a crucial architectural role in the higher-order multimerization of RdRp molecules and consequently in MNV replication inside the host. Here we propose a model of the VPg-mediated RdRp multimerization in MNV. A large ball-like RdRp hexamer is generated through dimeric and trimeric interactions among RdRp molecules. VPg augments the interaction between the RdRp hexamers, which can be closely packed into higher-order multimers or tubular fibrils in the presence of RNA. The construction of RdRp tubular fibrils with a uniform diameter (∼18 nm) leads to the spatial clustering of replication components. The ball-like hexameric RdRp can provide a large inner space where single-stranded RNA can be sequestered. Both the RNA and VPg are required for higher ordered multimer formation of RdRp, which strongly enhances cooperative RNA binding as well as highly efficient replication. RdRp has a high tendency to readily form aggregates at high concentrations (∼100 μM). In contrast, the concentration of RdRp upon multimeriztion by VPg was much lower (∼2 μM). The crystal structure of the MNV RdRp-VPg(1-73) complex showed a more tightly packed arrangement of RdRp molecules than that of RdRp molecules themselves; a hexamer of RdRp molecules was surrounded compactly by six neighboring hexamers and this was mediated by specific binding of VPg(1-73). Considering the higher affinity of VPg(1-124) than that of VPg(1-73) to RdRp (8.9 nM vs. 17 nM), RdRp molecules might be even more compactly multimerized in the presence of VPg(1-124), thereby leading to different higher-order structures. In fact, the binding of VPg(1-124) to RdRp resulted in a higher degree of multimerization of RdRp than the VPg(1-73), suggesting a potential role of the C-terminal region of VPg in RdRp binding. However, the affinity difference between the full length and the truncated VPgs might be due to simple conformational change of VPg after substantial deletion of the protein, and the C-terminal region of VPg may not play any role in the RdRp--VPg interaction. It is noteworthy that the C-terminal region of VPg is involved in the recruitment of translation initiation protein, eIF4G ([@B10]). In this regard, the C-terminal region of VPg may hinder the formation of compact structure of RdRp-VPg multimers by bringing other cellular proteins *in vivo*. Alternatively, such interaction between the C-terminal region of VPg with cellular translational machinery may happen only after VPg is linked to viral genome, so it may not affect the formation of RdRp-VPg multimers during replication. Collectively, although we cannot exclude the possibility that the VPg(1-124) might have an alternative way of binding to RdRp, we speculate that the disordered C-terminal region of VPg might further strengthen the interaction of VPg with the large ball-like RdRp hexamers. The replication of +RNA viruses was proposed to be facilitated by higher-order structure formation of viral RdRps that are immobilized on membranous structures ([@B36]; [@B26]; [@B3]). Such facilitation might be mediated through augmented avidity of participating polymerases as a group for their substrate RNAs and/or retention of intermediate products for sequential reactions. The formation of planar or tubular oligomeric arrays of RdRp molecules correlate with cooperative RNA binding and elongation and a high efficiency of viral replication ([@B7]; [@B36]; [@B38]; [@B37]). Likewise, the VPg-mediated formation of tubular fibrils or higher-order multimers of MNV RdRp molecules may increase the clustering of replication components and consequently enhance the replication of MNV. Interestingly, the recombinant MNV with the VPg-binding defective RdRp mutants, D331A or L354D, showed no sign of productive replication in a cell culture system. These data suggest that the defective VPg-binding ability of these RdRp mutants led to impaired multimerization of RdRps and consequently faulty replication of MNV, indicating the necessity of the VPg-mediated higher-order multimerization of RdRp for MNV replication in cells. Considering the decreased yet still substantial binding affinities of the D331A and L354D RdRp mutants to the full length VPg, this halted replication of the MNV mutants was unexpected and striking. Such a dramatic phenotype warrants subsequent studies to investigate the involved molecular mechanism. Therefore, although the current study is based on MNV and may not be applicable to HuNV, these data may contribute to developing a novel target of therapeutic intervention to inhibit the replication of NoV. Accession Numbers ================= Atomic coordinates and structure factors for the RdRp-VPg(1-73)-RNA complex have been deposited in the Protein Data Bank ([www.rcsb.org](http://www.rcsb.org)) under the identification code PDB ID 5Y3D. Author Contributions ==================== KK, SH, MC, and HK conceived and analyzed the experimental data. J-HL, BP, IA, and JS solved the crystal structure. J-HL and SK conducted TEM. KH, J-HL, SB, and JC conducted the *in vitro* assay and cell-based *in vivo* assay. SH and KK wrote manuscript. Conflict of Interest Statement ============================== The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. **Funding.** This study was supported by grants from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare and Family Affairs (A085119), the National Research Foundation funded (NRF) by the government (MSIP) (NRF-2016R1E1A1A01942558), the BK21+ program, and Basic Science Research Programs through NRF funded by the Ministry of Education (NRF-2013R1A1A2064940, J-HL and NRF-2016R1A6A3A01012238, KH). KH and J-HL are recipients of postdoctoral fellowship from the BK21+ program. J-HL was supported by Korean-EU (ERC) researchers exchange program (NRF-2015K2A7A1064286) and thank Professor James Naismith at University of St Andrews for giving a comment of crystal structure during visiting scholarship. SB was supported (in part) by NIH T32 GM007183. SH was supported by NIH grants R01AI127518 and DP2CA225208, the Brinson Foundation Junior Investigator Grant, the University of Chicago Comprehensive Cancer Center Support Grant (P30 CA14599), the Cancer Center Core facilities, and the National Center for Advancing Translational Sciences of the National Institutes of Health (UL1 TR000430). We thank the staff operating the beamlines at Pohang Light Source (5C) and Photon Factory (BL-17A) synchrotrons for their help with the data collection. Biacore2000 was used at the Korea Basic Science Institute, Seoul Branch. <http://www.pymol.org/> Supplementary Material ====================== The Supplementary Material for this article can be found online at: <https://www.frontiersin.org/articles/10.3389/fmicb.2018.01466/full#supplementary-material> ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. [^1]: Edited by: Alfredo Berzal-Herranz, Instituto de Parasitología y Biomedicina "López-Neyra", Spain [^2]: Reviewed by: José-Antonio Daròs, Consejo Superior de Investigaciones Científicas, Spain; Thierry Michon, INRA, France [^3]: ^†^These authors have contributed equally to this work. [^4]: ^‡^Present address: Kang R. Han, Konkuk University, Seoul, South Korea Intekhab Alam, Case Western Reverve University, Cleveland, OH, United States [^5]: This article was submitted to Virology, a section of the journal Frontiers in Microbiology

1. Introduction {#s0005} =============== Low levels of physical activity (PA) is a major health problem in industrialized countries around the world. ([@b0005]) In North America, \<35% of children and youth are achieving the recommended 60 min of moderate to vigorous PA (MVPA) per day. ([@b0010], [@b0015]) This figure has remained consistent over the past ten years. ([@b0020], [@b0015]) Increasing the proportion of children meeting the MVPA guidelines of 60 min of MVPA per day is imperative, as higher levels of MVPA are linked to a decrease in chronic disease risk factors, such as obesity, high blood pressure, and waist circumference. ([@b0025], [@b0030], [@b0035]) Health researchers have used the socio-ecological model (SEM) to help develop an understanding of the factors that influence children\'s health behavior, ([@b0040]) including MVPA. ([@b0045]) This model offers researchers a framework to move beyond thinking about variables in isolation to an approach that tries to understand an individual's health behavior as a complex interaction among numerous variables. This comprehensive approach is beneficial as it can suggest multiple strategies to improve children's MVPA. Using the SEM allows researchers to examine how intrapersonal, interpersonal, physical environment, and policy factors interact to influence behavior at different time points. At the intrapersonal level, age typically has an inverse relationship with PA, ([@b0050], [@b0055]) boys are more active than girls, ([@b0050], [@b0055], [@b0060]) PA may vary with ethnicity, ([@b0065]) and research has shown that how children perceive their ability to do certain activities can have a positive relationship with PA. ([@bib276], [@b0055]) Variables at the interpersonal level are not always the clearest, but social, neighborhood, and safety barriers can impact children's PA, ([@b0075], [@b0080]) children from a two-parent household are more likely to participate in sports compared to other households, ([@b0085]) children who own a dog tend to accumulate more PA, ([@b0090], [@b0095]) parental support has shown a positive association on PA, ([@b0055], [@b0050], [@b0100]) and socioeconomic status (SES) can influence PA. SES has been measured through parental employment ([@b0110], [@b0115]) and median household income. ([@bib278], [@b0120]) At the policy level, girls who attend a school with a balanced school day are more active. ([@bib277]) Physical environment variables have generally shown a positive association with PA and are normally based on accessibility to features, such as distance to recreation facility, ([@b0130], [@b0075], [@b0135]) distance to school, ([@b0130], [@b0145], [@b0140]) and if a park is near you house based on a distance buffer. ([@b0130], [@b0075], [@bib278]) One part of the physical environment that is essential to examine is the general type of environment in which a child lives, specifically measured as the level of urbanicity. Urbanicity attempts to capture the characteristics of different environments including built forms and social norms that are inherent to different urbanicities. Some studies have suggested a difference in MVPA from rural to urban areas. ([@b0155], [@b0150]) However, these studies are limited, as it is difficult to determine if their samples only include rural areas that are near major population centers. This creates a gap in the research as it misses nuances that could exist between different levels of urbanicity between varying geographical areas. ([@b0160]) Using more discrete measures of urbanicity while including different geographical areas can further our understanding of how the general environment influences health-related outcomes. ([@b0165], [@b0080]) One factor from the SEM that is often omitted is the impact of time, but temporal changes can significantly impact children's PA. ([@b0170], [@b0175]) Specifically, research suggests that children are more active during the week than on weekends. ([@b0175]) Researchers have examined correlates of MVPA on weekdays and weekend days, ([@b0180]) but there is little research that takes a comprehensive approach to examining MVPA on weekdays and weekend days. There are two main gaps in the literature this paper is trying to address. First, there is a lack of understanding as to the factors that are related to children getting 60 min of MVPA on weekdays and weekend days that uses a comprehensive approach during the school year. Second, most researchers treat children living in urban, suburban, urban small towns, and rural areas the same or lack geographically separate places while research has shown that there are differences in the environments and the lives of children in these various urbanicities and locations. ([@b0185], [@b0160]) To address these gaps in the literature, this paper will address two research questions:(1)What factors at the intrapersonal, interpersonal, physical environment, and policy levels influence children's ability to get 60 min of MVPA on a weekday?(2)What factors at the intrapersonal, interpersonal, and physical environment levels influence children's ability to get 60 min of MVPA on a weekend day? By addressing these questions, this paper will be able to inform researchers and health promoters to create more targeted policies by furthering the understanding of the variables that influence MVPA at different time points. 2. Methods {#s0010} ========== Data were collected as part of the Spatial Temporal Environmental and Activity Monitoring (STEAM) project. A full description of the project is available elsewhere. ([@bib280], [@bib278]) The STEAM project examines health behaviors of 1,068 children in grades 4 to 8 (ages 8--14 years) from 33 elementary schools in Ontario, Canada. The elementary schools were located in two distinct geographical regions: 29 schools from Southern Ontario and four schools from Northern Ontario. The schools in Southern Ontario were selected from groups of schools stratified by neighborhood SES and urbanicity. The schools in Northern Ontario included four schools that were in a rural region of the Thunder Bay District. Students were invited to attend a presentation given by a member of the research team where a brief presentation about the project was given. Researchers discussed that each student has a different lifestyle, and all different lifestyles should participate in the study. To further encourage participation, a small incentive was given to students based on conversations with principals, ensuring that the sum would no induce participation. This study was carried out in accordance with the Declaration of Helsinki and was conducted with approval from the Non-Medical Research Ethics Board at Western University and all seven of the participating school boards. Before participating in this study, children were required to obtain parental consent and sign their own assent form. A mixed tool protocol was used to collect data on individual and family characteristics, PA, perceptions of the physical environment, and other health behaviors. Data for this study was collected over an eight-day period. Child participants and parents completed a survey with questions about demographics, PA, health-related quality of life, and perceptions of their neighborhood environments. These survey questions were based on the Neighborhood Environment and Walkability Survey, ([@b0190]) Pediatric Quality of Life Measurement Model (PedsQL), ([@b0195]) and other highly used surveys. ([@bib278]) Immediately after children completed the surveys, they were outfitted with a hip-worn accelerometer and a passive-GPS data logger that they wore for the duration of the study. The cross-sectional sample for this study includes the spring season from Southern Ontario (2009--2013) schools and the fall season of the Northern Ontario schools (2016) to control for general temperature differences. Historical weather data suggests the spring and fall seasons were closer in temperature compared to fall in both locations. ([@b0200]) The original sample of 1,068 children, was reduced after eliminating participants who did not meet the following inclusion criteria: 1) meet an accelerometer wear-time minimum of 10-h per day (see Outcome Variable); 2) have at least two valid weekdays and one valid weekend day 3) completed the child survey; and 4) have a valid home location identified by GPS. The final sample consisted of n = 532 cases*.* A chi-square test was performed on age and gender between the students included and excluded from the sample, and no significant differences were found. 2.1. Dependent Variable: PA {#s0015} --------------------------- This study has two dependent variables derived from objective measures of PA using an accelerometer: (1) a binary measure of whether a child had an average of at least 60 min of MVPA per day on weekdays; and (2) a binary measure of whether a child had an average of at least 60 min of MVPA per day on weekend days. MVPA was measured using an Actical® Z Accelerometer (Philips Respironics, Murrysville, PA, USA), a device worn around the hips sitting on either hipbone. Participants were instructed to wear the accelerometer for all waking hours, only removing it for sleep, bathing, and swimming. The accelerometers measured PA in 30-sec epochs, which is an epoch length used in this age group. ([@b0205]) The accelerometer records movement made by each participant in all directions, summed over one min (counts per min, or CPM). If the device had zero counts for 60 consecutive min that hour was considered invalid wear time ([@bib279]) and these methods have been used in other studies. ([@bib278]) A valid day was considered six hundred min of valid wear time each day (or 10 h). ([@b0215]) MVPA was considered to be at least 1,500 counts per min. ([@b0225], [@b0220]) For this study, children were included if they had at least two valid weekdays and one weekend day. An average of children's valid weekdays and weekend days were used to determine if children met the MVPA guidelines on weekdays and weekend days. These criteria allowed us to maintain a large enough sample size for parametric statistics. 2.2. Independent variables {#s0020} -------------------------- The independent variables used in this paper are fully described in [Table 1](#t0005){ref-type="table"}. Independent variables for the analyses came from those that are found significant in past research on MVPA of children, including factors at the intrapersonal, interpersonal, physical environment, and policy levels. Intrapersonal factors used in this model include age, gender, ethnicity, and physical functioning as measured using the PedsQL measured from self-reported questions on the child survey. Missing data for child age, gender, and ethnicity were derived from the parent survey. Interpersonal factors in this paper include children's perceptions of social, neighborhood, and safety barriers, dog ownership, family composition, and parental support from the child survey, maternal employment, from the parent survey, as well as the median household income of the child's neighborhood, which was derived from 2011 Census of Canada data at the Dissemination Area level. In cases where missing data could not be derived from the parent survey a separate category for missing data was created. The physical environment factors are represented by four variables, computed based on the child's precise home location: accessibility to a park (i.e., is there a park within a 500 m buffer \[y/n\]), accessibility to a child's school (shortest network path between home and school \[m\]), accessibility to a recreation building (shortest network path between home and closes recreation centre \[m\]), and urbanicity. Children were grouped into different urbanicities based on their precise home location. Urbanicity was created by the research team using information from Statistics Canada and city plans. Urban large city (geographic areas with greater than 100,000 people residing in defined city limits), suburban large city (surrounding larger geographic regions with greater than 100,000 residents), urban small town (regions with a population of 10,000 -- 99,999), and rural (population\<9,999). Children in rural areas were further subdivided based on if they lived in rural Southern or Northern Ontario. A critical geographical difference exists between the rural Southern and Northern Ontario communities as the rural Northern communities are located over 100 km \[62mi\] from its nearest metropolitan centre; whereas the rural towns in Southern Ontario are much closer and therefore much more influenced by larger urban centres. Finally, the policy factors are measured by the type of recess schedule at a child's school: traditional (two 15 min recesses and a 30 min lunch recess) or balanced (two 20 min recesses).Table 1Variables associated with children's PA by the level of the SEM.VariableSourceDescriptionIntrapersonalGenderChild survey (categorical) (boy/girl)Self reported gender as boy or girl ([@b0050], [@b0055], [@b0060])EthnicityChild survey (categorical) (Caucasian/other)Ethnicity coded as either Caucasian or other ([@b0065])Physical functioningChild survey PedsQL (categorical) (high/low)A categorical variable based on face validity from four questions based on how hard it was to do physical tasks ([@bib276], [@b0055])AgeChild survey (continuous)Age in years ([@b0050], [@b0055])InterpersonalParental supportChild survey (categorical) (agree/disagree)A categorical variable based on if children agree or disagree that their parents take part in activities with them ([@b0055], [@b0050], [@b0100])Maternal employmentParent survey (categorical) (unemployed/employed)Mother's employment status ([@b0110], [@b0115])Family compositionChild survey (categorical) (two parent/lone parent)Number of parents in the main household ([@b0085])Dog ownershipChild survey (categorical) (yes/no)Categorical variable on if a child's family owns a dog ([@b0090], [@b0095])Social barriersChild survey (composite score)Composite score of social barrier questions ([@b0080])Neighborhood barriersChild survey (composite score)Composite score of neighborhood barrier questions ([@b0080])Safety barriersChild survey (composite score)Composite score of safety barrier questions ([@b0080])EnvironmentUrbanicityGIS (categorical) (urban large city, suburban large city, urban small town, rural south, and rural north)Categorical variable on different levels of urbanicity ([@b0165])Park in 500 m bufferGIS (yes/no)If any section of a park was within a 500 m buffer of a child's home based on GPS ([@b0130], [@b0075], [@bib278])Home schoolGIS (continuous)Shortest distance along the street network between each child's home and the school they attended ([@b0130], [@b0145], [@b0140])Recreation facilityGIS (continuous)Shortest distance along the street network between each child's home and the nearest arena or public/private recreational facility ([@b0130], [@b0075], [@b0135])Census average median household income (continuous) was taken from the 2011 censusCensus 2011 (continuous)Census average median household income (continuous) was taken from the 2011 census ([@bib278], [@b0120])PolicySchool daySchool data (categorical) (balanced/traditional)Variable based on school policy ([@bib277]) 2.3. Statistical analyses {#s0025} ------------------------- Analyses were performed in STATA version 14 (StataCorp, College Station, TX, USA) in 2019. One multi-level logistic regression model and one logistic regression model were specified in this paper to answer the research questions: (1) children having an average of 60 min of MVPA on weekdays; and (2) children having an average of 60 min of MVPA on weekend days. First, variables at the intrapersonal, interpersonal, physical environment, and policy levels (i.e., only included during the weekday to account for school day differences) were entered into the model using a least absolute shrinkage and selection operator to make predictions about variables related to MVPA. Once variables were selected, a multilevel logistic regression that controlled for clustering at the school level using the weekday data was conducted and, since children do not attend school on the weekend, a logistic regression was conducted on weekend data. 3. Results {#s0030} ========== Descriptive statistics are presented in [Table 2](#t0010){ref-type="table"}. The sample has more girls (58%) than boys, the average age was 11 years, and around 75% of children were Caucasian. About one quarter of the children had a park within a 500 m buffer of home, on average their school was about 5 km away from home, and the average distance to the nearest recreation facility from a child's home was 5 km. During the week, nearly half the sample met the MVPA guideline (51%), while on the weekend only about one quarter of the children met the MVPA guideline (25%).Table 2Descriptive statistics of the variables of the child participants STEAM study.Variablen%Intrapersonal  Gender  Boy22341.9Girl30958.1Ethnicity  Caucasian39574.2Other13725.8Physical functioning, *mean (std dev)*85.615.7Age, *mean (std dev)*11.21.1Interpersonal  Parental support\*  Agree29254.9Disagree22141.5Maternal employment\*  Unemployed8215.4Employed33462.8Family Composition  Two parent household37770.9Lone parent household15529.1Dog ownership\*  Yes24445.9No25948.7Social score, *mean (std dev)*−0.70.7Safety score, *mean (std dev)*−1.00.9Neighborhood score, *mean (std dev)*−0.90.7Environment  Urbanicity  Urban large City5310.0Suburban large City23644.4Urban small town519.6Rural south13625.6Rural north5610.5Park in 500 m buffer  Yes13525.4No39774.6Home school (km) *mean (std dev)*5.38.3Closest rec. (km) *mean (std dev)*5.07.2Neighborhood income per 10 000 *mean (std dev)*6.92.7Organizational  School day\*  Balanced29856.0Traditional22542.3Outcome  MVPA weekday  Meet recommendations26950.6Do not meet recommendations26349.4MVPA weekend  Meet recommendations13124.6Do not meet recommendations40175.4[^1] The first model addressing research question 1 ([Table 3](#t0015){ref-type="table"}) examines the factors from the SEM that influences the odds of a child getting the recommended 60 min of MVPA on weekdays. The results of this analysis find that three intrapersonal variables and one interpersonal variable are significant. The results show that the odds of boys meeting the recommendations on weekdays are 4.652 times that of girls (*p* \< 0.001). Age is also found significant, with each additional year of age decreasing the odds of getting the recommended amount of PA by 0.758 (*p =* 0.013). Children with high self-reported physical functioning are 2.731 (*p* \< 0.001) times more likely of getting the recommended amount of PA as compared to children with low physical functioning. Finally, as children's perceptions of safety barriers increase, they are 1.244 (*p =* 0.046) times more likely to get the recommend amount of PA.Table 3Multi-level logistic regression of the association between SEM variables in children on weekday MVPA.VariableOdds Ratio*p*95% Confidence IntervalIntrapersonalBoy (ref: girl)4.652**\*\*\<0.001**3.0777.032Age0.758**\*0.013**0.6090.947Physical functioning -- high (ref: low)2.731**\*\*\<0.001**1.7954.157InterpersonalSafety barrier1.244**\*0.046**1.0031.544EnvironmentDistance to home school0.9870.3050.9631.012ConstantSchool0.2500.0750.831[^2] The second model addressing research question 2 ([Table 4](#t0020){ref-type="table"}), examines the factors from the SEM that influence the odds of a child getting 60 min of MVPA on weekend days. The results of this analysis find variables at both intrapersonal and physical environment levels of the SEM are related to children meeting the recommendations on weekend days. The only significant intrapersonal variable was gender, which found that the odds of boys meeting the recommendations are 1.683 that of girls (*p* = 0.014). The other significant variable is urbanicity. The urbanicity measure finds that children living in the rural Northern Ontario are significantly more likely to meet the MVPA guidelines on weekends than children living in urban areas (OR = 0.320, *p* = 0.019), suburban areas (OR = 0.395, *p* = 0.006), urban small towns (OR = 0.351, *p* = 0.022), or in rural Southern Ontario (OR = 0.347, *p* = 0.003).Table 4Logistic regression of the association between SEM variables in children on weekend day MVPA.VariableOdds Ratio*p*95% Confidence IntervalIntrapersonal    Boy (ref: girl)1.683**\*0.014**1.1112.545Age0.8720.1510.7231.051Physical functioning -- high (ref: low)1.2670.2900.8171.964Interpersonal    Social barrier0.7610.0950.5521.048Dog ownership (ref: no)1.3130.2180.8512.026Environment    Urbanicity (ref: rural north)    Urban large city0.320**\*0.019**0.1230.831Suburban large city0.395**\*\*0.006**0.2030.722Urban small town0.351**\*0.022**0.1430.860Rural south0.347**\*\*0.003**0.1740.693Closest rec. (km)1.0260.0890.9961.058[^3] 4. Discussion {#s0035} ============= The purpose of this paper was to use the SEM to examine what factors influence whether children achieve the recommended minutes of MVPA on weekdays and weekend days. This was done using logistic regression models, one to represent the weekday and one to represent the weekend day. Previous research has indicated that PA levels and correlates differ from weekday to weekend day and this paper contributes to the literature by taking a more comprehensive approach in identifying what specific factors influence the odds of meeting the MVPA guidelines on weekdays and weekends days. ([@b0175], [@b0180]) Researchers also identified that few studies included geographical setting variables that go beyond an urban/rural dichotomy/trichotomy or include geographically distant places. ([@b0165]) This led to one major finding as children living in rural Northern Ontario communities were more likely to meet the MVPA guidelines on weekends when compared to children from Southern Ontario in differing levels of urbanicity. The results of this study found that boys were more likely than girls to meet the MVPA guidelines on both weekdays and weekends, but the odds dropped from 4.652 on weekdays to 1.683 on weekends. Although gender is commonly found as a factor that influences MVPA, the difference in the strength of the association is an important finding. For example, a report from Statistics Canada found that boys were twice as likely to meet the MVPA guidelines when compared to girls. ([@b0230]) This suggests separating weekdays and weekend days provides valuable evidence for program leaders as something during the week is conducive to boys MVPA, but not girls. One potential program that has been successful with girls is offering children a free access pass to facilities (e.g., YMCA, Boys and Girls club, arenas, and pools) that include a wide variety of programs including dance, basketball, and free swimming. ([@b0235]) Another potential weekday intervention could target the school. Multi-component school interventions that include modified physical education lessons, more choice, and a focus on enjoyment has shown some success in increasing girls MVPA. ([@b0240]) On weekdays the children who reported high physical functioning and perceived more safety barriers were more likely to meet the MVPA guidelines. The physical functioning variable was based on four questions that asked children how difficult it is to walk, run, participate in sports, or lift something heavy essentially their perceived competence in different domains of PA. Previous research has shown that psychological variables based on competence have been positively related to PA. ([@bib276], [@b0055]) In this study we found that this variable was only significant during the weekday. Previous research also indicates that certain psychological correlates of PA are context specific. ([@b0245]) This suggests that there is something about children who scored higher on this scale and the weekday context that makes them more active. Similarly, children's perceptions of safety barriers were only significant during the weekday. Although some research suggests barriers make children less active ([@b0080]) in this case stronger perceptions of safety barriers meant children were more likely to reach the MVPA guidelines. This could be explained by more active children spending more time exploring their neighbourhood and encountering safety barriers. Further research is needed to explore the difference in strengths of relationships on weekdays and weekend days. At the physical environment level, children from rural Northern Ontario were more likely to meet the MVPA guidelines on the weekend compared to suburban, urban, small town, and rural children from Southern Ontario. This suggests that there is something about the North that increases the chances of children getting the recommended amount of MVPA on weekends, not necessarily the level of urbanicity. As research has only touched on rural children's PA, especially rural children in a Northern setting, it is difficult to determine why these differences exist. ([@b0255], [@b0250]) One potential explanation is that our Northern Ontario study area is more geographically isolated providing children more freedom to explore their environment and be active. Children in the rural Northern Ontario sample lived in a sparsely populated area over 100 km \[62mi\] away from the closest major city; most people in the these rural Northern communities know one another, potentially making parents feel safer about letting their children out to explore their natural environment. ([@b0260]) Another potential factor could be the community norms. This study took place in the fall, which aligns with hunting season. On weekends children could be out hunting, accumulating higher levels of MVPA as certain types of hunting require hours of walking through difficult terrain. With a substantial portion of the North America population living in rural areas, it is essential to study the variables that influence PA in rural children in different geographic areas. ([@b0265], [@b0270]) 5. Limitations {#s0040} ============== A limitation of this study is that two weekdays and one weekend day were used as inclusion criteria. Some other researchers have used a minimum of four valid days. ([@b0275]) Using more valid days could help improve the overall accuracy of the model as more days used helps capture a better overall average estimate of a child's PA levels. Another limitation is this study did not examine summer MVPA when children are out of school and researchers could not account for MVPA during water-based activities. 6. Conclusion {#s0045} ============= This paper identified that different factors influence whether children meet the MVPA guidelines during different timepoints, weekdays compared to weekends. Finding different factors at different timepoints can help recreation programmers as they can use this information to create more targeted programs. Conceptually, this study has important implications for how researchers think about the SEM and predictors of PA. In some children's PA research, the temporal realm is either omitted or inadequately explained even though it is considered part of the model. ([@b0170]) If researchers ignore temporal differences and use an average value which lumps together weekdays and weekend days, some nuances are lost, and there is a possibility that factors that influence MVPA during the weekday are driving the overall significance of that variable. Another important finding from this study is that children in Northern Ontario were significantly more likely to meet the MVPA recommendations than those in Southern Ontario on weekend days. Research needs to further investigate these regional differences in MVPA, especially on weekends when children have more time to access community based features and are potentially more influenced by community norms. Allowing program leaders to focus their efforts on smaller time points, specific groups, and specific regions could lead to more efficient and cost-effective interventions for improving children's PA levels. CRediT authorship contribution statement {#s0055} ======================================== **Brenton L.G. Button:** Methodology, Formal analysis, Writing - original draft, Project administration. **Andrew F. Clark:** Methodology, Data curation, Writing - review & editing, Project administration. **Jason A. Gilliland:** Conceptualization, Methodology, Resources, Writing - review & editing, Supervision, Funding acquisition. Declaration of Competing Interest ================================= The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Appendix A. Supplementary data {#s0065} ============================== The following are the Supplementary data to this article:Supplementary data 1 The STEAM study was jointly-funded by 10.13039/501100000024Canadian Institutes of Health Research and the 10.13039/100004411Heart and Stroke Foundation of Canada, with seed funding from the 10.13039/501100000155Social Sciences and Humanities Research Council of Canada. Additional support was provided by the 10.13039/100012422Children's Health Research Institute and the Children's Health Foundation. We thank the students, parents, teachers, principals, and school research boards. We would also like to acknowledge the dozens of research assistants from the Human Environments Analysis Lab who helped with the STEAM project. Supplementary data to this article can be found online at <https://doi.org/10.1016/j.pmedr.2020.101145>. [^1]: \* Does not add up to 100% to account for missing data. [^2]: Boldface indicates statistical significance (\*p \< 0.05, \*\*p \< 0.001) [^3]: Boldface indicates statistical significance (\*p \< 0.05, \*\*p \< 0.01)

1. Introduction {#sec1-ijerph-14-01544} =============== 1.1. Background {#sec1dot1-ijerph-14-01544} --------------- Non-communicable diseases (NCDs), also known as chronic diseases (CD), such as asthma, chronic obstructive pulmonary disease (COPD), diabetes, heart failure and hypertension, affect millions on a global basis \[[@B1-ijerph-14-01544]\]. The number of NCD deaths has increased worldwide, from 38 million in 2012 and is projected to reach 52 million by 2030 \[[@B2-ijerph-14-01544]\]. On a global basis, the toll of diabetes alone accounted for 1.5 million deaths. It was the eighth leading cause of death for both genders and the fifth leading cause of death in women in 2012. It is estimated by the WHO, that 422 million adults aged over 18 years were living with diabetes in 2014. Approximately half the diabetes cases in the world can be found in South East Asia \[[@B3-ijerph-14-01544]\]. In Taiwan, the 10 leading causes of death in 2015 were NCDs: for which diabetes and hypertension were listed as the fifth and eighth most frequent causes of death \[[@B4-ijerph-14-01544]\]. The annual medical expense incurred by patients with NCD in Taiwan is almost NT\$70 billion and continues to rise. Between 2004 and 2014, the annual medical expense for treating diabetes patients increased by approximately NT\$6.2 billion \[[@B4-ijerph-14-01544]\]. Patients with NCD require long-term, continuous care. The existing equipment, treatment procedures, and short-term care approaches employed by the medical system for acute diseases do not meet the requirements of patients with NCD \[[@B5-ijerph-14-01544]\]. The existing care approaches for patients with NCD require significant improvements. In response to the multifaceted care requirements of patients with NCD, the medical personnel and scholars from the U.S. MacColl Institute co-developed a chronic care model. It proposes six mandatory elements for long-term care: (1) the organization of health care; (2) delivery system design; (3) decision support; (4) clinical information systems; (5) self-management support; and (6) resources and policies \[[@B6-ijerph-14-01544]\]. Particularly vital for improving the health management and self-care capabilities of patients with NCD, is self-management support. Patients with NCD require long-term and continuous medical intervention and assistance from others. Additionally, due to the changes in traditional Asian familial social structure and the limited number of hospitalization days covered under the National Health Insurance (NHI) system, NCD patients are often discharged early and forced to rely on their communities or home for further health care \[[@B7-ijerph-14-01544]\]. Therefore, patients with NCD living at home, in communities, or in institutions must implement appropriate self-care and health management practices to effectively control their diseases and prevent comorbidity \[[@B8-ijerph-14-01544]\]. Telecare is defined as care practiced at a distance. It is an effective strategy for improving the self-care capabilities of patients with NCDs. With the advent of information communication technology utilized to provide patients with NCD medical services; technologies such as telephones, mobile phones, emails, instant messages, and online video calls are used for service delivery or communication between medical professionals and NCD patients \[[@B9-ijerph-14-01544]\]. Currently six telecare systems exist: (1) home tele-monitoring; (2) clinical decision-making and instant warning response; (3) NCD self-management education; (4) periodic care services; (5) resource links and referrals to other medical teams or institutes; and (6) remote video conferences. Home tele-monitoring, the most prevalently applied telecare form, involves installation of self-monitoring devices in the homes of patients with NCDs. This allows the medical team to monitor the patients' physiological, psychological, or behavioral indicators and transmit patient data to remote monitoring centers. This data enables health professionals in evaluating patient health statuses and allows for rapid medical response \[[@B10-ijerph-14-01544]\]. Thus, NCD patients enable the monitoring of their own physiological indicators, self-manage their health, and acquire appropriate health education. A literature review revealed that most studies report that telecare had positive effects and almost none reported negative effects on CD management \[[@B11-ijerph-14-01544]\]. Telecare led to patients and families taking a more active role in disease self-management. Patients monitored by telecare showed significant improvement in glycemic control in type 2 diabetes when compared with those monitored by routine follow-ups \[[@B12-ijerph-14-01544]\]. Telecare profoundly benefits patients with NCDs, by contributing toward their health management, enabling them to preemptively identify and mitigate their diseases, reducing the frequencies of clinical visit, and shortening the time of their clinical visits. As a result of these benefits, Telecare acts to reduce the cost of healthcare \[[@B13-ijerph-14-01544]\]. These benefits demonstrate the effectiveness and importance of telecare for patients with NCDs. Currently, most studies on telecare in Taiwan have focused on the establishment of information platforms and systems or scales measuring the effectiveness of Telecare: few have explored the intent to use telehealth (IUT) from the patients' perspective. Patients' requirements for and acceptance of Telecare has been explored in the research of Tai et al. \[[@B5-ijerph-14-01544]\] and Lin et al. \[[@B14-ijerph-14-01544]\], along with the benefits of these services. It is a strongly recommended care model for patients with NCD in Eastern Taiwan, which is characterized by its rough terrain and remote mountain areas. 1.2. Purpose {#sec1dot2-ijerph-14-01544} ------------ The purpose of this research was to explore the IUT for in home diabetes and/or hypertension patients. The resulting data was analyzed to determine intent to use of Telehealth and any correlation between the IUT and the self-care behaviors of in-home CD patients. The focus of the purposive sampling within the domain of NCD to satisfy the goals of the research and hospital program effectiveness was diabetes and hypertension as both diabetes and hypertension may be monitored accurately with technology currently available to patients. 2. Methods {#sec2-ijerph-14-01544} ========== 2.1. Study Design {#sec2dot1-ijerph-14-01544} ----------------- This was a cross-sectional research. Through purposive sampling, patients with diabetes and/or hypertension who had used the health management platform, Personal Health Records (PHRs), from the telecare center of a hospital in Eastern Taiwan were selected. Linking the telecare medical unit (Gateway, Yi Yuan Medical Technology Company, Taipei, Taiwan) to the existing landline telephone or other network allowed for convenient integration of the health care equipment within the patient's home environment. Telecare as a long distance intervention allows for managing patients PHR, monitoring medical treatment, and health education, while tracking patients' statuses of their disease self-care management. Autonomy of the participant in controlling their state of health is promoted through telephone communication from a member of the medical staff at the PHR. A structured questionnaire, using purposive sampling was conducted to examine the IUT of the telecare service and correlations between the IUT and self-care behaviors was examined to further improve future development of telecare services. Additionally, specific benefits of the telecare intervention were investigated through analysis of the patients' physiological and psychological indicators. This study was conducted from 1 April 2014 to 31 March 2015. 2.2. Participants {#sec2dot2-ijerph-14-01544} ----------------- Participants were purposively selected from a telecare center of an Eastern Taiwan hospital. Research subjects were requested to participate in the PHR by the diagnosing doctor, as most of these patients live in remote or extremely rural areas. Selection criteria was as follows: (a) hypertension, diabetes, or both diseases were diagnosed; (b) not younger than 20 years old and (c) communicate in Mandarin Chinese or Taiwanese. The exclusion criteria were as follows: (a) having a psychiatric or other disease that could affect cognitive capabilities, or prevent him or her from answering the questionnaires; (b) refusal to participate in the study. This study received approval from the Institutional Research Board (IRB14-07-012) after adjustments were made to the questionnaire according to their guidelines. Consent was requested and obtained from all participants in written form. Privacy of the participants, and any data collected in this study are treated as strictly confidential and used for academic research purposes only. 2.3. Data Collection {#sec2dot3-ijerph-14-01544} -------------------- The structured questionnaires employed in this study were the telecare usage intention scale (TUS), the self-care behavior scale (SCS), and participant demographic information. Face-to-face interview was the data collection method of preference. In addition, the physiological indicators included glycated hemoglobin (HbA1c), pre-meal glucose levels pre- and monthly blood pressure (BP) checks. Prior to tele-monitoring, the participants' glucose levels and BP were measured by medical professionals through the rural medical service or outpatient clinic. ### 2.3.1. Telecare Usage Intention Scale {#sec2dot3dot1-ijerph-14-01544} The TUS was a revision of the translated telecare scale formulated by Tsai and Chuang \[[@B15-ijerph-14-01544]\]. This scale was designed to assess the usage intention of telehealth of patients. The modified scale utilized in this study included 15 items, which covered three dimensions: (1) personal benefits gained from using telecare; (2) the activeness of an individual in using telecare; and (3) the individual perception of telecare. Each dimension comprised five items, which contained three answers, disagree (=1), neutral (=2), and agree (=3). A higher score indicated that the IUT of a participant was greater. The Cronbach's α value was 0.795. ### 2.3.2. Self-Care Behavior Scale {#sec2dot3dot2-ijerph-14-01544} The self-care behavior scale was a revision of the self-care behavior scale created by Tseng \[[@B16-ijerph-14-01544]\] for elderly people with CDs It was designed to evaluate self-care behaviors of participants at home. This modified scale comprised 13 items, covering three dimensions: (1) medication compliance behaviors (four items); (2) self-care behaviors (five items); and (3) health promotion behaviors (four items). Each item contained five answers: never (=1), rarely (=2), occasionally (=3), frequently (=4), and always (=5). The maximal and minimal scores of the scale were 65 and 13 points respectively; the higher the score the more satisfactory the self-care behavior of the participant. The Cronbach's α value was 0.873. ### 2.3.3. Demographic Information {#sec2dot3dot3-ijerph-14-01544} The demographic information investigated in this study included the patients' sex, age, marital status, education level, occupation, area of residence, CD history, and medication status. ### 2.3.4. Physiological Indicators {#sec2dot3dot4-ijerph-14-01544} The physiological indicators examined in this study were the HbA1c and AC glucose levels and the monthly BP check. Physiological data was collected pre and post one month of the study's completion to allow for analysis of change in physiological parameters. 2.4. Data Analysis {#sec2dot4-ijerph-14-01544} ------------------ The Statistical Package for the Social Sciences Version 21.0 for Windows (IBM, Armonk, NY, USA) was employed for data archiving, descriptive and inferential statistical analyses. The data was converted into percentages to clarify the structural distribution of the participants' demographics. Descriptive statistics were applied to describe the IUT by the participants. Pearson correlation analysis was conducted to explore the correlation between the self-care behaviors of the participants and their IUT. A general linear model was used to examine the factors that affected the IUT of the participants. A paired *t* test was used to explore the changes in the participants' physiological indicators before and after they used the telecare services. The Cronbach's α value was 0.05; *p* \< 0.05 indicated a statistically significant difference. 3. Results {#sec3-ijerph-14-01544} ========== A total of 100 patients with CD who had used PHR from a hospital in Eastern Taiwan were surveyed. There were three patients who were unable to answer the questionnaires, two patients that died, three patients who moved to other areas, and 10 who refused to participate in this study were excluded. As such, a total of 82 patients were recruited. Their data analysis is described as follows. 3.1. Demographics {#sec3dot1-ijerph-14-01544} ----------------- The demographics of the patients' included: sex, age, marital status, education level, occupation, area of residence, CD history, and medication status (see [Table 1](#ijerph-14-01544-t001){ref-type="table"}). 3.2. Descriptive Data {#sec3dot2-ijerph-14-01544} --------------------- ### 3.2.1. Analysis of the IUT of the Participants {#sec3dot2dot1-ijerph-14-01544} More than half of the participants stated that the telecare services were beneficial and accepted the use of the services. However, the participants were less inclined to upload or employ the platform information; more than half of the participants did not proactively upload their dietary (73.2%) and exercise records (75.6%). Additionally, most of the participants (52.4%) felt that telecare services should be paid for by the National Health Insurance (NHI) ([Table 2](#ijerph-14-01544-t002){ref-type="table"}). ### 3.2.2. Analysis of the Correlation between the Self-Care Behaviors of the Participants and their IUT {#sec3dot2dot2-ijerph-14-01544} The correlations among the self-care behaviors of the participants and their IUT are listed in [Table 3](#ijerph-14-01544-t003){ref-type="table"}. The self-care behaviors of the participants were positively correlated with both their IUT (*p* \< 0.01) and the three dimensions of IUT. Those dimensions defined as: (1) the personal benefits gained from using the telecare services (*p* \< 0.05); (2) cooperation in uploading or using the platform information (*p* \< 0.05); and (3) the participants' perception of the Telecare services (*p* \< 0.05). Consequently, a higher score for self-care behaviors indicated that the IUT of a participant was greater. Additionally, the health promotion behaviors of the participants were positively correlated with the personal benefits gained from using the *t*elecare services (*p* \< 0.01) and the participants' teamwork in uploading and employing the platform information (*p* \< 0.01). Correspondingly, the higher a participant scored on health promotion behaviors, the more the participant acknowledged the benefits that the *t*elecare services provided. 3.3. Outcome Data {#sec3dot3-ijerph-14-01544} ----------------- ### 3.3.1. Factors that Affected the IUT of the Participants {#sec3dot3dot1-ijerph-14-01544} A general linear model was incorporated to investigate the factors that affected the IUT of the participants. The results indicated that the self-care behaviors of the patients were significantly correlated with their IUT (*p* = 0.004) but not with their demographic characteristics. Of the factors that affected each dimension of the IUT, the self-care behaviors of the participants significantly influenced their personal benefits gained from using the Telecare services (*p* = 0.028), their cooperation in uploading or employing platform information (*p* = 0.005), and their perception of the telecare services (*p* = 0.017). The participants' monthly BP checks significantly influenced their perceived personal benefits gained from using the telecare services (*p* = 0.003). The marital status of the participants significantly affected their consistency in uploading and employing platform information (*p* = 0.028). The results indicate it may require a longer time interval to motivate and empower the majority of patients. The mere provision of telecare services to CD patients is insufficient to maximize its' potential. The mechanism of empowerment remains a psychologically complex problem. Multiple improvements to telecare services are required as part of continued development. If implemented correctly this should have a positive effect on patient IUT. The correlation analyses support this supposition. ### 3.3.2. Results of the Physiological Indicators {#sec3dot3dot2-ijerph-14-01544} The physiological indicators examined in this study were the participants' HbA1c and AC glucose levels as well as monthly BP recording. The analysis indicated that the HbA1c and AC glucose levels of the participants substantially improved after their use of the telecare platform (*p* \< 0.005), and their frequencies of BP measurement also increased (*p* \< 0.001). See [Table 4](#ijerph-14-01544-t004){ref-type="table"} for further details. 4. Discussion {#sec4-ijerph-14-01544} ============= 4.1. Effect of Demographic Characteristics on the IUT {#sec4dot1-ijerph-14-01544} ----------------------------------------------------- Of the participants that were surveyed in this study, 40 were men and 42 were female. The age range of telecare users was 40--64 years old (63.4%) and 30% were older than 65 years. This was consistent with the findings by Lin et al. \[[@B14-ijerph-14-01544]\] The usage rate for the over 65 year old participants was much lower than that of the 40 to 64 year old group. This may be due to lack of familiarity using advanced technology products. This finding supports the research of \[[@B17-ijerph-14-01544]\] which indicated that the age of patients with CD significantly influenced their IUT of telecare. The bulk of telecare users were married (80.5%) which is consistent with the findings of Lu et al. \[[@B18-ijerph-14-01544]\] Married patients with CD used telecare more frequently than did unmarried patients with CD as spouses encouraged and supervised each other's use of Telecare. Most of the telecare users did not have education greater than high school (85.4%). This was consistent with the findings by Hong and Deci \[[@B19-ijerph-14-01544],[@B20-ijerph-14-01544]\], in which the proportions of CD users with education levels no higher than high school were 83.3% and 74--87% respectively. This indicates low education levels amongst most telecare users researched to date. This attributes to the negative correlation between the education levels of the participants and the number of CDs diagnosed (*r* = −0.238, *p* \< 0.05). Essentially, as education level lowers, the number of diagnosed CDs increases, which indirectly furthers the related IUT. This suggests that health conditions of CD patients are a factor that affects the healthcare service usage of patients. The greater the CDs a patient has, the higher the rate of the patient's healthcare service usage. The demographic characteristics of the participants affects observed telecare usage behaviors. This is explained through the use of the health service utilization model devised by Deci \[[@B21-ijerph-14-01544]\] According to this model, three factors affect the healthcare service usage behaviors of patients with CD: (1) predisposing characteristics (e.g., age, sex, marital status, education level, occupation, and religious belief); (2) enabling characteristics (e.g., sufficiency and accessibility of medical resources and the travel time for clinical visits); and (3) needs characteristics (e.g., self-perceived health status and disease status). According to Deci \[[@B21-ijerph-14-01544]\], although predisposing characteristics do not directly affect the healthcare service usage behaviors of patients with CD, they directly influence the intentions of the patients to use the services. The usage behaviors of patients vary with patient demographic characteristics. Therefore, on the basis of the analysis in this study, Telecare services should be provided to patients with CD based on their IUT. 4.2. Effect of Telecare on in-Home CD Patients {#sec4dot2-ijerph-14-01544} ---------------------------------------------- Almost 70% of in-home patients with CD, affirmed the beneficial effect of telecare on their individual health management. This was consistent with the findings of Andersen \[[@B22-ijerph-14-01544]\], in which nearly 90% of patients with hypertension at home expressed satisfaction with the convenience and benefits of Uei; Tsai et al. \[[@B13-ijerph-14-01544]\] indicated that patients diagnosed with metabolic syndromes were extremely satisfied with the decrease in their health expenditures, revealing a considerably favorable perception of Telecare by patients with CD at home. The IUT of at-home patients with CD was positively correlated with their self-care behaviors as well as blood glucose and hypertension measurement behaviors. This was consistent with the findings of Andersen \[[@B22-ijerph-14-01544]\], in which approximately 80% of telecare users affirmed the benefits of telecare: It was concluded that telecare improved their daily living habits, care knowledge, mitigated worry of personal health status, and promoted interaction with medical professionals. In addition, the participants were more cooperative regarding checking their BP toward the conclusion of the IUT study. This is consistent with the findings of Hong et al. \[[@B19-ijerph-14-01544]\], in which hypertension patients uploaded their data more frequently after they began using telecare. Additionally, cognitive, attitudinal, and self-care behaviors improved substantially amongst participants. Similarly, participants showed increased awareness and cooperation regarding monitoring their BP. This study incorporated portable PHRs, assisting patients in monitoring their daily physiological changes and their own health status. The case managers of the patients then further assessed the health status of each patient and promptly provided health education feedback accordingly; thus improving the patients' intention and sense of responsibility in caring for their own health. Even though medical professionals reduced the frequency of their interactions with patients, they remained successful in promoting the self-care behaviors of the in-home CD patients. Telecare motivates patients with CD to learn how to manage their diseases, practice self-care behaviors, become responsible for their own health, and thereby successfully perform self-management of their health. The study of Chen et al. \[[@B23-ijerph-14-01544]\] addressed three major tasks of CD self-management: (1) medical management (i.e., taking prescribed medication, exercising, and changing diets according to physicians' instructions); (2) role management (i.e., adjusting daily living lifestyle to maintain the integrity of the patient's own daily roles); and (3) emotional management (i.e., learning to manage the psychological stress and negative emotions caused by diseases). Telecare empowers in-home CD patients to alter their behavior, as it makes them aware of their health status and enhances their intrinsic motivation to self-care. The physical presence of the medical technology at their fingertips may act as a visual stimuli to remind patients of their responsibility to themselves. This is consistent with the perspective of self-determination theory \[[@B24-ijerph-14-01544]\], in which human behaviors are generated from intrinsic motivation, and that the expression of these behaviors is affected by extrinsic factors. Unfortunately, the participants showed low cooperation in uploading their dietary and exercise records or in using the health education information from the platform. This may be due to the fact that 95% of the participants used cable telephones for the telecare services, which compromised the ability of uploading dietary and exercise data. Likewise, the health education was primarily provided through telephone inquiries by the case managers. The study by Lo et al. \[[@B25-ijerph-14-01544]\] indicated that patients with CD are typically concerned about privacy, telecare usage capabilities, and budget deficiencies, which inhibits their use of telecare services. In this study, 52.4% of the patients felt that telecare should be paid for by the NHI, revealing budgetary considerations as a key factor affecting the future prevalence of telecare services. Telecare mitigates the inequality in medical care caused by distance and time constraints. It also improves the timeliness of health self-management, yet comprehensive consideration is required to maximize the benefits of telecare. Factors such as: (1) suitability of users; (2) costs; (3) service convenience; (4) demands on self-care capabilities; (5) learning motivations of users; and (6) user privacy guarantees, must be incorporated to develop effective Telecare systems within varying local conditions. Due to the cost of telecare, a minority of the participants would not use it, as they feel it should be covered under NHI. This does not suggest that they would reject telecare outright, as the "unwillingness to pay" may be outweighed by the need receive timely medical attention. In accordance with existing laws, the home-telecare model developed in this study maximized the healthcare services of patients with CD through professional spatial integration. However, numerous limitations exist: (1) the timeliness of home care services; (2) the support and connection of long-term care resources; (3) the medical referral process in remote areas; (4) the integration of the information environment; and (5) expenses not covered by National Health Insurance (NHI), all impede telecare service implementation in Taiwan. However, adopting telecare services could improve healthcare availability for NCD patients, thus reducing the medical costs and improving the accessibility of healthcare resources. 4.3. Limitations {#sec4dot3-ijerph-14-01544} ---------------- This study suffers from several limitations: (a) Scope of research: This study investigated in-home NCD patients that had used telecare services from a hospital in Eastern Taiwan. Due to the remoteness and cultural peculiarity of Eastern Taiwan, the results of this study may not accurately reflect other regions. (b) Sample size and selection bias: The participants were limited to patients with CD who had received telecare services from a hospital in Eastern Taiwan as such, the selection bias existed due to purposive sampling and critical case sampling due to the nature of the disease under study. (c) Geographic factors: Over half of the participants lived in mountainous areas which presented mobility and geographical constraints. Consequently, some of the participants were unable to participate in face to face interviews, and telephone communication was substituted, thus potentially affecting the survey results. (d) No pre-post surveys were done to better assess IUT. Future studies focusing on recruiting participants from several geographical areas and with multiple forms of CDs would add useful information to the telecare development data base. The mechanism of empowerment and motivation require deeper examination and an augmented questionnaire with respect to psychological focus. The NHI Research Database should be examined for historical data analyses and comparison of previous to present costs in medical care comparisons. 5. Conclusions {#sec5-ijerph-14-01544} ============== This study examined the IUT of in-home patients with CD in Eastern Taiwan. The results indicated a high IUT and positive perception of the Telecare services from the patients. The IUT of the patients was positively correlated with self-care behaviors and physiological indicators. Telehealth is a new service designed to improve the health self-management and preventive care of patients with CD in the comfort of their home. Telehealth is not designed to replace professional medical care, but to act as an adjunct to disseminating timely health care to patients in need in a cost effective and efficient manner. At the same time, the implementation of telehealth, while reducing overall costs to NHI, will in the long term reduce the cost of health care to NHI. Telehealth appears to empower in-home CD patients to alter their behavior, as they are made aware of their health status, which enhances their intrinsic motivation to self-care. This research was supported by grants (TWNA-1041002) from Taiwan Nurses Association. And the authors thank Mennonite Christian hospital for assistance in collecting the data for this study. Shu-Lin Uei conceived and designed the study; Shu-Lin Uei and Yu-Lun Kuo performed the experiments; Yu-Ming Kuo analyzed the data; Chung-Hung Tsai gave advice about study; Yu-Lun Kuo wrote the paper. The authors declare no conflict of interests. ijerph-14-01544-t001_Table 1 ###### Demographics (N = 82). Variable Frequency/Mean (SD) Percentage (%) Variable Frequency Percentage (%) ------------------------------------ -------------------------------------------------------------------- ---------------- ---------------------------- ----------- ---------------- Sex Area of Residence Male 40 48.8 Hualien City 46 56.1 Female 42 51.2 Outside Hualien City 36 43.9 Average Age (Years)--60.55 (±12.2) Distribution of the Residences ≤40 5 6.1 City plain region 47 57.3 41--64 52 63.4 Aboriginal mountain region 35 42.7 ≥65 25 30.5 History of Diseases Marital Status Hypertension 9 11.0 Diabetes 29 35.3 Unmarried 16 19.5 Both 44 53.7 Married 66 80.5 Medication Status Education Level Single Medication (antihypertensive drugs or hypo-glycemic agents) 40 48.8 High School or Lower 70 85.4 Both 42 51.2 Junior College/or Higher 12 14.6 Employment Status Employed 37 45.1 Homemaker/Retired 45 54.9 ijerph-14-01544-t002_Table 2 ###### The IUT of the participants (N = 82). Variable Disagree Number and Percentage (%) Neutral Number and Percentage (%) Agree Number and Percentage (%) ---------------------------------------------------------------------------------------------------------------------------- ------------------------------------ ----------------------------------- --------------------------------- ------ ---- ------ Personal benefits gained from using the telecare services Participating in the telecare services enabled me to control my hypertension, diabetes, and/or hyperlipidemia. 4 4.9 6 7.3 72 87.8 Participating in the telecare services enabled me to control my body weight. 11 13.4 29 35.4 42 51.2 I believe that the health records distributed through the telecare platform enhanced the doctors' management of my health. 24 29.3 8 9.8 50 61.0 The telecare services shortened the time, I spend on acquiring medical and healthcare services. 21 25.6 14 17.1 47 57.3 The telecare services reduced my medical and healthcare expenses. 26 31.7 15 18.3 41 50.0 Activeness in uploading or in using the platform information I actively measured my physiological indicators by using the telecare platform. 23 28 5 6.1 54 65.9 I actively uploaded my dietary records to the telecare platform. 60 73.2 8 9.8 14 17.1 I actively uploaded my exercising records to the telecare platform. 62 75.6 9 11.0 11 13.4 I actively used the health education information provided by the telecare platform. 59 72.0 5 6.1 18 22.0 I actively discussed my health problems with my telecare case manager. 38 46.3 20 24.4 24 29.3 Individual perception of the telecare services The telecare services motivated me to be responsible for managing my hypertension, diabetes, and/or hyperlipidemia. 4 4.9 19 23.2 59 72 The telecare services clarified my health status. 20 24.4 5 6.1 57 69.5 I believe that the telecare services will be prevalently used in the future. 5 6.1 7 8.5 70 85.4 The telecare services should be paid for by the individuals requiring it rather than by the NHI. 43 52.4 21 25.6 18 22.0 I am willing to present my health data to the healthcare team approved by me through the telecare platform. 5 6.1 6 7.3 71 86.6 ijerph-14-01544-t003_Table 3 ###### Correlation between the self-care behaviors of the participants and their IUT (N = 82) via Pearson's correlation coefficient. Variable Total Score for Self-Care Behaviors Medication Compliance Behaviors Self-Care Behaviors Health Promotion Behaviors Total Score for Usage Intention of Telehealth Personal Benefits Gained from Using Telecare Activeness in Uploading and Employing Platform Information Individual Perception of the Telecare ------------------------------------------------------------ ------------------------------------- --------------------------------- --------------------- ---------------------------- ----------------------------------------------- ---------------------------------------------- ------------------------------------------------------------ --------------------------------------- Total score for self-care behaviors 1 Medication compliance behaviors 0.511 \*\* 1 Self-care behaviors 0.805 \*\* 0.297 \*\* 1 Health promotion behaviors 0.673 \*\* −0.019 0.247 \* 1 Total score for usage intention of telehealth 0.327 \*\* 0.017 0.257 \* 0.325 \*\* 1 Personal benefits gained from using telecare 0.247 \* −0.045 0.192 0.285 \*\* 0.834 \*\* 1 Activeness in uploading and employing platform information 0.256 \* −0.043 0.203 0.289 \*\* 0.773 \*\* 0.485 \*\* 1 Individual perception of the telecare 0.279 \* 0.147 0.249 \* 0.160 0.789 \*\* 0.662 \*\* 0.489 \*\* 1 \*\*: Reached a statistical significance when α = 0.01 (2-tailed); \*: Reached a statistical significance when α = 0.05 (2-tailed). ijerph-14-01544-t004_Table 4 ###### Physiological indicators of the participants. Indicator *n* Before Use of the Telecare Services After Use of the Telecare Services *t* *p* ------------------------------------- ----- ------------------------------------- ------------------------------------ ------- ---------- HbA1c (%) 66 8.7 8 −3.78 0.000 \* AC sugar 66 182.7 159.7 −3.14 0.003 \* Monthly frequency of BP measurement 75 3.08 6.68 4.39 0.000 \* \* Reached a statistical significance when *p* \< 0.05.

Introduction {#sec1-1} ============ Spinal cord injuries result in complete or partial loss of sensation and/or mobility and affect the quality of life of patients\[[@ref1][@ref2][@ref3][@ref4][@ref5]\]. Severe spinal cord injury often causes paralysis and loss of sensation and reflex function below the site of injury, as well as impairing autonomic activity, such as breathing, and other functions, such as bowel and bladder control. Stem cell therapy has considerable therapeutic potential in spinal cord injury\[[@ref6][@ref7]\]. A recent study has shown that canine bone marrow mesenchymal stem cells exhibit various features *in vitro* that make them suitable for spinal cord reconstruction, such as their ability to proliferate as undifferentiated spheres, and under appropriate stimuli, to differentiate into neurons, astrocytes and oligodendrocytes. Because these cells can form neurosphere-like clumps and differentiate into neuron-like cells expressing neuronal markers\[[@ref8][@ref9]\], they hold great potential for nerve repair. However, mesenchymal stem cell transplantation alone is not sufficient for spinal cord repair because the majority of the mesenchymal stem cells engrafted into the spinal cord phenotypically differentiate into glial lineages and rarely survive\[[@ref10]\]. The microenvironment of the injured spinal cord is thought to play a crucial role in the differentiation and survival of engrafted mesenchymal stem cells\[[@ref11]\]. The neurite growth inhibition mediated by the Nogo-66 receptor\[[@ref12]\] is a major factor affecting the efficacy of mesenchymal stem cell transplantation. In this study, we used RNA interference to silence Nogo-66 receptor gene expression in mesenchymal stem cells. Our findings demonstrate the effectiveness of this strategy for enhancing mesenchymal stem cell transplantation for spinal cord injury. Results {#sec1-2} ======= Morphology of bone marrow mesenchymal stem cells {#sec2-1} ------------------------------------------------ The numbers of bone marrow stromal cells and colonies were significantly increased at 5 days of culture. Cells at passages 1--3 proliferated actively, and the majority of cells were seen to adhere as a monolayer. These cells were either spindle-shaped, oval-shaped, flat-shaped, triangular or irregular, and very strongly refractive, with more than two processes, some of which formed connections with each other. These cells had a visible nucleus and nucleolus, and when confluent, they grew in a parallel or spiral arrangement (Figure [1A](#F1){ref-type="fig"}--[C](#F1){ref-type="fig"}) . Flow cytometry showed that these cells were positive for CD29, CD44, CD105 and CD166, and negative for CD34, CD80 and CD86. The bone marrow mesenchymal stem cells were quite homogeneous, with a purity above 96%. {#F1} Nogo-66 receptor expression was reduced in siRNA-transfected bone marrow mesenchymal stem cells {#sec2-2} ----------------------------------------------------------------------------------------------- RT-PCR and western blot assay showed that Nogo-66 receptor gene and protein expression in siRNA-transfected bone marrow mesenchymal stem cells were significantly decreased compared with cells transfected with a control siRNA ([Figure 1D](#F1){ref-type="fig"}). Transplantation of Nogo-66 receptor-silenced bone marrow mesenchymal stem cells improved the morphology of the injured spinal cord {#sec2-3} ---------------------------------------------------------------------------------------------------------------------------------- At 4 weeks after transection injury, spinal cord tissue breakage, scars, and structural disorder were visible at the affected site in the model group, and a cavity was clearly visible ([Figure 2A](#F2){ref-type="fig"}). In the bone marrow mesenchymal stem cell group, astrocytes aggregated at the edge of the affected site and formed scars at the junction of the intact and damaged spinal cord. The cavity was smaller than in the model group, but larger than in the Nogo-66 receptor gene silencing group ([Figure 2B](#F2){ref-type="fig"}). In the Nogo-66 receptor gene silencing group, astrocytes exhibited reactive hypertrophy, aggregated and formed scars at the edge of the affected site. Some cells were spindle-shaped, forming a dense network with their processes, but the cavity disappeared ([Figure 2C](#F2){ref-type="fig"}). Immunohistochemical staining showed that the number of BrdU-positive cells increased in rats transplanted with Nogo-66 receptor-silenced bone marrow mesenchymal stem cells ([Figure 3](#F3){ref-type="fig"}), indicating improved survival of the transplanted bone marrow mesenchymal stem cells at the site of injury. {#F2} {#F3} Transplantation of Nogo-66 receptor-silenced bone marrow mesenchymal stem cells promoted the growth of nerve fibers after spinal cord injury {#sec2-4} -------------------------------------------------------------------------------------------------------------------------------------------- By horseradish peroxidase retrograde nerve tracing, only a few horseradish peroxidase-positive nerve fibers were visible at the T~8~ and higher segments in the model group ([Figure 4A](#F4){ref-type="fig"}). The number of horseradish peroxidase-positive nerve fibers in the bone marrow mesenchymal stem cell group was less than in the Nogo-66 receptor gene silencing group, but more than in the model group ([Figure 4B](#F4){ref-type="fig"}). The Nogo-66 receptor gene silencing group showed a large number of horseradish peroxidase-positive nerve fibers in the spinal cord ([Figure 4C](#F4){ref-type="fig"}). The number of horseradish peroxidase-positive nerve fiber bundles is shown in [Figure 4D](#F4){ref-type="fig"}, demonstrating significant differences among the three groups at 8 weeks post-injury (*P* \< 0.01). {#F4} Effects of Nogo-66 receptor-silenced bone marrow mesenchymal stem cell transplantation on tissue ultrastructure in the injured spinal cord {#sec2-5} ------------------------------------------------------------------------------------------------------------------------------------------ Transmission electron microscopy showed glial scarring and a small number of myelinated nerve fibers, macrophage phagocytosis, degeneration and necrotic myelinated nerve fibers in the model group ([Figure 5A](#F5){ref-type="fig"}). Massive myelinated nerve fibers and non-myelinated nerve fibers could be seen in the Nogo-66 receptor gene silencing group, with more axons and intact myelin ([Figure 5B](#F5){ref-type="fig"}). The numbers of myelinated nerve fibers and non-myelinated nerve fibers at the injury site in the bone marrow mesenchymal stem cell group were greater than in the model group, but less than in the Nogo-66 receptor gene silencing group ([Figure 5C](#F5){ref-type="fig"}). {#F5} Nogo-66 receptor-silenced bone marrow mesenchymal stem cell transplantation improved behavioral performance in rats with spinal cord injury {#sec2-6} ------------------------------------------------------------------------------------------------------------------------------------------- ### Neurological function {#sec3-1} After injury, rats manifested paraplegia, no activity of the hind limb or tail, and urinary dysfunction, but without defecatory dysfunction. Hind limb movement recovered at 2 and 4 weeks postinjury and became more coordinated at 6 weeks, and urinary function was partially restored, but residual urine was still visible in the bladder. The three groups exhibited similar changes after injury. Basso, Beattie and Bresnahan scores in the bone marrow mesenchymal stem cell and Nogo-66 receptor gene silencing groups were higher than in the model group. Moreover, Basso, Beattie and Bresnahan scores were higher in the Nogo-66 receptor gene silencing group than in the bone marrow mesenchymal stem cell group (*P* \< 0.01, *P* \< 0.05; [Figure 6A](#F6){ref-type="fig"}). {#F6} ### Inclined plate test {#sec3-2} At 4 weeks post-injury, scores in the inclined plate test were higher in the bone marrow mesenchymal stem cell and No-go-66 receptor gene silencing groups than in the model group. Moreover, scores in the inclined plate test were higher in the Nogo-66 receptor gene silencing group than in the bone marrow mesenchymal stem cell group (*P* \< 0.01, *P* \< 0.05; [Figure 6B](#F6){ref-type="fig"}). Nogo-66 receptor-silenced bone marrow mesenchymal stem cell transplantation reduced mortality in rats with spinal cord injury {#sec2-7} ----------------------------------------------------------------------------------------------------------------------------- At 8 weeks after injury, mortality was substantially lower in the bone marrow mesenchymal stem cell and Nogo-66 receptor gene silencing groups than in the model group (*P* \< 0.05). Mortality was lower in the Nogo-66 receptor gene silencing group than in the bone marrow mesenchymal stem cell group (*P* \< 0.05; [Figure 6C](#F6){ref-type="fig"}). Discussion {#sec1-3} ========== With the continued increase in vehicle use, the incidence of spinal cord injury has risen in tandem. Spinal cord injury has become one of the main causes of morbidity and mortality. Current treatment methods including surgery, medication and physiotherapy have limited efficacy\[[@ref13][@ref14][@ref15][@ref16][@ref17][@ref18][@ref19][@ref20][@ref21][@ref22][@ref23][@ref24][@ref25][@ref26][@ref27][@ref28][@ref29]\]. In recent years, mesenchymal stem cell transplantation for the treatment of neurological diseases has shown considerable therapeutic potential\[[@ref30][@ref31][@ref32]\]. Mesenchymal stem cells have many advantages, including easy collection, advanced methods for separation, culture, amplification and exogenous gene transfection, feasibility of autologous implantation following *in vitro* amplification or genetic modification, low risk of immune rejection, and fewer ethical considerations. Mesenchymal stem cell transplantation has been shown in a variety of experimental studies to be able to treat nervous system injury. The mechanisms of action are complex. Mesenchymal stem cells show a high expansion potential, genetic stability, and stable phenotype, they can be easily collected and shipped from the laboratory to the bedside, and they are compatible with different delivery methods and formulations\[[@ref33]\]. In addition, mesenchymal stem cells have two other extraordinary characteristics; they are able to migrate to sites of tissue injury and have strong immunosuppressive properties that can be exploited for successful autologous as well as heterologous transplantation without requiring pharmacological immunosuppression\[[@ref34][@ref35]\]. Furthermore, mesenchymal stem cells are capable of differentiating into neurons and astrocytes *in vitro* and *in vivo*\[[@ref36]\]. Recently, mesenchymal stem cell injection has shown promise for amyotrophic lateral sclerosis treatment in humans\[[@ref37]\]. They are able to improve neurological deficits and promote the restoration of functional synaptic transmission when transplanted into animal models of neurological disorders\[[@ref38]\]. Mesenchymal stem cells have been observed to migrate to the injured tissues and mediate functional recovery following brain, spinal cord and peripheral nerve lesions. However, bone marrow mesenchymal stem cell transplantation alone is not sufficient for spinal cord repair, because the majority of the mesenchymal stem cells implanted into the spinal cord have been shown to differentiate into a phenotype that is restricted to glial lineages and they rarely survive. The microenvironment of the injured spinal cord is believed to play a crucial role in inducing the differentiation and survival of the grafted mesenchymal stem cells. Growth inhibitory factors associated with the myelin sheath limiting axonal regeneration are a major impediment to adult mammalian regeneration in the central nervous system. A variety of factors isolated from central nervous system myelin have been shown to inhibit nerve regeneration, including the protein Nogo, one of the most important growth inhibitory factors. Guo et al.\[[@ref39]\] reported that Nogo protein gene expression was elevated after central nervous system injury in rats. Jiang et al.\[[@ref40]\] showed that Nogo protein, Nogo-66 receptor and RhoA expression in the brain tissues of rats with focal cerebral infarction began to increase at 6 hours, reached its peak at 24 hours, and lowered back to normal levels at 96 hours. Nogo protein may lead to growth cone collapse and inhibit neurite extension. The Nogo monoclonal antibody can neutralize the inhibitory activity of the protein. *In vitro* cultured oligodendrocytes also exhibit an inhibitory effect on axonal extension. Similarly, Yang et al.\[[@ref41]\] showed that Nogo neutralizing anti-body could promote recovery in rats with spinal cord injury. The inhibitory effect of Nogo protein is mediated by the No-go-66 receptor\[[@ref42],\[\],[@ref43][@ref44][@ref45][@ref46][@ref47]\]. In this study, gene silencing was used to suppress Nogo-66 receptor expression in mesenchymal stem cells, thereby promoting neurite growth following mesenchymal stem cell differentiation, and improving the efficacy of mesenchymal stem cell transplantation for repairing damage following central nervous system injury. Nogo-66 receptor gene knockout can result in permanent Nogo-66 receptor gene silencing, but the physiological function of the Nogo-66 receptor gene remains unclear, and permanent Nogo-66 receptor gene silencing may lead to unexpected consequences. RNAi is a very convenient and effective method of gene silencing, which is usually maintained for 3--5 days. This method is ideal for our study because high Nogo-66 receptor expression is observed following traumatic brain injury\[[@ref41][@ref48]\]. To avoid the drawbacks of permanent gene silencing, RNAi silencing combined with mesenchymal stem cell transplantation is the most promising treatment strategy for spinal cord injury. Recent *in vivo* and *in vitro* studies in non-neuronal and neuronal tissues have shown that different pathways of macrophage activation result in cells with different properties. Interleukin-6 triggers the classically activated inflammatory macrophages (M1 phenotype), whereas the alternatively activated macrophages (M2 phenotype) are anti-inflammatory. In this study, mesenchymal stem cells were subjected to Nogo-66 receptor gene silencing before transplantation, which may result in better repair of the damaged brain tissue, and promote mesenchymal stem cell proliferation and differentiation in the grafted area after injury. Our results showed that mesenchymal stem cell transplantation, after Nogo-66 receptor gene silencing, is greatly superior to simple mesenchymal stem cell transplantation in the treatment of spinal cord injury in rats, in terms of histological and functional outcomes. Tissue repair was better in the Nogo-66 receptor gene silencing group compared with the model and bone marrow mesenchymal stem cell groups. Immunohistochemical staining demonstrated a significant difference in the number of BrdU-positive cells and horseradish peroxidase-positive nerve fibers at the site of spinal cord injury, as follows: Nogo-66 receptor gene silencing group \> bone marrow mesenchymal stem cell group \> model group. In summary, the Nogo-66 receptor gene in mesenchymal stem cells can be silenced using the RNAi approach. The mesenchymal stem cells can be transplanted into the site of spinal cord injury *via* the tail vein. The transplanted mesenchymal stem cells better survive, proliferate, differentiate and migrate at the site of injury, and promote the recovery of nerve function after spinal cord injury. Our findings provide support for the use of this novel approach for the clinical treatment of spinal cord injury. Materials and Methods {#sec1-4} ===================== Design {#sec2-8} ------ A randomized, controlled, animal experiment. Time and setting {#sec2-9} ---------------- This experiment was performed at Hebei Medical University in China from May 2010 to May 2011. Materials {#sec2-10} --------- Sixty-four clean, healthy Wistar rats of both genders, aged 2 months and weighing 250--300 g, were purchased from the Chinese Academy of Medical Sciences Animal Laboratory (license No. SCXK20060008). All experimental procedures were performed in accordance with Chinese National Natural Science Foundation animal research regulations and the animal care guidelines of the National Institutes of Health. Methods {#sec2-11} ------- ### Bone marrow mesenchymal stem cell isolation and culture {#sec3-3} Bone marrow was harvested aseptically from the tibias of rats at the age of approximately 2 months. Nucleated cells were isolated by density gradient centrifugation using Percoll (1.073 g/mL) and were plated in growth medium consisting of Dulbecco\'s modified Eagle\'s medium/F12 (Hyclone, Logan, UT, USA) supplemented with 20% fetal bovine serum (Sigma, St. Louis, MO, USA) and benzylpenicillin (1 × 10^5^ U/mL)\[[@ref10][@ref49]\]. The mesenchymal stem cells were isolated in the medium by their tendency to adhere to plastic\[[@ref50][@ref51][@ref52]\]. After 3 days, the dishes were washed twice with PBS to remove nonadherent cells. The remaining cells were fed every third day. Nonadherent cells were removed and adherent cells were expanded until subconfluence and processed through sequential passages. Most contaminating hematopoietic stem cells were progressively lost, and after the second passage, cultures contained a morphologically homogenous cell population designated bone marrow mesenchymal stem cells. This was confirmed by fluorescence-activated cell-sorting analysis showing a lack of expression of the typical hematopoietic cell surface markers, including CD45, CD34 and CD14, and positivity for CD71, CD105 and CD44. Cells between passages 3 and 6 were used for our experiments. They were labeled using medium containing BrdU\[[@ref10][@ref52]\]. ### RNAi-transfected Nogo-66 receptor-silenced bone marrow mesenchymal stem cells {#sec3-4} The two target sequences of the rat Nogo-66 receptor mRNA were as follows: 5′-UGC AGU ACC UCU ACC UAC AAG ACA A-3′, 5′-UUG UCU UGU AGG UAG AGG UAC UGC A-3′. The siRNA template was synthesized by Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. in Shanghai, China. According to the *in vitro* transcription kit (Silencer™ siRNA Construction Kit, Ambion) instructions, 1 mL of culture medium containing 1 × 10^9^ mesenchymal stem cells was added into a centrifuge tube and centrifuged at 800 r/min (radius = 16 cm) for 5 minutes. After discarding the supernatant, 900 μL culture medium without antibiotics was added, and cells were resuspended. Fifty pmol siRNA was diluted with 50 μL Opti-MEM, and 1 μL Lipofectamine 2000 was diluted with 50 μL Opti-MEM, mixed and incubated at room temperature for 15 minutes. The two solutions were then gently mixed and incubated for 15 minutes at room temperature. The mixture was added into the mesenchymal stem cell suspension, placed at 37°C in a 5% CO~2~ saturated humidity incubator, and 72 hours later, Nogo-66 receptor expression was assessed. We isolated total RNA from the injured spinal cord (4 mm long) using the RNeasy Kit (Qiagen), and obtained cDNA using reverse transcription. Primers were as follows: Nogo-66 receptor sense, 5′-GGG CAA CCT CAC GCG CAT CT-3′ and anti-sense, 5′-CGG GCA AAG TCC CAA AT-3′; β-actin sense, 5′-GTC CCT GTA TGC CTC TGG TC-3′ and anti-sense, 5′-GGT CTT TAC GGA TGT CAA CG-3′. All primers were synthesized by Sangon. The β-actin gene was amplified for 24 cycles, and the others were amplified for 28 cycles. The reverse transcription-PCR product was subjected to 1.3% agarose gel electrophoresis and processed with a gel imaging system (Beijing Seclaser Technology Co., Ltd., Beijing, China), and the ratio of Nogo-66 receptor absorbance to β-actin absorbance was used as an index of Nogo-66 receptor mRNA expression level. ### Western blot assay for detecting the effectiveness of Nogo-66 receptor transfection {#sec3-5} The undifferentiated mesenchymal stem cell suspension in the control group and the mesenchymal stem cell suspension at 72 hours after siRNA transfection were centrifuged at 800 r/min (radius = 16 cm) for 5 minutes. The cells were collected. Supernatant was discarded. Four hundred μL protein extraction solution was added, and proteins were extracted. Protein concentration was determined using the Bradford method. The samples were subjected to SDS-PAGE, blotted onto a membrane, and incubated with rabbit anti-mouse Nogo-66 receptor gene antibody (1:800; Sigma) at 37°C on a shaker for 2 hours. The membrane was washed with Tris-buffered saline containing Tween-20 for 5 minutes (four times). The blots were incubated with goat anti-rabbit antibody (1:700; Sigma) at 37°C for 1.5 hours, washed with Tris-buffered saline containing Tween-20 for 5 minutes (four times), and reacted with 3,3′-diaminobenzidine. The experiment was repeated three times. Quantity one image (BioRad, Hercules, CA, USA) analysis was conducted. The absorbance value ratios of the target and β-actin bands were assessed and expressed as protein expression levels. ### Preparation of animal models of spinal cord injury {#sec3-6} Sixty-three adult Wistar rats were anesthetized with a 4% chloral hydrate solution (360 mg/kg intraperitoneally). Body temperature was monitored and maintained using a thermal blanket. Using the aseptic surgical technique, a dorsal incision was made from the middle to lower thoracic regions. Using a microscope, T~9--10~ laminectomy was performed, and the spinal cord was exposed. The spinal cord was transected at the level of T~9--10~, leaving a 2-mm gap between the proximal and distal ends of the resected cord\[[@ref20]\]. ### Transplantation of mesenchymal stem cells {#sec3-7} Sixty-three model rats were equally and randomly divided into three groups. Six hours after injury, bone marrow mesenchymal stem cells pre-labeled with bromodeoxyuridine were transplanted *via* tail vein. The model group was injected with 1 mL of stem cell-free culture medium. The bone marrow mesenchymal stem cell group was injected with 1 mL of bone marrow mesenchymal stem cells (5 × 10^9^/L). The Nogo-66 receptor gene silencing group was injected with 1 mL (5 × 10^9^/L) Nogo-66 receptor gene-silenced bone marrow mesenchymal stem cells. Intraperitoneal injection of gentamicin 2,000 U served as antibiotic treatment. Rats were fed in separate cages. ### Histological examination {#sec3-8} At 4 weeks after injury, the rats were randomly selected from each group and killed under anesthesia. Specimens were harvested for histological examination to determine the degree of recovery. The dissected spinal cord tissues were postfixed for 3 hours in 4% paraformaldehyde, soaked overnight in 10% followed by 30% sucrose, and cut into 15-mm-thick sagittal and parasagittal sections using a cryostat. Hematoxylin-eosin staining was carried out for general histological examination. ### Immunocytochemistry for BrdU expression in cells {#sec3-9} Four weeks after operation, two rats were randomly taken from each group. Immunocytochemistry for the detection of BrdU requires a pretreatment of tissue sections to denature DNA. All staining was done on free-floating 40-μm sections. A mouseanti-BrdU monoclonal antibody (1:100; Boehringer Mannheim, Ingelheim am Rhein, Germany) was used in combination with avidin biotin complex and a horse anti-mouse IgG antibody conjugated with biotin (1:167; Vector Laboratories, Burlingame, CA, USA). Primary and secondary antibodies were incubated at 37°C. Ten fields were randomly selected from each slice under the light microscope at a magnification of × 200 (Sigma). The number of BrdU-positive cells was calculated in each field of vision, and the mean value was obtained. ### Retrograde tracing with horseradish peroxidase {#sec3-10} Eight weeks after operation, two rats were randomly taken from each group. After surgery, the spinal cord was exposed at T~12~ and 1 μL aqueous suspension of 30% horseradish peroxidase (Sigma) was injected bilaterally 1 mm into the spinal dorsal vein. After injection, the incision was closed, and the animals were maintained for 36 hours before being perfused with buffered 1% paraformaldehyde and 1.25% glutaraldehyde. The spinal cord was removed and stored in 20% sucrose in 0.1 mol/L PBS at 4°C overnight. The spinal cord was dissected, and 10 fields were randomly selected from each slice under the light microscope at × 200. The number of horseradish peroxidase-labeled nerve fibers was calculated in each field, and the mean value was obtained. ### Electron microscopy {#sec3-11} Eight weeks after operation, two rats were randomly selected from those subjected to labeling from each group, and perfused intracardially with saline\[[@ref17][@ref18][@ref19][@ref20]\], followed by 2% glutaraldehyde and 4% paraformaldehyde in 0.1 mol/L sodium cacodylate buffer, pH 7.4. Immediately after perfusion, the spinal cords were removed and postfixed in the same fixative overnight at 4°C. The spinal cord segment at the injury site was sliced into 1-mm pieces, postfixed for 2 hours in 1% OsO~4~ in 0.1 mol/L cacodylate buffer, dehydrated in graded ethanol solutions, and embedded in Epon-812. Semi-thin plastic sections (1 μm) were cut and stained with 1% toluidine blue before examination with a Nikon Eclipse TE300 microscope equipped with a Spot RT Color CCD camera. For electron microscopy, blocks were trimmed and sections were cut at 100-nm thickness, mounted on copper grids, stained with uranyl acetate and lead citrate, and viewed with a JEOL Jem 1200 EX transmission electron microscope (JEOL Ltd., Tokyo, Japan). ### Evaluation of functional recovery {#sec3-12} Two types of functional tests were used to assess functional recovery. Each test was observed by two independent investigators. The test was performed at 1, 2, 4, 6 and 8 weeks post-operation. Basso, Beattie and Bresnahan test: the open-field locomotion test assesses movement, weight support and coordination. It was scored using the standardized Basso, Beattie and Bresnahan locomotor scoring system. Basso, Beattie and Bresnahan scores range from 0 (flaccid paralysis) to 21 (normal gait). Rats were acclimated to the testing environment (90-cm diameter plastic wading pool; 4-cm height) prior to testing. Basso, Beattie and Bresnahan scores were averaged for each group. Inclined plane test: this test evaluates the maximum angle on the inclined plane upon which each animal can maintain a stable position for 5 seconds. Rats were placed on a board that was incrementally raised to increasing angles\[[@ref17][@ref18][@ref19][@ref20]\]. Angle scores were averaged for each group. Mortality: the mortality of each group was evaluated at 8 weeks post-injury. Statistical analysis {#sec2-12} -------------------- Data were expressed as mean ± SD, and analyzed using SPSS 17.0 software (SPSS, Chicago, IL, USA). Intergroup comparison was done using one-way analysis of variance. Paired comparison was done using Dunnett\'s t-test. A value of *P* \< 0.05 was considered statistically significant. **Conflicts of interest:** *None declared.* **Peer review:** *This study used gene-silenced bone marrow mesenchymal stem cell transplantation in animal models of spinal cord injury, provided new ideas and experimental evidence for the treatment of spinal cord injury in the clinic. This method can restore neurological function in patients with spinal cord injury and improve their quality of life.* Copyedited by Patel B, Frenchman B, Yu J, Qiu Y, Li CH, Song LP, Zhao M [^1]: **Author contributions:** *All authors designed, implemented, evaluated the study, and approved the final version of the paper.*

Introduction {#sec1} ============ The synthesis of defined polysilanes in which more than five silicon atoms are connected is challenging. The standard approaches for such polysilanes are Wurtz-type coupling^[@ref1]−[@ref3]^ or Lewis acid catalyzed rearrangement reactions.^[@ref4]^ These two methods generally give rise to structurally simple polysilanes with a low set of functionalities for further derivatization, which prevent the construction of molecules of even moderate complexity. A potent strategy for the construction of structurally more challenging silicon frameworks is the use of di- or multifunctionalized starting materials such as α,ω-dianions. Gilman^[@ref5],[@ref6]^ and Hengge^[@ref7],[@ref8]^ were pioneers in this area and developed the cleavage of strained cyclosilanes to obtain dianions. Sekiguchi,^[@ref9]^ Tokitoh,^[@ref10]^ Kira,^[@ref11]^ and Apeloig^[@ref12]^ also contributed with their groups to this research field and prepared some previously unknown 1,1-, 1,2-, and 1,4-dilithiooligosilanes. Marschner and Baumgartner, who introduced KO*t*Bu into the field of polysilane chemistry, achieved a milestone in polysilane synthesis. Consequently, the construction of relative complex polysilanes could be accomplished in a straightforward way.^[@ref13]−[@ref16]^ Recently, Klausen and co-workers established new phenyl-substituted dianions.^[@ref17]^ These dianions were used as building blocks for the formation of defined polysilanes as well as for the synthesis of heteroelement substituted polysilanes.^[@ref18]−[@ref20]^ Scheschkewitz et al. treated their hexasilabenzene with lithium naphthalenide and obtained a novel dianionic silicon cluster. This dianion turned out to be a valuable synthon for the generation of unprecedented molecular heterosiliconoids with boron and phosphorus directly incorporated into the cluster scaffold.^[@ref21]^ In addition, we just published a paper about the synthesis of a mixed substituted dianion, which allows straightforward access to a hitherto unknown tricyclic polysilane (see [Chart [1](#cht1){ref-type="chart"}](#cht1){ref-type="chart"}).^[@ref22]^ Nevertheless, the synthesis of mixed functionalized disilanides has not been reported so far, although these substances would represent ideal building blocks for highly complex silicon frameworks. {#cht1} As we have reported earlier, it is possible to synthesize and characterize cyclic silenolates as well as cyclic germenolates and convert them with suitable electrophiles in order to gain a new set of differently substituted acylsilanes as well as acylgermanes (see [Chart [2](#cht2){ref-type="chart"}](#cht2){ref-type="chart"}). {#cht2} Furthermore, we could show that the reaction of these enolates with chlorosilanes ClSiR~3~ allowed straightforward access to silenes and germenes with exocyclic structures.^[@ref23],[@ref24]^ Due to the straightforward accessibility of these cyclic enolates, we saw the potential to investigate their chemical behavior in greater depth. In this context, we have established a novel synthetic strategy for the synthesis of previously unknown dianionic cyclic silenolates and germenolates. The aim of this work is to investigate the spectroscopic properties and the reactivity of these new types of dianions with selected examples of electrophiles. Results and Discussion {#sec2} ====================== Synthesis of Dianionic Silenolates {#sec2.1} ---------------------------------- The reaction of the acylcyclohexasilanes **1a**,**b** with 2 equiv of KO*t*Bu led to the formation of compounds **2a**,**b**, whereby two different functionalized anionic silicon atoms were incorporated into one molecule ([Scheme [1](#sch1){ref-type="scheme"}](#sch1){ref-type="scheme"}). {#sch1} To the best of our knowledge, **2a**,**b** represent the first examples of dianionic polysilanes bearing a silyl anion and a silenolate fragment in one molecule. The dianionic compounds **2a**,**b** were formed in the same fashion as previously described for the corresponding silenolates. Two major differences are worth mentioning. First, the use of an appropriate solvent is highly important. We observed the formation of **2a**,**b** only in DME, Et~2~O, and toluene. In the case of THF, no product was formed, probably due to the reaction of **2a**,**b** with THF leading to degradation. This was also described in the case of α,ω-oligosilyl dianions by Marschner et al.,^[@ref14]^ who observed that stable dianionic species were only formed with the use of DME or benzene/toluene with the addition of crown ethers. Second, the reaction is characterized by a two-step reaction sequence. The first 1 equiv of KO*t*Bu is consumed immediately (approximately 10 min), yielding the silenolates **S**~**1**~**a**,**b**. The second abstraction of the trimethylsilyl group is much slower and takes place within approximately 18 h. For isolation, **2a**,**b** were crystallized from Et~2~O/18-cr-6 at room temperature to give orange crystals of the 1:2 18-cr-6 adducts, which were obtained in isolated yields of \>90%. After filtration, the crystals can be stored at −30 °C in the absence of air even for prolonged periods. **2a**,**b** afforded crystals of sufficient quality for single-crystal X-ray crystallography. The molecular structures are depicted in [Figures [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"} and [2](#fig2){ref-type="fig"}; selected bond lengths and the sums of valence angles are summarized in [Table [1](#tbl1){ref-type="other"}](#tbl1){ref-type="other"}. {#fig1} {#fig2} ###### Selected Bond Lengths *d* (Å) and Sum of Valence Angles ∑αSi~(1)~, ∑αSi~(6)~, and ∑αC~(1)~ (deg) for **2a**,**b**   **2a** **2b** ---------------------- -------- -------- *d*(Si~(1)~--C~(1)~) 1.892 1.916 *d*(Si~(1)~--K~(1)~) 5.102 5.215 *d*(C~(1)~--K~(1)~) 3.843 3.672 *d*(C~(1)~--O~(1)~) 1.254 1.254 *d*(K~(1)~--O~(1)~) 2.614 2.579 *d*(Si~(6)~--K~(2)~) 3.441 3.458 *d*(Si~(1)~--K~(2)~) 4.143 7.361 ∑αSi~(1)~ 312.5 314.4 ∑αSi~(6)~ 307.8 306.1 ∑αC~(1)~ 359.7 360.0 On the basis of the observed structural features, **2a**,**b** are best described as acyl silyl anions (keto form) with Si--C single bonds, C=O double bonds, and markedly pyramidal central Si~(1)~ atoms. Interestingly in **2a** the K~(2)~^+^ cation coordinates simultaneously to Si~(1)~ and Si~(6)~. This is probably caused by a packing phenomenon. Furthermore, this simultaneous coordination is also the reason for **2a** to adopt the half-boat conformation, while **2b** and **4b** (**4b** is the dianionic germenolate and will be introduced in the next section) adopt chair conformations. Additionally **2a** shows short Si~(2)~--CH contacts which are less than the van der Waals radii of silicon and hydrogen. This can also explain its half-boat coordination. A similar result in terms of Si--CH contacts was obtained by the Klausen group.^[@ref17]^ Synthesis of Dianionic Germenolates {#sec2.2} ----------------------------------- The straightforward synthesis of **2a**,**b** encouraged us to expand our new methodology to other starting materials. As we have reported previously, it is possible to synthesize cyclic acylgermanes **3a**,**b**.^[@ref24]^ The reaction of these cyclic acylgermanes with 2 equiv of KO*t*Bu led to the formation of dianionic germenolates **4a**,**b** ([Scheme [2](#sch2){ref-type="scheme"}](#sch2){ref-type="scheme"}). Again, the reaction is characterized by a two-step reaction sequence with reaction rates similar to those of the corresponding acylsilanes. {#sch2} For isolation, **4a**,**b** were crystallized from Et~2~O/18-cr-6 at room temperature to give orange crystals of the 1:2 18-cr-6 adducts, which can be stored after filtration at −30 °C in the absence of air even for prolonged periods. **4b** afforded crystals of sufficient quality for single-crystal X-ray crystallography. The molecular structure is depicted in [Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}; selected bond lengths and the sums of valence angles are summarized in [Table [2](#tbl2){ref-type="other"}](#tbl2){ref-type="other"}. {#fig3} ###### Selected Bond Lengths *d* (Å) and Sum of Valence Angles ∑α(Ge1), ∑α(Ge2), and ∑α(C1) (deg) for **4b** ---------------------- -------- *d*(Ge~(1)~--C~(1)~) 2.047 *d*(Ge~(1)~--K~(1)~) 3.427 *d*(C~(1)~--K~(1)~) 3.335 *d*(C~(1)~--O~(1)~) 1.248 *d*(K~(1)~--O~(1)~) 2.760 *d*(Ge~(2)~--K~(2)~) 3.635 *d*(Ge~(1)~--K~(2)~) 6.671 ∑αGe~(1)~ 314.5° ∑αGe~(2)~ 298.2° ∑αC~(1)~ 359.6° ---------------------- -------- On the basis of the observed structural features, **4b** is best described as an acyl germyl anion (keto form) with a Ge--C single bond, a C=O double bond, and markedly pyramidal central Ge~(1)~ and Ge~(2)~ atoms. NMR Spectroscopy of **2a**,**b** and **4a**,**b** {#sec2.3} ------------------------------------------------- NMR data also supported that the dominant structure of **2a**,**b** and **4a**,**b** in solution is the keto form. Very similar ^13^C chemical shifts were observed for the carbonyl C atom of the two compounds between δ 266.6 and 280.9 ppm in a typical range for carbonyl groups. Furthermore, **2a**,**b** and **4a**,**b** exhibit only two sharp SiMe~2~ resonance lines in the ^29^Si NMR, which clearly suggest free rotation around the Si~(1)~--C~(1)~ bond ([Table [3](#tbl3){ref-type="other"}](#tbl3){ref-type="other"}). It was not possible to use THF-*d*~8~ for **2b** and **4b** because a detectable degradation was found within minutes at room temperature. ###### Selected ^13^C and ^29^Si NMR Chemical Shifts for the Silenolates **2a**,**b** and **4a**,**b**[a](#t3fn1){ref-type="table-fn"}   **2** (ppm) **4** (ppm) ---------------------- ------------- ------------- --------- --------- δ~^13^C~(*C*=O) 266.63 273.96 281.01 280.92 δ~^29^Si~(*Si*Me~3~) 0.05 --1.14 0.93 0.36 δ~^29^Si~(*Si*Me~2~) --27.69 --26.48 --27.69 --23.45   --32.37 --31.31 --29.35 --25.40 δ~^29^Si~(*Siq*) --67.70 --87.33       --189.08 --192.42     δ values relative to external TMS. In THF-*d*~8~ at 25 °C. In C~6~D~6~ at 25 °C. UV--Vis Spectroscopy and TDDFT-PCM Calculations {#sec2.4} ----------------------------------------------- Toluene was used as a solvent to determine the charge transfer behavior for the longest wavelength absorption band.^[@ref25]^[Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"} depicts the measured UV--vis spectra of **2a**,**b** and **4a**,**b** in toluene together with their calculated frontier Kohn--Sham orbitals. ).](om0c00385_0006){#fig4} In order to examine the differences between aromatic and saturated substituents at the carbonyl moiety, the mesityl- and adamantyl-substituted derivatives **2a**,**b** and **4a**,**b** were investigated. All UV--vis calculations were performed on the geometry-optimized X-ray crystal structures *via* TDDFT-PCM in toluene at the CAM-B3LYP/6-31+G(d,p) level of theory.^[@ref26]^ Noteworthy, CAM-B3LYP achieved a better consistency for dianions **2a**,**b** and **4a**,**b** in calculated vertical excitations in comparison to B3LYP, which was previously applied to UV--vis calculations on silenolates **S**~**1**~**a**,**b** and germenolates **S**~**2**~**a**,**b**.^[@ref24],[@ref27]^ The silenolates **2a**,**b** exhibit intense absorption maxima in the range between 433 and 450 nm, which are red-shifted in the order **2b** → **2a**. The same bathochromic trend **4b** → **4a** also applies to the germenolates **4a**,**b** with absorption maxima between 420 and 447 nm. The acyl substituent (aryl vs alkyl) significantly affects the HOMO orbital density and hence its shape, which ultimately leads to different reaction centers in conversion with electrophiles (see section below). The HOMO-1 and HOMO of **2a** ([Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}) correspond to the p~*z*~ orbital of the silenolate with a significant part of the corresponding silanide mixed in, respectively. This contribution makes the silanide equally nucleophilic regarding reactions of cyclic silenolates with aromatic acyl substituents. In contrast, the HOMO of **2b** only exhibits the p~*z*~ character of the silenolate, whereas the HOMO-1 of **2b** shows the silanide orbital alone, allowing a site-specific functionalization. In addition, the energy difference between the HOMO-1 and the HOMO is in **2a** significantly larger than in **2b** (0.28 eV vs 0.09 eV). Similar observations were made with the dianionic germenolates **4a**,**b**. Upon excitation, electron density is displaced into the π\* orbital of the corresponding carbonyl orbitals. In the corresponding LUMOs of the aryl-substituted species **2a** and **4a**, our calculations additionally showed considerable conjugation of the carbonyl group and the aromatic π systems, which is not possible for the alkyl-substituted silenolate **2b** and germanolate **4b**. As a consequence of this, the empty orbitals are energetically stabilized in the order **2b** → **2a**. This stabilization results in smaller excitation energies and in the observed bathochromic shifts of the corresponding absorption bands. The obtained experimental and computational data are summarized in [Table [4](#tbl4){ref-type="other"}](#tbl4){ref-type="other"} and show reasonable agreement. ###### Experimental and TDDFT-PCM(toluene) CAM-B3LYP/6-31+G(d,p)//B3LYP/6-31+G(d,p) Calculated Absorption Bands λ in Toluene and Extinction Coefficients ε with Respect to Oscillator Strengths *f* for **2a**,**b** and **4a**,**b**   λ~max,exp~ (nm) ε (L mol^--1^ cm^--1^) λ~max,calc~[a](#t4fn1){ref-type="table-fn"} (nm) *f* assignment -------- ----------------- ------------------------ -------------------------------------------------- -------- ----------------------- **2a** 450 6602 441 0.2102 p~*z*~ → π\*(CO/aryl) **2b** 433 3730 428 0.1131 p~*z*~ → π\*(CO) **4a** 447 5753 435 0.1591 p~*z*~ → π\*(CO/aryl) **4b** 420 2178 418 0.0858 p~*z*~ → π\*(CO) λ~max,calc~ values are corrected by a factor of 5% due to a consistent overestimation of excitation energies with CAM-B3LYP. Reactivity of **2a**,**b** versus Selected Examples of Chlorosilanes {#sec2.5} -------------------------------------------------------------------- The reactivity of **2a**,**b** versus chlorosilanes parallels the observed reactivities for silenolates and silyl anions. The same reactivity was found by Ohshita and Ishikawa, by Marschner et al., and by our group.^[@ref13]−[@ref15],[@ref23],[@ref28],[@ref29]^ Thus, **2b** with an alkyl group attached to the carbonyl moiety reacted with an equimolar amount of tetramethyldichlorodisilane (ClSiMe~2~SiMe~2~Cl) at 0 °C in THF with formation of the acyl bicyclo\[2.2.2\]octasilane **6**. **6** was obtained in nearly quantitative yield (95% yield). The asymmetrically substituted acylsilane **6** exhibits two ^29^Si resonance lines for the SiMe~2~ groups near −37 ppm, which are not significantly influenced by the nature of the adamantoyl group. The two δ~^29^Si~ values for the bridgehead Si atoms were measured near −131 ppm (characteristic for tetrasilyl-substituted silanes) and −77 ppm (characteristic for acyl-substituted quaternary silanes). The same tendencies were found earlier by Stueger et al. during the synthesis of a series of substituted bicyclo\[2.2.2\]octasilanes.^[@ref30]^ The aryl-substituted compound **2a** exclusively afforded the *O*-silylated silene **5** under the same conditions (compare [Scheme [3](#sch3){ref-type="scheme"}](#sch3){ref-type="scheme"}). NMR spectral data of **5** (see the [Experimental Section](#sec4){ref-type="other"}) are also typical for a Brook-type silene. ^13^C and ^29^Si signals characteristic for Si=C were observed at δ~^29^Si~ 32.8 ppm and δ~^13^C~ 198.9 ppm, respectively. **5** was obtained in a good yield (64% yield). {#sch3} Reactivity of **4a**,**b** versus Selected Examples of Chlorosilanes {#sec2.6} -------------------------------------------------------------------- Furthermore, the reactivity of **4a**,**b** versus chlorosilanes was investigated and parallels that previously observed for germenolates and germyl anions.^[@ref24],[@ref31]^ The reaction of **4a** with 2 equiv of ClSi*i*Pr~3~ afforded the formation of the *O*-silylated germene **7** in excellent yields (compare [Scheme [4](#sch4){ref-type="scheme"}](#sch4){ref-type="scheme"}). NMR spectral data of **7** (see the [Experimental Section](#sec4){ref-type="other"}) are again typical for a Brook-type germene. A ^13^C signal characteristic for Ge=C was observed at δ~^13^C~ 210.17 ppm. Interestingly, the reaction of **4b** with an equimolar amount of ClSiMe~2~SiMe~2~Cl did not lead to the formation of the expected product **8**; instead, an undefined polymeric material was formed. {#sch4} The unsuccessful derivatization of **4b** with ClSiMe~2~SiMe~2~Cl encouraged us to investigate the reactivity of **4b** with 2 equiv of ClSi*i*Pr~3~. Again, no expected product formation was observed ([Scheme [5](#sch5){ref-type="scheme"}](#sch5){ref-type="scheme"}). The same experiment was further repeated with **2b** and gave also rise to an undefined polymer. Therefore, we reasoned that ClSi*i*Pr~3~ is too sterically demanding to allow M--Si (M = Si for **2b** and M = Ge for **4b**) bond formation in the presence of an adamantoyl group. {#sch5} Reactivity of **2a**,**b** and **4a**,**b** versus Carbon-Centered Electrophiles {#sec2.7} -------------------------------------------------------------------------------- We selected MeI as a carbon-centered electrophile, because it represents a benchmark reagent with numerous examples found in the literature.^[@ref27],[@ref29],[@ref32]^ In the reaction of **2a**,**b** and **4a**,**b** with MeI, the same reactivities in terms of reaction sites were observed. In all cases, alkylation of the negatively charged silicon as well as germanium atoms were found in nearly quantitative yields ([Scheme [6](#sch6){ref-type="scheme"}](#sch6){ref-type="scheme"}). Again, the same tendency was reported in the case of acyclic silenolates and silanides by Ohshita, Ottosson, and Marschner earlier.^[@ref14],[@ref15],[@ref28],[@ref29],[@ref32]^ The methylated silicon derivatives **9a**,**b** and germanium derivatives **10a**,**b** were obtained as *cis*/*trans* mixtures. The silicon atoms of **9a**,**b** undergo a significant low-field shift from −70 to 45 ppm (in the case of the acyl-substituted silicon atom) and from −131 to −84 ppm (for the silyl-substituted silicon atom). This is caused by the lower shielding of the methyl group in comparison to the trimethylsilyl group (see the [Experimental Section](#sec4){ref-type="other"}). {#sch6} Competitive Reactivity of the Silyl Anion and the Silenolate {#sec2.8} ------------------------------------------------------------ Finally, we investigated which silanide is more nucleophilic, the silyl anion or the silenolates. Therefore, we reacted **2a**,**b** with 1 equiv of trimethylchlorosilane (ClSiMe~3~) at −30 °C. The outcome of the reaction was again strongly dependent on the substituent at the carbonyl moiety and reflected our predictions from the computational analyses. **2a** with an aryl group attached to the carbonyl moiety reacted with an equimolar amount of ClSiMe~3~ to form the cyclic silenolate **S**~**1**~**a**, making the silanide the more nucleophilic reaction center. **2b**, on the other hand, reacted with an equimolar amount of ClSiMe~3~ to give the bicyclic oxahexasilabicyclo\[3.2.1\]octan-8-ide **11**, which clearly showed that the silenolate is more nucleophilic than the silyl anion in the case of an alkyl substitution. The formation of **11** can be rationalized by assuming the intermediate formation of the silanide **12**, which subsequently rearranged to give the bicyclic carbanion **13** by an intramolecular sila-Peterson alkenation. Apparently, **13** is very unstable, losing its intense red color within minutes, presumably by the abstraction of one proton from the surrounding media to give **11** as the final product. Analytical and spectroscopic data (see the [Experimental Section](#sec4){ref-type="other"}) clearly supported the bicyclic structure of **11** (see [Scheme [7](#sch7){ref-type="scheme"}](#sch7){ref-type="scheme"}). Moreover, this compound was obtained by a previous study by our group.^[@ref27]^ {#sch7} Conclusion {#sec3} ========== In summary, we synthesized the first examples of mixed functionalized compounds **2a**,**b** and **4a**,**b**, which represent ideal building blocks for highly complex silicon frameworks. These dianions are easily accessible, can be isolated, and were fully characterized. Silenolates **2a**,**b** as well as the germenolates **4a**,**b** adopt the keto form in solution, irrespective of the nature of the R group attached to the carbonyl moiety. Furthermore, the reactivity of **2a**,**b** and **4a**,**b** versus chlorosilanes was investigated as an example of a silicon-centered electrophile. **2a** and **4a** reacted with ClSi*i*Pr~3~ to give new examples of a polysilane and a polygermane with an exocyclic double bond. The reaction of **2b** with ClSiMe~2~SiMe~2~Cl led to the formation of the acyl bicyclo\[2.2.2\]octasilane **6**. Moreover, the reaction of **2a**,**b** and **4a**,**b** with MeI, as a carbon-centered electrophile, led to selective alkylation reactions at the negatively charged silicon and germanium atoms. The methylated structures **9a**,**b** and **10a**,**b** were formed in nearly quantitative yields. Finally, we examined the competitive reactivity of the silyl anion and the silenolate toward 1 equiv of ClSiMe~3~. The outcome of the reaction was strongly influenced by the substituent at the carbonyl moiety, which was in alignment with our computational analysis. **2a** reacted with 1 equiv of ClSiMe~3~ to give the corresponding cyclic silenolate **S**~**1**~**a** and demonstrated that the silyl anion is more nucleophilic than the silenolate. In contrast to that, **2b** reacted with 1 equiv of ClSiMe~3~ to give the bicyclic compound **11***via* an intramolecular sila-Peterson alkenation reaction. This observation clearly showed that the alkyl-substituted silenolate is more nucleophilic than the silyl anion. Further studies to probe the scope of these new dianions are currently in progress. Experimental Section {#sec4} ==================== All experiments were performed under a nitrogen atmosphere using standard Schlenk techniques. Solvents were dried using a column solvent purification system.^[@ref33]^ ClSiMe~3~ (95%), KO*t*Bu (\>98%), ClCOMes (99%), ClCOAd (98%) and 18-cr-6 (99%), were used without any further purification. ^1^H, ^13^C, and ^29^Si NMR spectra were recorded on a Varian INOVA 300 spectrometer in C~6~D~6~, THF-*d*~8~, or CDCl~3~ solutions and were referenced versus TMS using the internal ^2^H-lock signal of the solvent. HRMS spectra were obtained on a Kratos Profile mass spectrometer. Infrared spectra were obtained on a Bruker Alpha-P Diamond ATR spectrometer from the solid sample. Melting points were determined using a Stuart SMP50 apparatus and are uncorrected. Elemental analyses were carried out on a Hanau Vario Elementar EL apparatus. UV absorption spectra were recorded on a PerkinElmer Lambda 5 spectrometer. Synthesis of **2a** {#sec4.1} ------------------- A 500 mg portion of acylcyclohexasilane **1a** (0.76 mmol) and 423 mg of 18-cr-6 (1.60 mmol) were dissolved in 20 mL of Et~2~O. The solution was then cooled to −70 °C, and 180 mg (1.60 mmol) of KO*t*Bu was added. During the addition, the reaction mixture turned from yellow to dark orange and an orange precipitate began to form. The reaction mixture was warmed to room temperature and stirred overnight for 15 h. The orange precipitate was isolated and washed with Et~2~O (3 × 5 mL). The orange powder was dried under vacuum (not longer than 5 min; otherwise a slow degradation process occurs) to give **2a**. Yield: 775 mg (91%) of analytically pure **2a** as an orange powder. Data for **2a** are as follows. Mp: 158--159 °C. Anal. Calcd for C~45~H~92~K~2~O~13~Si~7~: C, 48.43; H, 8.31. Found: C, 48.68; H, 8.45. ^29^Si NMR (THF-*d*~8~, TMS, ppm): 0.05 (*Si*Me~3~); −27.69, −32.37 (*Si*Me~2~); −67.70 (*Si*COAryl); −189.08 (*Si*(SiMe~3~)). ^13^C NMR (THF-*d*~8~, TMS, ppm): 266.63 (*C*=O); 154.00, 132.80, 132.50, 128.18 (Aryl-*C*); 71.03 ((−*C*H~2~*C*H~2~O−)~6~); 21.61, 21.29 (Aryl-*C*H~3~); 8.92 (Si(CH~3~)~3~); 4.33, 1.30 (Si(CH~3~)~2~). ^1^H NMR (THF-*d*~8~, TMS, ppm): 6.57 (s, 2H, Mes-*H*); 3.61 (s, 48H, (−C*H*~2~C*H*~2~O−)~6~); 2.38 (s, 6H, Mes-C*H*~3~); 2.15 (s, 3H, Mes-C*H*~3~); 0.12, 0.05 (s, 33H, Si(C*H*~3~)~2~ and Si(C*H*~3~)~3~). UV--vis: λ \[nm\] (ε \[L mol^--1^ cm^--1^\]) 450 (6602). Synthesis of **2b** {#sec4.3} ------------------- A 500 mg portion of of acylcyclohexasilane **1b** (0.74 mmol) and 413 mg of 18-cr-6 (1.60 mmol) were dissolved in 20 mL of Et~2~O. The solution was then cooled to −70 °C, and 175 mg (1.60 mmol) of KO*t*Bu was added. During the addition, the reaction mixture turned from colorless to dark orange and a yellow precipitate began to form. The reaction mixture was warmed to room temperature and stirred overnight for 15 h. The yellow precipitate was isolated and washed with Et~2~O (3 × 5 mL). The yellow powder was dried under vacuum (not longer than 5 min; otherwise a slow degradation process occurs) to give **2b**. Yield: 801 mg (95%) of analytically pure **2b** as a yellow powder. Data for **2b** are as follows. Mp: 155--157 °C. Anal. Calcd for C~46~H~96~K~2~O~13~Si~7~: C, 48.81; H, 8.55. Found: C, 48.92; H, 8.63. ^29^Si NMR (C~6~D~6~, TMS, ppm): −1.14 (*Si*Me~3~); −26.48, −31.31 (*Si*Me~2~); −87.33 (*Si*COAryl); −192.42 (*Si*(SiMe~3~)). ^13^C NMR (C~6~D~6~, TMS, ppm): 273.96 (*C*=O); 70.14 ((−*C*H~2~*C*H~2~O−)~6~); 50.79 (Ad-*C*-CO); 40.36, 38.46 (Ad-*C*H~2~); 30.17 (Ad-*C*H); 9.36 (Si(CH~3~)~3~); 5.21, 4.11, 2.50 (Si(CH~3~)~2~). ^1^H NMR (C~6~D~6~, TMS, ppm): 3.34 (s, 48H, (−C*H*~2~C*H*~2~O−)~6~); 2.34, 2.05, 1.96 (bs, 15H Ad-*H*); 0.95, 0.86, 0.79 (bs, 33H, Si(C*H*~3~)~2~ and Si(C*H*~3~)~3~). UV--vis: λ \[nm\] (ε \[L mol^--1^ cm^--1^\]) 433 (3730). Synthesis of **4a** {#sec4.5} ------------------- A 500 mg portion of cyclic acylgermane **3a** (0.67 mmol) and 372 mg of 18-cr-6 (1.41 mmol) were dissolved in 20 mL of Et~2~O. The solution was then cooled to −70 °C, and 158 mg (1.41 mmol) of KO*t*Bu was added. During the addition, the reaction mixture turned from yellow to dark orange and an orange precipitate began to form. The reaction mixture was warmed to room temperature and stirred overnight for 15 h. The orange precipitate was isolated and washed with Et~2~O (3 × 5 mL). The orange powder was dried under vacuum (not longer than 5 min; otherwise a slow degradation process occurs) to give **4a**. Yield: 688 mg (85%) of analytically pure **4a** as an orange powder. Data for **4a** are as follows. Mp: 90--92 °C. Anal. Calcd for C~45~H~92~Ge~2~K~2~O~13~Si~5~: C, 44.85; H, 7.70. Found: C, 44.96; H, 7.96. ^29^Si NMR (THF-*d*~8~, TMS, ppm): −0.93 (*Si*Me~3~); −27.69, −29.35 (*Si*Me~2~). ^13^C NMR (THF-*d*~8~, TMS, ppm): 281.01 (*C*=O); 154.14, 133.42, 131.23, 129.20, 128.57 (Aryl-*C*); 71.19 (−*C*H~2~*C*H~2~O−)~6~; 21.35, 21.16 (Aryl-*C*H~3~); 8.86 (Si(CH~3~)~3~); 4.54, 1.93 (Si(CH~3~)~2~). ^1^H NMR (THF-*d*~8~, TMS, ppm): 6.54 (s, 2H, Mes-*H*); 3.62 (s, 48H, (−C*H*~2~C*H*~2~O−)~6~); 2.30 (s, 6H, Mes--C*H*~3~); 2.14 (s, 3H, Mes--C*H*~3~); 0.16 (s, 9H, Si(C*H*~3~)~3~); 0.08 (s, 24H, Si(C*H*~3~)~2~); UV--vis: λ \[nm\] (ε \[L mol^--1^ cm^--1^\]) = 447 (5753). Synthesis of **4b** {#sec4.7} ------------------- A 500 mg portion of cyclic acylgermane **3b** (0.66 mmol) and 365 mg of 18-cr-6 (1.38 mmol) were dissolved in 20 mL of Et~2~O. The solution was then cooled to −70 °C and 155 mg (1.38 mmol) of KO*t*Bu was added. During the addition, the reaction mixture turned from colorless to dark orange and a yellow precipitate began to form. The reaction mixture was warmed to room temperature and stirred overnight for 15 h. The yellow precipitate was isolated and washed with Et~2~O (3 × 5 mL). The yellow powder was dried under vacuum (not longer than 5 min; otherwise a slow degradation process occurs) to give **4b**. Yield: 688 mg (88%) of analytically pure **4b** as yellow powder. Data for **4b** are as follows. Mp: 190--192 °C. Anal. Calcd for C~46~H~96~Ge~2~K~2~O~13~Si~5~: C, 45.25; H, 7.92. Found: C, 45.30; H, 7.77. ^29^Si NMR (C~6~D~6~, TMS, ppm): 0.36 (*Si*Me~3~); −23.45, −25.40 (*Si*Me~2~). ^13^C NMR (C~6~D~6~, TMS, ppm): 280.92 (*C*=O); 70.16 ((−*C*H~2~*C*H~2~O−)~6~); 52.11 (Ad-*C*-CO); 39.78, 38.43 (Ad-*C*H~2~); 30.06 (Ad-*C*H); 9.74 (Si(CH~3~)~3~); 5.73, 5.64, 4.23, 2.35 (Si(CH~3~)~2~). ^1^H NMR (C~6~D~6~, TMS, ppm): 3.35 (s, 48H, (−C*H*~2~C*H*~2~O−)~6~); 2.30, 2.08, 1.95 (m, 15H Ad-*H*); 1.01, 0.90, (s, each 12H, Si(C*H*~3~)~2~); 0.83 (s, 9H, Si(C*H*~3~)~3~). UV--vis: λ \[nm\] (ε \[L mol^--1^ cm^--1^\]) 420 (2178). Synthesis of **5** {#sec4.9} ------------------ A 500 mg portion (0.76 mmol) of **1a** was dissolved in 20 mL DME and cooled to −30 °C, and 180 mg (1.60 mmol) of KO*t*Bu was added. After it was stirred for an additional 30 min, the mixture was warmed to room temperature and finally stirred for an additional 14 h. At this time, reaction control by ^29^Si NMR showed that the dianionic species **2a** was completely formed. Subsequently, the reaction mixture was cooled to −30 °C and 309 mg (1.60 mmol) of ClSi*i*Pr~3~ was added dropwise. The red solution immediately turned yellow. After removal of the volatile components under vacuum, the remaining yellow solid was dissolved in heptane, the solution was filtered through dry Celite, and the solvent was stripped off again. Recrystallization from Et~2~O afforded 405 mg (64%) of the analytically pure silene **5** as yellow crystals. Data for **5** are as follows. Mp: 155--157 °C. Anal. Calcd for C~39~H~86~OSi~9~: C, 56.86; H, 10.52. Found: C, 56.90; H, 10.25. ^29^Si NMR (C~6~D~6~, TMS, ppm): 32.83 (*Si*=C); 13.15 (Si*i*Pr~3~); −9.07 (*Si*Me~3~); −33.90, −34.19, −35.68, −35.84 (*Si*Me~2~); −128.70 (*Si*(SiMe~3~)(Si*i*Pr~3~)). ^13^C NMR (C~6~D~6~, TMS, ppm): 199.38 (Si=*C*); 143.16, 137.21, 137.09, 136.92, 128.85 (Aryl-*C*); 21.50, 21.21 (Aryl-*C*H~3~); 20.83, 18.54 (CH(*C*H~3~)~2~); 15.36, 14.18 (*C*H(CH~3~)~2~); 5.35 (Si(*C*H~3~)~3~); 0.95, 0.50, 0.36, −0.77, −0.92, −2.03, −2.36 (Si(*C*H~3~)~2~). ^1^H NMR (C~6~D~6~, TMS, ppm): 6.80 (s, 2 H, Mes-*H*); 2.61 (s, 6H, Mes-*CH*~3~); 2.12 (s, 3H, Mes-*CH*~3~); 1.25--1.07 (m, 42 H, C*H*(C*H*~3~)~2~); 0.78, 0.74 (s, 3H each, Si(C*H*~3~)~2~); 0.52 (s, 6H each, Si(C*H*~3~)~2~); 0.43--0.42 (s, 15H, Si(C*H*~3~)~2~ and Si(C*H*~3~)~3~); 0.07, 0.02 (s, 3H each, Si(C*H*~3~)~2~). IR (neat): ν(Si=C) 1159 (s) cm^--1^. HRMS: calcd for \[C~39~H~86~OSi~9~\]^•+^ (M^+^), 822.4602; found, 822.4610. Synthesis of **6** {#sec4.11} ------------------ A 500 mg portion (0.74 mmol) of **1b** was dissolved in 20 mL of DME and cooled to −30 °C, and 175 mg (1.60 mmol) of KO*t*Bu was added. After it was stirred for an additional 30 min, the mixture was warmed to room temperature and finally stirred for an additional 14 h. At this time, reaction control by ^29^Si NMR showed that the dianionic species **2b** was completely formed. Subsequently, the reaction mixture was cooled to −30 °C and 146 mg (0.74 mmol) of ClSiMe~2~SiMe~2~Cl was added dropwise. The red solution immediately turned colorless. After aqueous workup with 10 mL of 3% sulfuric acid, the organic layer was separated and dried over Na~2~SO~4~ and the solvent was stripped off with a rotary evaporator. Drying under vacuum and crystallization from acetone solution at −30 °C afforded 454 mg (95%) of the analytically pure acylbicyclo\[2.2.2\]octasilane **6** as colorless crystals. Data for **6** are as follows. Mp: 242--244 °C Anal. Calcd for C~26~H~60~OSi~9~: C, 48.68; H, 9.43. Found: C, 48.75; H, 9.55. ^29^Si NMR (C~6~D~6~, TMS, ppm): −6.11 (*Si*Me~3~); −37.79--38.37 (*Si*Me~2~); −77.76 (*Si*C=O); −131.12 (*Si*(q)). ^13^C NMR (C~6~D~6~, TMS, ppm): 246.07 (*C*=O); 51.15 (Ad-*C*-CO); 37.43, 37.10 (Ad-*C*H~2~); 28.57 (Ad-*C*H); 3.75 (Si(*C*H~3~)~3~); −0.79, −1.31 (Si(*C*H~3~)~2~). ^1^H NMR (CDCl~3~, TMS, ppm): 1.95, 1.80, 1.64 (15H, Ad-H); 0.50, 0.39 (18H each, s, Si(C*H*~3~)~2~); 0.31 (9H, s, Si(C*H*~3~)~3~). IR (neat): ν(C=O) 1618 (m) cm^--1^. HRMS: calcd for \[C~26~H~60~OSi~9~\]^•+^ (M^+^), 640.2568; found, 640.2590. Synthesis of **7** {#sec4.13} ------------------ A 500 mg portion (0.67 mmol) of **3a** was dissolved in 20 mL of DME and cooled to −30 °C, and 158 mg (1.41 mmol) of KO*t*Bu was added. After it was stirred for an additional 30 min, the mixture was warmed to room temperature and finally stirred for an additional 14 h. At this time, reaction control by ^29^Si NMR showed that the dianionic species **4a** was completely formed. Subsequently, the reaction mixture was cooled to −30 °C and 272 mg (1.41 mmol) of ClSi*i*Pr~3~ was added dropwise. The red solution immediately turned yellow. After removal of the volatile components under vacuum, the remaining yellow solid was dissolved in heptane, the solution was filtered through dry Celite, and the solvent was stripped off again. Recrystallization from Et~2~O afforded 558 mg (91%) of the analytically pure germene **7** as yellow crystals. Data for **7** are as follows. Mp: 108--110 °C. Anal. Calcd for C~39~H~86~Ge~2~OSi~7~: C, 51.31; H, 9.50. Found: C, 51.45; H, 9.70. ^29^Si NMR (C~6~D~6~, TMS, ppm): −4.38 (*Si*Me~3~); −23.98, −24.83, −29.76, −30.03 (*Si*Me~2~). ^13^C NMR (C~6~D~6~, TMS, ppm): 210.17 (Ge=*C*); 145.26, 137.00, 135.80, 135.67, 128.82 (Aryl-*C*); 21.57, 21.53 (Aryl-*C*H~3~); 20.75, 18.51 (CH(*C*H~3~)~2~); 15.63, 14.12 (*C*H(CH~3~)~2~); 5.74 (Si(*C*H~3~)~3~); 1.41, 1.14, 1.05, 1.00, 0.11, −1.34, −1.36, −1.51 (Si(*C*H~3~)~2~). ^1^H NMR (C~6~D~6~, TMS, ppm): 6.79 (s, 2 H, Mes-*H*); 2.62 (s, 6H, Mes-*CH*~3~); 2.13 (s, 3H, Mes-*CH*~3~); 1.38--1.07 (m, 42H, C*H*(C*H*~3~)~2~); 0.84, 0.82 (s, 3H each, Si(C*H*~3~)~2~); 0.56 (s, 6H each, Si(C*H*~3~)~2~); 0.46--0.45 (s, 15H, Si(C*H*~3~)~2~ and Si(C*H*~3~)~3~); 0.14, 0.12 (s, 3H each, Si(C*H*~3~)~2~). IR (neat): ν(Si=C) 1245 (s) cm^--1^. HRMS: calcd for \[C~39~H~86~Ge~2~OSi~7~\]^•+^ (M^+^), 914.3503; found, 914.3509. Synthesis of **8** {#sec4.15} ------------------ A 500 mg portion (0.66 mmol) of **3b** was dissolved in 20 mL of DME and cooled to −30 °C, and 155 mg (1.38 mmol) of KO*t*Bu was added. After it was stirred for an additional 30 min, the mixture was warmed to room temperature and finally stirred for an additional 14 h. At this time, reaction control by ^29^Si NMR showed that the dianionic species **4b** was completely formed. Subsequently, the reaction mixture was cooled to −30 °C and 123 mg (0.66 mmol) of ClSiMe~2~SiMe~2~Cl was added dropwise. The red solution immediately turned colorless. After aqueous workup with 10 mL of 3% sulfuric acid, the organic layer was separated and dried over Na~2~SO~4~ and the solvent was stripped off with a rotary evaporator. Drying under vacuum and subsequent NMR measurement showed complete degradation to an uncharacterizable polymer. Synthesis of **1c** {#sec4.16} ------------------- A 500 mg portion (0.74 mmol) of **1b** was dissolved in 20 mL of DME and cooled to −30 °C, and 175 mg (1.60 mmol) of KO*t*Bu was added. After it was stirred for an additional 30 min, the mixture was warmed to room temperature and finally stirred for an additional 14 h. At this time, reaction control by ^29^Si NMR showed that the dianionic species **2b** was completely formed. Subsequently, the reaction mixture was cooled to −30 °C and 287 mg (1.49 mmol) of ClSi*i*Pr~3~ was added dropwise. The red solution immediately turned colorless. After aqueous workup with 10 mL of 3% sulfuric acid, the organic layer was separated and dried over Na~2~SO~4~ and the solvent was stripped off with a rotary evaporator. Drying under vacuum and subsequent NMR measurement showed complete degradation to an uncharacterizable polymer. Synthesis of **4c** {#sec4.17} ------------------- A 500 mg portion (0.66 mmol) of **3b** was dissolved in 20 mL of DME and cooled to −30 °C, and 155 mg (1.38 mmol) of KO*t*Bu was added. After it was stirred for an additional 30 min, the mixture was warmed to room temperature and finally stirred for an additional 14 h. At this time, reaction control by ^29^Si NMR showed that the dianionic species **4b** was completely formed. Subsequently, the reaction mixture was cooled to −30 °C and 267 mg (1.38 mmol) of ClSi*i*Pr~3~ was added dropwise. The red solution immediately turned colorless. After aqueous workup with 10 mL of 3% sulfuric acid, the organic layer was separated and dried over Na~2~SO~4~ and the solvent was stripped off with a rotary evaporator. Drying under vacuum and subsequent NMR measurement showed complete degradation to an uncharacterizable polymer. Synthesis of **9a** {#sec4.18} ------------------- A 500 mg portion (0.76 mmol) of **1a** was dissolved in 20 mL of DME and cooled to −30 °C, and 180 mg (1.60 mmol) of KO*t*Bu was added. After it was stirred for an additional 30 min, the mixture was warmed to room temperature and finally stirred for an additional 14 h. At this time, reaction control by ^29^Si NMR showed that the dianionic species **2a** was completely formed. Subsequently, the reaction mixture was cooled to −30 °C and an excess of MeI was added dropwise. The red solution immediately turned yellow. After aqueous workup with 10 mL of 3% sulfuric acid, the organic layer was separated and dried over Na~2~SO~4~ and the solvent was stripped off with a rotary evaporator. Drying under vacuum afforded 366 mg (89%) of the analytically pure cyclic acylsilane **9a** as a *cis*/*trans* mixture. The obtained product was recrystallized from acetone, giving yellow crystals of one isomer. Yield: 144 mg (35%) of analytically pure **9a** (isomer 1). Data for **9a** (isomer 1) are as follows. Mp: 124--126 °C. Anal. Calcd for C~23~H~50~OSi~7~: C, 51.23; H, 9.35. Found: C, 51.25; H, 9.45. ^29^Si NMR (CDCl~3~, TMS, ppm): −11.81 (*Si*Me~3~); −40.48 to −40.64 (*Si*Me~2~); −46.77 (*Si*C=O); −84.85 (Si(Me)). ^13^C NMR (CDCl~3~, TMS, ppm): 251.92 (*C*=O); 144.75, 137.94, 131.65, 128.82 (Aryl-*C*); 21.21, 19.48 (Aryl-*C*H~3~); 1.22 (Si(*C*H~3~)~3~); −3.00, −4.07, −4.87, −5.67 (Si(*C*H~3~)~2~); −11.24 (Si*C*H~3~). ^1^H NMR (CDCl~3~, TMS, ppm): 6.78 (s, 2 H, Mes-*H*); 2.26 (s, 3H, Mes-*CH*~3~); 2.14 (s, 6H, Mes-*CH*~3~); 0.29, 0.24, 0.18 0.17 (s, 6H each, Si(C*H*~3~)~2~); 0.19 (s, 9H each, Si(C*H*~3~)~3~); 0.30, 0.15 (s, 3H each, Si(C*H*~3~)). HRMS: calcd for \[C~23~H~50~OSi~7~\]^•+^ (M^+^), 538.2247; found, 538.2249. Data for **9a** (isomer 2) are as follows. ^29^Si NMR (C~6~D~6~, TMS, ppm): −12.48 (*Si*Me~3~); −41.59 to −42.55 (*Si*Me~2~); −46.69 (*Si*C=O); −86.18 (*Si*(Me)). ^13^C NMR (CDCl~3~, TMS, ppm): 251.86 (*C*=O). Synthesis of **9b** {#sec4.21} ------------------- A 500 mg portion (0.74 mmol) of **1b** was dissolved in 20 mL of DME and cooled to −30 °C, and 175 mg (1.60 mmol) of KO*t*Bu was added. After it was stirred for an additional 30 min, the mixture was warmed to room temperature and finally stirred for an additional 14 h. At this time, reaction control by ^29^Si NMR showed that the dianionic species **2b** was completely formed. Subsequently, the reaction mixture was cooled to −30 °C and an excess of MeI was added dropwise. The red solution immediately turned colorless. After aqueous workup with 10 mL of 3% sulfuric acid, the organic layer was separated and dried over Na~2~SO~4~ and the solvent was stripped off with a rotary evaporator. Drying under vacuum afforded 335 mg (81%) of the analytically pure cyclic acylsilane **9b** as a *cis*/*trans* mixture. Finally, this mixture of isomers was chromatographed on a precoated TLC SIL G-200 UV~254~ plate, with toluene/heptane (1/5) as eluent, to separate both isomers. Yield: 145 mg (35%) of analytically pure **9b** (isomer 1). Yield: 103 mg (25%) of analytically pure **9b** (isomer 2). Data for **9b** (isomer 1) are as follows. Mp: 180--183 °C. Anal. Calcd for C~24~H~54~OSi~7~: C, 51.91; H, 9.80. Found: C, 51.82; H, 9.85. ^29^Si NMR (CDCl~3~, TMS, ppm): −9.70 (*Si*Me~3~); −38.87 to −39.75 (*Si*Me~2~); −43.96 (*Si*C=O); −83.27 (*Si*(Me)). ^13^C NMR (CDCl~3~, TMS, ppm): 249.15 (*C*=O); 51.93 (Ad-*C*-CO); 36.95, 36.80 (Ad-*C*H~2~); 28.17 (Ad-*C*H); 1.08 (Si(*C*H~3~)~3~); −3.40, −4.42, −4.39, −4.57 (Si(*C*H~3~)~2~); −4.02, −11.21 (Si*C*H~3~). ^1^H NMR (CDCl~3~, TMS, ppm): 2.04, 1.71, 1.70 (15H, Ad-H); 0.29 (6H each, s, Si(C*H*~3~)~2~); 0.23, 0.19, 0.17, 0.15, 0.13 (24H, s, Si(C*H*~3~)~2~ and Si(C*H*~3~)~3~); 0.55, 0.11 (s, 3H each, Si(C*H*~3~)). HRMS: calcd for \[C~24~H~54~OSi~7~\]^•+^ (M^+^), 554,.2560; found, 554.2566. Data for **9b** (isomer 2) are as follows. Anal. Calcd for C~24~H~54~OSi~7~: C, 51.91; H, 9.80. Found: C, 51.96; H, 9.89. ^29^Si NMR (CDCl~3~, TMS, ppm): −9.70 (*Si*Me~3~); −38.85 to −39.72 (*Si*Me~2~); −43.93 (*Si*C=O); −83.21 (*Si*(Me)). ^13^C NMR (CDCl~3~, TMS, ppm): 249.12 (*C*=O); 51.94 (Ad-*C*-CO); 36.97, 36.82 (Ad-*C*H~2~); 28.19 (Ad-*C*H); 1.08 (Si(*C*H~3~)~3~); −3.40, −4.22, −4.39, −4.57 (Si(*C*H~3~)~2~); −4.00, −11.21 (Si*C*H~3~). ^1^H NMR (CDCl~3~, TMS, ppm): 2.05, 1.70 (15H, Ad-H); 0.29 (6H each, s, Si(C*H*~3~)~2~); 0.19, 0.17, 0.15, 0.14 (27H, s, Si(C*H*~3~)~2~ and Si(C*H*~3~)~3~); 0.55, 0.11 (s, 3H each, Si(C*H*~3~)). HRMS: calcd for \[C~24~H~54~OSi~7~\]^•+^ (M^+^), 554.2560; found, 554.2568. Synthesis of **10a** {#sec4.24} -------------------- A 500 mg portion (0.67 mmol) of **3a** was dissolved in 20 mL of DME and cooled to −30 °C, and 158 mg (1.41 mmol) of KO*t*Bu was added. After it was stirred for an additional 30 min, the mixture was warmed to room temperature and finally stirred for an additional 14 h. At this time, reaction control by ^29^Si NMR showed that the dianionic species **4a** was completely formed. Subsequently, the reaction mixture was cooled to −30 °C and an excess of MeI was added dropwise. The red solution immediately turned yellow. After aqueous workup with 10 mL of 3% sulfuric acid, the organic layer was separated and dried over Na~2~SO~4~ and the solvent was stripped off with a rotary evaporator. Drying under vacuum afforded 392 mg (93%) of the analytically pure cyclic acylgermane **10a** as a *cis*/*trans* mixture. Data for **10a** (*cis*/*trans* mixture) are as follows. Mp: 134--138 °C. Anal. Calcd for C~23~H~50~Ge~2~OSi~5~: C, 43.97; H, 8.02. Found: C, 43.85; H, 8.05. ^29^Si NMR (CDCl~3~, TMS, ppm): −2.84/--3.78 (*Si*Me~3~); −29.19 to −31.29/--30.96, −32.06 (*Si*Me~2~). ^13^C NMR (CDCl~3~, TMS, ppm): 250.52 (*C*=O); 145.12/144.47, 137.75, 131.03/130.84, 128.71 (Aryl-*C*); 21.18, 19.30 (Aryl-*C*H~3~); 1.95/1.74 (Si(*C*H~3~)~3~); −2.19/--2.60, −3.47/--3.42, −3.95/--3.92, −4.86/--4.13, (Si(*C*H~3~)~2~); −5.25/--5.99, −11.78/--12.17 (Ge*C*H~3~). ^1^H NMR (CDCl~3~, TMS, ppm): 6.78 (s, 2 H, Mes-*H*); 2.27 (s, 3H, Mes-*CH*~3~); 2.15 (s, 6H, Mes-*CH*~3~); 0.41, 0.39, 0.35, 0.32, 0.29, 0.27, 0.26, 0.25, 0.23 (s, 39H each, Si(C*H*~3~)~2~, Si(C*H*~3~)~3~ and Ge(C*H*~3~)). HRMS: calcd for \[C~23~H~50~Ge~2~OSi~5~\]^•+^ (M^+^), 630.1132; found, 628.1140. Synthesis of **10b** {#sec4.26} -------------------- A 500 mg portion (0.66 mmol) of **3b** was dissolved in 20 mL of DME and cooled to −30 °C, and 155 mg (1.38 mmol) of KO*t*Bu was added. After it was stirred for an additional 30 min, the mixture was warmed to room temperature and finally stirred for an additional 14 h. At this time, reaction control by ^29^Si NMR showed that the dianionic species **4b** was completely formed. Subsequently, the reaction mixture was cooled to −30 °C and an excess of MeI was added dropwise. The red solution immediately turned colorless. After aqueous workup with 10 mL of 3% sulfuric acid, the organic layer was separated and dried over Na~2~SO~4~ and the solvent was stripped off with a rotary evaporator. Drying under vacuum afforded 360 mg (85%) of the analytically pure cyclic acylgermane **10b** as a *cis*/*trans* mixture. Finally, this mixture of isomers was chromatographed on a precoated TLC SIL G-200 UV~254~ plate, with toluene/heptane (1/5) as eluent, to separate both isomers. Yield: 80 mg (29%) of analytically pure **10b** (isomer 1). Yield: 60 mg (14%) of analytically pure **10b** (isomer 2). Data for **10b** (isomer 1) are as follows. Mp: 173--178 °C. Anal. Calcd for C~24~H~54~Ge~2~OSi~5~: C, 44.74; H, 8.45. Found: C, 44.90; H, 8.52. ^29^Si NMR (CDCl~3~, TMS, ppm): −3.63 (*Si*Me~3~); −31.45 to −31.79 (*Si*Me~2~). ^13^C NMR (CDCl~3~, TMS, ppm): 247.31 (*C*=O); 52.14 (Ad-*C*-CO); 37.00, 36.95 (Ad-*C*H~2~); 28.23 (Ad-*C*H); 1.78 (Si(*C*H~3~)~3~); −2.47, −3.30, −3.46, −4.06 (Si(*C*H~3~)~2~); −3.99, −12.05 (Ge*C*H~3~). ^1^H NMR (CDCl~3~, TMS, ppm): 2.05, 1.69 (m, 15H, Ad-*H*); 0.60 (3H, s, Ge(C*H*~3~)); 0.33 (6H, s, Si(C*H*~3~)~2~); 0.23, 0.21 (m, 30H each, Si(C*H*~3~)~2~, Si(C*H*~3~)~3~ and Ge(C*H*~3~)). HRMS: calcd for \[C~24~H~54~Ge~2~OSi~5~\]^•+^ (M^+^), 646.1445; found, 644.1452. Data for **10b** (isomer 2) are as follows. Anal. Calcd for C~24~H~54~Ge~2~OSi~5~: C, 44.74; H, 8.45. Found: C, 44.97; H, 8.59. ^29^Si NMR (CDCl~3~, TMS, ppm): −2.86 (*Si*Me~3~); −29.53, −30.96 (*Si*Me~2~). ^13^C NMR (CDCl~3~, TMS, ppm): 247.73 (*C*=O); 52.57 (Ad-*C*-CO); 37.42, 37.37 (Ad-*C*H~2~); 28.65 (Ad-*C*H); 2.21 (Si(*C*H~3~)~3~); −2.04, −2.87, −3.03, −3.63 (Si(*C*H~3~)~2~); −3.57, −11.63 (Ge*C*H~3~). ^1^H NMR (CDCl~3~, TMS, ppm): 2.06, 1.70 (15H, Ad-H), 0.49 (3H, s, Ge(C*H*~3~)); 0.32, 0.28, 0.26, 0.23, 0.21 (36H, m, Si(C*H*~3~)~2~, Si(C*H*~3~)~3~ and Ge(C*H*~3~)). HRMS: calcd for \[C~24~H~54~Ge~2~OSi~5~\]^•+^ (M^+^), 646.1445; found, 644.1442. Competitive Reactivity of **1a** {#sec4.29} -------------------------------- A 500 mg portion (0.76 mmol) of **1a** was dissolved in 20 mL of DME and cooled to −30 °C, and 180 mg (1.60 mmol) of KO*t*Bu was added. After it was stirred for an additional 30 min, the mixture was warmed to room temperature and finally stirred for an additional 14 h. At this time, reaction control by ^29^Si NMR showed that the dianionic species **2a** was completely formed. Subsequently, the reaction mixture was cooled to −30 °C and 83 mg (0.76 mmol) of ClSiMe~3~ was added dropwise. The reaction mixture was warmed to room temperature and stirred for an additional 30 min. At this time, reaction control by NMR showed that the cyclic silenolate **S**~**1**~**a** was completely formed. Data for **S~1~a** are as follows. ^29^Si NMR (THF-*d*~8~, TMS, ppm): −9.00 (*Si*Me~3~); −29.61 to −36.72 (*Si*Me~2~); −78.00 (*Si*C=O); −131.17 (*Si*(q)). ^13^C NMR (THF-*d*~8~, TMS, ppm): 263.96 (*C*=O); 151.90, 132.71, 131.65, 127.38 (Aryl-*C*); 20.13, 20.10 (Aryl-*C*H~3~); 3.42 (Si(*C*H~3~)~3~); −0.26, −1.17 (Si(*C*H~3~)~2~). ^1^H NMR (THF-*d*~8~, TMS, ppm): 6.62 (s, 2 H, Mes-*H*); 2.35 (s, 6H, Mes-*CH*~3~); 2.16 (s, 3H, Mes-*CH*~3~); 0.22 (18H, s, Si(C*H*~3~)~3~), 0.11, 0.10, 0.07, 0.05, 0.04 (24H, m, Si(C*H*~3~)~2~). Competitive Reactivity of **1b** {#sec4.31} -------------------------------- A 500 mg portion (0.74 mmol) of **1b** was dissolved in 20 mL of DME and cooled to −30 °C, and 175 mg (1.60 mmol) of KO*t*Bu was added. After it was stirred for an additional 30 min, the mixture was warmed to room temperature and finally stirred for an additional 14 h. At this time, reaction control by ^29^Si NMR showed that the dianionic species **2b** was completely formed. Subsequently, the reaction mixture was cooled to −30 °C and 81 mg (0.74 mmol) of ClSiMe~3~ was added dropwise. The reaction mixture was warmed to room temperature and stirred for an additional 30 min. At this time, reaction control by NMR showed that **9** was completely formed. After aqueous workup with 100 mL of 3% sulfuric acid, the organic layer was separated and dried over Na~2~SO~4~ and the solvent was stripped off on a rotary evaporator. Finally, the crude material was chromatographed on a precoated TLC SIL G-200 UV~254~ plate, with with heptane as eluent, to give 268 mg (60%) of **11**. Data for **11** are as follows. Mp: 218--222 °C. Anal. Calcd for C~25~H~58~OSi~8~: C, 50.09; H, 9.75. Found: C, 50.17; H, 9.68. ^29^Si NMR (C~6~D~6~, TMS, ppm): 8.57 (O*Si*Me~2~); 6.81 (*Si*q-OSiMe~2~); −7.36, -- 16.85 (*Si*Me~3~); −39.43, −43.33, −48.13 (*Si*Me~2~); −79.34 (*Si*q). ^13^C NMR (C~6~D~6~, TMS, ppm): 47.01, 37.53, 36.50, 29.49 (Ad-*C*); 35.34 (*C*H-Ad); 2.91, 0.32 (Si(*C*H~3~)~3~); 3.15, 2.55, −1.25, −2.27, −2.34, −2.70, −5.52, −6.55 (Si(*C*H~3~)~2~). ^1^H NMR (CDCl~3~, TMS, ppm): 1.97--1.58 (m, 16H, Ad-*H* and C*H*-Ad); 0.49, 0.42, 0.39, 0.36, 0.28, 0.26, 0.25, 0.17 (s, 3H each, Si(C*H*~3~)~2~); 0.34, 0.32 (s, 9H each, Si(C*H*~3~)~3~). HRMS: calcd for \[C~25~H~58~OSi~8~\]^•+^ (M^+^), 598.2642; found, 598.2646. The Supporting Information is available free of charge at [https://pubs.acs.org/doi/10.1021/acs.organomet.0c00385](https://pubs.acs.org/doi/10.1021/acs.organomet.0c00385?goto=supporting-info).NMR spectra, crystallographic details, and computational section ([PDF](http://pubs.acs.org/doi/suppl/10.1021/acs.organomet.0c00385/suppl_file/om0c00385_si_001.pdf))Cartesian coordinates of the calculated structures ([XYZ](http://pubs.acs.org/doi/suppl/10.1021/acs.organomet.0c00385/suppl_file/om0c00385_si_002.xyz)) Supplementary Material ====================== ###### om0c00385_si_001.pdf ###### om0c00385_si_002.xyz CCDC 1964268--1964270 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge *via*[www.ccdc.cam.ac.uk/data_request/cif](http://www.ccdc.cam.ac.uk/data_request/cif), or by emailing <[email protected]>, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033. The authors declare no competing financial interest. We thank the FWF (Wien, Austria) for financial support (project number P32606-N).

[^1]: Li Ji, Chunyan Cao, Yuman Li. These authors contributed equally to this manuscript.

1. Introduction {#sec1-microorganisms-08-00299} =============== *Candida* species are associated with a range of clinical manifestations, including mucosal and invasive bloodstream infections \[[@B1-microorganisms-08-00299]\]. Among the *Candida* species, *Candida albicans* is a leading cause of bloodstream infections, although the incidence of infections caused by non-*albicans Candida* species is increasing \[[@B1-microorganisms-08-00299]\]. Moreover, because there are limited drug classes available for the treatment of *Candida* infections, and due to the overuse of antifungals, the emergence of drug resistance is becoming a significant concern in clinical settings \[[@B2-microorganisms-08-00299],[@B3-microorganisms-08-00299]\]. Antimicrobial peptides (AMPs) have been identified in virtually all organisms and have diverse structures and functions, such as antimicrobial and immunomodulatory activities \[[@B4-microorganisms-08-00299],[@B5-microorganisms-08-00299],[@B6-microorganisms-08-00299],[@B7-microorganisms-08-00299],[@B8-microorganisms-08-00299]\]. Because AMPs exhibit broad-spectrum activity against microorganisms and insusceptibility to conventional drug resistance mechanisms, AMPs are promising candidates for the development of new antifungal drugs \[[@B9-microorganisms-08-00299],[@B10-microorganisms-08-00299],[@B11-microorganisms-08-00299],[@B12-microorganisms-08-00299],[@B13-microorganisms-08-00299]\]. Human histatin 5 (Hst 5) is a naturally occurring protein found in human saliva that exhibits potent antifungal activity. P-113, a peptide containing 12 Hst 5 amino acid residues, retains full candidacidal activity and has had no adverse effects in clinical trials \[[@B14-microorganisms-08-00299],[@B15-microorganisms-08-00299]\]. However, the efficacy of P-113 is significantly reduced in the presence of high salt concentrations and at pH 4.5 \[[@B16-microorganisms-08-00299],[@B17-microorganisms-08-00299],[@B18-microorganisms-08-00299]\]. In our previous study, novel P-113 derivatives, such as P-113Tri (a tandem arrangement of three P-113 repeats) were synthesized and characterized \[[@B19-microorganisms-08-00299]\]. P-113Tri contained significant fractions of an α--helical conformation and was more resistant to high salt and low pH than P-113 \[[@B19-microorganisms-08-00299]\] and [Figure S1](#app1-microorganisms-08-00299){ref-type="app"}. Moreover, compared to P-113, P-113Tri exhibited increased antifungal activity against planktonic cells, biofilm cells, and clinical isolates of *C. albicans* and non-*albicans Candida* species \[[@B19-microorganisms-08-00299]\]. However, the detailed mechanism by which P-113Tri functions differently from P-113 in its anti-*C. albicans* activity is still unknown. In this work, we aim to study the difference between P-113Tri and P-113. We showed that P-113 rapidly gains access to the cells where it accumulates. However, although small amounts of P-113Tri slowly gained access to the cells, most of the P-113Tri remained associated with the *C. albicans* cell surface. Particularly, P-113Tri interacted with the glycan components of the cell wall. In addition, the interaction between P-113Tri and the cell wall carbohydrates was somehow correlated with the candidacidal activity of P-113Tri. These results enhance our understanding of how an AMP attacks *C. albicans* through its interaction with the glycans present in fungal pathogens. Moreover, our findings suggest the potential use of P-113Tri as a new therapeutic agent that can target the cell wall carbohydrates of fungal pathogens. 2. Materials and Methods {#sec2-microorganisms-08-00299} ======================== 2.1. Antifungal Peptides and Reagents {#sec2dot1-microorganisms-08-00299} ------------------------------------- P-113, P-113Tri, fluorescein isothiocyanate (FITC)-P-113, and FITC-P-113Tri were synthesized by Mission Biotech System (Taipei, Taiwan). FITC is conjugated to the N-terminus of the peptides. The purities of these peptides were analyzed by reversed-phase high-performance liquid chromatography and mass spectrometry to be \>95% pure. All reagents were obtained from Sigma-Aldrich unless indicated otherwise. 2.2. C. albicans Strains and Growth Media {#sec2dot2-microorganisms-08-00299} ----------------------------------------- All *C. albicans* strains used in this study are listed in [Table S1](#app1-microorganisms-08-00299){ref-type="app"}. Cells were routinely grown in YPD medium (2% glucose, 1% yeast extract, and 2% peptone). Plates were prepared with 1.5% agar. For the minimum inhibitory concentration (MIC) assay, LYM broth (5.4 mM KCl, 5.6 mM Na~2~HPO~4~, 0.5 mM magnesium sulfate, 1.0 mM sodium citrate, 0.4 mg of ZnCl~2~, 2.0 mg of FeCl~3~·6H~2~O, 0.1 mg of CuSO~4~·5H~2~O, 0.1 mg of MnSO~4~·H~2~O, and 0.1 mg of Na~2~B~4~O~7~·10H~2~O, 2% glucose, amino acid mixture and a vitamin mixture, all per liter of medium) was used \[[@B14-microorganisms-08-00299]\]. The amino acid mixture and vitamin mixture were purchased from Thermo Fisher Scientific (Waltham, MA, USA). 2.3. C. albicans Killing Assay {#sec2dot3-microorganisms-08-00299} ------------------------------ The killing assays were performed as previously described \[[@B19-microorganisms-08-00299]\]. Briefly, *C. albicans* cells were grown overnight in YPD medium at 30 °C with shaking, subcultured into fresh YPD and further grown to the exponential phase (\~5 h). Then, the cells were treated with or without AMPs for 1 h. The number of viable cells after peptide treatment were normalized to those of control cells (no peptide treatment) and are reported as percentages. 2.4. Measurement of Minimum Inhibitory Concentrations (MICs) {#sec2dot4-microorganisms-08-00299} ------------------------------------------------------------ The MICs for each peptide were determined using the Clinical and Laboratory Standards Institute (CLSI) method M27-A3 \[[@B20-microorganisms-08-00299]\] with some modifications. Cells were inoculated into each well of 96-well plates in LYM broth (4 × 10^4^ cells/mL) with or without different concentrations of peptides. The cells were grown at 37 °C for 48 h and examined by visual estimation and spectrophotometric determination. The MIC~90~ values of the AMPs were defined as the concentrations of the peptides that caused a 90% reduction in cell growth relative to the control cells without peptide treatment. The assays were performed independently at least three times for each experimental group. 2.5. Confocal Scanning Laser Microscopy {#sec2dot5-microorganisms-08-00299} --------------------------------------- To examine the cellular localization of the peptides, *C. albicans* cells were grown to the exponential phase, harvested by centrifugation, and resuspended in 12.5 mM sodium acetate to reach a concentration of 2.4 × 10^8^ cells/mL. Then, cells were added to 25 μM CellTracker Blue 7-amino-4-chloromethylcoumarin (CMAC; Invitrogen) or 8 μg/mL calcofluor white (CFW; Sigma-Aldrich F-3543) and further grown at 30 °C for 30 min in the dark. CellTracker Blue CMAC and CFW were used to stain yeast vacuoles \[[@B21-microorganisms-08-00299]\] and cell wall chitin, respectively. After centrifugation (12,000× *g*, 5 min) to remove the unbound dyes, cell pellets were collected, washed three times with 12.5 mM sodium acetate, and resuspended in 12.5 mM sodium acetate. Finally, FITC-conjugated peptides were added to the cells, as indicated. The colocalization of FITC-conjugated peptides to the vacuoles or cell walls was examined using a Zeiss LSM 800 Airyscan confocal microscope. The images were processed and analyzed using Zen software (Zeiss). 2.6. Binding of P-113 and P-113Tri to C. albicans Cells {#sec2dot6-microorganisms-08-00299} ------------------------------------------------------- The binding of AMPs to *C. albicans* was determined as previously described \[[@B22-microorganisms-08-00299]\]. Briefly, *C. albicans* (6 × 10^7^ cells) were incubated with Zymolyase-20T (2.5 mg/mL) in SCE buffer (1 M Sorbitol, 10 mM sodium citrate buffer \[pH 6.0\] and 1 mM EDTA) at 37 °C for 1 h, followed by washing twice with SCE buffer. To remove the carbohydrates from the cell wall, cells were treated with concanavalin A (100 μg/mL in 12.5 mM sodium acetate) at 30 °C for 1 h and then washed twice with 12.5 mM sodium acetate. For metaperiodate treatment, cells were suspended in 50 mM sodium acetate (pH 4.5) containing 100 mM metaperiodate and incubated at 4 °C for 30 min in the dark and then washed twice with 12.5 mM sodium acetate. For α1-2,3,6 mannosidase treatment, cells were inoculated in GlycoBuffer 4 (New England Biolabs, Madison, WI) containing 5 U of α-mannosidase, incubated at 37 °C for 16 h, and washed twice with 12.5 mM sodium acetate. Then, *C. albicans* cells (2.4 × 10^8^ cells/mL) were treated with FITC-P-113 and FITC-P-113Tri (0.6 μg/mL in 12.5 mM sodium acetate) for 2 min. Subsequently, an Accuri C6 flow cytometer (BD Biosciences) was used to determine the extent of the peptides bound to *C. albicans* cells by calculating the mean fluorescence intensity (MFI) from 10,000 cells per sample. The relative peptide binding to the cells is presented as a percentage by dividing the MFI of the cells treated with peptide by that of the control without peptide treatment. Finally, the cells from the peptide binding assay described above were also treated with 20 μg/mL propidium iodide (PI) to determine the extent of membrane disruption upon peptide treatment, as previously described \[[@B23-microorganisms-08-00299]\]. 2.7. β-Glucan Staining {#sec2dot7-microorganisms-08-00299} ---------------------- To stain for total β-Glucan, cells were incubated with aniline blue fluorochrome (Wako; 500 μg/mL) for 5 min in a black 96-well microplate. Aniline blue fluorescence intensity was measured using a VICTOR3 Multilabel Plate Reader fluorescence spectrophotometer (PerkinElmer) with excitation and emission wavelengths of 405 nm and 460 nm, respectively. 2.8. Competition Assays {#sec2dot8-microorganisms-08-00299} ----------------------- For the competition assays, P-113 and P-113Tri (12 μg/mL) were preincubated with 1, 2, 4, and 8 mg/mL mannan and laminarin at 4 °C for 30 min, followed by mixing with cells and incubating at 37 °C for 1 h. Then, the numbers of viable cells after peptide treatment were normalized to those of the control cells (no peptide treatment) and are reported as percentages. All carbohydrate stock solutions were prepared in 12.5 mM sodium acetate. Mannan from *Saccharomyces cerevisiae* and Laminarin from Laminaria digitata were purchased from Sigma-Aldrich (catalog numbers M7504 and L9634). 2.9. Measurement of the Dissociation Constants for the Peptide/Glycan Complexes {#sec2dot9-microorganisms-08-00299} ------------------------------------------------------------------------------- Dissociation constants (K~D~) for the peptide/glycan complexes were measured using isothermal titration calorimetry (ITC). Thermodynamic analysis of peptide binding to mannan and laminarin was performed using a MicroCal iTC200 calorimeter (MicroCal, Northampton, MA) as previously described \[[@B24-microorganisms-08-00299]\]. All experiments were performed at 25 °C using P-113 (25 μM), P-113Tri (25 μM), mannan (116 μM) and laminarin (130 μM). The P-113 or P-113Tri solution was placed in a calorimeter cell, and the mannan or laminarin solution was loaded into the syringe injector. In an individual titration, the autocontrolled microsyringe injected 2 μL of mannan or laminarin solution into the peptide solution over an interval of 120 s. The integrated heat change was analyzed by means of nonlinear regression using MicroCal Origin software, and the dissociation constant (K~D~) was obtained from a single sigmoidal titration curve. The effect of the dilution of the mannan or laminarin solution in the titration cell was removed by subtracting the calorimetric data for a blank titration, which consisted of the titration of the mannan or laminarin solution into sodium acetate. 2.10. Glycan Microarray Analysis {#sec2dot10-microorganisms-08-00299} -------------------------------- Glycan array screening was carried out using a rapid, nonwashing, solution carbohydrate array as previously described \[[@B25-microorganisms-08-00299]\]. Briefly, donor beads (500 ng/well) and biotin-polyacrylamide (PAA)-sugars (20 ng/well) (GlycoTech, Gaithersburg, MD, USA) mixed with FITC-P-113Tri (40 ng/well) were incubated for 1 h (a total of 15 μL of reaction solution). A mixture of acceptor beads (500 ng/well), mouse anti-FITC antibody (50 ng/well) and rabbit anti-mouse IgG antibody (25 ng/well) (Zymed, San Francisco, CA) was added to the reaction to reach a final volume of 25 μL. All reactions were performed in the dark. After incubation for 2 h, the peptide binding signals were measured from a PerkinElmer EnVision instrument and analyzed using the AlphaScreenTM detection program. The results are expressed as the fluorescence intensities. 2.11. Statistical Analysis {#sec2dot11-microorganisms-08-00299} -------------------------- Data were assessed for statistical significance by the two-tailed Student's *t*-test. 3. Results {#sec3-microorganisms-08-00299} ========== 3.1. P-113Tri Directly Interacts with the Cell Surface of C. albicans {#sec3dot1-microorganisms-08-00299} --------------------------------------------------------------------- In our previous study, P-113Tri showed overall higher anti-candidacidal activity than its parental compound P-113 \[[@B19-microorganisms-08-00299]\]. To further investigate the mechanisms of P-113Tri against *C*. *albicans*, the interaction between the cells and peptides conjugated with fluorescein isothiocyanate (FITC) was determined using confocal microscopy. After cell incubation with peptides for approximately 5 min, P-113 readily gained access into the cells, whereas P-113Tri remained on the cell surface ([Figure 1](#microorganisms-08-00299-f001){ref-type="fig"}A). However, after incubation for approximately 1 h, although small amounts of P-113Tri were present intracellularly, the majority of P-113Tri was still retained on the cell surface ([Figure 1](#microorganisms-08-00299-f001){ref-type="fig"}B). To verify this finding, the cellular localization of FITC-conjugated peptides was determined by cell staining with calcofluor white (CFW) and CellTracker Blue CMAC. CFW and CellTracker Blue CMAC are fluorescent dyes that selectively stain the cell wall and the lumen of yeast vacuoles, respectively. [Figure 1](#microorganisms-08-00299-f001){ref-type="fig"}C,D show that P-113Tri mainly interacted with the cell wall, although small amounts of P-113Tri gained intracellular entry, as exemplified by its colocalization with vacuoles. These results suggest that P-113Tri binds to the outer surface of *Candida* cells. 3.2. P-113Tri Binding to C. albicans Cell Wall Carbohydrates Is Related to the Candidacidal Activity of the Peptide {#sec3dot2-microorganisms-08-00299} ------------------------------------------------------------------------------------------------------------------- The cell wall is the outermost layer of *C. albicans* and plays a key role in interacting with the environment and host cells. Carbohydrates are the major components of the *C. albicans* cell wall, comprising 80% to 90% of the wall, and contain mannans, β-1,6-glucan, β-1,3-glucan, and chitin. Mannan is commonly the glycoprotein carbohydrate portion of the cell wall found with the following three structures: Linear *O*-linked mannan, highly branched *N*-linked mannan, and phosphomannan \[[@B26-microorganisms-08-00299]\]. The glucan and chitin layers of the *C. albicans* cell wall are buried beneath a thin but electron dense mannan layer \[[@B27-microorganisms-08-00299]\]. Because the peptides colocalized with the cell wall ([Figure 1](#microorganisms-08-00299-f001){ref-type="fig"}A−D), it raises the possibility that P-113Tri interacts with components of the cell wall. To test this hypothesis, different parts of the cell wall carbohydrates were removed. Concanavalin A (ConA) specifically binds to the α-D-glucose and α-D-mannose residues of cell wall glycoproteins, and α1-2,3,6 mannosidase is an exoglycosidase that can degrade the mannan network. As shown in [Figure 2](#microorganisms-08-00299-f002){ref-type="fig"}A, the amount of FITC-P-113Tri bound to the ConA-treated *C*. *albicans* cells was significantly reduced compared to that in the controls without ConA treatment or with P-113 treatment. Moreover, the FITC-conjugated P-113 and P-113Tri bound to cells treated with α1-2,3,6 mannosidase were decreased by 40% and 80%, respectively, compared to the untreated controls ([Figure 2](#microorganisms-08-00299-f002){ref-type="fig"}B). These results confirm the interaction between the peptides we tested and mannan in the *Candida* cell wall. To further examine the peptides and cell wall carbohydrate interactions, Zymolyase and sodium metaperiodate were also used. Zymolyase (or laminaripentaohydrolase) can hydrolyze the β-1,3-glucan layer, and sodium metaperiodate oxidizes cleaves the glycan chains of β-1,6-glucan and mannan. The extent of cell wall disruption by Zymolyase was assessed by aniline blue staining \[[@B28-microorganisms-08-00299]\]. After deglycosylation by Zymolyase, the amount of cell-surface-bound aniline blue decreased by \~50%, representing the removal of \~50% of the cell wall glucan ([Figure S2](#app1-microorganisms-08-00299){ref-type="app"}). Interestingly, the binding of FITC-conjugated P-113 and P-113Tri to the metaperiodate-treated cells decreased by 30% and 70%, respectively ([Figure 2](#microorganisms-08-00299-f002){ref-type="fig"}C). Similarly, the fluorescence intensity of FITC-conjugated peptides detected by a confocal microscope indicated that the binding of both FITC-conjugated peptides P-113 and P-113Tri to the Zymolyase-treated cells was significantly reduced compared to their binding to cells without peptide treatment ([Figure 2](#microorganisms-08-00299-f002){ref-type="fig"}D,E). In addition, cells treated with Zymolyase were more resistant to the peptides compared to the untreated cells, as determined by PI staining ([Figure 2](#microorganisms-08-00299-f002){ref-type="fig"}F). The dissociation constant (K~D~) for the carbohydrate/peptide complexes was further determined using isothermal titration calorimetry (ITC). The K~D~ values for the complexes of laminarin/P-113 and laminarin/P-113Tri were 33.2 μM and 11.33 μM, respectively ([Figure 3](#microorganisms-08-00299-f003){ref-type="fig"}A). Laminarin is a polysaccharide of glucose and a representative of β-1,3-glucan. The K~D~ values for the complex of mannan/P-113 and mannan/P-113Tri were 2.51 μM and 1.07 μM, respectively. Moreover, the K~D~ values of the laminarin/peptide complex are much higher than those for the mannan/peptide, suggesting that the peptides preferentially bind to *C. albicans* mannan rather than glucan. Finally, to correlate the specific peptide--carbohydrate binding to the candidacidal activity, a competition assay was performed. Mannan and laminarin were premixed with the peptides at 4 °C for 30 min, followed by incubation of the peptide/carbohydrate complex with *Candida* cells. As shown in [Figure 3](#microorganisms-08-00299-f003){ref-type="fig"}B, although the laminarin and mannan concentrations varied, these polysaccharides rescued the cells from the candidacidal effect of the peptides. Taken together, our results indicate that the peptides interact with mannan and β-1,3-glucan of the cell wall, and this carbohydrate--peptide binding is related to the anti-*Candida* activity of the tested peptides. 3.3. Peptides Bind to N-Linked Mannan But Not O-Linked Mannan and Play a Partial Role in Candidacidal Activity {#sec3dot3-microorganisms-08-00299} -------------------------------------------------------------------------------------------------------------- To identify potential cell wall targets of the peptides, we prescreened 560 strains from *C. albicans* deletion mutant libraries \[[@B29-microorganisms-08-00299],[@B30-microorganisms-08-00299]\]. *C*. *albicans* cells were incubated in 12.5 mM sodium acetate with or without P-113 (16 μM) for 1 h. The *C*. *albicans* wild type strains (SC5314 and SN250) were sensitive to P-113, whereas 15 deletion mutants were resistant to P-113. Among these resistant strains, many of them lack genes encoding cell wall proteins ([Table 1](#microorganisms-08-00299-t001){ref-type="table"}), for example, the *och1*-deletion mutant. The *OCH1* gene encodes an α-1,6-mannosyltransferase that initiates elongation of the *N*-linked mannan outer chain of the *C*. *albicans* cell wall \[[@B26-microorganisms-08-00299]\]. [Figure 4](#microorganisms-08-00299-f004){ref-type="fig"}A shows that the *och1*-deletion mutant was more resistant to P-113 compared to the wild-type SN250 strain. Interestingly, the candidacidal activity of P-113Tri to the *och1*-deletion mutant seems to be even better than P-113 ([Figure 4](#microorganisms-08-00299-f004){ref-type="fig"}A). These results further suggest that the mannosylation of cell wall proteins is related to the activity of the peptides. To further reveal the relationship between mannosylation-defective mutants and FITC-conjugated peptides, binding assays were performed by flow cytometry. The *C*. *albicans* cells were treated with 0.6 μg/mL FITC-P-113 and FITC-P-113Tri for 2 min. The mean fluorescence intensity (MFI) was measured and compared among the cells treated with these two peptides. As shown in [Figure 4](#microorganisms-08-00299-f004){ref-type="fig"}B, peptide binding to the NGY152 strain was defined as 100%. Mnt1 and Mnt2 are α-1,2-mannosyl transferases that add mannose residues in *O*-glycosylation. The binding of peptides to the *mnt1*-, *mnt2*-, and *mnt1mnt2*-deletion mutants was similar to that of the parental NGY152 strain. Among the other *C. albicans* enzymes related to *N*-mannosylation, Mnt4 and Mnt5 participated in *N*-mannan branching \[[@B31-microorganisms-08-00299]\]. The binding of P-113 and P-113Tri to the *mnt4mnt5*-deletion mutants decreased by 56% and 40%, respectively, compared to the parental NGY152 strain. Och1 is responsible for adding the first α-1,6-mannose to the inner core, and inactivation of Och1 blocks the elongation of *N*-mannans \[[@B32-microorganisms-08-00299]\]. Moreover, Mnn4, Mnt3, and Mnt5 mediate the addition of β-1,2-linked and α-1,6-linked mannose residues during *N*-mannosylation \[[@B31-microorganisms-08-00299],[@B33-microorganisms-08-00299],[@B34-microorganisms-08-00299]\]. Mnt, Mnn, and Och1 all require Mn^++^ for their functions and therefore depend on the Golgi Mn^++^ transporter Pmr1. The *pmr1*Δ (NGY355) mutant was defective in *N*-linked and *O*-glycosylation \[[@B35-microorganisms-08-00299]\]. The overall peptide binding to the *mnt3mnt5*-, *pmr1*-, *och1*-, and *mnn4*-deletion mutants were largely decreased compared to the parental NGY152 strain ([Figure 4](#microorganisms-08-00299-f004){ref-type="fig"}B). The binding of the FITC-conjugated peptide to the mannosylation-defective mutants was also examined using confocal microscopy ([Figure 4](#microorganisms-08-00299-f004){ref-type="fig"}C). We found that P-113 and P-113Tri can bind to NGY337 (*mnt1*Δ*mnt2*Δ), NGY516 (*mnt4*Δ*mnt5*Δ), and NGY1227 (*mnt3*Δ*mnt5*Δ). Notably, binding of the peptides to CDH15 (*mnn4*Δ) and NGY357 (*och1*Δ) mutants was almost undetectable. Together, these results suggest that peptides bind to *N*-linked mannan but not *O*-linked mannan. To further reveal the relationship between the activity of the peptides and protein mannosylation, minimum inhibitory concentrations (MICs) of the peptides were determined using *C*. *albicans* mannosylation-defective mutants ([Table 2](#microorganisms-08-00299-t002){ref-type="table"}). For *O*-mannosylation, mutant strains of the protein mannosyltransferase (PMT) family, Mnt1 and Mnt2, were tested. The MICs of P-113 and P-113Tri were 6 and 1.5 μg/mL, respectively, for the CAF2-1 strain. In addition, the MIC values of the *pmt2*-, *pmt4*-, and *pmt6*-deletion mutants were similar to that of the parental CAF2-1 strain. Notably, the *pmt5*-deletion mutant was more resistant to P-113 (MIC = 12 μg/mL) than the CAF2-1 strain. Pmt1, Pmt2, Pmt4, Pmt5, and Pmt6 are members of the PMT family that are required for the initiation of *O*-glycosylation. The MICs of P-113 and P-113Tri for the control NGY152 strain were 12 and 3 μg/mL, respectively. The *mnt1*-deletion mutant was slightly more sensitive to P-113 and P-113Tri compared to its parental NGY152 strain. However, the *mnt2*-deletion mutant was more resistant to P-113, and the *mnt1*Δ/*mnt2*Δ double mutant was more resistant to P-113Tri compared to the NGY152 strain ([Table 2](#microorganisms-08-00299-t002){ref-type="table"}). For *N*-mannosylation, mutant strains of Mnt3, Mnt4, Mnt5, Och1, and Mnn4 were tested. In [Table 2](#microorganisms-08-00299-t002){ref-type="table"}, the *mnt3*Δ*mnt5*Δ (NGY1227), *mnt4*Δ*mnt5*Δ (NGY516), *och1*Δ (NGY357), and *mnn*4Δ (CDH15) mutants were more resistant to P-113 (MIC ≥ 24 μg/mL) compared to their parental NGY152 strain (MIC = 12 μg/mL). Finally, both *och1*Δ and *mnn4*Δ mutants were resistant to P-113Tri. Moreover, the *pmr1*Δ (NGY355) mutant was more sensitive to P-113 than the NGY152 strain ([Table 2](#microorganisms-08-00299-t002){ref-type="table"}). Together, the peptides binding to mannan, particularly *N*-linked mannan, have a partial effect on their activity against *C*. *albicans*. 3.4. Binding to Phosphomannan Also Plays a Profound Role in The Activity of The Peptides {#sec3dot4-microorganisms-08-00299} ---------------------------------------------------------------------------------------- The *N*-linked mannan contains an α-1,6-linked polymannose backbone attached with side chains (consisting of α-1,2- and α-1,3-linked oligomannosides and β-1,2-linked mannose residues), and a mannosylphosphate-containing fraction is attached to the side chains via phosphodiester bonds \[[@B36-microorganisms-08-00299]\]. Moreover, approximately 20% of the cell wall of phosphomannan has also been found to attach to the *O*-linked mannan \[[@B37-microorganisms-08-00299]\]. Interestingly, several studies indicate that the binding of antifungal proteins and synthetic peptides is reduced to glycosylation mutants with the loss of phosphomannan and concomitant reduction in cell surface negative charge \[[@B36-microorganisms-08-00299],[@B38-microorganisms-08-00299],[@B39-microorganisms-08-00299]\]. The reduced binding in turn enhances cellular resistance to antifungal proteins and peptides, suggesting that these antifungal agents can recognize the cell surface patterns of the pathogens to maximize their efficacy. Therefore, it is interesting to determine the possible binding of our peptides to phosphomannan and the impact of this binding on the anti-*Candida* activity of the tested peptides. In [Figure 4](#microorganisms-08-00299-f004){ref-type="fig"}B, the ratio of P-113 binding to various mutants is presented with shades of blue. The deep blue represents the strongest P-113 binding to the mutant strains, and the white represents the weakest binding of P-113. [Figure 4](#microorganisms-08-00299-f004){ref-type="fig"}B shows that the binding of P-113 and P-113Tri to *pmr1*Δ, *mnt3*Δ*mnt5*Δ, *mnn4*Δ, and *och1*Δ were largely decreased compared to the parental NGY152 strain. Importantly, all of these mutant strains also play an important role in phosphomannan biosynthesis. Therefore, these data in [Figure 4](#microorganisms-08-00299-f004){ref-type="fig"}B suggest that P-113 and P-113Tri can also bind to phosphomannan. To further determine the possible differences of P-113 and P-113Tri binding to negatively charged phosphate, we tested the effect of exogenous glucosamine 6-phosphate, monosaccharide mannose 6-phosphate-BSA, trisaccharide mannose 6-phosphate-BSA, and pentasaccharide mannose 6-phosphate-BSA on the antifungal efficacy of P-113 and P-113Tri. As shown in the MIC assay ([Table 3](#microorganisms-08-00299-t003){ref-type="table"}), 5 or 10 mM glucosamine 6-phosphate had no significant adverse effects on the efficacy of P-113Tri to inhibit *C*. *albicans* cell growth compared to the controls without glucosamine 6-phosphate. However, glucosamine 6-phosphate significantly reversed the efficacy of P-113 to inhibit *C*. *albicans* ([Table 3](#microorganisms-08-00299-t003){ref-type="table"}). Interestingly, 5 mg/mL monosaccharide mannose 6-phosphate-BSA, trisaccharide mannose 6-phosphate-BSA, and pentasaccharide mannose 6-phosphate-BSA significantly reversed the inhibition of *C. albicans* growth caused by P-113 and P-113Tri. These results indicated that the negative charge on the glycan also contributes to the efficacy of the peptides against *C*. *albicans*. 3.5. Screening the Potential Glycan Targets of P-113Tri by Glycan Array {#sec3dot5-microorganisms-08-00299} ----------------------------------------------------------------------- To further understand the carbohydrate/P-113Tri interaction, we determined whether P-113Tri can recognize and bind to specific glycans that commonly do not exist in *C*. *albicans* using a homogeneous solution carbohydrate array \[[@B25-microorganisms-08-00299]\]. In this array, there were 106 synthetic glycans that represent the terminal sequences found on *N*-glycans, *O*-glycans, and glycosphingolipids of different microbial cells and mammalian tissues. [Figure 5](#microorganisms-08-00299-f005){ref-type="fig"} shows that the 40 most common glycans were bound by P-113Tri. For example, P-113Tri bound to α-mannose monohydrate with a fluorescence intensity of \~15,000 ([Figure 5](#microorganisms-08-00299-f005){ref-type="fig"}). This result further verified that P-113Tri can recognize and interact with the α-mannose moiety on the *C. albicans* cell surface. In addition, P-113Tri can strongly interact with the glycans designed as Le^d^ (H type 1), Le^b^, Le^y^, and 6GlcNAc-HSO~3~-SiaLe^x^, and 6Gal-HSO~3~-SiaLe^x^, which are generally found to localize in the terminal structure of *N*-linked glycans of mammalian tissues and are commonly used as cancer markers. In addition, P-113Tri bound to the glycans α-L-rhamnose, Galα1-4Galβ1-4Glcβ, (NeuAcα2-8)~3~, and (NeuAcα2-8)~5−6~, which are found to generally localize on the bacterial cell surface. Interestingly, P-113 did not bind to any glycans we tested ([Figure 5](#microorganisms-08-00299-f005){ref-type="fig"}). The results suggest the differences in P-113Tri and P-113 binding glycans with different specificities. 4. Discussion {#sec4-microorganisms-08-00299} ============= In this study, we investigated the difference between P-113Tri and P-113 in their anti-*C*. *albicans* activity. P-113 was found to translocate rapidly through the cell surface and accumulate intracellularly. However, although small amounts of P-113Tri slowly gained access to the cells, most of the P-113Tri remained associated with the *C*. *albicans* cell surface ([Figure 1](#microorganisms-08-00299-f001){ref-type="fig"}A--D), particularly the carbohydrates of the cell wall ([Figure 2](#microorganisms-08-00299-f002){ref-type="fig"}A−E and [Figure 3](#microorganisms-08-00299-f003){ref-type="fig"}B). Importantly, this carbohydrate/peptide interaction is related to the candidacidal activity of P-113Tri ([Figure 2](#microorganisms-08-00299-f002){ref-type="fig"}F). The impacts of microbial cell wall components on the activity of cationic AMPs have been reported in several studies \[[@B40-microorganisms-08-00299],[@B41-microorganisms-08-00299],[@B42-microorganisms-08-00299],[@B43-microorganisms-08-00299]\]. For example, *Staphylococcus aureus* mutants defective in teichoic acids, a major cell wall component, have increased sensitivity to defensins, protegrins, and other AMPs \[[@B42-microorganisms-08-00299]\]. Human β-defensin-3 and α-defensin-1 exert antibacterial activity by binding to lipid II of Gram-positive bacteria \[[@B44-microorganisms-08-00299],[@B45-microorganisms-08-00299]\]. Moreover, aculeacin A and nikkomycin Z inbibit fungal 1,3-β-glucan synthase and chitin synthases, respectively \[[@B46-microorganisms-08-00299],[@B47-microorganisms-08-00299]\]. Moreover, Hst 5 binding to the β-glucans of the cell wall is required for candidacidal activity \[[@B48-microorganisms-08-00299]\]. A previous study also showed that a specific sequences of P-113 can be recognized by transporter (i.e., independent of cell wall binding) for intracellular translocation \[[@B17-microorganisms-08-00299]\]. In the present study, after cell incubation with peptides for \~5 min, most P-113 readily gained access into the cells ([Figure 1](#microorganisms-08-00299-f001){ref-type="fig"}) and this result is somehow consistent with Jang et al. \[[@B17-microorganisms-08-00299]\]. Interestingly, P-113Tri largely remained on the cell surface even after incubation for \~5 min and \~ 1 h ([Figure 1](#microorganisms-08-00299-f001){ref-type="fig"}). One possible explanation for these results is that the high net charge and alpha-helical content of P-113Tri \[[@B19-microorganisms-08-00299]\] may contribute to the enhanced interaction between P-113Tri and cell surface, leading to retain on the cell wall. Another possible explanation is that the conformation changes of P-113Tri (compared to P-113) somehow interfere the peptide--cell surface interaction. Moreover, we are not sure whether transporter(s) are involved in the transportation of P-113Tri. Further study is needed to address these questions. In this work, Zymolyase and sodium metaperiodate were used to remove β-1,3-glucan and β-1,6-glucan from the cell wall, respectively. The cells treated with Zymolyase showed a significant reduction in their interaction with the peptides compared to the cells treated with metaperiodate ([Figure 2](#microorganisms-08-00299-f002){ref-type="fig"}C and 2D). In addition, cells preincubated with laminarin (mainly consisting of β-1,3-glucan) can decrease the binding of the peptides to *C*. *albicans* cells ([Figure 3](#microorganisms-08-00299-f003){ref-type="fig"}A). Moreover, cells treated with Zymolyase showed lower sensitivity to the peptides compared to the cells without peptide treatment ([Figure 2](#microorganisms-08-00299-f002){ref-type="fig"}F), and adding laminarin can rescue the cells from the candidacidal effect of the peptides ([Figure 3](#microorganisms-08-00299-f003){ref-type="fig"}B). Therefore, our results suggest that the binding of peptides to the β-1,3-glucan layer plays an important role in the activity of AMPs. These results are consistent with Han et al. \[[@B23-microorganisms-08-00299]\], who demonstrated that the antimicrobial activities of Hst 5 and P-113 were drastically decreased in cells treated with laminarin but not pustulan (mainly consisting of β-1,6-glucan). Interestingly, the binding of P-113Tri to ConA-treated cells also decreased ([Figure 2](#microorganisms-08-00299-f002){ref-type="fig"}A). This result is in agreement with the glycan array screening ([Figure 5](#microorganisms-08-00299-f005){ref-type="fig"}), in which P-113Tri bound to α-mannose monohydrate ([Figure 5](#microorganisms-08-00299-f005){ref-type="fig"}). Moreover, the binding of P-113 and P-113Tri was reduced in cells treated with α1-2,3,6 mannosidase, which can remove the mannan layer of the cell wall ([Figure 2](#microorganisms-08-00299-f002){ref-type="fig"}B), and the competition assay using mannan also showed a decrease in the activity of the peptides ([Figure 3](#microorganisms-08-00299-f003){ref-type="fig"}A). Therefore, our results further indicate that the mannan layer plays an important role in mediating the function of the peptides. To further investigate the influence of mannan on the activity of peptides, *C*. *albicans* mutant strains defective in *O*-linked and *N*-linked mannan were used. We found that *N*-linked mannan and phosphomannan, but not *O*-linked mannan, on the cell surface can affect P-113Tri binding ([Figure 4](#microorganisms-08-00299-f004){ref-type="fig"}B,C). However, the candidacidal activity of P-113Tri is not correlated with the binding ability of P-113Tri to *N*-linked mannan and phosphomannan. In [Table 2](#microorganisms-08-00299-t002){ref-type="table"}, *och1*Δ (NGY357) and *mnn*4Δ (CDH15) mutants were more resistant to P-113Tri compared to the control NGY152 strain, which were correlated to the binding ratio of P-113Tri. However, the *mnt3*Δ*mnt5*Δ (NGY1227), *mnt4*Δ*mnt5*Δ (NGY516), and *pmr1*Δ (NGY355) mutants were more sensitive or similar to P-113Tri compared to the control NGY152 strain. These data suggest that the binding of P-113Tri to the *N*-linked mannan and phosphomannan on the *Candida* surface is not the only mechanism for the killing ability of P-113Tri. Because the cell wall is the outmost layer of *C. albicans* cells, we only focused on the cell wall--peptide interaction in this study and found that specific cell wall glycans are related to the candidacidal activity of P-113 and P-113Tri. In our previous study \[[@B49-microorganisms-08-00299]\], we found that the peptides can somehow also target to the mitochondria. However, the mechanism through which the peptides cross the cell surface and affect the mitochondrial functions is still under investigation. It is worth noting that only cells that have totally lost phosphomannan, mannan, and glucan (*och1*Δ and *mnn*4Δ mutants, and Zymolyase-treated cells) showed the reduced candidacidal activity from P-113Tri. Moreover, previous studies showed that physiological conditions, such as blood or serum, decreased the structural complexity of mannan in the *C*. *albicans* cell wall \[[@B50-microorganisms-08-00299]\]. In addition, *Candida* clinical isolates also showed increased glucan exposure through the loss of the acid-labile mannan structure \[[@B51-microorganisms-08-00299]\]. Therefore, P-113Tri can specifically and strongly bind to *N*-linked mannan and phosphomannan and can have potent antifungal activity against *C. albicans* with a reduced mannan content. Together, these data suggest that P-113Tri can be a potential antifungal drug in the future. In a previous study, the loss of negatively charged *N*-linked phosphomannans from the cell wall enhanced *C. albicans* resistance to the AMP dermaseptin by reducing peptide binding and entry from the cell surface \[[@B36-microorganisms-08-00299]\]. In addition, exogenous phosphosugars were able to reduce the efficacy of P-113 and P-113Tri ([Table 3](#microorganisms-08-00299-t003){ref-type="table"}). Moreover, it seems that the binding of cationic peptides to the *C. albicans* cell wall is not only related to the net charge of the cell surface but may also involve pattern recognition of *C*. *albicans* by the AMPs. This possibility requires further investigation. Using a glycan array, P-113Tri can bind to carbohydrates commonly found on the bacterial cell wall and on human cells, including some cancer glycan epitopes ([Figure 5](#microorganisms-08-00299-f005){ref-type="fig"}). For example, P-113Tri has a strong binding to Le^y^, Le^b^, Le^a^, Le^x^, and sLe^x^ \[[@B48-microorganisms-08-00299]\]. The Le^y^ epitope is found in lung, urinary bladder, prostate, ovarian, head, and neck and thymus cancer cells, whereas the Le^b^ epitope exists in urinary bladder, and endometrial cancer cells. Moreover, the Le^a^ epitope is present in stomach, pancreatic, lung, and endometrial cancer cells, whereas the Le^x^ is found in pancreatic, lung, kidney, urinary bladder, breast, head, and neck cancer cells. Finally, sLe^x^ has been identified in stomach, pancreatic, lung, prostate, and breast cancer cells \[[@B52-microorganisms-08-00299]\]. Interestingly, the binding of P-113 to all the glycans in the tested array was not detectable ([Figure 5](#microorganisms-08-00299-f005){ref-type="fig"}). These results suggest that P-113Tri and its parental P-113 have a fundamental difference in their carbohydrate recognition and binding, and P-113Tri can possibly bind to different cancer cells to facilitate its anticancer activity. Further study is required to determine the potential of P-113Tri as an anticancer agent. Although AMPs bind to carbohydrates, we cannot exclude that the peptides may also interact with cell wall proteins that consist of 20%--30% (in mass) of the fungal cell wall \[[@B27-microorganisms-08-00299]\]. In particular, mannan is covalently bound to proteins (mannoproteins), and this form of mannan accounts for 40% of the total cell wall polysaccharides on the *C. albicans* exterior \[[@B53-microorganisms-08-00299]\]. Mannoproteins are involved in cell--cell recognition and trigger immune responses \[[@B54-microorganisms-08-00299]\]. Several studies have shown that AMPs function via their interactions with microbial cell wall proteins. For example, *C*. *albicans* Ssa1/2 proteins are required for Hst 5 binding \[[@B55-microorganisms-08-00299]\]. The *C. albicans* proteins possibly targeted by P-113Tri and the significance of such AMP/protein interactions are currently under investigation. Moreover, in our previous study \[[@B19-microorganisms-08-00299]\], P-113Tri showed an overall higher efficiency to kill various *C. albicans* and non-albicans *Candida* clinical isolates than P-113. However, all the *C. tropicalis* reference strain ATCC 13803 and three clinical isolates (YH50007, YH50013 and YH50114) were sensitive to both P-113 and P-113Tri (Table 2 of \[[@B19-microorganisms-08-00299]\]). Recently, Navarro-Arias et al. compared the content of chitin, mannan, glucan, and phosphomannan on *C. tropicalis* and *C. albicans* cell wall and found that there is no significant difference in these two species \[[@B56-microorganisms-08-00299]\]. However, the porosity of *C. tropicalis* cell wall is higher than *C. albicans* (63% vs. 28%) \[[@B56-microorganisms-08-00299]\], which raises a possibility that P-113 is much easier to gain access into the *C. tropicalis* cells than P-113Tri due to the small size of P-113 and has a higher activity against *C. tropicalis* than *C. albicans*. Further study is needed to test this possibility. Many studies have revealed that peptides with higher net charge and alpha-helical content are generally prone to have strong antimicrobial activity \[[@B43-microorganisms-08-00299]\]. For example, Ma et al. showed that a peptide with a tandem arrangement of two leucine-rich repeats (LRRs) forms alpha-helical structure and possesses a higher antibacterial activity than that containing only one LRR with a random coil structure \[[@B57-microorganisms-08-00299]\]. Moreover, this study also suggests that the secondary structure plays a more vital role in antimicrobial activity of peptides than the primary sequence of peptides. For the peptides that we studied, using circular dichroism (CD), and the beta-structure selection method, the alpha-helical content of P-113Tri and P-113 is 21.4% and 2.9%, respectively \[[@B19-microorganisms-08-00299]\]. In addition, the net charges of P-113Tri and P-113 are +15 and +5, respectively. Therefore, other than the secondary structure, the higher net charge of P-113Tri may also contribute to the higher activity of P-113Tri compared to P-113. The relationship between the net charge and secondary structure of these peptides to the selective glycan binding is required to be further analyzed using a molecular dynamic (MD) simulation and/or nuclear magnetic resonance (NMR) spectroscopy. In addition, *C. albicans* is recognized by the pattern-recognition receptors (PRRs) of host monocytes, macrophages, dendritic cells, and neutrophils through their interaction with different pathogen-associated molecular patterns (PAMPs) on the fungal cell wall. These PAMPs include *N*-linked mannans, *O*-linked mannans, phosphomannan, and β-glucans \[[@B58-microorganisms-08-00299]\]. The PRR/PAMP interaction induces pro- and anti-inflammatory cytokines, phagocytosis-mediated fungal killing, and represents a crucial mechanism that allows the innate immune system to combat *Candida* infections. Interestingly, neutrophils can secrete a range of AMPs, such as LL-37 and HNP 1-3, to kill microorganisms \[[@B59-microorganisms-08-00299]\]. Intriguingly, our results imply that not only have immune cells evolved to recognize cell-surface molecular patterns of the fungal pathogens but also AMPs can recognize the same or similar molecular patterns to maximize their efficacy and specificity for pathogen killing. In summary, the cell wall is the first contact point between fungal pathogens and their environments. Therefore, the composition and structure of the fungal cell wall are important for immune recognition. AMPs are key components of the innate host defense, and the interaction between AMPs and fungal cells is complex because of their association with different components of the cell wall. Our findings here highlight that the tested peptides, particularly P-113Tri, bind to specific glycans (*N*-linked mannan and phosphomannan of the *C. albicans* cell wall) and suggest that these peptides can be modified in future motifs to enhance the interaction between the peptide and the microbial cell surface for potential antifungal therapy. We thank Neil A. R. Gow (University of Exeter, UK) and Joachim F. Ernst (Heinrich-Heine-Universität, Germany) for generously providing strains. We thank for Yuan-Chuan Lee (Johns Hopkins University, USA) providing saccharides and valuable comments. We also thank the support from the confocal imaging core in National Tsing Hua University (sponsored by MOST 108-2731-M-007-001), and Mr. Che-Kang Chang for technical assistance in this study. We appreciate the structural bioinformatics service provided by the BP Bioinformatics Core ([http//:www.tbi.org.tw](http//:www.tbi.org.tw)), funded by National Core Facility for Biopharmaceuticals (NCFB), MOST 108-2319-B-400-001. The following are available online at <https://www.mdpi.com/2076-2607/8/2/299/s1>, Figure S1. 3D structure of P-113 and P-113Tri predicted by I-TASSER (iterative threading assembly refinement). Figure S2. Aniline blue binding to zymolyase-treated *C. albicans*. Table S1. *C. albicans* strains used in this study. ###### Click here for additional data file. Conceptualization, G.-Y.L.; Data curation, G.-Y.L. and C.-F.C.; Formal analysis, G.-Y.L.; Funding acquisition, C.-Y.L.; Investigation, G.-Y.L. and C.-F.C.; Methodology, G.-Y.L. and C.-F.C.; Validation, G.-Y.L.; Writing---original draft, G.-Y.L. and C.-Y.L.; Writing---review & editing, G.-Y.L. and C.-Y.L. All authors have read and agreed to the published version of the manuscript. This work was supported by grants MOST108-2311-B-007-005 and MOST105-2311-B-007-007-MY3 (to CYL). GYL was supported by a doctoral student fellowship from the Ministry of Education, Taiwan. The funders had no role in study design, data collection and analysis, or the decision to submit the work for publication. The authors declare no conflict of interest. {#microorganisms-08-00299-f001} {#microorganisms-08-00299-f002} {#microorganisms-08-00299-f003} {#microorganisms-08-00299-f004} {#microorganisms-08-00299-f005} microorganisms-08-00299-t001_Table 1 ###### *C. albicans* deletion mutants that were P-113 resistant. No Systematic Name Gene Name Description -------- ----------------- ----------- ---------------------------------------------------- **1** orf19.7590 Putative NADH-ubiquinone oxidoreductase **2** orf19.7247 *RIM101* Transcription factor **3** orf19.7391 *OCH1* α-1,6-mannosyltransferase **4** orf19.13191 *SNF4* Putative subunit of the AMP-activated Snf1p kinase **5** orf19.287 *NUO2* NADH-ubiquinone oxidoreductase subunit **6** orf19.1625 Putative ubiquinone oxidoreductase **7** orf19.1710 *ALI1* Putative NADH-ubiquinone oxidoreductase **8** orf19.2570 *MCI4* Putative NADH-ubiquinone dehydrogenase **9** orf19.2821 Protein of unknown function **10** orf19.4758 Putative reductase or dehydrogenase **11** orf19.5547 Protein of unknown function **12** orf19.3995 *RIM13* Protease of the pH-response pathway **13** orf19.4755 *KEX2* Subtilisin-like protease **14** orf19.5068 *IRE1* Putative protein kinase **15** orf19.6293 *EMP24* COPII-coated vesicle component microorganisms-08-00299-t002_Table 2 ###### The minimum inhibitory concentrations (MICs) of P-113 and P-113Tri against *C. albicans* glycosylation mutants. MIC~90~ (μg/mL) ^a^ in LYM broth ---------------------------------------------- ---------------------------------- -------- ***O*-linked Glycosylation Mutants** CAF2-1 (Control) 6 1.5 SPCa2 (Pmt1) ND ^b^ ND ^b^ SPCa4 (Pmt2) 6 1.5 SPCa6 (Pmt4) 6 3 SPCa8 (Pmt6) 6 3 SPCa10 (Pmt5) 12 3 NGY152 (Control) 12 3 NGY145 (Mnt2) 24 3 NGY158 (Mnt1) 6 1.5 NGY337 (Mnt1Mnt2) 12 6 ***N*-linked glycosylation mutants** NGY152 (Control) 12 3 NGY516 (Mnt4Mnt5) 24 1.5 NGY1227 (Mnt3Mnt5) \>24 1.5 NGY357 (Och1) \>24 12 CDH15 (Mnn4) 24 \>12 ***O* and *N*-linked glycosylation mutants** NGY355 (Pmr1) 3 3 ^a^ The MIC~90~ was measured after the cells were incubated with each peptide for 48 h. ^b^ ND: Not determined. microorganisms-08-00299-t003_Table 3 ###### Exogenous phosphosugars reduce the anti-*C. albicans* activity of the peptides. ------------------------------------------------------------------------- MIC~90~ (μg/mL) ^a^ in LYM broth ------------------------------- ---------------------------------- ------ Control 3 0.75 5 mM Glucosamine 6-phosphate 6 1.5 10 mM Glucosamine 6-phosphate 12 1.5 5 mg/mL monosaccharide\ \>24 12 mannose 6-phosphate-BSA 5 mg/mL trisaccharide\ 24 6 mannose 6-phosphate-BSA 5 mg/mL pentasaccharide\ \>24 6 mannose 6-phosphate-BSA ------------------------------------------------------------------------- ^a^ The MIC~90~ was measured after the cells were incubated with each peptide for 48 h.

Abbreviations used: Ado, adenosine; ChIP, chromatin immunoprecipitation; ENT, equilibrative nucleoside transporter; HIF-1, hypoxia inducible factor 1; HMEC, human microvascular endothelial cells; HRE, hypoxia-responsive element; MPO, myeloperoxidase. Limited oxygen availability (hypoxia) is central to the pathogenesis of many cardiovascular, infectious, and inflammatory diseases. Pathophysiologic changes related to hypoxia include changes in tissue permeability, accumulation of inflammatory cells, and transcriptional induction of proinflammatory cytokines ([@bib1], [@bib2]). Recent studies have also identified hypoxia-elicited factors that counterregulate such proinflammatory circuit, thereby functioning as endogenous antiinflammatories ([@bib3]). Central to this latter pathway is extracellular adensoine (Ado), which through activation of surface-expressed receptors on a variety of cells, dampens ongoing inflammation ([@bib2], [@bib4]--[@bib6]) and promotes wound healing ([@bib7]). At present, the exact metabolic steps for generation of extracellular Ado in hypoxia are not well characterized, but likely involve increased enzymatic phosphohydrolysis from precursor adenine nucleotides (ATP, ADP, and AMP). For instance, we recently demonstrated that hypoxia coordinates both transcriptional and metabolic control of the surface ectonucleotidases CD39 and CD73 ([@bib8]--[@bib10]), and thereby amplifies extracellular accumulation of Ado. Additional mechanisms also exist to amplify Ado signaling during hypoxia, including coordinate changes at the Ado receptor level. For instance, the vascular endothelial Ado receptor subtype AdoRA~2B~ is selectively induced by hypoxia and such increases in receptor density are associated with increased vascular barrier responses to Ado ([@bib8]). Once generated into the extracellular milieu, Ado is rapidly cleared through passive or active uptake by nucleoside transporters, termed equilibrative nucleoside transporters (ENTs) and concentrative nucleoside transporters, respectively, expressed on a variety of cell types ([@bib11], [@bib12]). Recent studies suggest that the predominant functional nucleoside transporters in the vascular endothelium are ENT1 and ENT2, with a minimal contribution by centrative nucleoside transporters ([@bib13]), wherein ENT1 and 2 are bidirectional transporters functioning as diffusion-limited channels for transmembrane nucleoside flux. Previous studies have suggested that vascular Ado transport during hypoxia is predominantly inward ([@bib14]), thereby terminating extracellular Ado signaling. However, more recent studies show that the expression of ENT1 may be transcriptionally regulated by hypoxia ([@bib15], [@bib16]), thereby functioning to fine tune extracellular levels of Ado. Therefore, we examined the influence of hypoxia on endothelial and epithelial Ado transport. Results from these studies revealed that ENT1 and ENT2 gene expression and function are attenuated by hypoxia, and that this regulatory circuit maps to hypoxia inducible factor 1 (HIF-1)--mediated repression of ENT expression. These studies provide new molecular insight into endogenous mechanisms of tissue protection during hypoxia. Results ======= Increased extracellular Ado half-life in posthypoxic endothelia --------------------------------------------------------------- Recent studies suggest that hypoxia promotes a cellular phenotype that supports high capacity for rapid nucleotide phosphohydrolysis and enhanced Ado signaling ([@bib8], [@bib9]). As an extension of these studies, we addressed whether mechanisms of extracellular Ado clearance were different in posthypoxic endothelial cells. Initial studies indicated that hypoxia significantly decreased endothelial uptake of exogenous Ado ([Fig. 1](#fig1){ref-type="fig"} a), thereby increasing Ado half-life. Indeed, these studies indicated that human microvascular endothelial cells (HMEC)-1 subjected to hypoxia (pO~2~ 20 torr, 48 h) and examined for their capacity to transport extracellular Ado (Ado~EC~, 50 μM final concentration), had significantly attenuated ability to transport Ado (Ado~EC~ 19.2 ± 0.6 μM at 2 h) compared with normoxic controls (Ado~EC~ 1.3 ± 1.1μM at 2 h, P \< 0.01). Control experiments performed at 4°C or in the presence of the ENT-inhibitor dipyridamole (10 μM) revealed significant attenuation of Ado uptake (Ado~EC~ 40 ± 1.6 and 33 ± 2.1 μM concentrations, respectively; P \< 0.001), indicating that endothelial Ado uptake requires metabolism and depends on dipyridamole-sensitive carriers. Subsequent studies addressing the hypoxia time course ([Fig. 1](#fig1){ref-type="fig"} b) and dose response ([Fig. 1](#fig1){ref-type="fig"} c) revealed a time-dependent (P \< 0.01) and dose-dependent (P \< 0.05) decrease in transport of Ado~EC~. {#fig1} To confirm that the observed decreases in Ado~EC~ reflects transport and to rule out other potential mechanisms of Ado loss (e.g., metabolism by the ecto-Ado deaminase) ([@bib17]), we determined the kinetics of ^14^C-Ado~EC~ transmembrane transport ([Fig. 1](#fig1){ref-type="fig"} d). These studies confirmed our studies with native Ado and revealed that ^14^C-Ado~EC~ transport was 63 ± 6% decreased in posthypoxic endothelia relative to normoxia (P \< 0.001). In parallel, ^14^C-Ado~EC~ transport at 4°C or in the presence of dipyridamole revealed significant attenuation of endothelial uptake (P \< 0.001). Together, these data suggest an additional mechanism to elevate Ado~EC~ involves diminished active uptake through endothelial membrane transporters. Hypoxia rapidly represses expression of endothelial and epithelial ENT1 and ENT2 mRNA ------------------------------------------------------------------------------------- Prompted by these results, recent findings that ENT1/2 predominate in the vascular endothelium ([@bib13]) and that hypoxia down-regulates dipyridamole-sensitive Ado transporters ([@bib15], [@bib16]), we examined the influence of hypoxia on expression of ENT1 and ENT2 mRNA. Initial insight gained by microarray analysis revealed an 85 ± 5 and 78 ± 4% inhibition of ENT1 and ENT2 mRNA expression, respectively (18 h hypoxia, pO~2~ 20 torr). As shown in [Fig. 2](#fig2){ref-type="fig"} a, real-time PCR analysis confirmed our microarray results and revealed a time-dependent loss of both ENT1 (95 ± 8% loss at 24 h, P \< 0.01) and ENT2 (81 ± 7% loss at 24 h of hypoxia, P \< 0.01). In addition, we found that expression levels of ENT1 mRNA in HMEC-1 were approximately fourfold higher compared with ENT2 mRNA. {#fig2} Similar results of ENT1 and ENT2 repression were found when human saphenous vein tissues were subjected to hypoxia, indicating that such findings are not limited to cultured endothelium ([Fig. 2](#fig2){ref-type="fig"} b, P \< 0.001). Additionally, ENT1 and ENT2 expression were decreased in T84 cells and intestinal epithelial cell line ([Fig. 2](#fig2){ref-type="fig"} c, P \< 0.001), further suggesting that these findings are not limited to endothelial cells. We extended these mRNA findings to examine ENT1 protein expression in cultured HMEC-1. As shown in [Fig. 2](#fig2){ref-type="fig"} d, Western blot analysis of lysates derived from HMEC-1 subjected to hypoxia (pO~2~ 20 torr for 6--36 h) revealed a dramatic loss of ENT1 with increasing time in hypoxia. Protein repression was maximal at 36 h time periods of hypoxia (no additional loss was evident at 48 h). ENT1 repression mediates attenuated endothelial Ado uptake during hypoxia ------------------------------------------------------------------------- We next determined whether inhibition of ENT gene expression was required for the observed functional differences in Ado uptake between posthypoxic and postnormoxic endothelia. Here, we used siRNA-mediated repression in HMEC-1 of ENT1 or ENT2 for gaining insight into relative contributions of these individual gene products on Ado~EC~ transport. As a first step, we generated siRNA double-stranded oligoribonucleotides specific for ENT1 and ENT2 and profiled ENT expression by PCR ([Fig. 3](#fig3){ref-type="fig"}, a and b). As control siRNA, we used a single-stranded forward oligoribonucleotide, which did not influence ENT1 or ENT2 mRNA expression ([Fig. 3](#fig3){ref-type="fig"} c), suggesting that the aforementioned findings of decreased ENT1 or ENT2 expression ([Fig. 3](#fig3){ref-type="fig"}, a and b) were specific for targeted gene knock-down by the chosen siRNA molecules. As additional control, we used double-stranded siRNA directed against the 5′-ectonucleotidase (CD73), an endothelial surface enzyme that facilitates conversion of Ado monophosphate to Ado. As shown in [Fig. 3](#fig3){ref-type="fig"} (d and e), siRNA repression of CD73 did not influence expressional levels of ENT1 or ENT2. After showing that siRNA-mediated repression of ENT1 and ENT2 was specific and highly efficient using the aforementioned conditions (81 ± 8% inhibition of ENT1 mRNA expression, P \< 0.01, and 79 ± 10% inhibition of ENT2, P \< 0.01), we measured endothelial Ado uptake using ^14^C-Ado. As shown in [Fig. 3](#fig3){ref-type="fig"} f, whereas ^14^C-Ado uptake was not influenced by siRNA repression with control siRNA (5 ± 1% decrease compared with mock-loaded cells, p-values were not significant) or siRNA directed against ENT2 (14 ± 7% decrease compared with mock-loaded cells, p-values were not significant), Ado uptake was decreased after siRNA-mediated repression of ENT1 (76 ± 7% decrease compared with mock-loaded cells, P \< 0.01) or simultaneous ENT1 and ENT2 repression (81 ± 5% decrease compared with mock-loaded cells, P \< 0.01). Together, these results implicate ENT1 as the prevailing mechanism for the observed changes in endothelial Ado. {#fig3} Decreased endothelial Ado uptake promotes endothelial barrier function in vitro ------------------------------------------------------------------------------- We next determined whether hypoxia-mediated repression of ENT1 might enhance signaling mediated by Ado~EC~. Here, we compared Ado responses of normoxic and posthypoxic endothelia using a previously described in vitro model of endothelial barrier ([@bib8], [@bib18]). As reported previously ([@bib8]), we observed that barrier function (i.e., flux 70-kD FITC-dextran) in response to Ado was enhanced to a greater extent in posthypoxic HMEC-1 than in normoxic HMEC-1 (Ado EC~50~\'s of ∼30 μM and ∼1 μM for normoxia and posthypoxia, respectively; [Fig. 4](#fig4){ref-type="fig"} a). To define the contribution of hypoxia-mediated repression of ENT1/2, we repeated this experiment in the presence of the ENT inhibitor dipyridamole (10 μM). As shown in [Fig. 4](#fig4){ref-type="fig"} b, in the presence of dipyridamole, the dose response to Ado became indistinguishable between hypoxia and normoxia (Ado EC~50~ of ∼0.1 μM for both), implicating hypoxia-mediated repression of ENT1/2 in functional Ado responses. {#fig4} We next defined the contribution of ENT1 or ENT2 to such changes in Ado-elicited barrier responses of the endothelium. To do this, we used the aforementioned siRNA repression approach. As shown in [Fig. 4](#fig4){ref-type="fig"} c, siRNA repression of ENT1 was associated with an increase of Ado elicited barrier responses (P \< 0.01). In contrast, loss of ENT2 was not associated with a measurable change in endothelial Ado response ([Fig. 4](#fig4){ref-type="fig"} d). These data reveal that inhibition of Ado uptake, either via hypoxia inhibition of ENT gene expression, pharmacological intervention, or siRNA repression of ENT1, represents an adaptive mechanism to elevate extracellular Ado levels, and thereby prolong signaling mediated by this nucleoside. HIF-1**α**--dependent repression of ENT1 promoter ------------------------------------------------- In an attempt to gain specific insight into the molecular mechanisms of ENT1 repression by hypoxia, we profiled promoter activity in HMEC-1 transfected with luciferase promoter constructs expressing varying length 5′-truncations (225, 282, 344, and 756 bp relative to the major transcription start site). Highest basal expression (i.e., normoxia) was observed in the 756-bp construct (15 ± 2.1-fold over background) and graded decreases in baseline activity were observed with increasing 5′-truncation (6.8 ± 1.7, 3 ± 1.1, and 5 ± 0.6-fold over background for 344-, 282-, and 225-bp truncations, respectively). Comparison of promoter activity in HMEC-1 exposed to hypoxia or normoxia revealed that, whereas other constructs were expressed at or near the level of the normoxia control, expression of the 756-bp fragment was repressed by 59 ± 6% compared with normoxia controls ([Fig. 5](#fig5){ref-type="fig"} a, P \< 0.01). Such analysis indicates that the region spanning −344 to −756 of the ENT1 promoter confers hypoxia-repressive activity. {#fig5} In the course of our experiments, we identified two potential HIF binding sites oriented on the antisense strand of the ENT1 gene promoter (DNA consensus motif 3′-CCGTG-5′ and 3′-CCGTG-5′ located at positions from −653 to −657 and −717 to −721, respectively, relative to the major transcription start site). Because HIF can mediate transcriptional induction or repression ([@bib19], [@bib20]), we determined if either of these putative HIF-1--binding sites conferred hypoxia repression of ENT1 in HMEC-1. First, we examined the kinetics of HIF-1α expression in our HMEC-1 cultures. As shown in [Fig. 5](#fig5){ref-type="fig"} b, Western blot analysis of HIF-1α revealed minimal protein in hypoxia, and time-dependent induction over a 24-h period of hypoxia. Such kinetics are consistent with the loss of ENT1 protein during hypoxia ([Fig. 2](#fig2){ref-type="fig"} d). Second, HIF-1α--binding site mutations (Δ657 and Δ721) were introduced in the 756-bp construct, and as shown in [Fig. 5](#fig5){ref-type="fig"} c, mutation of the −717 to −721 site, but not the −653 to −657 site, reversed hypoxia repression of this promoter construct, suggesting that this region of the promoter, at least in part, confers hypoxia repressor activity. Third, we determined whether this region of the ENT1 promoter binds HIF-1α. For these purposes, we used chromatin immunoprecipitation (ChIP) to analyze HIF-1α binding in live cells. As shown in [Fig. 5](#fig5){ref-type="fig"} d, ChIP analysis of nuclei derived from HMEC-1 cells revealed a prominent band of 192 bp in hypoxic but not normoxic samples. No bands were evident in control IgG immunoprecipitates, and input samples (preimmunoprecipitation) revealed the predictable 192-bp band under conditions of both hypoxia and normoxia. Such results indicate that hypoxia induces HIF-1α binding to the distal 192-bp region of the ENT1 promoter. Together, these results provide strong evidence for a functional hypoxia repressor activity, mediated by HIF-1, in the distal 5′-region the ENT1 promoter. Role of HIF-1**α** in epithelial ENT1 regulation ------------------------------------------------ We next pursued the finding that epithelial ENT expression, like that in endothelia, is repressed by hypoxia ([Fig. 2](#fig2){ref-type="fig"} c). To probe the role of HIF-1α in an epithelial model, we generated a T84 cell line expressing oxygen-stable HIF-1α (T84ΔODD) ([@bib21]) via lentiviral transduction ([Fig. 6](#fig6){ref-type="fig"}). As shown in [Fig. 6](#fig6){ref-type="fig"} a, using this lentiviral vector-expressing GFP, \>90% of T84 cells were stably transduced by this method. Analysis of the T84ΔODD by Western blot ([Fig. 6](#fig6){ref-type="fig"} b) and by hypoxia-responsive element (HRE) luciferase ([Fig. 6](#fig6){ref-type="fig"} c) revealed increased HIF function and activity in both normoxia and hypoxia, releative to the control T84-GFP cell line. The further increase of HIF function and activity with hypoxia most likely reflects the presence of oxygen-stable and oxygen-unstable HIF-1α. {#fig6} Using the T84ΔODD cell line, we addressed whether ENT1 expression was differentially regulated. As shown in [Fig. 7](#fig7){ref-type="fig"} a, ENT1 mRNA expression in normoxia was decreased by \>80% in the T84ΔODD relative to the control T84-GFP cell line (P \< 0.01). Likewise, enhanced repression of ENT1 was observed in T84ΔODD compared with control T84-GFP cells at 6, 12, and 24 h of hypoxia (P \< 0.025 for each). Additionally, comparison of promoter activity in T84ΔODD and T84-GFP cell lines exposed to hypoxia or normoxia revealed enhanced repression of the 756-bp fragment in cells expressing oxygen-stable HIF-1α ([Fig. 7](#fig7){ref-type="fig"} b). Such findings support the hypothesis that ENT1 repression is proportional to HIF-1α activity. {#fig7} In vivo analysis of ENT function on the vasculature --------------------------------------------------- As proof of principle for these in vitro and ex vivo findings of hypoxia-induced ENT repression, we extended these findings to an in vivo model. We and others have shown that hypoxia promotes vascular leak and tissue PMN accumulation in vivo ([@bib1], [@bib8], [@bib10], [@bib22]--[@bib24]). Thus, we pursued pharmacological inhibition of Ado uptake in a previously described murine hypoxia model ([@bib8], [@bib10], [@bib22]). As first step, we administered the ENT inhibitor dipyridamole (10 mg/kg i.p. and 10 mg/kg s.c.) or PBS and subjected animals to either normoxia or hypoxia (8% O~2~ and 92% N~2~) for 4 h. Plasma levels of dipyridamole (determined by HPLC) ranged between 25- and 110-nm concentrations during the course of the experiment ([Fig. 8](#fig8){ref-type="fig"} a), consistent with previous dosing studies with dipyridamole and dipyridamole analogues ([@bib25]). In previous in vivo hypoxia experiments, we observed the lungs as particularly prone to developing hypoxia-induced vascular leak and pulmonary edema ([@bib10]). Therefore, we assessed pulmonary edema by determining lung water content (wet/dry ratio). As shown in [Fig. 8](#fig8){ref-type="fig"} b, hypoxia increased lung water content from 3.81± 0.42 mg/mg dry tissue to 5.83 ± 0.54 (P \< 0.05) in PBS treated mice. In contrast, lung water was decreased in dipyridamole-treated animals by 16 ± 2% under normoxic conditions, and by 21 ± 3% (P \< 0.025) under hypoxic conditions (P \< 0.05). These results suggest that pharmacological inhibition of Ado uptake is associated with a decrease in lung water. {#fig8} To assess vascular barrier function of other organs, we administered the albumin marker Evan\'s blue before hypoxia. Comparative analysis in normoxia revealed decreased Evan\'s blue concentrations in all examined organs (colon, lung, liver, and kidney) of dipyridamole-treated mice compared with PBS controls. Comparison of vascular permeability in dipyridamole or PBS-treated mice subjected to normobaric hypoxia (8% O~2~, 92% N~2~ for 4 h) revealed significantly increased vascular leak in all animals, however to smaller degree in dipyridamole-treated animals (P \< 0.05 for all organs; [Fig. 8](#fig8){ref-type="fig"} c). These findings support our hypothesis that decreased Ado uptake by the endothelium is a protective cellular strategy to dampen changes in vascular barrier function, both at baseline and during hypoxia-induced barrier dysfunction. We next examined whether a reduction of Ado uptake at the vascular surface may also function as antiinflammatory response during hypoxia, particularly given the antiinflammatory properties of Ado ([@bib4], [@bib6]). To test this hypothesis, we screened multiple mucosal organs (colon, lung, liver, and kidney) for PMN accumulation after administration of dipyridamole ([Fig. 8](#fig8){ref-type="fig"} d). This analysis revealed decreased myeloperoxidase (MPO) activity in all organs of dipyridamole-treated animals. Consistent with previous studies ([@bib26]), MPO levels were significantly increased after hypoxia exposure in all animals, however to a much lesser extend in all organs of dipyridamole-treated animals compared with PBS controls (P \< 0.05 for all organs). These in vivo findings support the hypothesis that Ado uptake via dipyridamole-sensitive carriers is a critical control point in the regulation of PMN accumulation within normoxic and hypoxic tissues and suggest ENT down-regulation as innate antiinflammatory adaptation. It is known that the two nucleoside transporters ENT1 and ENT2 differ in their sensitivity to inhibition by the inosine analogue NBTI ([@bib27], [@bib28]). NBTI is a highly potent inhibitor of ENT1 (IC~50~ ∼0.4--8 nM), whereas it is only a moderate inhibitor of ENT2 (IC~50~ ∼3 μM). Based on pharmacokinetic studies of NBTI in mice, we used an NBTI dose that would result in estimated plasma levels between 1 and 10 nM ([@bib29]). To pursue this, we administered NBTI (1 mg/kg i.p.) or vehicle (DMSO) and subjected animals to either normoxia or normobaric hypoxia. As shown in [Fig. 9](#fig9){ref-type="fig"} a, hypoxia significantly increased lung water content in vehicle-treated animals (P \< 0.05). In contrast, lung water was decreased in NBTI-treated animals under normoxic or hypoxic conditions. Measurement of vascular barrier function revealed significantly increased vascular leak with hypoxia in all animals, however to a smaller degree in NBTI treated animals (P \< 0.05 for all organs; [Fig. 9](#fig9){ref-type="fig"} b). Finally, we also screened multiple mucosal organs for PMN accumulation ([Fig. 9](#fig9){ref-type="fig"} c). Consistent with the aforementioned results with dipyridamole, we found decreased MPO activity in all organs of NBTI-treated animals after hypoxia exposure (P \< 0.05 for all organs). These in vivo results suggest that inhibition of ENT1 contributes to the in vivo resuscitation of vascular leak syndrome by nucleoside transport inhibitors. {#fig9} Role of HIF in epithelial ENT1 expression in vivo ------------------------------------------------- We extended these findings of HIF-1--mediated repression of ENT1 expression into a genetic in vivo model. Here, we examined baseline expression and the influence of hypoxia on mENT1 mRNA levels in intestinal epithelia derived from conditional Hif1α knockout mice, in which intestinal epithelia from these mice lack detectable Hif1α expression in \>70% of cells ([@bib3]). As shown in [Fig. 10](#fig10){ref-type="fig"}, ENT1 levels in mice expressing wild-type Hif1α showed a normal pattern of hypoxia-associated ENT repression (68 ± 3% decrease after hypoxia for 6, P \< 0.025). Consistent with our hypothesis that HIF-1 transcriptionally represses ENT1, real-time PCR analysis revealed a 37 ± 6.8-fold increase in intestinal epithelial ENT1 expression in Hif1α mutant animals ([Fig. 10](#fig10){ref-type="fig"}), relative to their littermate controls (P \< 0.01). Exposure of HIF1α mutants to hypoxia decreased ENT1 expression (P \< 0.05), but to a far lesser extent than in wild-type animals. These latter findings likely reflect the remaining ∼30% wild-type Hif1α expression in these mice ([@bib3]). Together, such findings support our in vitro findings and indicate the likelihood that HIF-1 directly regulates murine ENT1 expression. {#fig10} Discussion ========== Ado exerts paracrine and autocrine functions on most cell types. Pathophysiologic conditions of hypoxia/ischemia result in numerous adenine nucleotide metabolic changes, and targets for extracellular Ado signaling is now an area of much interest. In the present studies, we explored the mechanisms of extracellular Ado accumulation during hypoxia. For these studies, we used vascular endothelial and mucosal epithelial cells, which both lie anatomically positioned to function as determinants of the inflammatory response. Our results revealed that hypoxia increases extracellular Ado half-life. This response is mechanistically determined, at least in part, by HIF-1--regulated ENT1 repression. The results from the present study are consistent with previous findings that hypoxia inhibits intracellular metabolism of Ado to AMP. Decking et al. showed, by measuring Ado and AMP concentrations in the coronary sinus and coronary arteries of isolated guinea pig hearts, that hypoxia is associated with a functional inhibition of the Ado kinase ([@bib30]). The authors propose that hypoxia-associated inhibition of the Ado kinase leads to increased intracellular Ado levels. It is known that during hypoxia, Ado flux is predominantly directed from the extracellular to the intracellular space ([@bib14]). Therefore, two cellular strategies with regard to Ado transport may play a role to elevate extracellular Ado levels during hypoxia. First, as described by Decking et al., increased intracellular Ado levels due to inhibition of Ado kinase--dependent metabolism of Ado to AMP would decrease the transcellular Ado gradient, thereby decreasing flux through bidirectional ENTs and, thus, elevating extracellular Ado levels during hypoxia ([@bib30]). Second, transcriptional repression of ENTs, as we demonstrate in the present work, decreases overall equilibrative Ado transport capacities, thereby decreasing intracellularly directed Ado transport. It should be noted that we have not observed a direct change in ENT function by hypoxia per se. Rather, our studies addressed function as it related to expression levels of ENT. From this standpoint, it is likely that inhibition of adenine nucleotide metabolism (e.g., inhibition of Ado kinase) could serve as an immediate response to hypoxia (within minutes), whereas our proposed transcriptional mechanism serves as a more adaptive phenotype (within hours). Therefore it is reasonable that both mechanisms could function during hypoxia and, from this perspective, contribute synergistically to elevate extracellular Ado during hypoxia. Consistent with recent work in cardiac myocytes ([@bib16]), the present studies in vascular endothelia and mucosal epithelia identified transcriptional repression of ENT as a mechanism for such decreased Ado uptake during hypoxia. Given the temporal and robust hypoxia response observed in the repression of ENT1, a candidate regulator was HIF-1, a heterodimeric transcription factor whose activation is dependent on stabilization of an O~2~-dependent degradation domain ([@bib31]). It is recently appreciated that HIF can function as both a transcriptional activator as well as a repressor ([@bib19]). Therefore, a search of the cloned ENT1 gene promoter identified two potential HIF-1--binding DNA consensus motifs located in the distal 5′ region of the ENT1 promoter. However, the existence of a HIF-1α--binding consensus is not evidence for HIF-1α--mediated response; instead, the HRE is defined as a cis-acting transcriptional regulatory sequence located within 5′-flanking, 3′-flanking, or intervening sequences of target genes ([@bib32], [@bib33]). Therefore, two strategies were used to define the role of HIF-1 in repression of ENT1. First, mutational analysis of ENT1 promoter constructs defined a functional HRE located in the distal 5′region of the ENT1 promoter (−717 to −721 relative to the major transcription start site). Second, ChIP analysis confirmed HIF-1α binding to the ENT1 promoter. At present, we do not know if this binding is direct or indirect, and we do not know the nature of HIF-mediated transcription. While previous work with peroxisome proliferator-activated receptor α (PPAR-α) gene implicated potential repressor activity with HIF-1α binding sites oriented on the antisense strand ([@bib20]), implying some degree of transcriptional directionality, there is not direct evidence for such a mechanism. Moreover, while a recent study comparing transcriptional responses between hypoxia and constitutively active HIF1-1α identified a large cohort of transcriptionally repressed genes ([@bib19]), no unique patterns of HRE expression were noted. Thus, more work will be necessary to define the nature of HIF-mediated repression. The present study also identified ENT1 as a potential physiologic target in vivo. Indeed, using the ENT inhibitor dipyridamole, we demonstrated that blockade of ENT1 prevents vascular leakage associated with hypoxia, presumably through accumulation of vascular Ado. Several lines of evidence support the presumption that elevated Ado attenuates vascular leakage. First, Nozik-Grayck determined that dipyridamole effectively attenuates edema in an isolated perfused lung model of hyperoxia ([@bib34]). Second, in murine models of hypoxia, decreased vascular Ado is associated with substantially increased vascular leakage ([@bib8], [@bib10], [@bib22]). In particular, these observations have been made in animals lacking the ability to metabolize adenine nucleotide precursors into Ado, including *cd39* and *cd73*-null animals ([@bib8], [@bib10], [@bib22]). Third, the administration of Ado analogues in the form of Ado receptor A2a or A2b agonists, as well as the nonspecific analogue 5′-(N-ethylcarboxamido)-Ado (NECA), protect the vasculature during hypoxia-induced leakage ([@bib10]). This hypothesis was also tested in a conditional *Hif1*α mutant mice, a mouse line that has revealed a protective role for murine HIF-1α in a mucosal inflammation model ([@bib3]). Using this same mouse line, we confirmed our hypothesis that HIF transcriptionally represses ENT1. Indeed, our findings revealed that colonic epithelial cells lacking *Hif1*α express increased ENT1 transcript. Of particular interest, analysis of unmanipulated *Hif1*α mutant mice (i.e., normoxia condition) revealed substantial increases in ENT1. Such findings suggest, as previously speculated ([@bib3], [@bib9]), that HIF may regulate homeostatic gene expression (i.e., "physiologic hypoxia") in tissues with normally low pO~2~ values (e.g., colon) ([@bib35]). These results identify ENT repression as a mechanism for attenuating both inflammatory responses and vascular leakage during hypoxia. Interaction of HIF-1 with the ENT1 promoter region is central to the observed inhibition of ENT1 gene expression. Extensions of these findings will determine whether ENT regulation via HIF-1 might function as a pathway for development of therapies for disorders involving vascular leak syndromes or excessive inflammatory responses. Materials and Methods ===================== Cell culture ------------ HMEC-1 were a gift from F. Candal (Centers for Disease Control, Atlanta, GA) ([@bib36]) and were cultured as described previously ([@bib37]). For preparation of experimental HMEC-1 monolayers, confluent endothelial cells were seeded at ∼10^5^ cells/cm^2^ onto either permeable polycarbonate inserts or 100 mm Petri dishes. Endothelial cell purity was assessed by phase microscopic "cobblestone" appearance and uptake of fluorescent acetylated low-density lipoprotein. Where indicated, T84 cells were cultured as described previously ([@bib9], [@bib38]). Measurement of Ado in supernatants ---------------------------------- HMEC-1 were grown to full confluency and exposed as indicated to normoxia or different degrees of hypoxia. To develop a model for kinetics of endothelial Ado uptake, we first measured Ado uptake in the supernatant of HMEC-1 monolayers with different Ado concentrations (1--500 μM) and found optimal resolution of Ado uptake when using 50 μM Ado concentrations. These conditions were used throughout for further measurements. After washing in HBSS and addition of 50 μM Ado, samples from the supernatant were collected at indicated time points, and Ado concentrations were resolved by HPLC with a pump P680 and an UVD 170 detector on a reverse-phase column (Grom-Sil 120-ODS-ST-5μ; 150 × 3 mm Grom) using a mobile phase gradient from 0 to 25% acetonitrile/0.3 mM KH~2~PO~4~ (pH 5) in 5 min. Ultraviolet absorption spectra were measured at 260 nm with a retention time for Ado of 2.8 min. Measurement of Ado uptake. -------------------------- HMEC-1 were grown to full confluency and exposed as indicated to normoxia or hypoxia. To measure endothelial Ado uptake, 10 nCi of 8-^14^C-Ado (Sigma-Aldrich) was added to the supernatant, in addition to a 50 μM concentration of "cold" Ado. After indicated time periods, cells were washed with HBSS and lysed in cold H~2~O. Assessment of intracellular Ado uptake was done by measuring the radioactivity within the lysates by directly diluting the lysate into the scintillation mixture (Ultima Gold HFL-LSC-Cocktail; Packard) and counting decays per minute (Liquid Scintillation Counter Packard 1600). Transcriptional analysis ------------------------ Conventional PCR was used to verify endothelial ENT1 and ENT2 mRNA regulation, as described previously ([@bib38]). The PCR reaction contained 1 μM each of the sense primer 5′-AAGGCACCTGGTTTCTGTC-3′ and the antisense primer 5′-CTTGGGCTTGGAGAACAC-3′ for ENT1, and 5′-CTTCCATACCCACTCTCTCACC-3′ and 5′-GAGAGAGAGGGGATTGGGTC-3′ for ENT2, respectively. The primer set was amplified using increasing numbers of cycles of 94°C for 1 min, 58°C for 2 min, 72°C for 4 min, and a final extension of 72°C for 7 min. The PCR transcripts were visualized on a 1.5% agarose gel containing 5 μg/ml of ethidium bromide. Human β-actin (sense primer, 5′-TGACGGGGTCACCCACACTGTGCCCATCTA-3′ and antisense primer, 5′-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3′) in identical reactions was used to control for the starting template. In subsets of experiments, the transcriptional profile of endothelial cells subjected to normobaric hypoxia (12 h) was compared in RNA derived from control or hypoxic endothelia using quantitative genechip expression arrays (Affymetrix, Inc.) as described before ([@bib9]). Where indicated, mRNA was also quantified by real-time PCR (iCycler; Bio-Rad Laboratories Inc.) as described previously ([@bib9]). The primer sets contained 1 μM sense and 1 μM antisense containing SYBR green I (GE Healthcare) in the reaction mixture. Transcript levels and fold change in mRNA were determined as described previously ([@bib39]). In addition, real-time PCR was performed from RNA isolations of human saphenous vein after ex vivo exposure to hypoxia. After approval by the institutional review board, saphenous vein material was obtained from patients undergoing aorta-coronary bypass surgery. Equal portions of dissected vein tissue were subjected to hypoxia (pO~2~ 20 torr) immediately after the operation for different time points (0, 2, 8, and 24 h). After hypoxia exposure, total RNA was isolated from the tissues and real-time PCR was performed as described before. Macromolecule paracellular permeability assay --------------------------------------------- Using a modification of methods previously described ([@bib18]), HMEC-1 were grown on polycarbonate permeable supports (0.4-μm pore, 6.5-mm diam; Costar Corp.) and studied 7--10 d after seeding (2--5 d after confluency). Inserts were placed in HBSS-containing wells (0.9 ml), and HBSS (alone or with indicated concentrations of Ado, with and without 10 μM concentration of dipyridamole) was added to inserts (100 μl). At the start of the assay (t = 0), FITC-labeled dextran 70 kD (concentration 3.5 μM) was added to fluid within the insert. The size of FITC-dextran, 70 kD, approximates that of human albumin, both of which have been used in similar endothelial paracellular permeability models ([@bib18]). Fluid from opposing well (reservoir) was sampled (50 μl) over 60 min (t = 20, 40, and 60 min). Fluorescence intensity of each sample was measured (excitation, 485 nm; emission, 530 nm; Cytofluor 2300; Millipore Corp., Waters Chromatography) and FITC-dextran concentrations were determined from standard curves generated by serial dilution of FITC-dextran. Paracellular flux was calculated by linear regression of sample fluorescence. Immunoblotting experiments -------------------------- HMEC-1 were grown to confluency on 100-mm dishes and exposed to indicated experimental conditions. The monolayers were lysed for 10 min in 300 μl lysis buffer (150 mM NaCl, 25 mM Tris, pH 8.0, 5 mM EDTA, 2% Triton X-100, and 10% mammalian tissue protease inhibitor cocktail; Sigma-Aldrich), scraped and collected into microfuge tubes. After spinning at 14,000 *g* to remove cell debris, the pellet was discarded. Proteins were solublized in nonreducing Laemmli sample buffer and heated to 90°C for 5 min. Samples were resolved on a 12% polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were blocked for 1 h at room temperature in PBS supplemented with 0.2% Tween 20 (PBS-T) and 4% BSA. The membranes were incubated in 5 μg/ml polyclonal rabbit anti-ENT1 in PBS-T for 1 h at room temperature, followed by 10 min washes in PBS-T. The membranes were incubated in 1:3,000 goat anti--rabbit IgG (ICN Biomedicals/Cappel), and conjugated to horseradish peroxidase for 1 h at room temperature. The wash was repeated and proteins were detected by enhanced chemiluminescence. To control for protein loading, blots were stripped and reprobed for β-actin using a mouse monoclonal anti--human β-actin antibody (Abcam Inc.). In subsets of experiments, identical conditions were used to examine HIF-1α protein levels in nuclear lysates derived from HMEC-1. For these purposes, 1 μg/ml of monoclonal mouse anti--HIF-1α (Novus Biologicals) was used. ENT1 and ENT2 suppression with RNA interference. ------------------------------------------------ HMEC-1 were either grown on inserts or in 60-mm Petri dishes. Different sets of siRNA directed against human ENT1 and ENT2 were designed using standard molecular tools, synthesized by Dharmacon, and tested with RT-PCR for their efficiency to suppress ENT1 and ENT2 expression at different concentrations. Highest efficiencies were found with 5′-CGGCCACUCAGUAUUUCACdTdT-3′ (sense strand) and 5′-GUGAAAUACUGAGUGGCCGdTdT-3′ (antisense strand) for ENT1 and 5′-CUCUCUCACCGAAGCCUAAdTdT-3′ (sense strand) and 5′-UUAGGCUUCGGUGAGAGAGdTdT-3′ (antisense strand) for ENT2. As nonspecific control, the sense strand of the ENT2 oligoribonucleotide was used under identical conditions. As additional control, knockdown with a set of siRNA directed against human 5′-ectonucleotidase (CD73) was performed using the sense sequence 5′-CUAUCUGGUUCACCGUGUAdTdT-3′ (sense strand) and 5′-UACACGGUGAACCAGAUAGdTdT-3′ (antisense strand). HMEC-1 loading was accomplished using standard conditions of Fugene6 (Roche Diagnostics Corp.), when cells had reached 40--60% confluence. After 48 h of loading, RNA was isolated as described before or functional assays (flux assays, Ado uptake) were performed. ENT1 reporter assays -------------------- Plasmids expressing sequence corresponding to full-length ENT1 (−756 to +1), or the following 5′ truncations: ENT1-344 (bp −334 to +1), ENT1-282 (bp −282 to +1), ENT1-225 (bp −225 to +1), have been previously characterized (unpublished data). As a control for hypoxia, cells were transfected with a PGL3-based HRE plasmid containing four tandem HIF-1 enhancer sequences from the 3′-region of the erythropoietin gene ([@bib40]). HMEC-1 were cotransfected with constitutively expressed Renilla plasmids using standard methods of overnight transfection using polyfect transfection reagent (QIAGEN). T84 cells were transfected using solution T and a modified form of electroporation (Amaxa Inc.). After transfection, cells were subjected to hypoxia or normoxia for 48 h. Luciferase activity was assessed (Turner Designs) using a luciferase assay kit (Promega). All firefly luciferase activity was normalized with respect to the constitutively expressed Renilla luciferase reporter gene. In subsets of experiments, HIF-1--binding site mutations were introduced in ENT1-756 plasmids using the GeneEditor in vitro site-directed mutagenesis system (Promega). In brief, mutations encoding two nucleotide mutations ENT1 HIF-1 binding sites at positions −721 and −657, respectively, as described previously. All mutations were confirmed by sequencing using pGL3-basic primers. Hypoxia inducibility in transient transfectants using such mutated luciferase constructs was exactly as described in the previous paragraph. ChIP assay ---------- ChIP assays were performed using HMEC-1 subjected to normoxia or hypoxia. In brief, 2 × 10^7^ cells were fixed with 1% paraformaldehyde for 10 min. Cross-linking was stopped by the addition of 125 mM glycine, and chromatin derived from isolated nuclei was sheared using a F550 micro-tip cell sonicator (Fisher Scientific). After centrifugation, supernatants containing sheared chromatin were incubated for 4 h with 5 μg of anti--HIF-1α antibody (Novus Biologics) or control IgG. Protein A sepharose was added and the incubation continued overnight at 4°C. Immune complexes were washed extensively and eluted from the protein A sepharose. The supernatants were transferred to a new tube, and 1 μg/μl of RNase was added and incubated for 5 h at 67°C. Samples were frozen at −80°C and 60 μg/μl proteinase K was added and incubated for 2 h at 45°C. Next, samples were diluted with TE containing 10 μg of tRNA followed by one extraction with phenol/chloroform and one extraction with chloroform. DNA was precipitated from the samples, washed, dried, and resuspended in 30 μl of autoclaved water. 2 μl of sample was used for each PCR reaction. The sequences of the ENT1 promoter-specific primers spanning the putative HIF-1--binding region were as follows: sense, 5′-GTGTCAGTGCACATCTGCCTGGC-3′ and antisense, 5′-CCTGTCCGCTTCCCCTTTCTAAG-3′. The size of the amplified product resulting from the use of this primer pair was 192 bp. Lentiviral vector design, production, and transduction in T84 cells. -------------------------------------------------------------------- The HIV-1 lentiviral vector used was based on a vector previously described in detail ([@bib41]). In short, the ΔODD variant of HIF-1α (containing a proline→alanine substitution at position 564; the ODD mutant HIF-1α plasmid was provided by H. Franklin Bunn \[Harvard Medical School, Boston, MA\]) ([@bib21]) was cloned into the Bam HI/ClaI sites of the lentiviral vector. Virus was produced by first transfecting 293T cells with 3.2 μg of vector, 4.0 μg of the packaging plasmid, and 0.4 μg of REV, TAT, and VSV-G and collecting and filtering the cell supernatant through a 0.45-μm filter. Viral titers were determined by FACS analysis and/or quantitative Southern blot analysis. T84 cells were plated at a density of 2 × 10^5^ cells/10cm^2^ dish 24 h before transduction. These cells were transduced using 2 × 10^6^ infectious U/ml (MOI = 10) of viral supernatant along with 5 μg/ml protamine sulfate for 3 h at 37°C. Cells were fed and propagated in standard T84 media. In vivo hypoxia model. ---------------------- C57BL/6/129 svj mice were matched according to sex, age, and weight. Total organ vascular permeability was quantified by intravascular administration of Evan\'s blue as described previously ([@bib42]). Animals were injected with dipyridamole (10 mg/kg i.p. and 10 mg/kg s.c) or of S-(4-nitrobenzyl)-6-thioinosine (NBTI, 1 mg/kg i.p.\[Sigma-Aldrich\]) or vehicle (DMSO). For the purpose of quantifying vascular permeability, 0.2 c.c. of Evan\'s blue (0.5% in PBS) were injected intravenously. Animals were exposed to normobaric hypoxia (8% O~2~, 92% N~2~) or room temperature air for 4 h (*n* = 6 animals per condition). After hypoxia/normoxia exposure, the animals were killed and the colon, kidney, liver, and lungs were harvested. Organ Evan\'s blue concentrations were quantified after formamide extraction (55°C for 2 h) by measuring absorbances at 610 nm with subtraction of reference absorbance at 450 nm. Pulmonary edema was assessed in additional experiments. For this purpose, lungs of the animals (*n* = 6) were collected, weighed, and dried by speed-vac (Eppendorf Vacufuge). Weight differences before and after drying were used to calculate lung water content. For the purpose of quantifying PMN tissue concentrations, the animals were exposed to normobaric hypoxia (8% O~2~, 92% N~2~) or room temperature air for 4 h (*n* = 4--6 animals per condition). After hypoxia/normoxia exposure, the animals were killed, organs were harvested, and the PMN marker MPO was quantified as previously described ([@bib43]). Plasma levels of dipyridamole were determined by HPLC with a pump P680 and a Hitachi Fluorescence Detector L-7480 on a reverse-phase column (Grom-Sil 120-ODS-ST-5μ; 150 × 3 mm Grom) using a mobile phase gradient from 0 to 33% acetonitrile/0.3 mM KH~2~PO~4~ (pH 5) in 10 min. Fluorescence was measured at 404 nm excitation and 480 nm emission wavelength, with a retention time for dipyridamole of 8 min. In subsets of experiments, colonic mucosal scrapings (enriched in epithelial cells) were obtained from 6--8-wk-old conditional *Hif1*α mutant mice or littermate controls, as described before ([@bib3]). Scrapings were homogenized in RNAlater (QIAGEN) using a 22-gauge syringe (Becton Dickinson) and Qiashredder column (QIAGEN). RNA extraction including DNase digestion was performed using the RNeasy kit (QIAGEN). Reverse-transcription was done using the iScript cDNA Synthesis Kit (BioRad Laboratories). Amplification was performed on an i-Cycler IQ real-time PCR detection system (BioRad Laboratories) using the gene-specific primers for ENT1 as described before. These protocols were in accordance with National Institutes of Health guidelines for use of live animals and were approved by the Institutional Animal Care and Use Committee at Brigham and Women\'s Hospital. Data analysis. -------------- Data were compared by two-factor analysis of variance, or by Student\'s *t* test where appropriate. Values are expressed as the mean ± SD from at least three separate experiments. The authors wish to thank Dr. P. Allen for advice on lentiviral construction and A. Mager, S. Zug, M. Faigle, and M. Hochgutbrot for technical assistance. This work was supported by Fortune grant no. 1319-0-0 and DFG grant no. EL274/2-2 (to H.K. Eltzschig), National Institutes of Health grant nos. HL60569 and DK50189 (to S.P. Colgan), and by grants from the NSERC and PREA (to I.R. Coe). The authors have no conflicting financial interests. [^1]: CORRESPONDENCE Sean P. Colgan: <[email protected]>

Introduction {#s1} ============ The thylakoid lipid bilayer formed by amphipathic glycerolipids serves as a matrix embedded with photosynthesis protein--cofactor complexes forming the electron transport chain. The lipid composition of the thylakoid membrane is highly conserved among cyanobacteria and plant chloroplasts, with glycolipids as major constituents (Mizusawa and Wada, [@B30]). The only major phospholipid in the thylakoid membrane is phosphatidylglycerol (PG), an anionic lipid with a negative charge in the phosphoglyceryl head group attached to the diacylglycerol backbone. Although PG accounts only for \~10 mol% of total thylakoid lipids, this lipid has crucial roles in oxygenic photosynthesis, as described later. In addition to having a role as building blocks of the thylakoid membrane, lipids serve as structural components of membrane protein complexes to support their structure and function. Many lipid molecules are present in photosystem I (PSI) and PSII complexes. Crystallography analysis of the PSII dimer complex from *Thermosynechococcus vulcanus* at 1.9-Å resolution identified 20 lipid molecules per monomer in the structure; 5 molecules were PG, which were buried near the reaction center with their head groups facing the cytoplasmic side (Umena et al., [@B43]). In the PSII complex, 3 PGs were located around the primary electron acceptor (Q~A~) binding site, one was present at the interface between D1 and CP43 and the other was located near the secondary electron acceptor (Q~B~) binding site. In the crystal structure of the PSI complex from *Thermosynechococcus elongatus* at 2.5-Å resolution, 4 lipid molecules were assigned per monomer, and 3 of the 4 molecules were PG (Jordan et al., [@B16]). One of the 3 PG molecules was located near the reaction center, and the other 2 located between the PsaB and PsaX subunits and near the monomer--monomer interface of the trimeric PSI complex, respectively. Moreover, x-ray crystallography analysis of the light-harvesting complex I (LHCI)-PSI complex from pea at 2.8 Å identified 6 PG molecules in addition to 4 molecules of other thylakoid lipids; 3 PGs were found in the PSI core and another 3 were in the LHCI complex (Qin et al., [@B35]). PG is also structurally involved in LHCII; in LHCII crystal structures from spinach at 2.7 Å (Liu et al., [@B27]) and pea at 2.5 Å (Standfuss et al., [@B39]), one PG molecule was buried at the monomer-monomer interface with the *trans*-Δ^3^-hexadecenoic acid at the *sn*-2 position penetrating the deep binding pocket of the trimer. The role of PG in photosynthesis was initially examined in phospholipase-treated thylakoid membranes in which PG was specifically degraded. Elimination of up to 70% of the original PG from pea thylakoid membranes disrupted electron transport in PSII without decreasing PSI activity (Jordan et al., [@B15]; Droppa et al., [@B5]). Moreover, the PSII dimer from spinach (Kruse et al., [@B25]) and LHCII trimers from pea (Nussberger et al., [@B33]) were dissociated into monomers by phospholipase A~2~ treatment, which suggests a requirement of PG for the structural organization of the PSII-LHCII complex. Likewise, phospholipase A~2~ treatment to thylakoid membranes from *Arabidopsis thaliana* inhibited the PSII electron transport at both donor and acceptor sides and disassembled the PSII--LHCII complexes into PSII and LHCII monomers (Kim et al., [@B18]). Reverse genetic approaches have greatly contributed to unraveling the functions of PG in oxygenic photosynthesis. Both in plants and cyanobacteria, PG is synthesized from phosphatidic acid through PG phosphate (PGP) as an intermediate (Mizusawa and Wada, [@B30]). In *Synechocystis* sp. PCC 6803 (hereafter *Synechocystis* 6803), a mutant of the *pgsA* gene encoding PGP synthase was made and characterized to study the role of PG (Hagio et al., [@B10]). The mutant was unable to synthesize PG but could grow photoautotrophically with PG exogenously supplemented to the growth media. After deprivation of PG from the growth media, the photosynthetic activity of the *pgsA* mutant rapidly decreased with concomitant decrease of PG content, mainly due to the severely impaired PSII function. In the mutant, deprivation of PG inhibited electron transport from Q~A~ to Q~B~ (Gombos et al., [@B8]; Itoh et al., [@B13]) and destabilized the Mn cluster of the oxygen-evolving complex by dissociating extrinsic proteins (PsbO, PsbV, and PsbU) from the PSII core (Sakurai et al., [@B36]). The activity of PSI was decreased in *pgsA* as well but only after a longer period of PG deficiency (Domonkos et al., [@B4]). Long-term PG deprivation induced accumulation of PSI monomer with a decrease in PSI trimer, which is consistent with the existence of a PG molecule in the monomer-monomer interface of PSI from *T. elongatus* (Jordan et al., [@B16]). In higher plants, PG is synthesized in plastids, mitochondria, and endoplasmic reticula (ER) membranes (Wada and Murata, [@B44]). In *A. thaliana*, two isoforms of PGP synthase (PGP1 and PGP2) have been identified; PGP1 is dual localized to plastids and mitochondria (Babiychuk et al., [@B3]), whereas PGP2 is in the ER (Tanoue et al., [@B41]). Knockout mutations of PGP1 decreased PG content in leaves by \~80% from wild-type levels and impaired thylakoid membrane development in chloroplasts (Hagio et al., [@B11]; Babiychuk et al., [@B3]). In a T-DNA insertional PGP1 knockout mutant (*pgp1-2*), amounts of PSI and PSII core proteins were substantially decreased with strong transcriptional downregulation of photosynthesis-associated genes (Kobayashi et al., [@B20]). The photochemical efficiency of PSII was greatly decreased in *pgp1-2*, and further depletion of PG in the mutant by phosphate starvation caused complete loss of the PSII activity (Kobayashi et al., [@B20]). Despite the severe perturbation of chloroplast functions in *pgp1-2*, the morphology of mitochondria was similar to that in the wild type (Hagio et al., [@B11]; Babiychuk et al., [@B3]). A double knockout mutant of PGP1 and PGP2 (*pgp1-2 pgp2*) has an embryonic lethal phenotype with complete loss of PG biosynthesizing activity (Tanoue et al., [@B41]), so PG biosynthesis by PGP2 at the ER can complement the loss of PGP1 activity in mitochondria but not in chloroplasts in the *pgp1-2* mutant. Studies in phospholipase-treated thylakoid membranes of plants and in cyanobacterial mutants deficient in PG biosynthesis have elucidated a special requirement of PG for photochemical and electron transport reactions particularly in PSII, as described earlier. Meanwhile, some parts of the PG functions can be substituted by sulfoquinovosyldiacylglycerol (SQDG), another anionic lipid in the thylakoid membrane (Güler et al., [@B9]; Essigmann et al., [@B7]; Yu and Benning, [@B46]). Moreover, as suggested by no or negligible photosynthetic defects in a PGP1 point mutant (*pgp1-1*) with an 80% reduction in the PGP1 activity (Xu et al., [@B45]; Yu and Benning, [@B46]), the effect of reduced PG content on photosynthesis may be alleviated to some extent by fine-tuning the photosynthetic systems and lipid metabolism during plant growth. By contrast, the effect of severe loss of plastidic PG biosynthesis on photosynthesis during plant growth is largely unknown. To gain deep insight into *in vivo* effects of PG biosynthesis on photosynthetic machinery during plant growth, we examined the activity and functionality of photosynthetic components in the PGP1-knockout *pgp1-2* mutant. Materials and methods {#s2} ===================== Plant materials and growth conditions ------------------------------------- The wild type and *pgp1-2* mutant (the KG10062 line; Hagio et al., [@B11]) were the Columbia ecotype of *A. thaliana*. Surface-sterilized seeds were cold-treated at 4°C for 3 days in the dark before germination. Unless otherwise stated, plants were grown on Murashige and Skoog medium (adjusted to pH 5.7 with KOH) containing 3% (w/v) sucrose solidified with 0.8% (w/v) agar in plates at 23°C under continuous white light (15 μmol photon m^−2^ s^−1^). Wild-type and *pgp1-2* plants were grown in a growth chamber (CLE-303, Tomy Seiko, Tokyo) for 14 and 21 days, respectively, to equalize their developmental stages. Pigment determination --------------------- Lipophilic pigments were extracted with 80% (v/v) acetone from leaves or thylakoid membrane fractions, and debris was removed by centrifugation at 10,000 × *g* for 5 min. The absorbance of the supernatant at 720, 663.2, 646.8, 645, and 470 nm was measured by using the V-730 BIO spectrophotometer (JASCO, Tokyo). The chlorophyll (chl *a* and *b*) and carotenoid contents were determined as described in Melis et al. ([@B28]) and Lichtenthaler ([@B26]), respectively. Imaging analysis of chlorophyll fluorescence -------------------------------------------- Seedlings grown on agar plates were dark-incubated for 15 min before measurement. Minimum chlorophyll fluorescence (Fo) and maximum quantum yield of PSII (Fv/Fm) were determined under a saturating pulse with the IMAGING-PAM MAXI chlorophyll fluorometer and the ImagingWin software (Heinz Walz, Effeltrich, Germany). Lowest intensity of measuring light was used to minimize the photosynthetic effect of measuring light. Photochemical efficiency analysis under light --------------------------------------------- Photochemical efficiency of PSII under actinic light in leaves was determined with the JUNIOR-PAM chlorophyll fluorometer and the WinControl-3 software (Heinz Walz). Light-response curves of effective quantum yield of PSII (Y~II~) and quantum yield of non-regulated energy dissipation (Y~NO~) were determined as described in Kobayashi et al. ([@B22]). To determine the contribution of photoinhibition-related non-photochemical quenching (qI) to total non-photochemical quenching (qN), leaves dark-adapted for 15 min were exposed to light stress (125 μmol photon m^−2^ s^−1^) for 10 min and total qN was measured at the end of the light stress. After relaxation of the rapidly reversible qN component with additional dark treatment for 15 min, the remaining qN component was determined as qI. Measurement of red-light induced photoinhibition ------------------------------------------------ Leaves detached from seedlings were vacuum-infiltrated with 150 mM sorbitol containing 0 or 1 mg/ml lincomycin and incubated under dim light at room temperature for 3 h. Then leaves were illuminated with or without red LED light (660 nm peak wavelength) (VBL-SL150-RB, Valore, Kyoto, Japan) for 1 h. Red light intensity was measured with the LI-250A light meter and the LI-190SA quantum sensor (LI-COR, Lincoln, USA). Fv/Fm in illuminated leaves was measured with the JUNIOR-PAM fluorometer under lowest measuring light intensity after incubation in darkness for 15 min. Electron paramagnetic resonance (EPR) spectroscopy -------------------------------------------------- Seedlings were incubated in a growth chamber at 23 or 45°C for 16 h before measurement. EPR spectra from Mn^2+^ were directly obtained from fresh leaves placed in a quartz EPR tube (Tokyo Chemical Industry, Tokyo) with a JES-TE300 X-band (9.2 GHz) spectrometer (JEOL, Tokyo) at room temperature. Measurement conditions were frequency = \~9.18 GHz, power = \~1.0 mW, center field = 325 mT, sweep width = 50 mT, sweep time = 4 min, modulation width = 2 mT, amplitude = 500--1000, time constant = 0.3 s. Signal intensity was normalized with fresh weight of leaf samples. Measurement of fast-induction kinetics of chlorophyll fluorescence in leaves ---------------------------------------------------------------------------- Leaves were treated with or without 40 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) in 150 mM sorbitol by vacuum infiltration, followed by dark-incubation for 5 min. Chlorophyll fluorescence transients in leaves were measured in a logarithmic time series between 30 μs and 20 s after the onset of strong actinic light (1650 μmol photon m^−2^ s^−1^) with a light-emitting diode pump-probe spectrometer (JTS-10, BioLogic, Claix, France). Measurement of decay kinetics of chlorophyll fluorescence in thylakoid membrane fractions ----------------------------------------------------------------------------------------- Thylakoid membrane fractions of 5 μg/ml chlorophyll concentration were prepared under dim light as described in Allahverdiyeva et al. ([@B1]), but with skipping the washing step with a low osmotic buffer. When required, 40 μM DCMU or 400 μM 1,4-benzoquinone (BQ) was added to the thylakoid fraction. We confirmed that 0.1 % (v/v) ethanol and 1% (v/v) dimethyl sulfoxide, the solvents used to dissolve DCMU and BQ, respectively, did not affect the kinetics. Decay kinetics of chlorophyll fluorescence following a single saturation flash was measured between 0.2 ms and 60 s with the FL3500 fluorometer (Photon Systems Instruments, Brno, Czech). Because small artificial signals were recorded even in the blank sample containing only water, they are subtracted from data as background noises. Fv/Fm values of thylakoid samples were calculated from the minimum (Fo) and maximum (Fm) yields of chlorophyll fluorescence measured before and just after the saturating excitation, respectively. Chlorophyll fluorescence analysis in thylakoid membranes at 77K --------------------------------------------------------------- To prepare stacked thylakoid fractions, seedlings were homogenized in a cold grinding buffer (0.4 M sucrose, 5 mM MgCl~2~, 10 mM NaCl, 2 mM EDTA, 2 g/L bovine serum albumin, 2 g/L ascorbic acid, HEPES-NaOH, pH 7.5) under dim light. The homogenates were filtered through a single layer of Miracloth (Merck Millipore, Darmstadt, Germany). After centrifugation at 6000 × *g* for 4 min at 4°C, the pellet was resuspended in a cold resuspension buffer (0.4 M sucrose, 5 mM MgCl~2~, 10 mM NaCl, HEPES-NaOH, pH 7.5) to obtain 1 μg/ml chlorophyll-containing membrane fractions. To prepare unstacked thylakoids, MgCl~2~ and NaCl were eliminated from both the grinding buffer and resuspension buffer. A restacked membrane fraction was obtained by adding 5 mM MgCl~2~ and 10 mM NaCl in a final concentration to the unstacked thylakoid samples, followed by incubation for 1 h at 4°C. Fluorescence emission spectra of chlorophyll proteins at 77K were obtained from thylakoid membrane fractions in liquid nitrogen by using the RF-5300PC spectrofluorometer (Shimadzu, Kyoto, Japan) under 435- nm excitation. Measurement of oxygen-evolving activity in thylakoid membrane fractions ----------------------------------------------------------------------- Thylakoid membranes were prepared as described in Allahverdiyeva et al. ([@B1]). Steady-state rate of oxygen evolution from thylakoids was measured as 23°C with a Clark-type oxygen electrode (Hansatech, King\'s Lynn, UK) using 1 mM 2, 6-dimethyl-1, 4-benzoquinone (DMBQ) under saturating white light. P700 absorbance measurement --------------------------- The light-response curves corresponding to the different redox states of P700 were determined in a batch of leaves at room temperature by measuring the absorbance change at 830--875 nm as a reference with a Dual-PAM-100 (Heinz Walz). Maximum P700 change (Pm) was determined under a saturating far-red light irradiation for 20 s without saturation pulse flash, because saturation pulses caused small artificial signals unaffected by both DCMU and methylviologen (MV) treatments, which was significant in the case of the *pgp1-2* leaves emitting only low signals. The minimum P700 level (Po) in the absence of light was taken as the baseline and the steady-state P700 level (P) was recorded under actinic light. Using these values the ratio of oxidized P700^+^ to total P700 under a given light treatment was calculated as (P-Po)/(Pm-Po). When required, 40 μM DCMU was used to block electron transfer from PSII. P700 oxidation-reduction kinetics in leaves was measured by absorbance changes at 705 nm under a weak far-red excitation with the JTS-10 spectrometer (BioLogic). When required, leaf samples were treated with 1 mM MV by vacuum infiltration before measurement to block cyclic electron transport around PSI. Results {#s3} ======= Impaired photosynthesis with decreased chlorophyll content in *pgp1-2* ---------------------------------------------------------------------- The *pgp1-2* mutant requires exogenous sugars for growth (Hagio et al., [@B11]). This mutant showed better growth with supplementation of 3% (w/v) sucrose than with 1% sucrose in the growth medium (Figure [1A](#F1){ref-type="fig"}). Even in the presence of 3% sucrose, the growth of *pgp1-2* was slower than that of wild type; 21-days-old *pgp1-2* seedlings were at a growth stage similar to that of 14-days-old wild-type plants (Figure [1B](#F1){ref-type="fig"}). Decreasing light intensity from about 15 μmol photon m^−2^ s^−1^ to \< 5 μmol photon m^−2^ s^−1^ did not alleviate the impaired leaf development, whereas stronger light intensity (\~75 μmol photon m^−2^ s^−1^) damaged the *pgp1-2* seedlings (Supplemental Figure [1](#SM1){ref-type="supplementary-material"}). Thus, we compared 21-days-old *pgp1-2* seedlings with 14-days-old wild-type seedlings under the 3% sucrose condition with 15 μmol photon m^−2^ s^−1^ continuous light for further experiments. {#F1} We previously reported that the *pgp1-2* mutation substantially decreased chlorophyll accumulation (Kobayashi et al., [@B20]). As represented by the pale yellow-green leaf color, chlorophyll content in the first and second true leaves of the *pgp1-2* mutant was only 3% of wild-type levels on a fresh weight basis even under the growth condition with 3% sucrose (Table [1](#T1){ref-type="table"}). Carotenoid content was also substantially decreased in *pgp1-2* leaves. In the mutant, ratios of chl *a* to chl *b* and to carotenoids were decreased as compared with the wild type (Table [1](#T1){ref-type="table"}), which suggests greater decrease of photosystem reaction centers than light-harvesting antennas in *pgp1-2*. ###### **Pigment composition in the first and second leaves of seedlings**. **Chl *a* nmol g^−1^ FW** **Chl *b* nmol g^−1^ FW** **Carotenoids μg g^−1^ FW** **Chl *a*/*b* mol/mol** **Chl *a*/Carotenoids mmol/g** ----------- --------------------------- --------------------------- ----------------------------- ------------------------- -------------------------------- Wild type 2276.6±117.1 734.2±37.0 463.8±27.0 3.10±0.01 4.91±0.22 *pgp1-2* 60.6±2.4 28.1±0.9 26.6±0.6 2.16±0.06 2.28±0.12 *Values are mean ± SD (n = 4). Chl, chlorophyll*. Unlike in the wild type, in the *pgp1-2* mutant, chloroplast development in leaves is non-uniform and is limited around vascular tissues in leaves (Hagio et al., [@B11]; Kobayashi et al., [@B20]). To examine a spatial pattern of photosynthetic activity in the *pgp1-2* mutant, we determined Fv/Fm in whole seedlings using an imaging pulse amplitude modulation fluorometer (Figure [1C](#F1){ref-type="fig"}). Consistent with our previous analysis that *pgp1-2* plants had reduced Fv/Fm levels in leaves (Kobayashi et al., [@B20]), the mutant showed very low Fv/Fm values in whole seedlings, with particularly low signals around the shoot apical meristem and petioles. The low Fv/Fm was attributed to high Fo values representing large emission of chlorophyll fluorescence in the dark-adapted state (Figure [1D](#F1){ref-type="fig"}). The higher Fo with lower Fv/Fm in *pgp1-2* mutant than the wild type was also confirmed in thylakoid membrane samples by single-turnover flash-induced chlorophyll fluorescence measurement (Table [2](#T2){ref-type="table"}). These data suggest that PG deficiency globally occurs in photosynthetic membranes and limits the photosynthetic electron transfer in the *pgp1-2* mutant. ###### **PSII activity in isolated thylakoid membranes**. **Fo (a.u.)** **Fm (a.u.)** **Fv/Fm** **O~2~ evolution** ----------- --------------- --------------- --------------- -------------------- Wild type 0.073 ± 0.009 0.266 ± 0.031 0.727 ± 0.006 190±16 *pgp1-2* 0.326 ± 0.031 0.385 ± 0.045 0.151 ± 0.024 22±4 *Thylakoid membrane samples corresponding to 5 μg chlorophyll ml^−1^ were used for both plants. Values are mean ± SD (n = 8 for fluorescence analysis and 3 for O~2~ evolution). Fo, minimum yield; Fm, maximum yield; Fv/Fm, maximum quantum yield of PSII. The steady-state rates of O~2~ evolution (μmol O~2~ mg chlorophyll^−1^ h^−1^) was measured in the presence of 1 mM DMBQ*. PG deficiency in *pgp1-2* plants increases the light susceptibility ------------------------------------------------------------------- To further characterize the functionality of photosynthetic machinery in the *pgp1-2* mutant, we analyzed the quantum yield of PSII (Y~II~) and non-regulated energy dissipation (Y~NO~) under increased actinic light intensity. Both wild-type and *pgp1-2* leaves showed a gradual decrease in Y~II~ in response to increased light intensity (Figure [2A](#F2){ref-type="fig"}). The Y~II~ values were substantially lower in the *pgp1-2* mutant than the wild type mainly due to the decreased intrinsic photochemical efficiency of PSII represented by low Fv/Fm (Figure [1C](#F1){ref-type="fig"}). Meanwhile, Y~NO~ values were consistently higher in *pgp1-2* plants than the wild type under all ranges of light intensity (Figure [2B](#F2){ref-type="fig"}). The higher proportion of light energy dissipation in a non-regulated form (Y~NO~) implies loss of photoprotective mechanisms in the *pgp1-2* mutant (Kramer et al., [@B23]). Although the level of non-photochemical quenching (qN) was similar in the *pgp1-2* mutant and the wild type, the proportion of the photoinhibitory component (qI) to total qN was substantially higher in the mutant; in fact, most of the qN was attributed to the qI component in the mutant (Figure [2C](#F2){ref-type="fig"}). {#F2} To investigate the photoinhibition mechanism in *pgp1-2* plants, we illuminated leaves with various intensities of red light in the presence or absence of lincomycin, an inhibitor of protein synthesis in chloroplasts, for 1 h and then measured Fv/Fm values in the leaves after dark adaptation for 15 min (Figure [3](#F3){ref-type="fig"}). In wild-type leaves, Fv/Fm was only slightly decreased in response to increased red light intensities even in the presence of lincomycin. The data were consistent with observations that red light has little photoinhibitory effect on undamaged leaves (Ohnishi et al., [@B34]; Sarvikas et al., [@B37]; Takahashi et al., [@B40]). By contrast, in the absence of lincomycin, Fv/Fm was strongly decreased in *pgp1-2* leaves in response to increased red light intensity, and the decrease was further enhanced by lincomycin treatment. These data indicate that photoinhibition of PSII is strongly enhanced particularly in the damage process in *pgp1-2* mutant. {#F3} Oxygen-evolving activity of PSII is abolished in *pgp1-2* --------------------------------------------------------- In the *Synechocystis* 6803 *pgsA* mutant, the Mn cluster of the oxygen-evolving complex is destabilized by PG deprivation with the extrinsic proteins being dissociated from the PSII complex (Sakurai et al., [@B36]). To examine the state of Mn in *pgp1-2* cells *in vivo*, we performed EPR spectroscopic analysis in intact leaves (Figure [4](#F4){ref-type="fig"}). The feature of the EPR-spectrum of protein-unbound Mn^2+^ is its sextet signals (Morsy and Khaled, [@B31]). In wild-type seedlings incubated at 23°C, no clear sextet lines from Mn^2+^ were detected. However, Mn^2+^ signals emerged with incubation at 45°C. Thus, in the wild type, most Mn^2+^ ions are integrated into protein systems but are dissociated from the complexes by heat treatment. In *pgp1-2* seedlings, fine sextet lines of Mn^2+^ were detected even at 23°C, so Mn^2+^ ions exist as a free form in the *pgp1-2* seedlings. {#F4} To directly determine the PSII activity in the *pgp1-2* mutant, oxygen-evolving activity was measured in the thylakoid membrane fraction in the presence of the artificial electron acceptor DMBQ (Table [2](#T2){ref-type="table"}). Consistent with the result in the *Synechocystis* 6803 *pgsA* mutant, oxygen-evolving activity was almost abolished in the *pgp1-2* mutant. Energy transfer from antenna pigments to the PSII reaction center is severely impaired in *pgp1-2* mutant --------------------------------------------------------------------------------------------------------- To evaluate electron transport activity in the *pgp1-2* mutant, we analyzed the induction kinetics of chlorophyll fluorescence in leaves in a logarithmic time series (Figure [5](#F5){ref-type="fig"}). The wild-type leaves showed a slow polyphasic increase of chlorophyll fluorescence after actinic light irradiation, which is explained by a stepwise retardation of the photosynthetic electron flow from primary photochemical reactions in PSII to later reduction processes at the acceptor side of PSI (Krause, [@B24]). In the presence of DCMU, which inhibits electron transport from Q~A~ to Q~B~, chlorophyll fluorescence rapidly peaked because reoxidation of Q${}_{\text{A}}^{-}$ was inhibited. In *pgp1-2* leaves, chlorophyll fluorescence quickly increased in the absence of DCMU. In the presence of DCMU, fluorescence increase was further accelerated, although the fluorescence decreased after peaking at about 5 ms. Very fast increase of chlorophyll fluorescence in the *pgp1-2* mutant suggests that the electron-transfer capability of PSII is very limited in the mutant. In *pgp1-2* leaves, particularly in the presence of DCMU, continuous irradiation of strong actinic light might severely damage PSII complexes and cause strong quenching of chlorophyll fluorescence. {#F5} Different characteristics of electron transfer at the PSII acceptor side between *pgp1-2* and *synechocystis* 6803 *pgsA* mutant -------------------------------------------------------------------------------------------------------------------------------- In the *Synechocystis* 6803 *pgsA* mutant, PG deprivation strongly impairs electron transfer from Q~A~ to Q~B~, particularly in the presence of artificial electron acceptors such as BQ (Hagio et al., [@B10]; Gombos et al., [@B8]; Itoh et al., [@B13]). To ascertain whether this also occurs in the *pgp1-2* mutant, we analyzed decay of single flash-induced chlorophyll fluorescence in thylakoid membrane fractions from wild-type and *pgp1-2* leaves (Figure [6](#F6){ref-type="fig"}). The decay profiles reflect reoxidation kinetics of Q~A~, which can be divided into 3 processes (Krause, [@B24]): (1) fast fluorescence decay (\< 1 ms) related to the electron transfer from Q${}_{\text{A}}^{-}$ to a plastoquinone bound to the Q~B~ pocket; (2) middle exponential decay (\~2 ms) related to the reoxidation of Q${}_{\text{A}}^{-}$ by a plastoquinone molecule moving from the plastoquinone pool to the empty Q~B~ pocket; and (3) slow decay (\~1 s) related to reoxidation of Q${}_{\text{A}}^{-}$ by charge recombination with donor-side components. {#F6} In the absence of BQ, fluorescence decay was slower for the *pgp1-2* mutant than the wild type through the fast to the middle phase (Figure [6A](#F6){ref-type="fig"}), which suggests that reoxidation of Q${}_{\text{A}}^{-}$ by Q~B~ and the plastoquinone pool is impaired in *pgp1-2* thylakoids. In the wild type, BQ treatment accelerated the decay of chlorophyll fluorescence particularly in the middle phase (Figure [6B](#F6){ref-type="fig"}). In *pgp1-2* leaves, the fluorescence decay was almost unchanged by BQ treatment, which contrasted with the strong impairment of Q${}_{\text{A}}^{-}$ reoxidation with BQ supplementation in the *pgsA* mutant (Itoh et al., [@B13]). In Figure [6C](#F6){ref-type="fig"}, the variable fluorescence decays with similar kinetics in wild type and the *pgp1-2* mutant. The slow fluorescence decay in the DCMU-treated sample mainly resulted from charge recombination between Q${}_{\text{A}}^{-}$ and the donor-side components. These data suggest that charge recombination with the donor-side components is not notably affected in the mutant but reoxidation of Q${}_{\text{A}}^{-}$ at the acceptor side is retarded independently of the effect of artificial quinones. Cyclic electron flow around PSI is dysfunctional in the *pgp1-2* mutant ----------------------------------------------------------------------- To assess whether the PSI activity is also affected by the *pgp1-2* mutation, we examined the redox state of the primary electron donor chlorophyll (P700) of the PSI reaction center under increased actinic light intensity (Figure [7A](#F7){ref-type="fig"}). In the presence of DCMU, P700 in both wild-type and *pgp1-2* leaves was mostly oxidized even under low actinic light conditions because of the blockage of electron transfer from PSII by DCMU. In the absence of DCMU, P700 in wild-type leaves was highly reduced under low actinic light mainly by linear electron transfer from PSII but was gradually oxidized in response to increased light intensity. By contrast, P700 in *pgp1-2* leaves was highly oxidized under low actinic light irradiation in the absence of DCMU. The data suggest that the electron donation from PSI to the acceptor side is more active than electron supply to PSI from the donor side. {#F7} To further assess the functionality of PSI in the *pgp1-2* mutant, kinetics of P700 oxidation by far-red light irradiation was measured in leaves by monitoring absorbance at 705 nm in the presence or absence of 1 mM MV (Figure [7B](#F7){ref-type="fig"}). MV with this high concentration efficiently accepts electrons from all PSI and thus abolishes the cyclic electron flow around PSI. In wild-type leaves treated with MV, rapid oxidation of P700 was observed as was reported previously (Joliot and Joliot, [@B14]). However, in the absence of MV, kinetics of P700 oxidation was slowed, which indicates reinjection of electrons to P700 via the cyclic pathway. In the *pgp1-2* leaves, P700 oxidation kinetics were similar with and without MV. The data suggest that cyclic electron flow is impaired in the *pgp1-2* mutant. Moreover, as compared with wild-type leaves treated with MV, P700 oxidation kinetics were slower in MV-treated *pgp1-2* leaves. PG deficiency in *pgp1-2* plants perturbs interaction between antenna complexes and reaction centers ---------------------------------------------------------------------------------------------------- Thylakoid membrane lipids greatly affect energetic interactions between reaction centers and antenna complexes. In fact, chlorophyll fluorescence at 77K, which reflects interaction states of photosystem--antenna complexes and structural organization of thylakoids (Krause, [@B24]; Andreeva et al., [@B2]; Kirchhoff et al., [@B19]), was remarkably changed in mutants defective in thylakoid lipid biosynthesis, including the *pgp1-2* mutant (Härtel et al., [@B12]; Kobayashi et al., [@B21], [@B20]). To gain insight into the effect of the lack of PG on protein arrangement in the thylakoid membrane, we examined the effect of cations on membrane organization in the *pgp1-2* mutant by measuring chlorophyll fluorescence spectra at 77K (Figure [8](#F8){ref-type="fig"}). The thylakoid membrane fraction from wild-type leaves showed two major peaks at 682 nm from the PSII-LHCII complex and at 731 nm from the PSI-LHCI complex. In the presence of cations (NaCl and MgCl~2~), LHCII was tightly connected with PSII in stacked grana regions (Kirchhoff et al., [@B19]), which resulted in high fluorescence emission from the PSII complexes (Figure [8](#F8){ref-type="fig"}). Depletion of cations in the buffer induced unstacking of the grana thylakoids and caused a spillover of excitation energy from LHCII to PSI as indicated by increased fluorescence emission from the PSI complex. Supplementation of cations to unstacked thylakoids induced restacking and decreased the emission from PSI probably due to a reduced energetic connection between PSI and LHCII (Kirchhoff et al., [@B19]). {#F8} As reported previously (Kobayashi et al., [@B20]), in *pgp1-2* thylakoids, emissions from PSII and PSI were both blue-shifted to 679 and 724 nm, respectively, which implies dissociation of antenna complexes from each reaction center. As observed in wild-type samples, fluorescence emission from PSI relative to that from PSII was increased in the *pgp1-2* mutant by preparing thylakoids in the absence of cations but was decreased with the addition of cations to the membrane samples (Figure [8](#F8){ref-type="fig"}). The changes in energy distribution between PSII and PSI did not accompany notable shifts of peak wavelength, which suggests that global rearrangement of the thylakoid membrane environment does not affect an intrinsic structure of photosystem core-antenna complexes in the mutant. Discussion {#s4} ========== Pleiotropic effects of the *pgp1-2* mutation on photosynthetic activities in the thylakoid membrane --------------------------------------------------------------------------------------------------- In this study, the *pgp1-2* mutant, which lacks the ability to synthesize PG in plastids (Hagio et al., [@B11]), showed various defects in photochemical and electron transport activities in the thylakoid membrane. The very high Fo levels in the mutant (Figure [1D](#F1){ref-type="fig"} and Table [2](#T2){ref-type="table"}) suggest that the energy transfer from antenna pigments to the open PSII reaction center is severely impaired, which results in loss of absorbed energy as fluorescence and decreased PSII photochemical efficiency. Dissociation of light-harvesting antenna complexes from the PSII core complex (Figure [8](#F8){ref-type="fig"}) may cause the energetic disconnection between the reaction center and antenna complexes in the mutant. Because high Fo is observed in mutants deficient in PSII (Meurer et al., [@B29]; Shikanai et al., [@B38]), PSII core components may be largely deficient in the *pgp1-2* mutant as suggested in the lack of oxygen evolution (Table [2](#T2){ref-type="table"}). Upon illumination, chlorophyll fluorescence from the mutant PSII immediately reached nearly the maximal level (Figure [5](#F5){ref-type="fig"}), which reflects very small electron-accepting capacity of Q~A~ and the plastoquinone pool. Consistent with a larger decrease in reaction center proteins than that in LHC proteins in *pgp1-2* seedlings (Kobayashi et al., [@B20]), the mutant showed lower ratio of chl *a* to chl *b* and to carotenoids (Table [1](#T1){ref-type="table"}), which may explain in part the decreased PSII capacity in *pgp1-2* plants. In addition, accumulation of oxidized P680, the primary electron donor of PSII, with the dysfunctional oxygen-evolving complex may inhibit repeated photochemical reactions in the mutant, thereby causing a rapid rise of chlorophyll fluorescence from the antenna systems. In the presence of DCMU, chlorophyll fluorescence from *pgp1-2* leaves was strongly quenched soon after peaking (Figure [5](#F5){ref-type="fig"}). Interestingly, similar kinetic profiles were observed in spinach monomeric PSII core complexes incubated with PG or SQDG in the absence of DCMU (Kansy et al., [@B17]). Excessive imbalance in the thylakoid lipid composition with more or less anionic lipids may strongly perturb the intrinsic PSII electron transport. *In vitro* biochemical analysis demonstrated that degradation of phospholipids in the pea thylakoid membrane did not decrease PSI activity (Jordan et al., [@B15]). Moreover, in the *Synechocystis* 6803 *pgsA* mutant, decreased PSI activity was observed only after a longer period of PG deprivation (\>2 weeks; Domonkos et al., [@B4]). These data suggest less importance for PG in the function of PSI. Indeed, compared to the crucial defects of the mutant PSII, PSI seemed relatively active in *pgp1-2* plants (Figure [7A](#F7){ref-type="fig"}). Considering the high structural stability of PSI, the interaction of PG molecules with PSI core proteins may be stronger than with PSII, so the effect of PG deprivation may be primarily observed in the PSII function. However, kinetic analysis of P700 oxidation showed that the cyclic electron pathway is dysfunctional in the *pgp1-2* mutant (Figure [7B](#F7){ref-type="fig"}). Moreover, the P700 oxidation kinetics in the presence of MV was slower in the mutant than the wild type, which may be attributed to an enhanced charge recombination within the PSI reaction center and/or inefficient energy transfer from the LHCI as suggested in the 77K chlorophyll fluorescence (Figure [8](#F8){ref-type="fig"}). X-ray crystallographic analyses revealed that PG molecules are structural components of the PSI complex in both cyanobacteria (*T. elongatus*; Jordan et al., [@B16]) and plants (*Pisum sativum*; Qin et al., [@B35]). Therefore, PG in the PSI complex, which may be resistant to phospholipase treatments (Jordan et al., [@B15]) and short-term PG deprivation (Domonkos et al., [@B4]), may also play an important role in the PSI activity. In *pgp1-2*, the PGP2 isoform is functional in ER (Tanoue et al., [@B41]). Although total PG content is substantially decreased in the mutant (Hagio et al., [@B11]; Kobayashi et al., [@B20]), whether PGP2 can provide a significant amount of PG for chloroplasts remains undetermined. We previously reported that phosphate starvation further decreased total PG levels in *pgp1-2* mutant, which resulted in complete loss of Fv/Fm despite of accumulation of thylakoid membrane glycolipids (Kobayashi et al., [@B20]). Thus, PG produced by PGP2 may be partially transported into chloroplasts and support the marginal photosynthetic activity in *pgp1-2* plants, but further depletion of PG by phosphate starvation may completely abolish the PSII photochemical activity. The *pgp1-2* mutation caused strong photodamage ----------------------------------------------- The impaired energy transfer from antenna pigments to the PSII reaction center and decreased electron-accepting capacity of PSII plastoquinones could increase non-photochemical dissipation of light energy within the antenna system. The very high Y~NO~ levels in *pgp1-2* mutant (Figure [2B](#F2){ref-type="fig"}) represent enhanced dissipation of absorbed light energy as a non-regulated form of fluorescence or heat, which indicates limited photoprotective capacity in the mutant. In fact, the non-photochemical quenching in the *pgp1-2* mutant was predominantly caused by photoinhibition of PSII (Figure [2C](#F2){ref-type="fig"}), which is consistent with the *pgp1-2* PSII being very susceptible to photodamage under red light irradiation (Figure [3](#F3){ref-type="fig"}). Recent studies have proposed that photodamage to PSII occurs in two steps: first, the Mn cluster of oxygen-evolving complex is damaged by UV or blue light presumably via direct excitation of Mn, and second, the photochemical reaction center of PSII is inactivated by photosynthetically active light absorbed by chlorophyll (Tyystjärvi, [@B42]; Murata et al., [@B32]). Irradiation of red light alone has little effect on the PSII photoinhibition in the wild type, as was reported previously (Ohnishi et al., [@B34]; Sarvikas et al., [@B37]; Takahashi et al., [@B40]), but it strongly inactivated the PSII photochemical reaction in the *pgp1-2* mutant (Figure [3](#F3){ref-type="fig"}). As suggested by the dissociation of Mn^2+^ ions from protein systems in the mutant leaves (Figure [4](#F4){ref-type="fig"}) and lack of oxygen-evolving activity in the thylakoids from the mutant (Table [2](#T2){ref-type="table"}), the Mn cluster in *pgp1-2* mutant may be dysfunctional, so the mutant PSII may be susceptible to red light. The finding in *Synechocystis* 6803 that PG is required for the stabilization of the Mn cluster in the oxygen-evolving complex (Sakurai et al., [@B36]) supports this hypothesis. Because the effects of the *pgp1-2* mutation on photosynthesis were pleiotropic, other defects such as impaired photochemical reactions or acceptor-side limitations might also be responsible for photoinhibition of the mutant PSII. The severe defects in leaf development with reduced mesophyll cells in *pgp1-2* plants (Hagio et al., [@B11]; Kobayashi et al., [@B20]) may be explained in part by photodamage of developing chloroplasts. Indeed, the growth of *pgp1-2* seedlings was damaged under continuous 75 μmol photon m^−2^ s^−1^ light (Supplemental Figure [1](#SM1){ref-type="supplementary-material"}). However, the development of *pgp1-2* leaves was perturbed even under dim light (\< 5 μmol photon m^−2^ s^−1^), which suggests a specific role of PG synthesized in chloroplasts in leaf development. Effect of the *pgp1-2* mutation on the acceptor side of PSII ------------------------------------------------------------ In *Synechocystis* 6803, deprivation of PG caused impaired electron transport from Q${}_{\text{A}}^{-}$ to Q~B~, and the impairment was further enhanced by BQ treatment (Gombos et al., [@B8]; Itoh et al., [@B13]). Retarded electron transfer at the acceptor side of PSII was also observed in the *pgp1-2* thylakoids (Figure [6A](#F6){ref-type="fig"}), consistent with the reports that phospholipase treatment inhibits electron transport at the Q~B~ site in thylakoid membranes isolated from plant leaves (Droppa et al., [@B5]; Kim et al., [@B18]). However, unlike in the *pgsA* mutant, in *pgp1-2* leaves, BQ treatment did not inhibit Q~A~ reoxidation in thylakoids. In *Synechocystis* 6803, PG deficiency may change the structure around the Q~B~ site so that it becomes inactivated by BQ (Itoh et al., [@B13]). Furthermore, with site-directed mutations in the *Synechocystis* 6803 D1 protein disrupting interactions with PG around the Q~A~ binding site, the Q~B~-related site was sensitive to BQ-mediated inactivation, which suggests PG molecules near the Q~A~ site are important for the function of the Q~B~ site (Endo et al., [@B6]). Therefore, the mode of interaction between PG and proteins in the PSII reaction center may differ between *A. thaliana* and *Synechocystis* 6803, although both organisms require PG to maintain the function of the acceptor side of PSII. Involvement of PG in organization of antenna-core complexes of photosystems --------------------------------------------------------------------------- Biochemical approaches with phospholipases have revealed the importance of PG for the assembly of PSII-LHCII complexes. Phospholipase A~2~ treatments disassembled the spinach PSII dimer into monomers (Kruse et al., [@B25]), pea LHCII trimers into monomers (Nussberger et al., [@B33]), and *A. thaliana* PSII-LHCII complexes into PSII and LHCII monomers (Kim et al., [@B18]). Consistent with these findings, the *pgp1-2* thylakoids at 77K emitted chlorophyll fluorescence at \~680 nm (Figure [8](#F8){ref-type="fig"}), which suggests accumulation of free LHCII uncoupled from PSII. Furthermore, 77K fluorescence emission at \~723 nm suggests dissociation of LHCI from the PSI core, which may cause slower P700 oxidation in the presence of MV by far-red light in *pgp1-2* mutant than the wild type (Figure [7B](#F7){ref-type="fig"}). In the crystal structure of the pea PSI-LHCI complex, 3 PG molecules were identified in LHCI in addition to one PG molecule locating between LHCI and the PSI core (Qin et al., [@B35]). Thus, PG molecules in the complex may be involved in the association of PSI with LHCI. Formation of the supramolecular networks of photosystems in grana thylakoids is self-organized and the balance between negative thylakoid surface charges and cations such as Mg^2+^ would play a crucial role (Kirchhoff et al., [@B19]). Low salt conditions cause an intermixing and randomization of the protein complex along with unstacking of the grana thylakoids, presumably by electrostatic repulsion of negative thylakoid surface charges. Such changes increase spillover of excitation energy from the PSII antenna system to the PSI (Figure [8](#F8){ref-type="fig"}; Kirchhoff et al., [@B19]). Incubation of the unstacked thylakoids with cations induces restacking of grana and rearrangement of PSII-LHCII supercomplexes, which leads to reduced energy spillover from PSII to PSI (Figure [8](#F8){ref-type="fig"}). As in wild-type thylakoids, *pgp1-2* thylakoids show an increase and decrease in the fluorescence emission from PSI in response to elimination and re-addition of cations, respectively. Thus, PG may not be needed for the self-organization of macromolecular protein networks formed in stacked grana thylakoids, although it is essential for the intrinsic assembly of antenna complexes with photosystem reaction centers. These results are consistent with the observation that enhanced biosynthesis of glycolipids including SQDG by phosphate starvation caused extensive stacking of the thylakoid membrane in the *pgp1-2* mutant but could not induce association of LHC antennas with PS core complexes (Kobayashi et al., [@B20]). We previously reported that the amount of PS core proteins was decreased more strongly than that of LHC proteins in *pgp1-2* plants, although downregulation of genes encoding PS core proteins (*psaA* and *psbA*) was weaker than that of *LHC* genes (*LHCA4* and *LHCB6*; Kobayashi et al., [@B20]). Considering the importance of PG for the assembly of PS-LHC complexes, PG in chloroplasts may be required for stabilizing the core proteins during translation or post-translational assembly in complexes. Conclusion {#s5} ========== Loss of plastidic PG biosynthesis by the *pgp1-2* mutation caused severely impaired electron transfer both in donor and acceptor sides of PSII and energetic uncoupling between PSII and LHCII, which would cause strong photodamage to PSII and presumably affected leaf development in part. Functionality of PSI was also affected by the mutation. By contrast, an 80% reduction in the PGP1 activity in *pgp1-1* mutant caused no remarkable defects in photochemical and electron transport activities, although it decreased chlorophyll content and mesophyll cell number (Xu et al., [@B45]; Yu and Benning, [@B46]). Thus, in the *pgp1-1* mutant, photosynthetic efficiency may be maintained by fine-tuning the quality and quantity of photosynthetic components to the reduced PG content and by increasing another anionic lipid, SQDG, to substitute for some functions of PG. However, in the *pgp1-2* mutant, the stress with critical loss of PG in chloroplasts might exceed the homeostatic capacity of plants and cause fatal damage to photosynthetic machinery directly and indirectly. Author contributions {#s6} ==================== HW directed the study. KK and HW designed the experiments. KK and KE performed the experiments and analyzed the data. KK wrote the manuscript. Conflict of interest statement ------------------------------ The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We thank Dr. Krishna K. Niyogi, Department of Plant and Microbial Biology, University of California, Berkeley, for providing access to equipment for photosynthetic measurements and valuable comments. We thank Dr. Atsushi Okazawa, Graduate School of Arts and Sciences, The University of Tokyo, for a technical support in the EPR analysis. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas (No. 24770055 and 26711016). Supplementary material {#s7} ====================== The Supplementary Material for this article can be found online at: <http://journal.frontiersin.org/article/10.3389/fpls.2016.00336> ###### Click here for additional data file. [^1]: Edited by: Julian Eaton-Rye, University of Otago, New Zealand [^2]: Reviewed by: Norihiro Sato, Tokyo University of Pharmacy and Life Sciences, Japan; Yuichiro Takahashi, Okayama University, Japan [^3]: This article was submitted to Plant Cell Biology, a section of the journal Frontiers in Plant Science

Nature Communications 7, Article number: 10945 10.1038/ncomms10945 (2016); Published: 03102016; Updated: 05042016 The author Hagai Cohen is incorrectly omitted from the list of corresponding authors. The corresponding authors are Peter Rez and Hagai Cohen. The correct information for correspondence is: 'Correspondence and requests for materials should be addressed to P.R. (<[email protected]>) or to H.C. (<[email protected]>).\'

Introduction ============ Liver cancer is a malignant tumor of the digestive system with the highest morbidity and mortality rate in China. Early symptoms are often not typical and clinical diagnoses usually occur during middle and advanced stages. Consequently, the surgical resection rate is extremely low at 5--20% ([@b1-ol-0-0-4180]). Local tumor intervention treatment has become a viable option for the treatment of liver cancer. In addition, ultrasound-guided percutaneous ethanol injection (PEI) in tumors and percutaneous radiofrequency ablation (RFA) in early stage and small hepatocellular carcinoma have become increasingly applied. These methods were shown to be effective for obtaining a comparable clinical effect compared to surgical resections ([@b2-ol-0-0-4180]), and also have other advantages such as negligible trauma, extremely low rates of complications and are readily accepted by doctors and patients. However, in cases of tumor ablation without the background of 'pseudocapsule' there is a risk of alcohol diffusion during the injection which may damage the normal liver tissue ([@b3-ol-0-0-4180]). RFA also has a few advantages including 'thermal subsidence' and 'three-dimensional leakage effect'. Nevertheless, it is not effective in cases of larger tumors and tumors located in particular areas of the body, such as the aorta, diaphragmatic surface, gall bladder and heart ([@b4-ol-0-0-4180]). Several studies on the application of PEI in combination with RFA for the treatment of small hepatocellular carcinoma and liver cancer are available ([@b5-ol-0-0-4180]). However, the application of PEI in combination with RFA to treat liver cancer in middle and advanced stages has yet to be examined. Patients and methods ==================== ### Patients Between March 2013 and March 2015, 100 patients diagnosed with stage III--IV (Union for International Cancer Control stages) liver cancer were selected. The study conformed to interventional treatment testimony. Approval was obtained from the Ethics Committee of Zhengzhou Central Hospital Affiliated to Zhengzhou University and informed consent was provided by patients and their families. The inclusion criteria for the study were: i) Patients were mentally conscious; ii) all tumors were \<8 cm in diameter and the total number of tumors was \<6; and iii) it was the first time patients were treated using PEI and RFA. The exclusion criteria for the study were: i) Patients with hepatic metastasis; ii) pregnant patients and those with serious coagulation mechanism disorders as well as those with medical histories of liver cancer resection and chemoradiotherapy and serious cachexia; iii) patients who had \<6 months survival expectancy; and iv) patients who were intolerant to surgery and those with interrupted treatments, and missed follow ups. According to the selective treatment plan, the patients were divided into 3 groups. In group A (n=35), treatment was initiated with PEI and after 1--2 weeks RFA was applied, while in group B (n=33) treatment was initiated with RFA and after 1--2 weeks PEI was applied. Patients in group C received PEI and RFA simultaneously. The clinical effects in the 3 groups were compared after 6 months. Group A, comprised 20 male and 15 female patients, with an age range of 56--72 years and an average age of 63.4±10.5 years. The mean tumor size was 4.6±1.3 cm (range, 3.0--5.5 cm) and the total number of tumors was 1--4 with an average of 2.7±1.3. There were 16 patients with liver function classification Child-Pugh B and 19 patients with Child-Pugh C, of which 5 cases had tumors in paricular regions and 30 cases had other conditions. In group B, there were 17 male and 16 female patients, with an age range of 55--71 years and an average age of 61.7±12.3 years. The mean tumor size was 4.3±1.4 cm (range, 2.8--5.6 cm) and the total number of tumors was 1--4 with an average of 2.3±1.5. In group B there were 18 patients with liver function classification Child-Pugh B and 15 patients with Child-Pugh C, of which 4 cases had tumors in particular areas and 29 patients had other conditions. Group C, comprised of 19 male and 13 female patients with an age range of 54--75 years and average age of 63.6±15.2 years. The mean tumor size was 4.7±1.2 cm (range, 3.5--6.0 cm) and the total number of tumors was 1--4 with an average of 2.3±1.5. There were 17 patients with liver function classification Child-Pugh B and 15 patients with Child-Pugh C of which 5 cases of tumors were in particular areas and 27 patients had other conditions. The comparisons of baseline information for the 3 groups showed that the differences were not statistically significant (P\>0.05). ### Treatments The patient was placed in supine position and PEI was performed under general anesthesia, using percutaneous puncture needling guided by ultrasound (Aloka SSD-1100 Color Doppler Ultrasound; Siemens AG, Munich, Germany). Local anesthesia was performed using lidocaine. A 22G EV needle (Hakko Co., Ltd., Tokyo, Japan) was entered into the tumor by percutaneous liver biopsy, no blood and gail were evident when the syringe was withdrawn. Using a 10 ml syringe, anhydrous alcohol was uniformly injected into the tumor within 30--60 sec. After the alcohol injection, a high echogenic mass was observed under ultrasound that spread from the pinpoint. Injections were stopped when the high echogenic mass fully covered the tumor and surpassed the edge by \>0.5 cm. The injection volume was calculated using the formula: V=4/3π (r+0.5)^3^ where V is total volume, and r is the radius of lesion. After completion of injection, the puncture needle was gradually withdrawn. Drug overflow was avoided as the procedure was observed under ultrasound. After withdrawal of the puncture needle, the puncture point was covered with gauze and pressured was applied on the dressing ([Fig. 1](#f1-ol-0-0-4180){ref-type="fig"}). RFA was performed under general anesthesia once the patient was in supine position, using percutaneous puncture needling guided by ultrasound (Aloka SSD-1100 Color Doppler Ultrasound; Siemens AG). Local anesthesia was performed using lidocaine. The cool-tip RF System (Valleylab, Boulder, CO, USA), uni-polar cold circulation system, mating electrode wire and plate electrode were used during the process. Output frequency was adjusted to (1±5%) 460 kHz, the maximum power was 150 W and the needle used was a 14 G trocar. A total of 7--12 fine needle electrodes were set on top of the inner needles, which formed a 5.0 cm spherical heat coagulation after stretching energization. The unit was set to 90 W for RFA actual burning power and the temperature was set at 100°C. After the temperature reached 100°C, it was maintained for 15 min. Following completion of a single RFA, the echo of the ablation area under the observation of ultrasound was higher, the position of the needle electrodes was adjusted according to the tumor size for repeated ablation, in order to cover the whole target tumor in the echo area, and the ablation area surpassed tumor lesions by 0.5--1 cm. Following completion of the treatment, the stretching electrodes were withdrawn, and the needles were withdrawn after burning of closed needles. The adjustments of radiofrequency power started from 60 W each time, and it were gradually elevated until impedance markedly increased and power was automatically reduced. The scope of vaporization of strong echo covering all boundaries of the tumors was used to detrermine when to terminate the treatment. To prevent hemorrhage and implantation metastasis, needles were heated under high temperature each time prior to use ([Fig. 2](#f2-ol-0-0-4180){ref-type="fig"}). ### Observation indices The volume of tumor ablation necrosis, volume after ablation and complete ablation rate, quantity of alcohol and radiofrequency energy used and liver function damage indices, including raising levels of glutamic-pyruvic transaminase and total bilirubin were recorded. Differences of survival rates were compared 6 months after follow-ups. To determine tumor volume the formula used was: V(cm^3^)=4/3 × r1(cm) × r2 (cm) × r3(cm), (r1=longest diameter/2, r2=shortest diameter/2, r3=height/2). The volume of tumor necrosis was calculated by subtracting the volume after ablation from the volume before ablation. A contrast-enhanced ultrasound examination was conducted two weeks after the treatment. Arterial portal and delayed phases of the ablation area with no contrast agent observed was regarded as complete ablation; radiofrequency energy (J)=watt (W) × curative time (sec). ### Statistical analysis SPSS 20.0 software (IBM SPSS, Armonk, NY, USA) was used for statistical analyses. Measurement data were presented by mean ± standard deviation. Analysis of variance was used for comparisons among the groups and enumeration data were presented by %, while the χ^2^ test was used for comparison among groups. P\<0.05 was considered to indicate a statistically significant difference. Results ======= ### Comparisons of ablation parameters The volume of tumor ablation necrosis in group A was significantly larger than that in groups B and C, while the volume was significantly smaller than that in groups B and C after ablation. The complete ablation rate in group A was significantly higher than that in groups B and C, and the differences were statistically significant (P\<0.05). The comparisons of quantity of alcohol used and radiofrequency energy in the three groups showed that the differences were not statistically significant (P\>0.05) ([Table I](#tI-ol-0-0-4180){ref-type="table"}). ### Comparisons of liver function damage indices The comparisons of glutamic-pyruvic transaminase and total bilirubin levels prior to the treatment in the 3 groups showed that the differences were not statistically significant (P\>0.05). By contrast, after the treatment, the raising levels of glutamic-pyruvic transaminase and total bilirubin in group A were significantly lower than that in groups B and C, and the differences were statistically significant (P\<0.05) ([Table II](#tII-ol-0-0-4180){ref-type="table"}). ### Comparisons of survival rates Eight patients succumbed in group A, of whom 2 had tumor recurrence *in situ*, 1 patient succumbed to tumor rupture hemorrhage, 3 succumbed to tumor emboli blocking the hepatic artery or portal vein hemorrhage, 3 patients succumbed to liver failure, and 1 patient had extrahepatic tumor metastasis. Mortalities occured 2 weeks to 6 months after the treatment (average, 4.2 months) and the survival rate was 77.1%. In group B, 17 patients succumbed of whom 5 patients had tumor recurrence *in situ*, 3 patients succumbed to tumor rupture hemorrhage, 7 patients to tumor emboli blocking the hepatic artery or portal vein hemorrhage, 5 due to liver failure, and 2 patients succumbed to extrahepatic tumor metastasis. In group B, mortalities occured 1 week to 5.5 months after treatment (average, 3.5 months), and the survival rate was 48.5%. In group C, 15 mortalities occured of whom 6 patients succumbed to tumor recurrence *in situ*, 4 to tumor rupture hemorrhage, 6 to tumor emboli blocking the hepatic artery or portal vein hemorrhage, 3 to liver failure, and 2 patients succumbed to extrahepatic tumor metastasis. Patients in group C succumbed to the disease 1.5 weeks to 5.6 months following treatment (average, 3.7 months), and the survival rate was 53.1%. The survival rate in group A increased significantly, and the differences were statistically significant (χ^2^=6.739, P=0.034). Discussion ========== Ultrasound guidance technology has the advantages of real-time monitoring, accurate guidance, negligible trauma, safety and effectiveness, easy operation and repeatability ([@b6-ol-0-0-4180]). These advantages have led this technology to be considered as one of the three major types of treatment for liver cancer together with surgery and regional vascular intervention ([@b7-ol-0-0-4180]). Ultrasound guidance technology aims to effectively treat tumors or cytoreduction, reducing symptoms, improving life quality and extending survival time. Ultrasound-guided local interstitial chemical ablation consists of injecting substances into tumors to induce necrosis. Frequently used injections include anhydrous alcohol, acetic acid, hot saline water or hot distilled water, radionuclide, biological agents and cod liver oil acid sodium anhydrous alcohol solution ([@b8-ol-0-0-4180]). Sugiura *et al* ([@b9-ol-0-0-4180]) clinically applied PEI in treating liver cancer for the first time, and now PEI is the most widely used method for chemical ablation. However, due to the disadvantages of non-uniform diffusion and uncontrollability of injected alcohol observed in numerous treatments of larger tumors, PEI is mainly applied in the treatment of small hepatocellular carcinoma. However, Livraghi *et al* ([@b10-ol-0-0-4180]) reported that PEI could be applied in ≥5 cm liver tumors. They showed that for the 1,066 patients participating in their study, the 3-, 5- and 7-year survival rates were 72.3, 43.2 and 27.0%, respectively. The ultrasound-guided local interstitial thermal ablation method consists of conducting energy into the tumor to inactivate tumor cells *in situ*. Thermal ablation includes radiofrequency, microwave, laser, refrigeration and focused ultrasound. Rossi *et al* ([@b11-ol-0-0-4180]) were the first group who reported treating small hepatocellular carcinoma using ultrasound-guided RFA, with comparable results compared to PEI. RFA can also be used as a supplementary method for the treatment of large liver tumors. RFA is also suitable for those patients that cannot tolerate surgery or those unwilling to undergo surgery. The results obtained from prior studies showed that in cancer tissues, HSP-70 expression level, AFP expression level, clinical staging, liver function classifications and the inactivated degree of tumors in liver cancer tissues, were are all closely associated with RFA treatment effects in large liver tumors ([@b12-ol-0-0-4180]). RFA was certified by the Food and Drug Association in early 1996 and currently is cosidered the most widely applied ablation method ([@b13-ol-0-0-4180]). There are some limitations associated with RFA such as high costs, limited popularity and technological difficulties. The application of PEI in combination with RFA for the treatment of large liver cancer and liver cancer of particular areas has become a hot topic of research. This method is designed to reduce the tumor size of through dehydration and solidification using anhydrous alcohol, and thus increase the success rate of RFA ablation and reduce the frequency of ablation ([@b14-ol-0-0-4180]). Multi-polar RFA treatment devices (RITA Medical System; Rita Medical, Mountain View, CA, USA; Radio-Therapeutics, Sunnyvale, CA, USA) have 3--5 electrodes that are similar to 'eagle claw' or 'umbrella'. The cold circulation in radiofrequency electrode is designed to prevent the ebullition and the formation of cavities of tissues around the pinpoint of the electrodes because of the heat ([@b15-ol-0-0-4180]). There are no absolute conclusions regarding whether PRI and RFA should be carried out seperately or simultaneously, and there are few studies on the quantitative comparison of the volume of alcohol usage and radiofrequency power. Through large numbers of clinical practices, it was identified that although the basis of liver cancer in middle and advanced stages is poor, survival time cannot be improved via conservative treatments ([@b16-ol-0-0-4180]). The clinical effects of simple PRI and RFA treatments are not substantial, however, the present study concluded that adimistering PEI treatment 1--2 weeks prior to RFA treatment can significantly improve the patients\' condition by intensification of the volume of tumor ablation necrosis and reduce tumor volume following ablation. This method can also improve complete the ablation rate, reduce the raising levels of glutamic-pyruvic transaminase and total bilirubin and improve the survival rate. The volume of alcohol usage and radiofrequency power was not increased in this method. Possible reasons for this include: i) The stress damage of anhydrous alcohol to organic bodies is higher than that of RFA, and PEI was used for the first time ([@b17-ol-0-0-4180]); ii) usually there is the existence of 'pseudocapsule' in primary liver cancer, and PEI being used for the first time, which influences the dispersion effects of anhydrous alcohol ([@b18-ol-0-0-4180]); iii) the working mechanism of RFA is to use ion in tissues to create vibrations of the same frequency around electrodes, which turns it into heat energy to produce coagulative necrosis. The pretreatment of anhydrous alcohol can make the frequency of tumor tissues more concentrated, which shortens the duration of RFA ([@b19-ol-0-0-4180]); and iv) the simultaneous implementations of PEI and RFA treatments cannot produce a superimposed effect, which in turn creates the phenomenon of offset ([@b20-ol-0-0-4180]). In conclusion, for patients with liver cancer in middle and advanced stages, the treatment method using PEI followed by RFA is more beneficial in terms of improving tumor ablation rate, alleviating liver damage and increasing survival rates. {#f1-ol-0-0-4180} {#f2-ol-0-0-4180} ###### Comparisons among different ablation parameters. Groups Cases Volume before ablation (cm^3^) Volume after ablation Volume of necrosis ablation Complete ablation rate (%) Usage volume of alcohol, ml Radiofrequency energy (×10^3^ J) ---------- ------- -------------------------------- ----------------------- ----------------------------- ---------------------------- ----------------------------- ---------------------------------- A 35 15.5±3.3 1.5±0.8 13.8±4.2 30 (85.7) 8.5±1.3 456.7±33.4 B 33 14.7±3.4 6.6±1.2 10.6±3.7 20 (60.6) 8.2±1.5 432.6±45.2 C 32 15.3±3.5 7.2±1.5 10.5±3.8 20 (62.5) 8.3±1.6 423.4±43.3 F (χ^2^) 0.632 6.926 6.524 6.360 0.423 0.938 P-value 0.425 0.032 0.038 0.042 0.725 0.546 Group A, PEI was administered first and then RFA; group B, RFA was administered first and then PEI; group C, PEI and RFA were administered simultaneously. PEI, percutaneous ethanol injection; RFA, radiofrequency ablation. ###### Comparisons among liver function damage indices. Groups Glutamic-pyruvic transaminase level before treatments, U/l Raising levels^[a](#tfn2-ol-0-0-4180){ref-type="table-fn"}^ Total bilirubin before treatments, µmol/l Raising levels^[a](#tfn2-ol-0-0-4180){ref-type="table-fn"}^ --------- ------------------------------------------------------------ ------------------------------------------------------------- ------------------------------------------- ------------------------------------------------------------- A 78.5±13.6 13.6±4.2 27.9±5.3 6.3±1.2 B 75.6±14.2 18.2±4.7 25.5±4.2 10.2±2.5 C 76.7±13.5 17.5±4.3 23.4±4.6 8.9±2.4 F-value 0.632 6.325 0.759 6.856 P-value 0.421 0.039 0.532 0.027 Before treatment - after treatment. [^1]: Contributed equally

1. Introduction {#sec1-ijms-18-00632} =============== Schizophrenia (SCZ) is a mental disorder characterized by symptoms that include delusions, hallucinations, and disorganized speech \[[@B1-ijms-18-00632]\]. Approximately, 30% of SCZ patients are treatment-resistant \[[@B2-ijms-18-00632]\], and the atypical antipsychotic clozapine has become the treatment of choice in this setting \[[@B3-ijms-18-00632],[@B4-ijms-18-00632]\]. Recently, we have demonstrated that cell adhesion molecules, which play an important role in brain development including in axonal/dendrite growth, synapse formation and plasticity \[[@B5-ijms-18-00632]\], were potential candidates for the molecular basis of clozapine response by conducting gene expression profiling using induced pluripotent stem (iPS) cell-based technology \[[@B6-ijms-18-00632]\]. However, the molecular and epigenetic mechanisms underlying the therapeutic efficacy of clozapine have not yet been fully elucidated. DNA methylation is an epigenetic modification that plays a critical role in brain function \[[@B7-ijms-18-00632],[@B8-ijms-18-00632]\]. DNA methylation mainly occurs at the 5′ position of the cytosine base followed by a guanine base that is called CpG \[[@B9-ijms-18-00632]\]. A number of studies have demonstrated aberrant DNA methylation in SCZ \[[@B10-ijms-18-00632],[@B11-ijms-18-00632],[@B12-ijms-18-00632],[@B13-ijms-18-00632],[@B14-ijms-18-00632],[@B15-ijms-18-00632],[@B16-ijms-18-00632],[@B17-ijms-18-00632],[@B18-ijms-18-00632]\]. Furthermore, growing evidence suggests that DNA methylation may be involved in the therapeutic efficacy of atypical antipsychotic drugs \[[@B19-ijms-18-00632],[@B20-ijms-18-00632],[@B21-ijms-18-00632],[@B22-ijms-18-00632],[@B23-ijms-18-00632],[@B24-ijms-18-00632]\]. With respect to clozapine, two animal studies have demonstrated that acute clozapine treatment induces DNA demethylation in the promoters of specific GABAergic and glutamatergic genes \[[@B25-ijms-18-00632],[@B26-ijms-18-00632]\]. However, to our knowledge, no study investigating comprehensive DNA methylation changes in SCZ patients treated with chronic clozapine has been reported. In the present study, we comprehensively analyzed changes in DNA methylation in peripheral leukocytes from treatment-resistant SCZ patients treated with clozapine in a longitudinal study. Next, we examined the correlation between changes in DNA methylation in response to clozapine and clinical improvements in treatment-resistant SCZ. 2. Results {#sec2-ijms-18-00632} ========== 2.1. Changes in DNA Methylation in Leukocytes after Clozapine Treatment {#sec2dot1-ijms-18-00632} ----------------------------------------------------------------------- Of the 350,142 CpG sites analyzed, significant changes in DNA methylation were observed at 29,134 sites when we compared samples from 21 patients collected before and after one year of treatment with clozapine. The top 100 CpG sites are shown in [Supplementary Table 1](#app1-ijms-18-00632){ref-type="app"}. Of the 29,134 CpG sites showing significant differences in methylation, clozapine treatment increased DNA methylation at 13,052 sites (44.8%) and decreased DNA methylation at 16,082 sites (55.2%). Classification of significant CpG sites based on their locations within genes revealed that 11,850 sites (40.7%) were located in promoter regions; 9479 sites (32.5%) were in gene bodies; 864 sites (3.0%) were in 3′-UTRs; the remainders were found in intergenic regions. Decreases in DNA methylation following clozapine treatment were more likely to occur in promoter regions than in other regions (60.5% in promoter regions vs. 51.6% in other regions; Fisher's exact test *p* = 7.00 × 10^−16^; [Figure 1](#ijms-18-00632-f001){ref-type="fig"}). Classification of CpG sites based on location relative to CpG content in the genes (CpG island (CGI), CGI shore, CGI shelf, and others) revealed that 7656 sites (26.3%) were located within CGIs; 7334 sites (25.2%) were in CGI shores; and 2846 sites (9.8%) were in CGI shelves. Sites of decreased DNA methylation following clozapine treatment were more likely to occur in CGI regions than in other regions (66.9% in CGI regions versus 51.0% in other regions; Fisher's exact test *p* = 3.83 × 10^−36^; [Figure 1](#ijms-18-00632-f001){ref-type="fig"}). Interestingly, significant CpG sites of decreased DNA methylation which were located in CGIs in promoter regions include several GABA, glutamate, and related SCZ susceptibility genes \[[@B27-ijms-18-00632]\], such as GAD1 (glutamate decarboxylase 1), GRIN2A (glutamate ionotropic receptor NMDA type subunit 2A), GRIN2D (glutamate ionotropic receptor NMDA type subunit 2D), and GRM7 (glutamate metabotropic receptor 7). A list of the CpG sites located in promoter regions with average DNA methylation differences (Δβ) greater than 0.05 and paired *t*-test *p*-values less than 0.001 is shown in [Table 1](#ijms-18-00632-t001){ref-type="table"}. Two of these CpG sites are located in the *TRIM15* (tripartite motif containing 15) gene, which has been implicated in SCZ ([Figure 2](#ijms-18-00632-f002){ref-type="fig"}) \[[@B28-ijms-18-00632]\]. Additionally, gene ontology (GO) analysis revealed that genes with DNA methylation changes following clozapine treatment were enriched for the "cell substrate adhesion" and "cell matrix adhesion" GO terms (False discovery rate (FDR) *q* \< 0.05). 2.2. Correlations between Changes in DNA Methylation in Leukocytes and Clinical Outcomes {#sec2dot2-ijms-18-00632} ---------------------------------------------------------------------------------------- Analysis of correlations between changes in psychotic symptoms (% Positive and Negative Syndrome Scale (PANSS)) and clinical variables (clozapine dose, duration of clozapine treatment, age of onset, gender, and use of mood stabilizers) revealed no significant correlations (*p* \> 0.05). Upon analysis of correlations between ∆β-values for the 29,134 CpG sites showing significant changes in DNA methylation after clozapine treatment and % PANSS, a CpG site associated with the *CREBBP* (CREB binding protein) gene, cg05151055, was the only site that was significantly correlated with changes in psychotic symptoms (FDR *q* \< 0.05; [Figure 3](#ijms-18-00632-f003){ref-type="fig"}). 3. Discussion {#sec3-ijms-18-00632} ============= To the best of our knowledge, this study represents the first comprehensive analysis of the effects of clozapine treatment on DNA methylation in leukocytes from treatment-resistant SCZ patients. We identified 29,134 CpG sites that showed significant changes in DNA methylation following chronic clozapine treatment. The proportion of CpG sites with decreased DNA methylation was higher than the proportion of sites with increased DNA methylation after clozapine treatment (55.2% vs. 44.8%, respectively). Consistent with this finding, we previously demonstrated that DNA hyper-methylation patterns frequently occurred in medication-free patients with SCZ, while hypo-methylation was more common in patients with SCZ treated with antipsychotics \[[@B12-ijms-18-00632],[@B14-ijms-18-00632]\]. We also found that decreased DNA methylation following clozapine treatment was more likely to occur at CpG sites located in CGIs in gene promoter regions. This trend is consistent with results reported in a previous study of the effects of blonanserin, another atypical antipsychotic \[[@B22-ijms-18-00632]\]. In this study, we found that genes that showed DNA methylation changes following clozapine treatment were enriched for the GO terms "cell substrate adhesion", which is essential for cells to interact with their environments \[[@B29-ijms-18-00632]\], and "cell matrix adhesion", which is essential for cell migration, tissues organization, and differentiation \[[@B30-ijms-18-00632]\]. Similarly, in a recent analysis of differential gene expression profiles in neurons from twins with treatment-resistant SCZ who had discordant responses to clozapine using pluripotent stem (iPS) cell based technology, we found that differentially expressed genes were enriched for "cell adhesion" and "biological adhesion" \[[@B6-ijms-18-00632]\]. Cell adhesion is essential for forming tissue and neuronal connections crucial for nervous system development \[[@B31-ijms-18-00632]\]. Animal studies suggest that dysfunction of neuronal cell adhesion molecules, which are expressed primarily in the central nervous system where they regulate synaptic signaling, may lead to impairment of memory and learning \[[@B32-ijms-18-00632],[@B33-ijms-18-00632]\]. Furthermore, pathways related to cell adhesion have been shown to contribute to SCZ susceptibility in a genome-wide association study \[[@B34-ijms-18-00632]\], and increased blood levels of neuronal cell adhesion molecules have been observed in SCZ \[[@B35-ijms-18-00632],[@B36-ijms-18-00632]\]. These results suggest that changes of neuronal cell adhesion-related molecules may be implicated in the pathophysiology of SCZ, and clozapine may exert its therapeutic effects by altering DNA methylation of genes encoding these molecules. We found that increases in DNA methylation of the *CREBBP* gene following clozapine treatment was significantly correlated with clinical improvements in treatment-resistant SCZ. This result suggests that epigenetic modification of the *CREBBP* gene in peripheral leukocytes can predict clinical responses to clozapine in treatment-resistant SCZ. CREBBP is a protein that possesses intrinsic histone acetyltransferase activity, in addition to acting as a scaffold to stabilize protein interactions within the transcription complex \[[@B37-ijms-18-00632]\]. Pathway analyses of results from genome-wide association studies have demonstrated that this gene is associated with SCZ \[[@B38-ijms-18-00632],[@B39-ijms-18-00632]\]. Furthermore, *CREBBP* variants have been associated not only with clinical symptoms, but also with cognitive phenotypes in SCZ \[[@B36-ijms-18-00632],[@B40-ijms-18-00632]\]. Our study has several limitations that should be noted. First, our sample size was small, and studies that replicate our findings in a larger cohort are needed. Second, patients enrolled in this study had been treated with various kinds of antipsychotics prior to treatment with clozapine. Third, the dosage and duration of clozapine treatment were not uniform across the patient cohort. Fourth, it is unclear whether changes in DNA methylation following clozapine treatment are normalized to control levels due to a lack of control data. Fifth, it is also unclear whether the DNA methylation changes following the treatment are specific to clozapine or are common among other antipsychotics. Direct comparisons to previous studies that examined the effects of antipsychotics on DNA methylation are hampered by the use of different study designs and analysis of different tissues \[[@B19-ijms-18-00632],[@B20-ijms-18-00632],[@B21-ijms-18-00632],[@B22-ijms-18-00632],[@B23-ijms-18-00632],[@B24-ijms-18-00632]\]. Further blood studies using other antipsychotics are needed to reveal differences between clozapine and other antipsychotics. Sixth, we did not investigate the relationship between DNA methylation and expression. Further functional studies, including transcriptome analysis and cell adhesion assays, are required to clarify the molecular mechanisms of clozapine action. Finally, it is important to note that the observed changes in DNA methylation after clozapine treatment were analyzed only in peripheral leukocytes, and studies in brain tissue are needed to confirm the mechanistic interpretation of our results. 4. Materials and Methods {#sec4-ijms-18-00632} ======================== 4.1. Subjects {#sec4dot1-ijms-18-00632} ------------- Twenty-one patients with SCZ (mean age: 42.1 ± 11.4 years; eight males and 13 females) were recruited from Kochi, Tokushima, and Osaka University Hospitals in Japan. All subjects were of Japanese origin and had been treated with various antipsychotic drugs before treatment with clozapine. Peripheral blood was collected twice from each patient, just before introduction of clozapine and after one year of treatment. The mean dose of clozapine was 473.8 ± 91.3 mg/day, and the mean duration of clozapine treatment was 340.8 ± 182.7 days. The psychotic symptoms of the patients were evaluated using PANSS at the time of peripheral blood sample collection. The mean PANSS score was 113.4 at baseline and 91.7 at the end of treatment, respectively. The clinical characteristics of the patients are summarized in [Supplementary Table 2](#app1-ijms-18-00632){ref-type="app"}. SCZ was diagnosed according to DSM-IV criteria by at least two expert psychiatrists on the basis of extensive clinical interviews and a review of medical records. All patients met the criteria for treatment-resistant SCZ and clozapine administration, as described in the clozapine drug information provided in Japan \[[@B41-ijms-18-00632],[@B42-ijms-18-00632]\]. No psychiatric comorbidities were present in any of the patients. The study protocol was approved by the institutional ethics committee of Tokushima University Graduate School (Project ID, H28-1; Date of approval, 23 June 2016), and all enrolled participants provided their signed, written, informed consent for participation. 4.2. Analysis of DNA Methylation {#sec4dot2-ijms-18-00632} -------------------------------- Genomic DNA was extracted from peripheral blood using the QIAamp DNA Blood Mini Kit (Qiagen, Germantown, MD, USA). Bisulfite conversion of 500 ng of genomic DNA was performed with the EZ DNA methylation kit (Zymo Research, Irvine, CA, USA). DNA methylation levels were assessed using Infinium^®^ HumanMethylation450 BeadChips (Illumina Inc., San Diego, CA, USA), which makes it possible to examine DNA methylation status at more than 485,000 CpG sites, according to the manufacturer's instructions, and the resulting data was analyzed using the methylation analysis module within the BeadStudio software (Illumina Inc.). The DNA methylation status of CpG sites (termed the β-value) was calculated based on the ratio of the signal from a methylated probe relative to the sum of the signal from both methylated and unmethylated probes. β values ranged from 0 (completely unmethylated) to 1 (fully methylated). For intra-chip normalization of probe intensities, colored balance and background corrections were performed for every set of 12 samples from the same chip using internal control probes. CpG sites used for statistical analyses met the following criteria: (1) β-values with detection *p*-values \< 0.05; (2) autosomal CpGs, with no missing values in any subjects; (3) no probe single nucleotide polymorphisms (SNPs) with minor allele frequencies ≥5% in the HapMap-JPT population; (4) no probe cross-reactivity; and (5) no SNPs at CpG sites and single-base extension sites detailed in a previous paper \[[@B43-ijms-18-00632]\]. The final data set for peripheral leukocytes included 350,142 sites. 4.3. Statistical Analysis {#sec4dot3-ijms-18-00632} ------------------------- A paired *t*-test was used to assess DNA methylation changes following clozapine treatment. *p*-values \< 0.05 were considered statistically significant. Gene ontology analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) \[[@B44-ijms-18-00632]\]. An FDR correction was applied at the 0.05 level for multiple comparisons. Pearson's correlations were performed to assess the effects of clinical variables, including clozapine dose, duration of clozapine treatment, age of onset, gender, and use of mood stabilizers on PANSS percentage changes (% PANSS; defined as (PANSS at the end of the study − PANSS at baseline)/PANSS at baseline). A univariate linear regression model was used to examine the relationship between β-values for probes with significant DNA methylation changes following clozapine treatment and % PANSS. An FDR correction was applied at the 0.05 level for multiple comparisons. 5. Conclusions {#sec5-ijms-18-00632} ============== We report a comprehensive analysis of DNA methylation changes in peripheral leukocytes from patients with treatment-resistant SCZ following clozapine treatment. We found that genes with clozapine-induced changes in DNA methylation were associated with cell substrate adhesion and cell matrix adhesion. These results provide insight into potential mechanisms of action of clozapine in treatment-resistant SCZ. Further functional studies are needed to clarify the molecular mechanisms of clozapine action. The authors would like to thank all the volunteers who understood our study purpose and participated in this study and the physicians who helped us to collect clinical data and blood samples. The authors would also like to thank Akemi Okada for her technical assistance. This manuscript has been edited by native English-speaking experts from BioMed Proofreading LLC. This work was supported in part by Japan Agency for Medical Research and development, AMED (Hidenaga Yamamori, Masataka Kikuchi, Hitoshi Hashimoto, Ryota Hashimoto, Shusuke Numata, Takanobu Nakazawa and Tetsuro Ohmori), Grant-in-Aid for Young Scientists (B) (No.16K19768) (Makoto Kinoshita), Grant-in-Aid for Scientific Research (C) (No. 16K07222) (Masataka Kikuchi and Akihiro Nakaya), SENSHIN Medical Research Foundation (Shusuke Numata), and the JSPS Program (No. S2603) (Hitoshi Hashimoto). Supplementary materials can be found at [www.mdpi.com/1422-0067/18/3/632/s1](www.mdpi.com/1422-0067/18/3/632/s1). ###### Click here for additional data file. Ryota Hashimoto and Shusuke Numata designed the study; Ryota Hashimoto, Shusuke Numata and Tetsuro Ohmori managed the research; Hidehiro Umehara, Hidenaga Yamamori, Michiko Fujimoto, Makoto Kinoshita, Ryota Hashimoto, Shusuke Numata, Shinji Shimodera, Shinya Watanabe, Tetsuro Ohmori and Yuka Yasuda collected samples and acquired the data; Atsushi Tajima, Issei Imoto and Makoto Kinoshita undertook the statistical analysis; Makoto Kinoshita and Shusuke Numata wrote the draft of this paper; Akihiro Nakaya, Hitoshi Hashimoto, Ryota Hashimoto, Masataka Kikuchi, Takanobu Nakazawa and Tetsuro Ohmori helped to interpret data and edited the manuscript. All authors have read and approved the final manuscript. The authors declare no conflict of interest. {#ijms-18-00632-f001} {#ijms-18-00632-f002} {#ijms-18-00632-f003} ijms-18-00632-t001_Table 1 ###### A list of the significant CpG sites with average Δβ \> 0.05 and paired *t*-test *p*-value \< 0.001 in the gene promoter regions. CGI, CpG island; UCSC, University of California Santa Cruz; \* Positions refer to Genome Research Consortium human genome build 37 (GRCh37/UCSC human genome 19 (hg19). Probe ID Average Beta Difference before Treatment of Clozapine Average Beta Difference after Treatment of Clozapine Average Beta Difference between Treatment of Clozapine *p*-Value Chromosome Position \* UCSC RefGene Name UCSC RefGene Group Relation to UCSC CpG Island ------------ ------------------------------------------------------- ------------------------------------------------------ -------------------------------------------------------- -------------- ------------ ------------- ------------------- -------------------- ----------------------------- cg15542713 0.427 0.496 0.070 1.1 × 10^−4^ 1 42385581 *HIVEP3* Promoter CGI shore cg02772121 0.570 0.624 0.054 3.2 × 10^−4^ 6 30130881 *TRIM15* Promoter Others cg10864200 0.608 0.557 −0.050 7.3 × 10^−4^ 4 720809 *PCGF3* Promoter CGI shelf cg12422154 0.601 0.652 0.050 2.5 × 10^−4^ 6 30130819 *TRIM15* Promoter Others

Introduction {#section1-0009922820941228} ============ In January 2020, a new coronavirus was found in Wuhan, Hubei, China. The coronavirus research group of the International Committee for the Classification of viruses named it severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).^[@bibr1-0009922820941228]^ The World Health Organization calls the pneumonia caused by the coronavirus COVID-19.^[@bibr2-0009922820941228]^ Due to the characteristics of human-to-human transmission, COVID-19 has become popular all over the world and caused tens of thousands of deaths. Now it is a public health emergency of international concern over the global outbreak of novel coronavirus.^[@bibr3-0009922820941228]^ At present, the National Health Commission of China has published the corresponding guidelines for diagnosis, treatment, prevention, and control of COVID-19.^[@bibr4-0009922820941228],[@bibr5-0009922820941228]^ It is mentioned that medical institutions should set up isolation wards. The suspected or confirmed patients of COVID-19 were placed in the isolation ward first, and then pathogen or serological examination was performed. If evidence of COVID-19 infection is found, the patient will be transferred to a targeted treatment unit to continue treatment. At present, COVID-19 mainly infects adults, especially some elderly men.^[@bibr6-0009922820941228],[@bibr7-0009922820941228]^ There are relatively few cases of infection in children. In this study, the clinical characteristics of children in isolation ward during the COIVID-19 epidemic were analyzed retrospectively, to provide basis for isolation and timely diagnosis of diseases in children. It is conducive to the scientific investigation of COVID-19 and provides an effective basis for COVID-19 prevention and control methods. Method {#section2-0009922820941228} ====== Because of the nature of this retrospective study, the Medical Ethics Committee of Hubei Maternal and Child Health Hospital approved the research plan and waived patient consent. The children in the pediatric isolation ward of our hospital from January 1, 2020, to February 29, 2020, were selected as the object of study. The basic data, epidemic history, symptoms, blood analysis, liver function, myocardial enzyme spectrum, chest computed tomography (CT) results, electrocardiogram (ECG) results, SARS-CoV-2 nucleic acid test results, hospitalization days, and hospitalization expenses of all the children in the isolation ward were collected. Data processing was done by SPSS 25 statistical software. The counting data are described statistically by n (%). The measurement data of nonnormal distribution are described statistically by median and quartile \[M(P~25~, P~75~)\]. Result {#section3-0009922820941228} ====== From January 1, 2020, to February 29, 2020, we collected data of 66 cases in the isolation ward. There were 37 males (56.1%) and 29 females (43.9%), and the male-to-female ratio was 1.28:1. There were 35 children without definite exposure history (53%). There were 17 children (25.8%) with suspected COVID-19 at home, and the children were in close contact with them. Only 14 patients (21.2%) had close contact with the confirmed COVID-19 patients. All the children in the isolation ward had different symptoms before admission. Among them, 55 cases (83.3%) had fever, 48 cases had cough (72.7%), 21 cases had stuffy nose (31.8%), and 24 cases had runny nose (36.4%; see [Table 1](#table1-0009922820941228){ref-type="table"}). ###### General Information of Children in Isolation Ward.  Number of cases Percentage (%) ------------------------------ ----------------- ---------------- Gender  Male 37 56.1  Female 29 43.9 No exposure history 35 53.0 Contact with suspected cases 17 25.8 Contact with confirmed cases 14 21.2 Symptoms  Fever 55 83.3  Cough 48 72.7  Stuffy nose 21 31.8  Runny nose 24 36.4 The age of the children ranged from 1 month to 13 years, with an average of 4.3 years. The number of children of school age was the largest, with 18 cases (27.3%). There were 17 cases (25.8%) of infancy. There were 14 cases of early childhood (21.2%), 11 cases of preschool age (16.6%), and 6 cases of puberty (9.1%; see [Table 2](#table2-0009922820941228){ref-type="table"}). ###### Age Distribution of Children in Isolation Ward.  Age stage Number of cases Percentage (%) ----------------- ----------------- ---------------- Infancy 17 25.8 Early childhood 14 21.2 Preschool age 11 16.6 School age 18 27.3 Puberty 6 9.1 Total 66 100 In the course of collecting case data, we found that not all children had data of the pathogen. Among them, the largest number of children were infected with *Mycoplasma pneumoniae*, 17 cases (25.8%), followed by SARS-CoV-2 infection, 14 cases (21.2%). There were 6 children with respiratory syncytial virus (9.1%). Only 2 cases (3%) were infected with Epstein-Barr virus (see [Table 3](#table3-0009922820941228){ref-type="table"}). ###### Children With a Clear Pathogen.  Number of cases Percentage (%) ----------------------------- ----------------- ---------------- *Mycoplasma pneumoniae* 17 25.8 Respiratory syncytial virus 6 9.1 Epstein-Barr virus 2 3 SARS-CoV-2 14 21.2 From the laboratory test data, we found that white blood cells level was normal in 44 cases (66.7%), increased in 15 cases (22.7%), and decreased in 7 cases (10.6%). The number of lymphocytes (LYM) was normal in 21 cases (31.8%), increased in 19 cases (28.8%), and decreased in 26 cases (39.4%). C-reactive protein level was increased in 23 cases (34.8%). In the liver function of children, alanine aminotransferase was increased in 11 cases (16.7%) and aspartate aminotransferase was increased in 25 cases (37.9%). In the myocardial enzymes, lactate dehydrogenase was increased in 37 cases (56.1%), CK-MB was increased in 36 cases (54.5%), and creatine kinase was increased in only 7 cases (10.6%). All the children in the isolation ward had tests for the detection of SARS-CoV-2 nucleic acid, and 14 of them were positive (21.2%; see [Table 4](#table4-0009922820941228){ref-type="table"}). ###### Laboratory Test Data of Children.  Number of cases Percentage (%) ---------------------------------- ----------------- ---------------- WBC  Increase 15 22.7  Normal 44 66.7  Decrease 7 10.6 LYM  Increase 19 28.8  Normal 21 31.8  Decrease 26 39.4 CRP increase 23 34.8 ALT increase 11 16.7 AST increase 25 37.9 LDH increase 37 56.1 CK increase 7 10.6 CK-MB increase 36 54.5 SARS-CoV-2 nucleic acid positive 14 21.2 Abbreviations: WBC, white blood cell; LYM, lymphocytes; CRP, C-reactive protein; ALT, alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase; CK, creatine kinase; CK-MB, creatine kinase isoenzyme. All the children in the isolation ward had had chest CT at the time of admission. Among them, there were 8 cases without obvious abnormality (12.1%), 28 cases (42.4%) had speckled or patchy shadow, 6 cases (9.1%) had strip shadow, and 19 cases (28.8%) had ground glass opacity (GGO). A total of 4 cases (6.1%) were found to have atelectasis, and only 1 case (1.5%) had pleural effusion (see [Table 5](#table5-0009922820941228){ref-type="table"}). ###### Chest CT Results of Children.  Number of cases Percentage (%) ------------------------ ----------------- ---------------- No obvious abnormality 8 12.1 Speckled or patchy 28 42.4 Stripe shape 6 9.1 GGO 19 28.8 Atelectasis 4 6.1 Pleural effusion 1 1.5 Abbreviations: CT, computed tomography; GGO, ground glass opacity. Through the study of the ECG of the children, it was found that 27 cases showed sinus rhythm (40.9%) and 28 cases showed sinus tachycardia (42.4%), but we found that 11 cases showed sinus arrhythmia (16.7%; see [Table 6](#table6-0009922820941228){ref-type="table"}). ###### ECG Results of Children.  Number of cases Percentage (%) ------------------- ----------------- ---------------- Sinus rhythm 27 40.9 Sinus tachycardia 28 42.4 Sinus arrhythmia 11 16.7 Abbreviation: ECG, electrocardiogram. Based on the statistical analysis of the hospitalization days of all the children in the isolation ward, it was found that the median and quartile of hospitalization days were 8 (7, 10) days, and the hospitalization cost was CNY 9269.32 (8168.2, 11204.33). Discussion {#section4-0009922820941228} ========== Coronavirus is a RNA virus that exists in nature. It can be divided into 4 subtypes: α, β, γ, and δ. SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), which can cause serious respiratory diseases, belong to type β coronavirus.^[@bibr8-0009922820941228]^ The genome sequence of SARS-CoV-2 was matched with that of type β coronavirus, but it was different from that of SARS-CoV and MERS-CoV. At present, some of the physical and chemical properties of SARS-CoV-2 refer to the studies of SARS-CoV and MERS-CoV. At present, our diagnosis of COVID-19 is mainly based on the Diagnosis and Treatment of New Coronavirus Pneumonia (Trial Version 7) issued by the National Health Committee of the People's Republic of China.^[@bibr4-0009922820941228]^ It is mentioned that the diagnosis of suspected cases requires any one of the epidemiological history and conforms to any 2 of the clinical manifestations. If there is no clear history of epidemiology, it is necessary to comply with 3 of the clinical manifestations. Due to the age limitation, infections in most of the children are spread by family gatherings, and some of them have no obvious exposure history, which may lead to misdiagnosis or missed diagnosis, and finally bring difficulties to epidemic prevention and control. Because COVID-19 is highly infectious, in order to screen suspected cases of COVID-19 and prevent the spread of the epidemic, the inpatient department has set up an isolation ward, which aims to make Infectious Diseases get early detection, early diagnosis, early isolation, and early treatment. Some patients with potential COVID-19 infection were further screened in the isolation ward in order to provide new clinical basis for the prevention and control of COVID-19 in the future. In this study, by summarizing the clinical characteristics of children in the isolation ward, it was found that the male-to-female ratio was 1.28:1 in the isolation ward, and the percentage of male was higher than that of female. However, there is no sufficient evidence to prove that male children are more susceptible, which needs to be confirmed by further epidemiological investigation. There are 31 cases of children who had a history of exposure (47%), but only 14 cases (21.2%) were in close contact with COVID-19 patients. However, we also found that some children with COVID-19 had no clear history of exposure. It is suggested that COVID-19 cannot be excluded completely in children without definite epidemic history, and it may be judged comprehensively by combining other symptoms or examination results. Most of the symptoms in the isolation ward were fever and cough, and some children had stuffy nose and runny nose.^[@bibr9-0009922820941228]^ But these symptoms have no obvious specificity compared with other pneumonia. At present, some of COVID-19 are asymptomatic infection,^[@bibr10-0009922820941228],[@bibr11-0009922820941228]^ which suggests that it is not only from the clinical symptoms to judge whether the children should be admitted to the isolation ward. We found that the children in the isolation ward had not only SARS-CoV-2 infection but also *Mycoplasma pneumoniae* and respiratory syncytial virus infection. Previous studies have shown that the differential diagnosis of COVID-19 is *Mycoplasma pneumonia* and bronchiolitis.^[@bibr12-0009922820941228]^ This is consistent with our findings. The number of LYM decreased in 39.4% of the children in the isolation ward. This may be related to the fact that the virus inhibits LYM production or kills LYM.^[@bibr13-0009922820941228]^ Lactate dehydrogenase and CK-MB were increased in more than half of the children, which may be caused by different degrees of myocardial cell damage caused by infection. This is similar to what Mugosa et al^[@bibr14-0009922820941228]^ found in his study of influenza A (H1N1) virus. A total of 21.2% of the children in the isolation ward were SARS-CoV-2 nucleic acid positive, and the infection rate was high, which also suggested the necessity of screening children for COVID-19 in the isolation ward. In the isolation ward, 87.9% of the patients had abnormal chest CT, including 28 cases with speckled or patchy shadow, 6 cases with stripe shape, and 19 cases with GGO. However, the current study found that the CT image of COVID-19 is complex and changeable, mainly manifested as multiple GGO of both lungs. This may be related to telangiectasia and hyperemia of alveolar septum, fluid exudation in alveolar cavity, and interstitial congestion of interlobular septum.^[@bibr15-0009922820941228]^ This shows that the abnormality of chest CT is an important reference for children to enter the isolation ward. Through the study of the ECG of the children, it was found that 16.7% of the ECG of the children showed sinus arrhythmia. This may also be related to cardiac hypoxia and cardiomyocyte damage caused by infection. This also suggests that we should pay attention to protect the heart function in the treatment of children in the isolation ward. When the children were hospitalized in the isolation ward, we found that the hospitalization days were relatively long and the hospitalization costs were relatively high. This may be related to the need for multiple SARS-CoV-2 nucleic acid tests. Therefore, if we can rule out the risk of infectious diseases in the isolation ward as soon as possible, we can transfer the child to the general ward for further treatment, which can shorten the hospitalization time and treatment cost of the child. Although nucleic acid detection is the gold standard for the diagnosis of COVID-19, due to the influence of many factors on the specimen, it is easy to have false negative.^[@bibr16-0009922820941228]^ Therefore, we can comprehensively judge some suspected children with COVID-19 by clinical symptoms, history of epidemiology, number of LYM, myocardial zymogram, chest CT, and ECG. We can first put the suspected children with COVID-19 into the isolation ward for treatment, and then transfer them to the general ward for treatment after excluding COVID-19. In this way, it can play a certain role in the prevention and treatment of COVID-19. As this study belongs to a single-center study, and the sample size is small, there are some limitations, which may not reflect the actual situation of the disease. Therefore, further research is needed with a larger sample size and at multiple locations. Author Contributions {#section5-0009922820941228} ==================== JZ contributed to acquisition, analysis, or interpretation of data, and drafted the manuscript. YW substantially contributed to conception or design and critically revised the manuscript for important intellectual content. All authors gave final approval, agree to be accountable for all aspects of the work in ensuring that questions relating to the accuracy or integrity of any part of the work are appropriately investigated and resolved. **Declaration of Conflicting Interests:** The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. **Funding:** The author(s) received no financial support for the research, authorship, and/or publication of this article. **Ethical Approval:** All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. **ORCID iD:** Jian Zhu  <https://orcid.org/0000-0003-1892-1660>

Introduction {#S1} ============ Neuromyelitis Optica (NMO) is an idiopathic, autoimmune and inflammatory disease of the central nervous system (CNS) that occurs in individuals of all ethnicities. NMO has long been considered as a variant of multiple sclerosis (MS) until the detection of the serum antibodies to the astrocytic water channel aquaporin-4 (AQP4) ([@B14]). AQP4 antibodies are detectable in 60--90% of the patients with NMO, but not in the serum of MS patients ([@B7]). This finding together with the foveal thinning reported in NMO patients without optic neuritis (ON) history ([@B20]; [@B37]), supports the concept of NMO to be a predominantly serum AQP4 IgG antibody-mediated astrocytopathy \[NMO spectrum disorder (NMOSD)\] that is distinct from MS ([@B36]). Recently, Müller glial dysfunction has been reported in the eyes of seropositive AQP4-IgG NMOSD patients support a subclinical retinal astrocytopathy in the disease ([@B39]). It has been reported that subtype of reactive astrocytes are neurotoxic, which may explain the severe axonal loss observed in NMOSD ([@B15]). MOG-antibody-positive NMOSD has been recently recognized as a possible new inflammatory disorder of the CNS ([@B9]), patients with positive MOG-IgG were thus excluded in this study. A higher frequency of familial NMO cases was observed in a previous study, suggested complex genetic susceptibility in NMOSD ([@B17]). Though several genes and genetic variants have been evaluated as contributors to NMOSD, the major genes that confer significant susceptibility are still unknown ([@B10]; [@B33]; [@B34]; [@B38]; [@B3]). Brain-derived neurotrophic factor (BDNF) has unique roles in neuronal growth, differentiation, distribution and survival ([@B23]; [@B5]). The genetic variation that is the focus of this study has a single nucleotide substitution from G to A (Val66Met, NCBI database dbSNP rs6265) in the pro-region of the BDNF gene. The BDNF Val66Met polymorphism has been associated with various neurodegenerative and psychiatric diseases ([@B19]; [@B28]). In a recent study, carriage of Met alleles was associated with lower rates of optic nerve damage and was reported to be reducing the long-term open-angle glaucoma (OAG) progression in female patients ([@B26]). However, it remains to be elucidated whether Val66Met is associated with neuroaxonal damage in NMOSD. Optical coherence tomography (OCT) has been used to examine rates of axonal damage through retinal nerve fibre layer (RNFL) and macular thickness in NMO and MS which could help distinguish the patterns of optic nerve damage in the two diseases ([@B18]; [@B25]). Multifocal visual evoked potentials (mfVEPs) provide functional assessments of axonal loss (amplitude) and demyelination (latency) in the visual pathway ([@B11]). NMOSD patients were suggested to have a more severe axonal loss in ON eyes by more significantly reduced mfVEP amplitude compared to MS ([@B27]). The aim of the present study was to clarify whether the carriage of Met allele is associated with axonal loss and demyelination in ON and non-ON (NON) eyes of NMOSD participants by using both structural (OCT) and functional (mfVEP) analysis. Methods {#S2} ======= Neuromyelitis optica spectrum disorder (*N* = 17) patients were recruited from four tertiary neuro-ophthalmology or neurology clinics in Sydney. The NMOSD diagnosis was made per the 2015 diagnostic criteria ([@B36]). Exclusion criteria are: patients tested as MOG-positive, retinal, optic nerve, or other neurologic diseases affecting the visual system, including any other types of optic neuropathy, optic atrophy with no known cause. Patients tested within 12 months of the acute ON were also excluded from the study to eliminate the decline of amplitude and prolongation of latency in mfVEP in a post-acute stage of ON ([@B11]). Two participants with visual acuities worse than hand movement were not able to complete mfVEP testing and were excluded from mfVEP analysis. Details on the recruitment, composition and selective attrition are described in detail elsewhere, blood samples from all but two participants with MOG antibodies in the previous study were obtained ([@B11]). The ethnic background of the participants is mainly Caucasian, except for one Asian subject. All participants underwent OCT scans for RNFL and ganglion cell-inner plexiform layer (GCIPL) thickness, multifocal VEP testing for amplitude and latency, and MRI scans for whole-brain (WB) lesion volume (LV) as previously reported. The study was conducted in accordance with the Declaration of Helsinki and approved by the Human Research Ethics Committee of the University of Sydney (Sydney, NSW, Australia). Written informed consent was obtained from all study participants. Genotyping {#S2.SS1} ---------- The genomic DNA was isolated from peripheral blood with a commercially available DNA extraction kit (Qiagen, Hilden, Germany). Quantification of isolated DNA was carried out with a spectrophotometer (Thermo Scientific, Rockford, IL, United States). The G → A nucleotide substitution, identifying the of BDNF Val66Met polymorphism, was assayed by polymerase chain reaction (PCR, Eppendorf, Hamburg, Germany). The primers used in this study were as follows: forward 5′ ACTCTGGAGAGCGTGAATGG 3′ and reverse 5′ TCCAGGGTGATGCTCAGTAGT 3′. The amplification conditions for PCR were initiated at 95°C for 5 min, followed by 30 cycles comprising of denaturation at 94°C for 1 min, annealing at 55°C for 30 s and extension at 72°C for 1 min, with a final extension step of 5 min at 72°C. The carriage of BDNF Val66Met polymorphism was determined by direct sequencing (both directions, Australian Genome Research Facility, Sydney, NSW, Australia) of genomic DNA. A detailed description of the genotyping procedure is provided elsewhere ([@B28]). Statistical Analysis {#S2.SS2} -------------------- Statistical analysis was performed using SPSS software version 22 (SPSS, Inc., Chicago, IL, United States). Data from both ON eyes and NON eyes were included for the comparison between two genotype groups using the generalised estimating equation model (GEE). The gender was included as a cofactor, and the age, disease duration and number of ON episodes of all patients were included as covariates in the GEE analysis. The correlation between OCT and mfVEP parameters were also examined using the GEE method with adjustment of intrasubject factors. A *P*-value of less than 0.05 was considered statistically significant, and a *P*-value between 0.05 and 0.1 was considered borderline significant. Results {#S3} ======= Demographic and clinical features of the participants included in the study are presented in [Table 1](#T1){ref-type="table"}. Twenty eyes of 17 patients with NMOSD had ON history and was included in the following GEE analysis as ON eyes. Fourteen eyes of the patients had no previous ON attacks and were included as NON-eyes in the statistical analysis. The mean age of the patients was 47.8 years (range, 23--78). No significant difference was found in sex ratio, AQP4 status, age, disease duration, BCVA or T2 WB LV between two genotype groups ([Table 1](#T1){ref-type="table"}). ###### Demographic and clinical characteristics of participants. **Total (*N* = 17)** **Val/Val (*N* = 9)** **Met carriers (*N* = 8)** ***P*-values** --------------------------------------- ---------------------- ----------------------- ---------------------------- ---------------- Eyes, *N* (%) 34 18 (52.9) 16 (47.1) n.a.  ON eyes, *N* (%)^a^ 20 (58.8) 10 (55.6) 10 (62.5) 0.68  NON-eyes, *N* (%)^a^ 14 (41.2) 8 (44.4) 6 (37.5) ON history, *N* (%) 13 (76.5) 6 (66.7) 7 (87.5) 0.31  Unilateral ON history, *N* (%)^a^ 6 (35.3) 2 (22.2) 4 (50.0) 0.39  Bilateral ON history, *N* (%)^a^ 7 (41.2) 4 (44.4) 3 (37.5) Female, *N* (%)^a^ 11 (64.7) 6 (66.7) 5 (62.5) 0.86 AQP4-IgG seropositive, *N* (%)^a^ 13 (76.5) 6 (66.7) 7 (87.5) 0.31 Age, years, mean (range)^b^ 47.8 (23--78) 42.8 (23--64) 53.4 (38--78) 0.14 Disease duration, years, mean (SD)^b^ 7.4 (10.1) 10.9 (13.2) 3.5 (1.9) 0.1 BCVA, LogMAR, mean (SD)^b^ 0.4 (0.8) 0.3 (0.8) 0.4 (0.9) 0.63 T2 WB LV, cm^3^, mean (SD)^c^ 860.2 (1858.0) 926.0 (1737.7) 786.3 (2104.2) 0.91 AQP4-IgG = astrocyte water channel aquaporin-4 immunoglobulin G; BCVA = best-corrected visual acuity; WB = whole brain; LV = lesion volume. Statistics: a chi-square test; b generalized estimating equation; c Mann--Whitney U test. Neuromyelitis optica spectrum disorder treatment: immunosuppressants ( n = 13, including azathioprine, mycophenolate, rituximab, and prednisolone), plasma exchange ( n = 3), and not receiving treatment ( n = 3). No parameter showed significant differences between two genotype groups. The genotype frequencies of Val66Met among the NMOSD cohort were 9 (52.9%) for genotype GG (Val/Val), 7 (41.2%) for GA (Val/Met), and 1 (5.9%) for AA (Met/Met). The allele frequency of the A allele (Met) was 26.5% in the study subjects. The OCT and mfVEP measures were compared between two genotype groups and are summarized in [Table 2](#T2){ref-type="table"}. In ON eyes, there were significant differences between the BDNF Val66Met polymorphism genotypes in averaged GCIPL thickness (*P* = 0.002) and global retinal nerve fiber layer (gRNFL) thickness (*P* \< 0.001). In parallel with the OCT changes, carriage of Met allele was also associated with significantly more prolonged latency (*P* = 0.008) in ON eyes. The reduction of mfVEP amplitude showed the borderline difference between Met and Val genotypes (*P* = 0.07) in ON eyes. In NON-eyes, no significant difference was observed in any of the parameters mentioned above ([Table 2](#T2){ref-type="table"}). As there are possibilities for the AQP4 seronegative patients to be affected by another disease ([@B6]), a sub-analysis was performed to validate the differences in ON eyes and excluded the potential influence of AQP4 serology. In AQP4 seropositive patients (16 eyes), significant differences were observed between genotype groups in GCIPL, gRNFL and mfVEP latency (*P* \< 0.001, *P* = 0.04, and *P* \< 0.001, respectively). There was also a borderline significant difference between Val/Val and Met carriers in mfVEP latency (*P* = 0.1). ###### Association between BDNF Val66Met polymorphism and optic nerve damage in NMOSD. **Parameters** **Val/Val** **Met carriers** **Difference** ***P*-values** ---------------- ------------- ------------------ ---------------- ---------------- -------- ----------------- ------------ ON eyes OCT GCIPL 62.19 59.43 2.76 0.97 to 4.55 0.002^∗^ gRNFL 82.23 58.84 23.39 20.54 to 26.25 \<0.001^∗^ mfVEP Amp 137.64 68.54 69.1 −4.67 to 142.86 0.07 Lat 152.13 163.39 −11.26 −19.61 to−2.91 0.008^∗^ NON-eyes OCT GCIPL 78.81 75.44 3.37 −9.13 to 15.88 0.6 gRNFL 98.98 95.2 3.78 −22.72 to 30.28 0.78 mfVEP Amp 171.45 184.71 −13.26 −46.57 to 20.06 0.44 Lat 148.91 149.87 −0.96 −7.32 to 5.40 0.77 ON = optic neuritis; OCT = optic coherence tomography; GCIPL = ganglion cell-inner plexiform layer; gRNFL = global retinal nerve fiber layer; mfVEP = multifocal visual evoked potential; NON = non-optic neuritis, n.s. = not significant. GEE models were applied assuming additive effect for the minor allele variant (Met carriers), adjusted for sex, age, disease duration, and ON episodes. Statistical significance was defined as P \< 0.05 an asterisk ( ∗ ). The correlation between GCIPL and gRNFL (*P* \< 0.001) was showed in [Figure 1A](#F1){ref-type="fig"} as data visualization. In addition, correlation analysis demonstrated strong positive correlations in NMOSD participants with increased GCIPL (*P* \< 0.001) and gRNFL (*P* = 0.001) thickness associated with higher mfVEP amplitude ([Figure 1B](#F1){ref-type="fig"}). There was a significant correlation between GCIPL and mfVEP latency (*P* = 0.03). A borderline significant correlation was also observed between gRNFL and mfVEP latency (*P* = 0.06). {#F1} Discussion {#S4} ========== This study suggests that BDNF Val66Met polymorphism may be associated with more severe ON attacks in NMOSD patients. In cross-sectional analysis, the Met carriers were found to have significantly more reduced GCIPL and gRNFL thickness, more diminished mfVEP amplitude (borderline significance) and significantly more delayed mfVEP latency than Val homozygotes in ON eyes. The genotypic effect of Val66Met on NMOSD remains positive even with adjustment for a range of cofactors and covariates, including sex, age, disease duration, and ON episodes. In contrast, no association was found between BDNF polymorphisms and changes in NON-eyes of patients. Correlation analysis suggested positive associations between structural and functional measurements in the NMOSD cohort. To our knowledge, this is the first study to suggest that the presence of Met alleles may convey higher rates of axonal damage in ON in NMOSD. The frequency of the BDNF Val66Met polymorphism varies among different ethnicity. In our cohort, the Met allele frequency is 26.5%, which is close to the frequencies previous reported in the United States (28.0%) ([@B22]) and Italian cohorts (29.7%) ([@B31]). OCT is an objective, simple and non-invasive method that allows *in vivo* measurement of the thickness of retinal nerve fibers. It has been reported that the actual RNFL thickness measured from histologic sections would be 4--12% larger than the corresponding OCT measurements of nerve fibers ([@B29]). Previous studies demonstrated that ON in NMO is associated with thinner RNFL, GCIPL, and mfVEP amplitude compared to MS ([@B18]; [@B27]). The mfVEP amplitude and latency were suggested to be highly correlated with RNFL thickness ([@B21]; [@B30]; [@B12]). The age distribution was suggested to influence the AQP4 autoimmunity where females aged 65 years above were more likely to be seropositive than other age groups, and the detection rate of AQP4-IgG rise exponentially in females after 50 years old ([@B24]). A recent study has also established that the gray matter atrophy and RNFL thinning were correlated with longer disease duration in NMOSD ([@B32]). There were no significant differences between two genotype groups in gender, age or disease duration in this study, and though all these parameters have been included and adjusted for as either cofactors or covariate, the results remained significant in ON eye analysis. It has been reported that BDNF is transported anterogradely and retrogradely, and BDNF secretion has a selective effect on the survival and maintenance of lesioned optic nerve axons ([@B35]; [@B4]). As the carriage of BDNF Val66Met polymorphism has been associated with reduced activity-dependent secretion of BDNF ([@B2]), the more severe axonal loss in ON eyes may be explained by less availability of BDNF within the retina and along the higher visual pathway. The results of the present study demonstrated a potential negative effect BDNF Val66Met polymorphism has on ON damage in NMOSD, which contrasts with the previous glaucoma study where the Met alleles exhibited protective effects on OAG progression in a gender-specific way ([@B26]). The opposite effect may suggest different mechanisms in the axonal loss in glaucoma and NMOSD. The precursor form of BDNF (pro-BDNF) was suggested to increase cell apoptosis, while in contrast, mature BDNF (mBDNF) enhances cell survival ([@B16]). In neurons, pro-BDNF is generally converted to mBDNF by proteases such as tissue plasminogen activator (tPA), and cerebral tPA expression was reported to decrease with age in an animal model ([@B1]). Thus, higher levels of BDNF secretion in Val/Val may not increase neuronal function unless there are enough cleavage molecules to convert BDNF from the precursor form to mature form. The mean age was approximately 70 for the participants in the glaucoma study, and only 47 in our NMOSD cohort, the significant age difference between the cohorts may contribute to differentiated tPA levels and opposite effects of Val66Met on RGC survival. In addition, it is well established that in glaucoma, elevated intraocular pressure is the main cause of RGC apoptosis and optic nerve head degeneration via several mechanisms ([@B13]), while in NMOSD, inflammation of the optic nerve and spinal cord were usually dominating the disease and causing axonal damage. It has been reported that BDNF has protective effects against ischemic insult by modulating local inflammation in the brain in an animal model ([@B8]). The Met allele may interact differently with acute optic nerve damage in inflammation and progressive glaucomatous optic neuropathy; however, future studies are warranted to investigate further the cellular mechanisms of the contrast effects BDNF Val66Met have in two diseases. There are several potential limitations to the current study. First, the sample size of NMOSD is relatively small due to the rarity of the disease in the Caucasian population. However, the genotype distribution was favorable with both eyes included in the analysis, which significantly increased the sample size and met the minimum requirement for desired statistical power. The *P*-values were especially small for both gRNFL and GCIPL thickness ([Table 1](#T1){ref-type="table"}), which were likely representing true differences between the two genotype groups. Second, the investigation was in the absence of a healthy control group. This study is also limited by the cross-sectional study design, future longitudinal study with larger sample size is necessary to validate the reproducibility of the results, especially a cohort with only AQP4 seropositive subjects to exclude the possibility of seronegative patients influenced by another disease. In addition, future research could also focus on the BDNF Val66Met polymorphism's effect on ON in other neuroinflammatory diseases such as MS. Conclusion {#S5} ========== Our study demonstrated significant associations between carriage of BDNF Met allele and more severe axonal damage in ON eyes in NMOSD patients. The difference was not found in NON-eyes of participants indicating the effect of BDNF Val66Met on RGC axonal loss in NMOSD is specifically related to acute inflammatory ON attacks. Data Availability Statement {#S6} =========================== The raw data supporting the conclusion of this manuscript will be made available from the corresponding author, without undue reservation, to any qualified researcher. Ethics Statement {#S7} ================ The studies involving human participants were reviewed and approved by Human Research Ethics Committee of the University of Sydney (Sydney, NSW, Australia). The patients/participants provided their written informed consent to participate in this study. Author Contributions {#S8} ==================== TS: acquisition, analysis and interpretation of data, and drafting and revising the work. VG: revising the work and acquisition of data for the work. CY: acquisition of data and design of the work. AK and YY: design of the work, revising the work, and provided approval for publication of the content. SG: revising the work and provided approval for publication of the content. Conflict of Interest {#conf1} ==================== The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. **Funding.** This study was supported by the National Health and Medical Research Council Australia, The Ophthalmic Research Institute of Australia (ORIA), National Multiple Sclerosis Society (NMSS), and Sydney Medical School Foundation. [^1]: Edited by: Christine Nguyen, The University of Melbourne, Australia [^2]: Reviewed by: Aline Silva Miranda, Federal University of Minas Gerais, Brazil; Friedemann Paul, Charité University Medicine Berlin, Germany [^3]: This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience

Introduction {#s1} ============ Schistosomiasis continues to be one of the major health problems in the developing world since the control strategy centered on mass chemotherapy has failed to effectively control this disease. Currently, more than 207 million people worldwide are infected with schistosomiasis [@pone.0049234-King1]. Therefore, new and more effective control strategies including vaccines are urgently needed. Toward this end, trying to clarify the molecular events involved in the interaction between schistosome and immunocytes is crucial. Most studies on the immunology of schistosome infection have focused on the molecular mechanisms of adaptive immune responses. In view of the important role of innate immunity in initiating and regulating acquired immunity [@pone.0049234-Takeda1], it is necessary to study the impact of innate immunity on schistosome infection, including the relationship between schistosome antigens and antigen-presenting cells. The expression of MHC class II is affected by infection. Many pathogens such as *Mycobacterium*, *Murine cytomegalovirus* and *Leishmania donovani* possess the ability to suppress MHC class II expression and use it as a means of evading the host's immunological attack [@pone.0049234-Wojciechowski1], [@pone.0049234-Heise1], [@pone.0049234-Reiner1]. Some studies suggested that several components of schistosome SEA or SEA-induced factors might play a regulatory role on the I-A expression in granuloma macrophages (GMs) [@pone.0049234-Stadecker1]. Based on the key role of MHC class II in presenting foreign antigen to T cells, the limitation of MHC class II expression might represent one of the most effective strategies for down-regulating immune responses leading to evasion of the parasite from host's immune attack. Following the deposition of schistosome eggs in the tissue affected and the massive release of egg antigen, intense immune response is aroused indicating the development of the acute phase of schistosomiasis [@pone.0049234-Borojevic1]. Despite the uninterrupted antigenic stimulation, a down regulation of the granulomatous response is observed in chronic schistosomiasis [@pone.0049234-Domingo1]. The response events involved in acquired immunity in this course have acquired a broad exploration but no more studies were focused on the MHC class II expression on antigen presenting cells regulated by schistosome-derived components. Macrophages are the main antigen presenting cells presenting in schistosome egg granulomas, account for about 30% of the total cells in egg granulomas [@pone.0049234-Schook1]. We thus selected macrophage cell line RAW 264.7 in this study for detail exploration of the regulatory effect of SEA on MHC class II expression. Results {#s2} ======= SEA Attenuated IFN-γ-induced MHC Class II Expression in RAW 264.7 Cells {#s2a} ----------------------------------------------------------------------- IFN-γ, which has multiple immunomodulatory roles in immune responses, is a very effective cytokine for up-regulating MHC class II expression and is necessary for the host's defense to pathogens [@pone.0049234-Schroder1]. Macrophages are one of the most important cells influenced by IFN-γ. IFN-γ can directly promote antigen processing and presenting ability of macrophages [@pone.0049234-Schroder1]. To evaluate the regulatory role of SEA on MHC class II, we selected IFN-γ as an inducer of MHC class II expression so that we can make a detailed observation of the effect of SEA on regulating MHC class II expression. To ascertain the appropriate concentration of IFN-γ used for up-regulation of MHC class II, RAW 264.7 cells were incubated with serially diluted IFN-γ for 48 h. 4 ng/ml IFN-γ (5×10^6^ IU/mg, PeproTech) was the lowest effective dose for significantly upregulating MHC class II in RAW 264.7 cells and was selected for use throughout the study (data not shown). Compared with MHC class II expression in RAW264.7 cells incubated with IFN-γ alone, 40 µg/ml SEA can significantly attenuated IFN-γ-induced MHC class II expression in RAW 264.7 cells (*P*\<0.01). ([Figure 1A, 1B](#pone-0049234-g001){ref-type="fig"}). Considering the amount of deposited eggs increased gradually with the elongation of the worms′life in vivo or with the development of schistosomiasis, we analyzed the ability of different concentrations (serially diluted from 40 µg/ml to 0.156 µg/ml) of SEA at attenuating IFN-γ-induced MHC class II expression. The results showed that SEA significantly attenuated IFN-γ-induced MHC class II expression in RAW 264.7 cells in a dose-dependent fashion between concentration changing from 40 µg/ml to 0.625 µg/ml ([Figure 1C](#pone-0049234-g001){ref-type="fig"}) (*P*\<0.01), and this effect was lost at concentrations of 0.313 µg/ml and 0.156 µg/ml. {#pone-0049234-g001} CIITA, the MHC class II transactivator protein, is involved in the inducible expression of class II genes upon IFN-γ treatment and is controlled and induced by IFN-γ [@pone.0049234-Steimle1]. To analyze whether MHC class II expression was attenuated by SEA at the transcriptional level, we tested the level of CIITA by real-time PCR. RAW 264.7 cells were primed with 0.65 µg/ml of SEA in the presence or absence of IFN-γ. Cells primed with IFN-γ or culture medium alone (untreated) were used as controls. CIITA mRNA transcriptional level in these cells were determined after 48 h of culture. The results indicated that the transcription of CIITA induced by IFN-γ was attenuated significantly by SEA ([Figure 1D](#pone-0049234-g001){ref-type="fig"}). This suggested that the attenuation of IFN-γ-induced MHC class II expression by SEA might happened at transcriptional level. To exclude the possible effects of contaminated endotoxin in the prepared SEA on MHC class II expression, we pretreated the SEA with Detoxi-Gel™ Endotoxin Removing Gel (Thermo Scientific Pierce) and then tested the activity of endotoxin contained in the treated SEA sample. The results showed that following the treatment with endotoxin removing gel, the activity of endotoxin contained in the prepared SEA is similar to the negative control (0 EU/ml). Then RAW 264.7 cells were treated with 10 ng/80 IU/ml IFN-γ (R&D) in presence or absence of 10 µg/ml SEA for 48 h at 37°C, 5% CO~2.~ Cells cultured in medium alone were used as untreated control. The results showed that these SEA still possesses the effects in significantly attenuating IFN-γ-induced MHC class II expression in RAW 264.7 cells ([Figure 1E and 1F](#pone-0049234-g001){ref-type="fig"}). The Production of IL-10 and IL-6 Induced by SEA from RAW 264.7 Cells were Promoted by IFN-γ {#s2b} ------------------------------------------------------------------------------------------- The granulomas formation around the deposited schistosome eggs in liver is dependent on type IV hypersensitivity reaction which depends on effective antigen presentation. Our results above showed that SEA could down-regulate the expression of MHC class II induced by IFN-γ ([Figure 1B](#pone-0049234-g001){ref-type="fig"}). IL-10 and IL-6 have been reported to possess the ability in down-regulating the expression of MHC class II through feedback effects [@pone.0049234-Redpath1], [@pone.0049234-Kincaid1] and are induced following the deposition of schistosome eggs in vivo [@pone.0049234-Dunne1]. Without IFN-γ, 40 µg/ml and 5 µg/ml of SEA induced significant up-regulation of IL-10 production in RAW 264.7 cells comparing to non-SEA treatment (*P*\<0.01, *P*\<0.05 respectively) ([Figure 2A and 2B](#pone-0049234-g002){ref-type="fig"}). SEA concentration lower than 2.5 µg/ml failed to induce IL-10 from RAW 264.7 cells ([Figure 2B](#pone-0049234-g002){ref-type="fig"}). When IFN-γ is presented, SEA significantly attenuate IFN-γ-induced MHC class II expression even when its concentration was decreased to 0.625 µg/ml ([Figure 1C](#pone-0049234-g001){ref-type="fig"}). {#pone-0049234-g002} SEA could significantly attenuate IFN-γ-induced MHC class II expression even when its concentration was decreased to 0.625 µg/ml ([Figure 1C](#pone-0049234-g001){ref-type="fig"}). To explore the underlying reasons responsible for attenuating IFN-γ-induced MHC class II expression by low concentration of SEA, we detected IL-10 production in supernatants of RAW 264.7 cells primed with SEA and IFN-γ simultaneously. Surprisingly, we found that although RAW 264.7 cells could only generate about 17 pg/ml of IL-10 with the stimulation of 5 µg/ml SEA alone for 48 h ([Figure 2B](#pone-0049234-g002){ref-type="fig"}), they could generate about 680 pg/ml of IL-10 when were stimulated simultaneously with 5 µg/ml SEA and IFN-γ for 48 h ([Figure 2C](#pone-0049234-g002){ref-type="fig"}). Moreover, SEA at 2.5, 1.25 and 0.65 µg/ml still possess the same effect as 5 µg/ml of SEA at inducing IL-10 production in the presence of IFN-γ ([Figure 2C](#pone-0049234-g002){ref-type="fig"}). When the concentration of SEA was decreased to 0.313 µg/ml, the IL-10 production induced by stimulation of SEA and IFN-γ simultaneously from RAW 264.7 cells was still kept in a relatively high level (about 450 pg/ml) although it showed a significant decrease when compared with that induced by 0.65 µg/ml of SEA and IFN-γ simultaneously (*P*\<0.01) ([Figure 2C](#pone-0049234-g002){ref-type="fig"}). These results suggested that IFN-γ could enhance the ability of SEA at inducing IL-10 from RAW 264.7 cells. Similarly, we tested the IL-6 production induced by high and low level of SEA in presence or absence of IFN-γ. 40 µg/ml of SEA induced significant up-regulation of IL-6 production from RAW 264.7 cells (*P*\<0.01) ([Figure 2D](#pone-0049234-g002){ref-type="fig"}). 0.65 µg/ml of SEA alone failed to induce IL-6 from RAW 264.7 cells. However, when RAW 264.7 cells were stimulated simultaneously with 0.65 µg/ml of SEA and IFN-γ, they could generate significantly increased production of IL-6 (*P*\<0.01) ([Figure 2D](#pone-0049234-g002){ref-type="fig"}). This suggested that IFN-γ could also enhance the ability of SEA at inducing IL-6 from RAW 264.7 cells. The Attenuation of SEA on IFN-γ-induced MHC Class II Expression was Mediated at least Partly by IL-10 and IL-6 {#s2c} -------------------------------------------------------------------------------------------------------------- To ascertain whether IL-10 and IL-6 induced by SEA from RAW 264.7 cells might mediate the inhibition of IFN-γ-induced MHC class II expression, we adopted antibody neutralization tests. In short, the supernatants of RAW 264.7 cells primed with 40 or 0.65 µg/ml of SEA and IFN-γ simultaneously were collected and then prepared for the antibody neutralization tests. The supernatants were then pretreated with anti-IL-10 or anti-IL-6 antibodies, or their isotype controls in the presence of IFN-γ for 2 h at 37°C. Fresh medium alone or fresh medium containing IFN-γ were used as control. Following, the pretreated supernatants and control medium were used for culturing RAW 264.7 cells for 48 h. At the end of culture, MHC class II expression was detected by flow cytometry. The results indicated that neutralization of IL-10 ([Figure 3A, 3B](#pone-0049234-g003){ref-type="fig"}) or IL-6 ([Figure 3C, 3D](#pone-0049234-g003){ref-type="fig"}) in the supernatants of RAW 264.7 cells primed with 40 µg/ml of SEA and IFN-γ simultaneously both resulted in significant up-regulation of MHC class II expression (*P*\<0.01). Neutralization of IL-10 ([Figure 3E, 3F](#pone-0049234-g003){ref-type="fig"}) but not IL-6 ([Figure 3G, 3H](#pone-0049234-g003){ref-type="fig"}) in supernatants of RAW 264.7 cells primed with 0.65 µg/ml of SEA and IFN-γ simultaneously led to significant up-regulation of MHC class II expression. These results suggested that IL-10 might play a more important role than IL-6 in mediating the suppression of MHC class II expression by SEA. {#pone-0049234-g003} SEA Attenuated MHC Class II Expression through TLR4 {#s2d} --------------------------------------------------- Pattern recognition receptors play an important role in regulating immune response through initiation of cytokines production following interaction with pathogen-associated molecular patterns [@pone.0049234-Takeda1]. TLR4 is expressed on cell surface and is involved in recognition of the constituents contained in schistosome SEA [@pone.0049234-Thomas1]. To understand the role of TLR4 on attenuating MHC class II expression in RAW 264.7 cells by SEA, we adopted an antibody blocking assay. In short, after being incubated with anti-TLR4 monoclonal antibody or the isotype-matched control for 30 min at 37°C, these RAW 264.7 cells were incubated with 40 µg/ml of SEA and IFN-γ simultaneously for 48 h. Cells cultured in medium alone were used as control. The expression of MHC class II in these RAW 264.7 cells was detected by flow cytometry and the level of IL-10 and IL-6 in supernatants was assayed by ELISA. The results indicated that blocking TLR4 in RAW 264.7 cells leads to significant increase of MHC class II expression (*P*\<0.01) ([Figure 4A, 4B](#pone-0049234-g004){ref-type="fig"}) and significant decrease in IL-10 ([Figure 4C](#pone-0049234-g004){ref-type="fig"}) and IL-6 ([Figure 4D](#pone-0049234-g004){ref-type="fig"}) production compared with the isotype control antibody treated one (*P*\<0.01). These results suggested that TLR4 might play an important role in mediating SEA-induced production of IL-10 and IL-6, and the suppression of MHC class II expression in RAW 264.7 cells through interaction with some components contained in schistosome SEA. It has been suggested that the expression of pattern recognition receptors might be regulated by cytokines [@pone.0049234-Mueller1], [@pone.0049234-Miller1]. In our experiments, we found that IFN-γ could up-regulate TLR4 expression in RAW 264.7 cells ([Figure 4E](#pone-0049234-g004){ref-type="fig"}). The increased expression of TLR4 in RAW 264.7 cells stimulated with IFN-γ might improve the ability of RAW 264.7 cells in recognizing the components contained in SEA and might represent a reasonable explanation for the significant increasing in IL-10 and IL-6 production in supernatants of RAW 264.7 cells stimulated with 0.65 µg/ml of SEA and IFN-γ simultaneously compared with that being stimulated with 0.65 µg/ml of SEA alone. {#pone-0049234-g004} SEA Attenuated IFN-γ-induced MHC Class II Expression in Mouse Peritoneal Macrophages {#s2e} ------------------------------------------------------------------------------------ To find out whether SEA attenuated IFN-γ-induced MHC class II expression can also happen in primary macrophages, we isolated mouse peritoneal macrophages and treated them with 10 ng/80 IU/ml IFN-γ (R&D) in presence or absence of 10 µg/ml SEA. The purity of mouse peritoneal macrophages reached 80% based on the results of flow cytometry tested with PE-conjugated rat anti-mouse F4/80 (data not shown). SEA significantly attenuated IFN-γ-induced MHC class II expression in mouse peritoneal macrophages ([Figure 5A and 5B](#pone-0049234-g005){ref-type="fig"}) (\**P\<*0.05). {#pone-0049234-g005} Discussion {#s3} ========== With the chronicity of schistosomiasis, host responsiveness to schistosome eggs is alleviated [@pone.0049234-Warren1], [@pone.0049234-Boros1]. It is manifested as the spontaneous diminution of the granuloma formation around the newly deposited schistosome eggs in the chronic stage of the disease, which was described as 'endogenous desensitization' in the 1960s [@pone.0049234-Domingo1]. Along with this, the host's immune responses shifts from Th1 to Th2 phenotype accompanied with the up-regulation of a lot of cytokines including IL-10, TGF-β and IL-6 [@pone.0049234-Dunne1]. The immune response elicited by SEA around deposited eggs belongs to the delayed-type, T cell-mediated hypersensitivity [@pone.0049234-Warren2], [@pone.0049234-Phillips1] which depends on MHC class II-mediated antigen presentation [@pone.0049234-Hernandez1] and IFN-γ signaling [@pone.0049234-Oliveira1]. Previous studies have suggested that the components contained in SEA might possess the ability to down-regulate immune responses. For example, egg-tolerized mice suffered high mortalities due to an excessive immune response that brought about lethal immune damage [@pone.0049234-Fallon1], SEA could successfully ameliorate and prevent the progression of experimental autoimmune encephalomyelitis (EAE) in mice [@pone.0049234-Zheng1]. By an *in vitro* analysis on RAW 264.7 cell line, our study contributes new information for understanding the negative regulation of SEA. We found that SEA could attenuate IFN-γ-induced MHC class II expression by inducing IL-10 and IL-6 production. Moreover, we found that SEA alone at a high level (40 µg/ml) could also induce high level of IL-10 and IL-6 from RAW 264.7 cells and that MHC class II expression in RAW 264.7 cells stimulated with 40 µg/ml SEA could be up-regulated significantly, which was much weaker than that induced by IFN-γ (data not shown). Based on the pivotal role of the expression of MHC class II in initiation and regulation of immune responses, we could deduce that schistosome might suppress the host's immune response through down-regulating MHC class II expression to evade immune attack. This finding supports a viewpoint derived from a study conducted in *Schistosoma mansoni* infection that showed I-A expression on granuloma macrophages was regulated by SEA or SEA stimulated factors [@pone.0049234-Stadecker1]. Antigen presenting cells recognize pathogen-associated molecular patterns through pattern recognition receptors, which resulted in inflammatory cytokines gene expression [@pone.0049234-Takeda1]. Phagocytosis-mediated antigen presentation together with TLR-mediated inflammatory cytokines expression instructs the development of antigen-specific adaptive immunity [@pone.0049234-Takeda1]. Our study showed that SEA down-regulated the expression of MHC class II in RAW 264.7 cells through IL-10 and IL-6 induction. These results supported that pathogens could down-regulate immune responses by inducing IL-10 [@pone.0049234-Redpath1] or IL-6 [@pone.0049234-Kincaid1] which could attenuate MHC class II expression through negative feedback effects. The ability of SEA to induce IL-10 was stronger than that of IL-6. Our results showed that the production of IL-10 from RAW 264.7 cells stimulated with 0.65 µg/ml of SEA and IFN-γ was nearly equal to that with 40 µg/ml of SEA. IL-10 play the major role in mediating the down-regulation of MHC class II expression in RAW 264.7 cells as our results showed that neutralization of IL-10 but not IL-6 in supernatants of RAW 264.7 cells stimulated with 0.65 µg/ml of SEA and IFN-γ simultaneously could lead to the up-regulation of MHC class II. IL-10 and TGF-β were both induced and both recognized as factors can promote host survival by suppressing pro-inflammatory cytokine production and ova-induced hepatotoxicity in the acute phase of schistosomiasis [@pone.0049234-Herbert1]. Anti-inflammatory effects of IL-10 are well known including down-regulation of IFN-γ-induced MHC class II expression [@pone.0049234-Redpath1], [@pone.0049234-Sendide1]. Although TGF-β was reported playing a role in suppressing both constitutive and cytokine-inducible MHC class II expression in macrophages [@pone.0049234-Delvig1], it could not play an important role in mediating the suppression of MHC class II expression in RAW 264.7 cells by SEA because IFN-γ show a suppressive effect on TGF-β induction as we observed in our study (data not shown). 40 µg/ml of SEA could induce 1800 pg/ml production of TGF-β from RAW 264.7 cells following the stimulation for 48 h (data not shown). However, TGF-β showed no significant change in supernatants of RAW 264.7 cells stimulated simultaneously with 0.65 µg/ml of SEA and IFN-γ compared with that produced by cells cultured with medium alone or stimulated with 0.65 µg/ml of SEA alone (data not shown). TGF-β production induced with 40 µg/ml of SEA and IFN-γ simultaneously from RAW264.7 cells decreased significantly compared with that were induced with 40 µg/ml of SEA alone (*P*\<0.05) (data not shown). So the immunosuppressive effect of TGF-β in controlling the pathogenesis of schistosomiasis could be exerted maily through induction of Foxp3+ Treg cell [@pone.0049234-Fu1] which was shown to be responsible for the down-regulation of granuloma inflammation [@pone.0049234-Hesse1], [@pone.0049234-Singh1]. In innate immunity, suppressor of cytokine signaling (SOCS) proteins inhibit JAK/STAT signaling by various mechanisms [@pone.0049234-Yoshimura1]. The biological effects of IFN-γ are mediated mainly through a pathway in which STAT1 is the predominant transcription factor [@pone.0049234-Hu1]. IL-10 and IL-6 engage receptors and then activate STAT3 and both induce the activation of SOCS3 which may differently modulate STAT3 activation induced by IL-10 and IL-6 [@pone.0049234-Niemand1]. In this paper, we showed that SEA can suppress the expression of MHC class II at transcriptional level and this effect is related with the IL-10 and IL-6 released from SEA activated macrophages. Whether *Schistosoma japonicum* SEA can result in transcriptional up-regulation of SOCS3 and thus inhibit IFN-γ-induced phosphorylation of STAT-1 and transcription of CIITA in SEA-stimulated macrophages via a mechanism involving SOCS3 need to be studied further. SOCS3 can be recruited by IL-6 signal transducer gp130 to its phosphotyrosine residues (Y759), but not by the phosphorylated tyrosine motifs of the IL-10R [@pone.0049234-Niemand1]. The difference between the IL-10 and IL-6 induced by SEA from macrophages might be related with the different sensitivities of IL-10 and IL-6 signaling toward mechanisms that inhibit the Janus kinase/STAT pathway. A major concern about the effect of SEA on MHC class II expression is the involvement of the possible contaminated endotoxin in SEA. In order to exclude the possible effect of endotoxin, we treated SEA repeatedly with endotoxin removing gel and then tested the endotoxin level in the prepared SEA by Tachypleus Amebocyte Lysate (TAL) kit. The results showed that following the treatments there was no contaminated endotoxin contained in the SEA used for our experiments. These observations could exclude the possibility that the effects of SEA on RAW 264.7 cells observed in our study was due to contaminated endotoxin. The expression of MHC class II might be regulated at multiple segments [@pone.0049234-Berger1]. As a master controller of MHC class II gene activation, the transactivator CIITA play a pivotal role in control of cellular immune responses through quantitative regulation of MHC class II expression [@pone.0049234-Steimle1]. Our study showed that SEA could significantly attenuate CIITA transcription induced by IFN-γ, suggesting that the attenuation of MHC class II expression by SEA might occur at transcriptional level. This result is supported by gene microarray information analysis based on an *in vivo* experiment by use of cells derived from *Schistosoma japonicum* infected mouse livers obtained from various stages of the infection. The transcription level of histocompatibility 2, class II antigen including H2-Aa, H2-Ab1, H2-Ob, H2-Eb and H2-Ea changed consistently with the development of mouse schistosomiasis from 0--18 weeks, that is, up-regulated at 6 weeks (acute stage) and then down-regulated until the 18^th^ week post-infection (chronic stage) (data not shown). These results strongly suggested that MHC class II expression was regulated in association with egg deposition and SEA release. Our experiments with mouse peritoneal macrophages showed that the attenuation on macrophages MHC class II expression level by SEA not only occured in RAW 264.7 cells but also occured in primary macrophages. These results strongly suggested that MHC class II expression was regulated in association with egg deposition and SEA release. Expression of class II molecules is exquisitely controlled at the transcriptional level. CIITA, which does not bind directly to the promoter, is a master controller among the large set of proteins interact with the promoters of class II genes [@pone.0049234-Ting1]. In our research, we found that 0.65 µg/ml of SEA completely suppressed IFN-γ induced CIITA expression ([Figure 1D](#pone-0049234-g001){ref-type="fig"}), however, in presence of 0.65 µg/ml of SEA, the IFN-γ induced MHC class II still showed an significant up-regulation compared with the untreated cells ([Figure 1C](#pone-0049234-g001){ref-type="fig"}). This suggested that after being stimulated with IFN-γ and 0.65 µg/ml of SEA simultaneously for 48 h, although CIITA has been suppressed completely on the transcriptional level, the protein of MHC class II expressed on the cell surface was still not experienced degradation totally. It was reported that in immature dendritic cells (DCs), peptide loaded MHC II (MHC II-p) is ubiquitinated after peptide loading and thus driving its sorting to the luminal vesicles of multivesicular bodies. These luminal vesicles and the MHC II-p they carry, are delivered to lysosomes for degradation. In DCs that are activated by pathogens or inflammatory stimuli, peptide loaded MHC II is inefficiently ubiquitinated and thus allowing its transfer to and stable expression at the plasma membrane [@pone.0049234-vanNiel1]. Our results suggested that MHC class II might be inefficiently ubiquitinated and thus allowing its transfer to and stable expression at the plasma membrane of RAW 264.7 cells activated with SEA and IFN-γ for 48 h just as what happened in DCs as reported [@pone.0049234-vanNiel1]. It has been shown that the expression of pattern recognition receptors might be regulated by cytokines [@pone.0049234-Mueller1], [@pone.0049234-Miller1]. In our study, we found that IFN-γ could up-regulate TLR4 expression level in RAW 264.7 cells. Helminth carbohydrate lacto-*N*-fucopentaose III expressed by *Schistosoma mansoni* eggs could drive DC2 maturation through signaling via TLR4 [@pone.0049234-Thomas1]. Taken together, our results suggested that SEA might down-regulate MHC class II expression in RAW 264.7 cells through TLR4 signaling and TLR4-mediated IL-10 and IL-6 production. These results supplied additional evidence that TLR4 might mediate immunosuppression although more studies suggested that TLR4 mediated pro-inflammatory immune responses [@pone.0049234-Rolland1], [@pone.0049234-Oliveira2]. Materials and Methods {#s4} ===================== Reagents {#s4a} -------- Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, NY) was supplemented with 4.5 g/L D-Glucose, 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum (PAA laboratories, Linz, Austria), 100 U of penicillin per ml, and 100 µg of streptomycin per ml (complete DMEM medium) before use. Recombinant mouse IFN-γ were purchased from PeproTech (London, UK; Cat. number is 315-05, 1 µg = 5×10^3^ IU) and R&D System (Cat. number is 485-MI, 1 µg = 8.43×10^3^ IU, which was only used in experiments including primary mouse peritoneal macrophages and RAW 264.7 cells stimulated with SEA pretreated with endotoxin removing gel). Phycoerythrin (PE)-conjugated rat anti-mouse MHC class II (I-A/I-E) (M5/114.15.2), PE-conjugated rat IgG2b isotype control, functional grade purified antibodies including anti-mouse interleukin-10 (IL-10) (JES5-2A5), anti-mouse interleukin-6 (IL-6) (MP5-20F3) and their isotype control rat IgG1, anti-mouse Toll-like receptor 4 (TLR4)/MD-2 (MTS510) and its isotype control rat IgG2a were all from eBioscience (San Diego, CA, USA). PE anti-mouse F4/80 antibody, FITC anti-mouse I-A/I-E antibody and FITC rat IgG2b isotype control were from Biolegend. ELISA kit including mouse IL-10 and mouse IL-6 were all from Bender MedSystems (Vienna, Austria). TRizol Reagent for preparing intact RNA were from Invitrogen (Carlsbad, CA, USA). Super RT Kit for cDNA first strand synthesis was from BioTeke (Beijing, China). SYBR Premix Ex Taq™ (Perfect Real Time) were from TaKaRa (Kyoto, Japan). Detoxi-Gel™ Endotoxin Removing Gel were purchased from Thermo Scientific Pierce. The endotoxin testing kit were from Zhanjiang Bokang Marine Biological Co., LTD (Guangdong, P.R.China). 10.1371/journal.pone.0049234.t001 ###### Primers used for real-time PCR. {#pone-0049234-t001-1} *Gene* *Primer Sequence* *Size (bp)* --------- ---------------------------- ------------- CIITA 5′-acacctggacctggactcac-3′ 229 5′-gctcttggctcctttgtcac-3′ β-actin 5′-ggaaatcgtgcgtgacatc-3′ 180 5′-aaggaaggctggaaaagagc-3′ Preparation of SEA {#s4b} ------------------ *Schistosoma japonicum* eggs were isolated from infected liver and intestine of rabbit. Purified eggs in PBS with protease inhibitor cocktail were sonicated three times on ice for 10 min. The suspension was freeze-thawed several times and centrifuged at 30,000 g for 30 min at 4°C. The supernatant was passed through a 0.22 µm filter and used as soluble egg antigen (SEA). Protein concentration was determined by BCA protein Assay Kit (Pierce Biotechnology, Inc., IL, USA) [@pone.0049234-AbdelHafeez1]. To exclude the effects of endotoxin, SEA was repeatedly treated with Detoxi-Gel™ Endotoxin Removing Gel (Thermo Scientific Pierce) before being used. Testing Endotoxic Activity by Gel-clot TAL Test {#s4c} ----------------------------------------------- Materials required for TAL test including TAL, control standard endotoxin (10 EU/ml), TAL reagent water, test tubes and pipette tips free of detectable endotoxin. The gel-clot TAL tests were preformed according to the method described in Chinese Pharmacopoeia 2010 edition. Briefly, 100 µl series dilutions of control standard endotoxin were mixed with 100 µl TAL reagents. The mixed solutions were incubated at 37°C for 60 minutes. The formation of the gel was scored by turning each test tube upside down. If the gel remained a piece, it is considered a solid gel formation. Otherwise, it is considered a failed gel formation. Cell Culture {#s4d} ------------ Murine RAW 264.7 macrophage cell line obtained from ATCC (ATCC TIB-71™, Manassas, VA) was maintained in complete DMEM medium. Cells were adjusted to a concentration of 1×10^6^/ml in complete medium for use in all of the cellular assays. To ascertain the appropriate concentration of IFN-γ used for up-regulation of MHC class II expression, RAW 264.7 cells were incubated with serially diluted IFN-γ for 48 h (4 ng/ml was selected as work concentration for IFN-γ from PeproTech Inc.). To observe the effect of SEA on MHC class II expression, RAW 264.7 cells were grown for 48 h in culture medium alone, SEA (40 µg/ml) or SEA (40 µg/ml) plus IFN-γ. To observe the dose-response relationship between SEA and MHC class II expression, RAW 264.7 cells were primed with SEA diluted serially from 40 µg/ml up to 0.156 µg/ml in presence of IFN-γ for 48 h. Cells cultured with medium alone or with IFN-γ were used as control. All these experiments were performed in 96-well plates, 200 µl per well. Cells were collected for detection of MHC class II expression after 48 h. In the experiments performed on RAW 264.7 cells with SEA pretreated with endotoxin removing gel or in the experiments performed on primary peritoneal macrophages, RAW 264.7 cells or primary macrophages were cultured with IFN-γ purchased from R?D System (10 ng/ml was selected as working concentration) with or without SEA (10 µg/ml was selected as working concentration, pretreated with endotoxin removing gel). For primary macrophages experiments, cells were plated in 24-well plate with complete medium, 6×10^5^per well. 12 hrs later, cells were respectively treated with 10 ng/ml IFN-γ (R&D System) in presence or absence of 10 µg/ml of SEA (pretreated with endotoxin removing gel) for 48 hrs. Cells cultured with medium alone were used as control. Preparation of Mouse Peritoneal Macrophages {#s4e} ------------------------------------------- C57BL/6 mice aged from 6 to 8 weeks were obtained from Shanghai Laboratory Animal Co. Ltd. (SLAC) (Shanghai, China). Peritoneal macrophages were recovered by peritoneal lavage with 5 ml of cold PBS and isolated by centrifugation. For adherent cells, cells (1×10^6^) were plated in 10 cm tissue culture dishes and cultured in RPMI 1640 medium containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin for 2 h at 37°C, 5% CO~2~. Then medium was removed, and the non-attached cells were washed away with PBS and the remaining attached cells were used as peritoneal macrophages. PE rat anti-mouse F4/80 antibody were used for confirming the purity of the peritoneal macrophages. Cell Surface Staining and Flow Cytometry {#s4f} ---------------------------------------- RAW 264.7 cells were treated as described above. After 48 h, cells were recovered by being treated with 0.25% trypsine-EDTA for 10 min, terminated with DMEM containing 10% heat-inactivated FBS, washed with cold PBS for three times, and centrifuged at 400 *g*. RAW 264.7 cells were incubated with 5% bovine serum albumin in PBS on ice for 10 min and then incubated with phycoerythrin-conjugated rat anti-mouse MHC class II or TLR4 or phycoerythrin-conjugated rat IgG2b or IgG2a isotype control for 30 min on ice. In the experiments performed on RAW 264.7 cells with SEA pretreated with endotoxin removing gel, or experiments performed on primary peritoneal macrophages, cells were collected following stimulation for 48 h and labeled with FITC rat anti-mouse I-A/I-E (RAW264.7 cells) or double labeled with FITC rat anti-mouse I-A/I-E and PE rat anti-mouse F4/80 antibodies (primary macrophages). Then the cells were fixed with 1% paraformaldehyde and analyzed with a FACScan flow cytometer (Becton Dickinson), using CellQuest software (Becton Dickinson). Ten thousand events were recorded. Antibody Blocking Assay {#s4g} ----------------------- The receptor responsible for recognition of SEA and mediate the production of IL-10 and IL-6 were detected using an antibody-blocking assay with anti-TLR4 antibody. RAW 264.7 cells were incubated in 96-well flat-bottom plates (2.0×10^5^ cells/well). For experiments blocking TLR4, adherent RAW 264.7 cells were treated with 50 µg/ml of anti-mouse TLR4 antibody or isotype control antibody IgG2a for 30 min at 37°C. Following incubation with antibodies, IFN-γ and SEA (40 µg/ml) were added and cells were incubated for 48 h at 37°C, 5% CO~2~. Then these cells were collected and assayed for MHC class II expression on cell surface. The culture supernatants were collected and assayed for IL-10 and IL-6 production. Cells cultured with medium alone were used as controls. Evaluation of Cytokines Production {#s4h} ---------------------------------- IL-10 and IL-6 levels in culture supernatants were determined by ELISA using paired cytokine-specific monoclonal antibodies according to the manufacturer's instructions. RNA Extraction and Reverse Transcription {#s4i} ---------------------------------------- RAW 264.7 cells were primed for 48 h with medium, SEA (0.65 µg/ml) in presence or absence of IFN-γ or IFN-γ alone in 60 mm dishes at 5.0×10^6^ cells/dish. Then total RNA was extracted according to the TRizol RNA isolation protocol. The extracted total RNA was dissolved in diethyl pyrocarbonate-treated water and subjected to reverse transcription using a Super RT kit, according to the manufacturer's instructions, to synthesize the cDNA templates for real-time PCR analysis. Briefly, 100 ng of total RNA and Moloney murine leukemia virus reverse transcriptase were used to reverse transcribe RNA into cDNA at 42°C for 40 min. Real-time PCR Analyses {#s4j} ---------------------- Real-time PCR was conducted using a commercially available SYBR Green I real-time PCR premix reagent containing Taq DNA polymerase and SYBR-Green I. cDNA templates and primers were added to the SYBR Green I PCR mix containing SYBR Premix Ex Taq, dNTP mixture, Mg^2+^ and SYBR Green I to give a total reaction volume of 25 µl. Real-time PCR reactions were then performed using an Applied Biosystems 7300 Real-time PCR System. Conditions were set to the following parameters: 30 s at 95°C, followed by 40 cycles at 95°C for 5 s, 60°C for 31 s; then 95°C for 15 s, 60°C for 1 min and 95°C for 15 s for the final dissociation stage to generate a melting curve for verification of amplification product specificity. The β-actin was used as an internal control under the same conditions. To obtain relatively accurate results, each template was processed in three tubes in the same PCR mixture. After the completion of PCR amplification, a melting curve analysis was performed. The 2^−ΔΔCt^ method [@pone.0049234-Livak1] was used to analyze the relative changes in target gene transcription. The primer pairs used in real-time PCR analysis are listed in [Table 1](#pone-0049234-t001){ref-type="table"}. Effect of IL-10 and IL-6 on MHC Class II Expression {#s4k} --------------------------------------------------- To observe the effect of endogenous IL-10 and IL-6 on MHC class II expression, antibody neutralization test to supernatants of macrophages primed with SEA (0.65 µg/ml or 40 µg/ml) and IFN-γ simultaneously were performed in 96-well cell culture plates according to the reagent instructions. In brief, cell culture supernatants were collected after 48 h. Functional grade anti-IL-10 and anti-IL-6 antibodies or their respective isotype controls were serially diluted in 50 µl complete DMEM medium by 2-fold in the plates. Supernatants and IFN-γ were added to the culture plates, 100 µl per well. Simple medium or 50 µl medium plus 100 µl supernatants in presence of IFN-γ were used as controls. Following, the culture plates were incubated at 37°C, 5% CO~2~ for 2 h. RAW 264.7 cells were collected and adjusted to a density of 4×10^6^/ml. 50 µl of cell suspension was added to each well and cultured at 37°C, 5% CO~2~ for 48 h. Then the cells were harvested to determine MHC class II expression by flow cytometry. The results were indicated as mean fluorescence intensity to assay the MHC class II expression on cell surface. Statistical Methods {#s4l} ------------------- Data analysis was performed using software package SPSS for windows version 11.5 (SPSS Inc., Chicago, U.S.A). Results were presented in histograms with arithmetic mean ± SD (standard deviation). Comparisons of arithmetic means between two groups were analyzed using Student's *t*-test. Analysis of variance (ANOVA test) was used to test the difference of arithmetic means among three or more groups. Least Significant Difference (LSD) method was used for multiple comparisons between groups. Linear regression was performed for evaluating dose-dependent effect. Differences between groups were considered to be statistically significant at the level of *P*\<0.05. In all figures, \* was used to indicate *P*\<0.05 and \*\* for *P*\<0.01. Data shown were from one representative experiment of three independent experiments. The results are comparable among the three experiments. We thank Dr. Yong Wang and his co-workers for their kind support for supplying gene microarray information and analysis based on an *in vivo* experiment. We thank Dr. Rahul Gaur for language editing. [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: GXT GLW. Performed the experiments: GXT HJZ JWX MJJ. Analyzed the data: GXT HJZ GLW JMX. Contributed reagents/materials/analysis tools: HJZ GLW. Wrote the paper: GXT GLW HWW. [^3]: Current address: The Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, Shanghai, China

Introduction {#S0001} ============ Ultrasonography has become one of the basic diagnostic tools for vascular diseases of the central nervous system (CNS). Due to the widespread availability of ultrasound, it seems necessary to define standards for equipment requirements, the scope of ultrasound and the experience of the person performing the procedure. The goal of this paper is to standardize the testing protocol in all neurosonology laboratories. We hope that the presented standards will prove useful in everyday patient management as well as will become the basis for discussion and comments to be taken into account in subsequent versions. The paper further describes different types of neurosonological tests, such as cerebral circulatory arrest or diagnostics of right-to-left shunts. The proposed diagnostic criteria should be standardized in all neurosonology laboratories due to differences between ultrasonographic devices, particularly in relation to velocity calibration. An assessment of the competence of the circle of Willis (The Matas Test) {#S0002} ======================================================================== The Matas test is performed in situations when it is necessary to determine the competence of the circle of Willis, e.g. in the case of necessary ligation of the internal carotid artery due to the presence of a large cavernous segment aneurysm. Equipment requirements {#S20003} ---------------------- The evaluation is performed using an ultrasound device equipped with a linear probe with a frequency of 7.5--13 MHz and a 2--3.5 MHz sector probe (transcranial color-coded duplex, TCCD) or an ultrasound device equipped with a linear probe with a frequency of 7.5--13 MHz and "blind" Doppler ultrasound device equipped with 2 MHz probe with pulse wave (PW) Doppler (transcranial Doppler, TCD). Patient preparation {#S20004} ------------------- The test is performed in a patient lying in supine position. Technique {#S20005} --------- The procedure is preceded by the examination of the extracranial carotid arteries, as in accordance with the protocol, and the exclusion of atherosclerotic plaques in the common and internal carotid arteries. It should not be performed if atherosclerotic plaques are present in the extracranial carotid arteries. The evaluation can be performed using transcranial Doppler ultrasound (TCD) or transcranial color-coded Doppler ultrasonography (TCDD). A firm compression of the ipsilateral common carotid artery, as far as possible from the bulb, should be performed during insonation of the internal carotid artery bifurcation (insonation depth: 60-- 65 mm). Under no circumstances should be the carotid artery compressed in the vicinity of the bulb due to the risk of cardiac arrest (irritation of the carotid body). If the anterior communicating artery (the most important route for collateral circulation within the circle of Willis) is patent, then a reversal of the flow in the anterior cerebral artery will occur after several seconds (there will be two TCD spectra directed towards the probe or the blue colored TCCD flow 'away from the probe' in the anterior cerebral artery will change into red colored flow directed towards the probe, and there will be two overlapping spectra for the flow towards the probe in spectral Doppler). The absence of a change in the direction of flow in the anterior cerebral artery and a reduction in blood flow velocity of \>30% in the middle cerebral artery as well as delayed systolic peak indicate very poor competence of the circle of Willis and the risk of ischemic complications in the case of ligation of the ipsilateral internal carotid artery^([@CIT0001]--[@CIT0004])^. Documentation {#S20006} ------------- The documentation should include the recorded flow spectra from the vicinity of intracranial internal carotid artery bifurcation at rest and after common carotid artery compression. Results {#S20007} ------- The results should contain the name of the method used (Duplex scan of the extracranial carotid arteries and TCD or TCCD), the name of the devices and the frequency of the probe. It is necessary to report the absence of atherosclerotic lesions in the compressed common carotid artery. If bilateral atherosclerotic lesions are found in the common carotid artery, this fact should be reported as the reason for withdrawing from further testing. It should also be reported whether a reversal in the direction of blood flow in the ipsilateral anterior cerebral artery occurred (good collateral circulation, competent circle of Willis) or not, or whether there was a reduction in blood flow velocity and a delayed systolic peak in the ipsilateral middle cerebral artery following common carotid artery compression (poor collateral circulation in the circle of Willis) ^([@CIT0004])^. Measurement of the cerebral vasomotor reserve {#S0008} ============================================= The measurement of vasomotor reactivity (VMR) is the most commonly used ultrasonography technique assessing the competence of mechanisms underlying the autoregulation of cerebral blood flow. VMR evaluation involves the measurement of changes in the mean blood flow velocity in the circle of Willis, usually in the middle cerebral artery, caused by changes in the volume of the vascular bed of the cerebral microcirculation (increased or decreased blood flow) induced by a vasoactive agent. Carbon dioxide is a substance that has the most profound effect on cerebral microcirculation. Physiological (respiratory tests) and pharmacological provocation can be used for the assessment. VMR evaluation is currently used mainly for scientific research as well as in clinical practice as a method assisting the qualification for carotid artery repair^([@CIT0002],\ [@CIT0003],\ [@CIT0005],\ [@CIT0006])^. Equipment requirements {#S20009} ---------------------- The study is performed using "blind" Doppler ultrasound equipped with 2 MHz probe with pulse wave Doppler. The use of a monitoring headband significantly facilitates the examination (it also allows to maintain a constant insonation angle), and the sensitivity may be increased by simultaneous bilateral monitoring, i.e. dual-channel monitoring (if the apparatus features this option). Dual-channel monitoring also allows to shorten scanning duration^([@CIT0002],\ [@CIT0006])^. Patient preparation {#S20010} ------------------- The test is performed in a patient in a lying supine or sitting position and wearing a monitoring headband. The test is performed under the same conditions (temperature, light, humidity), after a 10-minute rest period. The end-tidal CO~2~ concentration (~ET~CO~2~) should be monitored using capnograph (lateral stream measurement) and the blood pressure (BP) should be monitored, preferably using continuous non-invasive measurement techniques. Significant changes in the BP values should be avoided during the measurement^([@CIT0002],\ [@CIT0003],\ [@CIT0006])^. Technique {#S20011} --------- The examination is performed by insonating the middle cerebral artery at a depth of 50--60 mm through the temporal window. It is advisable to use a headband and set a dual-channel monitoring (bilateral monitoring of the middle cerebral arteries). Provocation tests are preceded by the measurement of the mean blood velocity at rest (V~mean\ rest~) (1-minute registration) in the middle cerebral artery. Further stages involve performing provocation tests and tracing provocation-induced changes in the mean blood flow velocity. Provocation methods include:Breath-holding test^([@CIT0001]--[@CIT0003],\ [@CIT0005],\ [@CIT0006])^The method allows to assess the degree of microcirculatory vasodilatation induced by hypercapnia. The patient is asked to hold their breath for 30 minutes to achieve hypercapnia (t~BH~ = 30 s). Breath hold should not be preceded by an additional, deeper inhalation, which may provoke the Valsalva mechanism. The maximum mean blood flow velocity (V~mean\ end\ BH~) is measured immediately after the end of breath-hold (a minimum of 4--5 full cardiac cycles with the highest blood flow velocity). The breath-holding index (BHI) is calculated from the following formula:$$\text{BHI} = \lbrack\left\{ (\text{V}_{\text{mean\ end\ BH}}–\text{V}_{\text{mean\ rest}})/\text{V}_{\text{mean\ rest}} \right\}/\text{t}_{\text{BH}}\rbrack \times 100$$\[V~mean\ end\ BH~ -- the mean middle cerebral artery (MCA) blood flow velocity at the end of breath-hold; V~mean\ rest~ -- the mean middle cerebral artery (MCA) blood velocity at rest; t~BH~ -- breath-hold duration in seconds\].The measurement of vasomotor reserve^([@CIT0001]--[@CIT0003],\ [@CIT0005],\ [@CIT0006])^Vasomotor reactivity reserve/range (VMRr) reflects the full range of changes in blood flow parameters: from resistance vessel stenosis due to hypocapnia to resistance vessel dilation induced by hypercapnia. It is measured based on hyperventilation provocation test (during which hypocapnia occurs). The patient should take regular, deep breaths with a full exhalation for 2 minutes at a rate of about 12 breaths per minute. The next stage involves a breath-hold (see above). The breathing tests are performed consecutively at 4-minute intervals, which are necessary for the normalization of blood flow parameters. The minimum mean blood flow velocity during hyperventilation (V~mean\ hyperventilation~) is calculated as a mean for the last three breathing cycles during hyperventilation. The VMRr is calculated from the following formula:$$\text{VMRr} = \left\{ (\text{V}_{\text{mean\ BH}}–\text{V}_{\text{mean\ hyperventilation}})/\text{V}_{\text{mean\ rest}} \right\} \times 100\%$$\[V~mean\ BH~ -- mean MCA velocity at end breath hold; V~mean\ hyperventilation~ -- mean MCA velocity during hyperventilation; V~mean\ rest~ -- mean MCA velocity at rest\].Ventilation with 5% CO~2~^([@CIT0003],\ [@CIT0005],\ [@CIT0006])^A test with carbogen ventilation may be performed to induce hypercapnia. A patient wearing a respiratory mask (half-open circuit) is exposed to a mixture of 5% CO~2~ and 95% O~2~ (carbogen) for 90 minutes. Optionally, closed-circuit ventilation using breathing bags may be used. The maximum mean blood flow velocity (V~mean\ post-ventilation~) is calculated as a mean value recorded during at least 5 seconds after the end of ventilation with the mixture. The VMR is calculated from the following formula:$$\text{VMR} = \left\{ (\text{V}_{\text{mean\ post\ ventilation}}–\text{V}_{\text{mean\ at\ rest}})/\text{V}_{\text{mean\ at\ rest}}) \right\} \times 100\%$$4) Pharmacological provocation methodsThe acetazolamide test^([@CIT0005],\ [@CIT0006])^:The method allows to assess the degree of microcirculatory vasodilatation. Acetazolamide, a carbonic anhydrase inhibitor, induces transient hypercapnia and, consequently, vasodilatation (blood flow maximization). The assessment of vasomotor reactivity based on the acetazolamide testing (e.g. Diamox 1 g -- 15 mg/kg body weight IV) involves the measurement (within 1 minute) of blood flow velocity at rest (V~mean\ rest~) and 10 minutes after infusion (a slow 2--5-minute IV infusion of solution of 5 mL of the drug) (V~mean\ ACE~).The range of VMR assessed based on acetazolamide testing is calculated from the following formula:$$\text{VMRr} = \left\{ (\text{V}_{\text{mean\ ACE}}–\text{V}_{\text{mean\ rest}})/\text{V}_{\text{mean\ rest}} \right\} \times 100\%$$L-arginine test^([@CIT0006])^Infusion of L-arginine, an amino acid involved in the endogenous nitric acid synthesis, induces transient vasodilation in the microcirculation. The mechanism allows for a selective measurement of endothelium-dependent vasomotor reactivity. The test involves an infusion of 30 g L-arginine (usually in 100 mL 0.9% NaCl solution) for 30 minutes. Registration of blood flow parameters begins 10 minutes before infusion and ends 10 minutes after infusion. VMRr is calculated from the following formula:$$\text{VMRr} = \left\{ (\text{V}_{\text{mean\ post-infusion}} - \text{V}_{\text{mean\ pre-infusion}})/\text{V}_{\text{mean\ pre-infusion}} \right\} \times 100\%$$ Documentation {#S20012} ------------- The documentation includes the recorded curve for the mean blood flow in the middle cerebral artery (or both middle cerebral arteries using dual-channel monitoring) during hypercapnia and hypocapnia testing. Results {#S20013} ------- The result should include the name of the device and the frequency of the probe used for testing, as well as a statement whether it was one or dual-channel examination and the applied method for the provocation of changes in carbon dioxide pressure. The result should further include the percentage range of changes in the vasomotor reserve (hyperventilation and breath-hold) or the BHI in the case of breath-hold testing. The range of vasomotor reserve is more than 80% in healthy individuals, and the normal BHI value is 1.2 (±0.4). VMR below 50%, and MHI below 0.6 (±0.1) are considered pathological^([@CIT0002],\ [@CIT0003],\ [@CIT0006])^. Substantia nigra hyperechogenicity assessment {#S0014} ============================================= Equipment requirements {#S20015} ---------------------- The test is performed using a high-resolution ultrasound device equipped with a sector probe with a frequency of 1.6--2.5 MHz. Patient preparation {#S20016} ------------------- The test is performed in a patient lying in supine position. Technique {#S20017} --------- The evaluation is performed through the temporal window, as in the case of TCCD, in an axial insonation plane. Ultrasound penetration depth of 14--16 cm, and a dynamic range of 45--55 dB should be set. The brightness of the image should be adjusted; low-echoic signal reduction should be used, if possible. Once a cross-sectional image of the brain stem at the level of midbrain (butterfly shaped) is obtained, 1.5--4 times magnification of the image is performed in the axial plane and a planimetric measurement of the area of potential hyperechogenicity is performed. Cut-off values should be defined in each laboratory and for each apparatus to determine hyper- and normal echogenicity of the substantia nigra. Then, the hyperechogenic area should be outlined manually and its area should be measured. An area of hyperechogenicity exceeding the 90th percentile in a healthy population is considered a significant hyperechogenicity of the substantia nigra (≥0,25 cm^2^ for most apparatuses). If the area of hyperechogenicity is between the 70^th^ and the 90^th^ percentile (≥0.2 and \<0.25 cm^2^ for most apparatuses), the hyperechogenicity is defined as moderate. An area of substantia nigra hyperechogenicity of up to 70^th^ percentile is considered a normal echogenicity. Although most authors report the lager of the two bilaterally measured substantia nigra hyperechogenicity areas, the mean value of the bilaterally measured hyperechogenic areas can also be reported. A uniform system of reporting results should be used in every laboratory^([@CIT0007],\ [@CIT0008])^. Documentation {#S20018} ------------- The documentation should include images of the brain stem at the level of the midbrain without measurements as well as images from the planimetric measurement of the outlined hyperechogenic area of the substantia nigra with values in cm^2([@CIT0008])^. Results {#S20019} ------- The results should contain the name of the device, the frequency of the probe as well as data on the echogenicity of substantia nigra (normal echogenicity, moderate hyperechogenicity, severe hyperechogenicity). The area of the potential hyperechogenicity and the ranges of norm/ pathology specific for a given laboratory and apparatus, should be reported^([@CIT0008])^. Conflict of interest {#S0020} ==================== Authors do not report any financial or personal connections with other persons or organizations, which might negatively affect the contents of this publication and/or claim authorship rights to this publication

{#sp1 .244} {#sp2 .245} {#sp3 .246} {#sp4 .247}

The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus. Introduction ============ Tuberculosis (TB) is a highly contagious infection caused by the aerobic, non-motile bacillus Mycobacterium tuberculosis (MTB) and is an important cause of morbidity and mortality worldwide. In New York City, there is a high prevalence of pulmonary TB, especially in the foreign-born population with approximately 27% of TB cases reported to be culture-negative (CX --ve) \[[@REF1]\]. In 2013, the Center for Disease Control (CDC) has reported a 23% CX -ve rate for TB cases in the United States \[[@REF2]\]. We report a case of a 26-year-old-female, a recent immigrant from Serbia, who was initially managed for community-acquired pneumonia but was later found to have tuberculosis with complicated pleural effusion, despite having multiple smear- and culture-negative sputum specimens. Case presentation =================  A 26-year-old-female, with no significant medical history and who had recently emigrated from Kosovo, Serbia, presented to the emergency department (ED) with complaints of a cough, chest pain, and shortness of breath for two weeks. She initially presented to urgent care, where she was diagnosed with pneumonia, prescribed azithromycin and sent home. Her symptoms did not resolve and she became more dyspneic despite oral antibiotics, which prompted her to come to the ED. Upon presentation, her vital signs were notable for a fever to 102 degrees Fahrenheit and a blood pressure of 92/60 mm Hg. The physical examination showed decreased breath sounds on the right on auscultation and dullness on percussion. The chest X-ray was notable for significant right-sided pleural effusion (Figure [1](#FIG1){ref-type="fig"}). Due to the size of the effusion and worsening respiratory status, emergent tube thoracostomy was performed, which drained the effusion significantly (Figure [2](#FIG2){ref-type="fig"}). {#FIG1} {#FIG2} The patient was started on ceftriaxone and doxycycline for presumed pneumonia. Pleural fluid showed lactate dehydrogenase (LDH) 314 IU/L (compared with serum LDH 129 IU/L), and protein 5.2 g/L (compared with serum protein 5.5 g/L), indicative of exudative pleural effusion. Adenosine deaminase level was 1.7 units/L (N: 0-9.4 units/L) and a white blood cell (WBC) count of 1673 with 61% lymphocytes. Due to suspicion for TB, three acid-fast bacilli (AFB) sputum smear and cultures, collected eight hours apart, with one early morning sample, were sent. All the three sputum specimens came back negative for AFB. Even the first sputum cultures after six weeks were negative. Three samples were sent for an MTB/RIF assay (real-time PCR), which were also negative. Repeat chest X-ray showed decreased opacity in the right mid to upper chest with an appearance of increased effusion at the right lateral base. The computed tomographic (CT) scan of the chest was positive for pleural thickening and multiloculated pleural effusion (Figure [3](#FIG3){ref-type="fig"}). {#FIG3} Due to high suspicion for TB, the patient underwent right video-assisted thoracoscopic surgery (VATS) with decortication and biopsy. The histology from the pleural rind and a pleural nodule showed necrotizing caseating granuloma. At this point, the data were consistent with a diagnosis of pulmonary TB and the patient was placed on airborne isolation and started on standard anti-tubercular therapy with isoniazid, rifampin, pyrazinamide, ethambutol, and pyridoxine. Liver function tests were normal before initiating the therapy. After a week of therapy, the patient showed significant improvement in her respiratory symptoms. The Department of Health (DOH) was notified. As per their recommendations, three additional sets of sputum were sent for AFB, all of which came back negative, including a negative second set of sputum cultures six weeks later. The patient was discharged per DOH guidance, as she was not contagious. She continued anti-tubercular therapy as per standard protocol with close monitoring by DOH and outpatient infectious disease follow-up. The pleural biopsy from VATS was initially negative for gram stain and later came back positive for Mycobacterium tuberculosis, confirming the diagnosis of tuberculosis. Discussion ========== Mycobacterium tuberculosis (MTB) can be transmitted through the generation of airborne droplets during coughing, sneezing, or even speaking by a person with pulmonary or laryngeal tuberculosis. These droplets are inhaled and subsequently phagocytosed by macrophages in the alveoli. This results in a cascade of events, leading to either the successful resolution of infection followed by latent TB or progression to active pulmonary disease. Pulmonary tuberculosis is most often associated with the reactivation of latent infection but can also occur as a manifestation of primary infection. Pulmonary TB can form subpleural caseous foci, which subsequently rupture and complicate the disease process, leading to the development of pleural tuberculosis \[[@REF3]\]. Pleural TB generally results from a late hypersensitive reaction caused by Mycobacterium tuberculosis antigen. Even a small number of bacilli in the pleural space can cause a significant inflammatory reaction. The mechanism is T-lymphocyte mediated, as these cells produce inflammatory cytokines that stimulate macrophages to form granulomas. This inflammatory process subsequently increases the vascular permeability and results in the formation of pleural exudates \[[@REF4]\]. Moreover, radiographic imaging, such as computed tomography, has demonstrated that 40% of pleural and pulmonary TB cases are concomitant lesions rather than separate disease entities \[[@REF5]\]. The diagnosis of pulmonary TB is classically made by sputum gram stain and culture along with the clinical and radiographic findings. Sputum smear is a rapid test to detect the presence of acid-fast bacilli (AFB) but a single sputum test lacks sensitivity. One of the reasons for low sensitivity is the fact that 10,000 microorganisms/ml are required for AFB to be seen on microscopy \[[@REF6]\]. Furthermore, sputum culture requires 10 to 100 AFB/ml to confirm the diagnosis of TB \[[@REF6]\]. The gold standard for the diagnosis of TB is culture or nucleic acid amplification assay. The Xpert MTB/RIF assay is a PCR test that can identify both Mycobacterium tuberculosis (MTB) and rifampicin resistance, with cultures typically being the more sensitive method for the diagnosis \[[@REF7]-[@REF8]\]. Culture-negative TB (CX -ve TB) is defined as no growth of MBT in the first three sputum samples, and it can be further investigated by the methods listed below in the diagram (Figure [4](#FIG4){ref-type="fig"}). {#FIG4} Studies have shown that combinations of diagnostic modalities can increase accuracy in diagnosing TB. Miro et al. found in a retrospective study that evaluated respiratory secretion samples collected by bronchoscopy for TB detection that adding the transbronchial biopsy increased diagnostic accuracy from 96% to 100% \[[@REF9]\]. While sputum culture is the gold standard for diagnosis, many developing countries still rely on sputum smear as the diagnosis of active TB. Inadequate case detection is one major reason for the high burden of TB, especially in developing countries, as many cases are smear-negative and hence go undetected, further increasing the burden of TB. Furthermore, smear- and culture-negative patients often present atypically, with less frequent cough, hemoptysis, and radiographic abnormalities compared to smear-positive or culture-positive patients \[[@REF10]\]. Another study showed that CX- TB patients were more likely to have a chest CT as compared to CX+ patients and that lung cavitation is less frequently observed in culture-negative patients \[[@REF11]\]. Importantly, a human immunodeficiency virus (HIV) infection dramatically changes the development of tuberculosis infection, as the risk of TB doubles within the first seven years of HIV infection due to a rapid decline in TB-specific T helper cells \[[@REF12]\]. HIV-infected patients are at increased risk of developing reactivation TB and accelerate the progression of TB following a subsequent exposure. Considering the high burden and dissemination of TB infection, an HIV test was performed in our patient, which was negative. Conclusions =========== Physicians should not rely only on sputum smears and culture-negative results to exclude the diagnosis of TB but also consider advanced investigative modalities in patients with a high clinical likelihood of TB. The authors have declared that no competing interests exist. Consent was obtained by all participants in this study

Introduction {#s1} ============ Glucosinolates are a diverse group of nitrogen- and sulphur-rich secondary metabolites characteristic of the order Capparales, which includes nutritionally important *Brassica* crops and the model plant *Arabidopsis thaliana* ([@CIT0017]; [@CIT0053]). Based on the precursor amino acid used, glucosinolates are broadly classified into three major groups namely aliphatic, indolic, and aromatic. The biosynthesis of glucosinolates can be divided into three phases: (i) recruitment of precursor amino acids and side-chain elongation, (ii) formation of the core glucosinolate structure, and (iii) side group modification ([@CIT0025]). Together with side-chain elongation of the R-group, side-chain modifications generate a wide variety of glucosinolate compounds, with more than 200 structures identified to date ([@CIT0013]). In recent years, glucosinolates and their breakdown products have been the subject of extensive studies due to their role in defence against pests and pathogens ([@CIT0025]). Some glucosinolates and their breakdown products have anti-nutritional and goitrogenic properties in seed meal, while others act as anti-carcinogenic compounds in mammals ([@CIT0018]; [@CIT0032]; [@CIT0008]; [@CIT0049]). Due to their diverse roles in plant metabolism, animal nutrition, and disease, glucosinolates are a potential target for genetic manipulation in crop improvement programmes. Our understanding about the genes controlling the complex trait of glucosinolate accumulation has been obtained from various molecular-genetic and 'omics' based studies on the model plant *A. thaliana* ([@CIT0025]; [@CIT0020]; [@CIT0045]). To date, more than 20 glucosinolate biosynthesis pathway genes have been identified in *Arabidopsis*. Recent reports have confirmed that glucosinolate levels are further controlled by at least six members of subgroup-12 of the R2R3-MYB superfamily. The *Arabidopsis AtMYB28*, *AtMYB29*, and *AtMYB76* genes act as transcriptional regulators of aliphatic glucosinolate biosynthesis ([@CIT0023], [@CIT0022]; [@CIT0029]; [@CIT0046], [@CIT0044]), whereas AtMYB34, AtMYB51, and AtMYB122 specifically regulate indolic glucosinolate formation ([@CIT0009]; [@CIT0021]). The crops belonging to the genus *Brassica* have been of great economical importance to mankind because of their potential use as vegetables, oilseeds, feed, condiments, fodder, green manure, and even medical treatments. In *Brassica* crops, glucosinolate content and profiles are highly variable and species-specific, with aliphatic glucosinolates (derived from methionine) being the predominant glucosinolates (up to 95% of the total glucosinolates) in seeds ([@CIT0043]). Over the past few decades, there have been ongoing breeding efforts towards the enrichment of beneficial aliphatic glucosinolates (e.g. glucoraphanin) and the reduction of anti-nutritional aliphatic glucosinolates (e.g. progotrin, gluconapin) in these crops. Genetic studies have shown that seed aliphatic glucosinolate content is a quantitative trait, controlled by a variable number of loci in both *B. napus* ([@CIT0048]; [@CIT0050]; [@CIT0030]; [@CIT0019]) and *B. juncea* ([@CIT0012]; [@CIT0043]; [@CIT0036]; [@CIT0039]; [@CIT0006]). Although these reports have identified few genomic regions (QTLs) that control the variability of glucosinolate contents and profiles across *Brassica* species, our understanding of the molecular-genetic mechanism controlling such an economically important trait in *Brassica* species is largely limited at present. Allopolyploidy is a condition in which a cell has two or more sets of chromosomes derived from two different species. For example, *B. juncea* (2*n*=4*x*=36) is an allotetraploid having a set of chromosomes derived from *B. rapa* (2*n*=2*x*=20) and a set from *B. nigra* (2*n*=2*x*=16). Because of the occurrence of polyploidy and genome-wide rearrangements, the regulation of aliphatic glucosinolate biosynthesis in *Brassica* species is expected to be highly complex compared with that in the closely related diploid *Arabidopsis*. Comparative mapping studies between *Brassica* species and *Arabidopsis* revealed the triplicate nature of diploid *Brassica* genomes and strongly suggested that the extant diploid *Brassica* species have evolved from a common hexaploid ancestor at about 11--12 MYA ([@CIT0038]). Further, genomes of the allotetraploid *B. napus* and *B. juncea* are even more complex and are known to retain up to six conserved segments/blocks of the ancestral genome ([@CIT0041]; [@CIT0037]). Thus, the existence of multiple homologues of each glucosinolate candidate gene in allotetraploid *Brassica* genomes is expected. Studies on the expression and functional variance of the homologous genes arising from polyploidy are therefore fundamentally important for a better understanding of the complex mechanisms controlling glucosinolate accumulation in these crops. Such an understanding would ultimately allow for the manipulation of specific genes for targeted engineering of glucosinolate accumulation without compromising overall plant fitness. To date none of the transcriptional regulators of aliphatic glucosinolate biosynthesis genes from any of the *Brassica* crops have been functionally characterized. The isolation of multiple *MYB28* gene homologues from an economically important oilseed crop of the *Brassica* genus, *B. juncea* (AABB genome) as well as from genomes of its two progenitors namely, *B. rapa* (AA genome) and *B. nigra* (BB genome) is reported here. The consequence of polyploidy on gene structure, phylogeny, and gene expression and function has been investigated in detail. Taken together, our results highlight the importance of *MYB28* homologues towards controlling complex glucosinolate traits in polyploid *B. juncea*, which could be utilized towards manipulating the pest resistance, anti-nutritional properties and health benefits of oilseed cultivars of this species. Materials and methods {#s2} ===================== Plant materials and growth conditions {#s3} ------------------------------------- High glucosinolate cultivars of *B. nigra* (cv. IC257), *B. rapa* (cv. YID1), and *B. juncea* (cv. Varuna) were grown in a growth chamber set at day (22 °C, 10h)/night (15 °C, 14h) cycle, 70% relative humidity, and light intensity \<250 µmol m^--2^ s^--1^. Different developmental stages, namely seedling, root, stem, leaf (primary leaf, young, mature, and flag), and silique (10, 20, and 30 d post-anthesis: dpa), were collected, frozen in liquid nitrogen and stored at --80 °C. *A.* *thaliana* wild-type (ecotype Col-0) and the *AtMYB28* (At5g61420) loss-of-function mutant, BRC_H161b ([@CIT0005]) were grown in a growth room set at 22 °C under 16/8h light/dark cycle and at 40% relative humidity. Isolation of genomic and cDNA sequences of *MYB28* homologues from *B. juncea* {#s4} ------------------------------------------------------------------------------ The full-length genomic sequences and the coding sequences of the *BjuMYB28* genes were isolated from *B. juncea* (cv. Varuna) as well as species representing its progenitor genomes namely, *B. nigra* (cv. IC257) and *B. rapa* (cv. YID1). Total RNA was isolated using the TRI-Reagent (Sigma). DNase (New England Biolabs) treatment was performed on total RNA and about two microgram of RNA was reverse transcribed into cDNA with oligo-dT primers using a first strand cDNA synthesis kit (Applied Biosystems). Full-length coding sequences were obtained by performing 5′ and 3′ RACE using gene-specific primers and SMART^TM^ RACE cDNA amplification kit (Clontech). For obtaining the genomic sequences, PCR amplification was performed on total genomic DNA using the gene-specific primers designed based on cDNA sequences obtained above. PCR products were cloned into pGEMT-Easy cloning vector (Promega), sequenced and analysed using DNASTAR software (Lasergene). A list of primers used in the current study is summarized in [Supplementary Table S1](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1) at *JXB* online. Quantitative RT-PCR (qRT-PCR) analysis {#s5} -------------------------------------- The relative expression of glucosinolate pathway genes was analysed by real-time qRT-PCR in an ABI 7900HT Fast Real-time PCR machine (ABI) using a SYBR green protocol. Approximately, two micrograms of total RNA were reverse-transcribed using high capacity first strand cDNA synthesis kit (ABI) according to the manufacturer's instructions. The *ACTIN2* and *GAPDH* genes were used as endogenous controls ([@CIT0010]). Data were analysed from at least three independent sets of biological replicates (separate plant/transgenic lines) with two technical replicates for each. Primers used for qRT-PCR analysis are tabulated in [Supplementary Table S1](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1) at *JXB* online. Subcellular localization of BjuMYB28 proteins {#s6} --------------------------------------------- To generate a Pro~CaMV35S~:BjuMYB28:YFP fusion construct, the coding sequence of *BjuMYB28-2* was cloned into a C-terminal YFP fusion vector pEarlygate101 ([@CIT0016]). The *AtMYB28* was used as a reference control. Constructs were transformed into onion epidermal cells through a particle delivery system (PDS 1000, Bio-Rad) according to the manufacturer's instructions. Localization of the BjuMYB28:YFP fusion protein was determined after 48h of incubation in the transformed cells under a confocal laser scanning microscope (Leica). Generation of plant transformation constructs {#s7} --------------------------------------------- For the development of *BjuMYB28* over-expression constructs, the coding sequences of the *BjuMYB28* genes were cloned into the pPZP200 binary vector ([@CIT0024]) under the control of a CaMV 35S promoter along with the *bar* gene as the plant selection marker. The *BjuMYB28* over-expression constructs were transformed into a homozygous loss-of-function mutant of *AtMYB28* (BRC_H161b) and the wild-type (Col-0) genetic background. For the generation of Pro~BjuMYB28~:*uidA* constructs, the promoter regions (\~1kb) of the four *BjuMYB28* genes were isolated from genomic DNA of *B. juncea* by a genome walking protocol (Universal Genome Walker Kit, Clontech) and cloned into the gateway binary vector pMDC164 upstream of the *uidA* gene. Histochemical analysis of GUS reporter protein in independent single-copy transgenic lines (in the T~3~ generation) was performed according to the protocol described by [@CIT0031]. The GUS staining patterns at different stages of development were recorded under a Zoom Stereo microscope (SMZ-U, Nikon). The different constructs used in the current study were transformed into *A. tumefaciens* strain GV3101 by the freeze--thaw method and subsequently into *A. thaliana* by the floral dip method ([@CIT0014]). **S**election of transgenic plants was performed on appropriate antibiotics/selection agents. Both mutant-complemented and over-expression lines were selected on the herbicide Basta (at 120mg l^--1^ of phosphinothricin, 3--4 sprays at 1 d intervals). The Pro~BjuMYB28~:*uidA* lines were selected on hygromycin (20mg l^--1^). HPLC analysis for glucosinolate estimation {#s8} ------------------------------------------ The extraction and quantification of glucosinolates was performed on leaves of 25-d-old *Arabidopsis* plants by HPLC as per the protocols described earlier by [@CIT0007]. Briefly, glucosinolates were extracted twice in 70% methanol after adding 6mM glucotropaeolin (Applichem) as the internal standard. Samples were loaded on DEAE Sephadex A25 columns and desulphated overnight using purified sulphatase (type H1 from *Helix pomatia*) prior to HPLC. Concentrations of individual glucosinolates were calculated in nmol mg^--1^ dry weight relative to the area of the internal standard peak using the respective response factors reported earlier ([@CIT0007]). For each *BjuMYB28* construct, at least two experimental replicates of two independent transgenic lines were used for the glucosinolate analyses. The following glucosinolates were detected; 4-methylsulphinylbutyl-glucosinolate (4MSOB), 3-methylsulphinylpropyl-glucosinolate (3MSOP), 5-methylsulphinylpentyl-glucosinolate (5MSOP), 4-methylthiobutyl-glucosinolate (4MTB), 7-methylsulphinylheptyl-glucosinolate (7MSOH), 8-methylsulphinyloctyl-glucosinolate (8MSOO), 6-methylsulphinylhexyl-glucosinolate (6MSOH), indol-3-ylmethyl-glucosinolate (I3M), 1-methoxyindol-3-ylmethyl-glucosinolate (1MOI3M), and 4-methoxyindol-3-ylmethyl-glucosinolate (4MOI3M). Sequence and phylogenetic analysis {#s9} ---------------------------------- The coding sequences and amino acids of the *BjuMYB28* genes were predicted using the software DNASTAR (Lasergene). Multiple sequence alignments of coding sequences and the deduced amino acid sequences of *MYB28* from *Arabidopsis* and *Brassica* species were performed using ClustalW. Percentage similarities among MYB28 homologues were calculated using the MegAlign module of DNASTAR. The BLASTX search of AtMYB28 and four BjuMYB28 coding sequences was performed on the publicly available database at NCBI (<http://blast.ncbi.nlm.nih.gov/Blast.cgi>) and phytozome (<http://phytozome.net>). Full-length coding sequences showing high percentage similarity scores and sequence coverage were obtained. Evolutionary history was inferred using the Maximum Likelihood method ([@CIT0040]) based on the JTT matrix-based model in MEGA5 ([@CIT0047]). MYB28 like sequences reported from the following genomes were used for the analysis: *Arabidopsis thaliana* (AtMYB28, AtMYB29, AtMYB76); *Arabidopsis lyrata* (Aly_950898, Aly_487604, Aly_892702); *Capsella rubella* (Cru_10027861m, Cru_10001328m); *Thellungiella halophila* (Tha_v10004407m, Tha_v10013952m); *Brassica rapa* (Bra035929, Bra012961, Bra029311), and *Brassica oleracea* (BolC.MYB28.1/ADK38583, BolC.MYB28.2/CBI71385). The deduced proteins from the *B. rapa* (BraA.MYB28.1, BraA.MYB28.2); *B. nigra* (BniB.MYB28.1, BniB.MYB28.2), and *B. juncea* (BjuMYB28-1, -2, -3, and -4) genomes were also used. Statistical analysis {#s10} -------------------- Data from different experimental sets were analysed for statistical significance using one-way ANOVA applying Fishers LSD or Tukey's *post hoc* test. A *P* value \<0.05 was considered as significant. Accession numbers {#s11} ----------------- The sequences isolated in the current study were submitted to GenBank. Accession nos. JQ666166 (BjuMYB28-1 CDS), JQ666167 (BjuMYB28-2 CDS), JQ666168 (BjuMYB28-3 CDS), JQ666169 (BjuMYB28-4 CDS), JQ700565 (BjuMYB28-1 full-length gene), JQ700566 (BjuMYB28-2 full-length gene), JQ700567 (BjuMYB28-3 full-length gene), JQ700568 (BjuMYB28-4 full-length gene), JX947841 (BniMYB28-1 CDS), and JX947842 (BniMYB28-2 CDS). Results {#s12} ======= Isolation of *MYB28* homologues from *B. juncea* and its progenitor species {#s13} --------------------------------------------------------------------------- Degenerate primers (see [Supplementary Table S1](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1) at *JXB* online) based on the reported sequence of *A. thaliana* AtMYB28 (At5g61420) protein were used to isolate partial cDNA sequences of *MYB28* genes from *B. juncea*. A total of four partial *MYB28*-like sequences were isolated from this polyploid. Using a combination of 5′ and 3′ RACE strategy, four full-length *BjuMYB28* sequences \[designated as *BjuMYB28-1* (Accession no. JQ666166), *BjuMYB28-2* (JQ666167), *BjuMYB28-3* (JQ666168), and *BjuMYB28-4* (JQ666169)\] were identified and confirmed with multiple amplifications from different tissue types ([Table 1](#T1){ref-type="table"}). The nucleotide sequences of these cDNAs showed 79.8--89.4% sequence identity in their coding regions (see [Supplementary Fig. S1](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1) and [Supplementary Table S2](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1) at *JXB* online). The open reading frames of the four *BjuMYB28* sequences varied from 1053--1095bp, encoding proteins of 351--365 amino acids ([@CIT0004]). ###### DNA sequence summary of the BjuMYB28 genes identified in the current study S. no. Gene Gene (bp) CDS (bp) Protein (aa) No. of exons (sizes in bp) No. of introns (sizes in bp) 3′UTR Promoter (5′GW) Progenitor *Brassica* genome -------- ------------------------------- ----------- ---------- -------------- ---------------------------- ------------------------------ ------- ----------------- ------------------------------ 1\. *AtMYB28* 1321 1101 367 3 (133, 130, 838) 2 (80, 140) 324 -- -- 2\. *BjuB.MYB28.1 (BjuMYB28-1)* 1630 1053 351 3 (133, 130, 790) 2 (72, 505) 180 1040 B 3\. *BjuB.MYB28.2* (*BjuMYB28-2*) 1315 1095 365 3 (133, 130, 832) 2 (82, 138) 180 1275 B 4\. *BjuA.MYB28.1* (*BjuMYB28-3*) 1508 1065 355 3 (133, 130, 802) 2 (79, 364) 240 1100 A 5\. *BjuA.MYB28.2* (BjuMYB28-4) 1350 1065 355 3 (133, 130, 802) 2 (89, 196) 174 2400 A To determine the genomic structure of the four *BjuMYB28* sequences, PCR amplification on total genomic DNA of *B. juncea* was performed using sequence-specific primers. The four genomic sequences varied in size, ranging from 1314--1630bp in length ([Table 1](#T1){ref-type="table"}; Accession nos. JQ700565--68). Comparison of the four *BjuMYB28* cDNAs with their corresponding genomic sequences showed that all four genes consisted of two introns and three exons, with introns found to be more divergent in size and composition as compared to the coding regions (see [Supplementary Fig. S2](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1) and [Supplementary Table S3](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1) at *JXB* online). *B. juncea* is a natural allopolyploid species (AABB genome) formed from hybridization of *B. rapa* (AA genome) and *B. nigra* (BB genome). In order to assign the progenitor sub-genome to each *BjuMYB28*, *MYB28* homologues from *B. rapa* and *B. nigra* were also isolated. Two *MYB28* sequences could be isolated from both *B. rapa* (named as *BraA.MYB28.1* and *BraA.MYB28.2*) and *B. nigra* (named as *BniB.MYB28.1* and *BniB.MYB28.2*), representing the paralogous gene pair from each progenitor genome. Sequence analysis confirmed that two *BjuMYB28* genes namely, *BjuMYB28-1* and *BjuMYB28-2* were similar to the B-genome paralogues (see [Supplementary Fig. S1](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1) at *JXB* online), and so will henceforth be referred to as *BjuB.MYB28.1* and *BjuB.MYB28.2*, respectively ([Table 1](#T1){ref-type="table"}). Similarly, the nucleotide sequences of the remaining two *B. juncea* genes (*BjuMYB28-3*, and *BjuMYB28-4*) were highly similar to those of A-genome paralogues, and so will be referred to as *BjuA.MYB28.1* and *BjuA.MYB28.2*, respectively. In general, the coding sequences of the *MYB28* genes isolated from the species containing the progenitor A- and B-genomes showed very high levels of sequence identity (99.7--99.9%) with their corresponding *MYB28* homologues from *B. juncea* (see [Supplementary Fig. S1](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1) and [Supplementary Table S2](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1) at *JXB* online). Our data are in accordance with the reported allopolyploid origin of *B. juncea* ([@CIT0037]), wherein both progenitor genomes have contributed a duplicated *MYB28* gene pair (paralogues). Sequence analysis and phylogeny of BjuMYB28 proteins {#s14} ---------------------------------------------------- In order to investigate the evolutionary origin of the four *BjuMYB28* genes further, an amino acid sequence alignment of the deduced BjuMYB28 proteins was created with the known MYB aliphatic glucosinolate regulators from *A. thaliana*. The biosynthesis of aliphatic glucosinolates in *A. thaliana* is regulated by three closely related members of subgroup-12 of the R2R3-MYB superfamily namely AtMYB28, AtMYB29 and AtMYB76 ([@CIT0023], [@CIT0022]; [@CIT0029]; [@CIT0046], [@CIT0044]). The deduced BjuMYB28 proteins shared maximum identity with the AtMYB28 protein (72.8--82.3%) among the three aliphatic glucosinolate regulators of *Arabidopsis* ([Table 2](#T2){ref-type="table"}). The deduced protein sequences of the four *BjuMYB28* sequences were 74.9--86.6% identical with each other. Amino acid sequence alignment confirmed the presence of two imperfect sequence repeats (R2R3 repeat) at the N-terminal region ([Fig. 1](#F1){ref-type="fig"}). Predicted signature sequences for both R2 (-W-X~19~-W-X~19~-W-) and R3 (-F-X~18~-W-X~18~-W-) repeats showed a high level of sequence conservation among BjuMYB28 proteins. In contrast to the highly conserved N-terminal DNA-binding MYB domains, the downstream C-terminal region of BjuMYB28 proteins showed homology only in patches ([Fig. 1](#F1){ref-type="fig"}). The structural divergence of BjuMYB28 proteins might have consequences for their differential gene function(s) leading to loss or silencing, maintenance of ancestral function, or functional divergence either through sub-functionalization or neo-functionalization ([@CIT0034]; [@CIT0001]). ###### Amino acid sequence identity (%) of BjuMYB28 proteins with known aliphatic glucosinolate regulators belonging to subgroup-12 of the R2R3-MYB superfamily of *A. thaliana* AtMYB28 BjuB.MYB28.1 BjuB.MYB28.2 BjuA.MYB28.1 BjuA.MYB28.2 AtMYB29 AtMYB76 --------------------------- --------- -------------- -------------- -------------- -------------- --------- --------- AtMYB28 \*\*\* 72.8 82.3 74.9 80.4 59.1 55.9 BjuB.MYB28.1 \*\*\* 80.1 86.6 74.9 57.1 55.9 (BjuMYB28-1) BjuB.MYB28.2 \*\*\* 77.5 81.1 65.2 59.8 (BjuMYB28-2) BjuA.MYB28.1 \*\*\* 76.6 59.5 55.9 (BjuMYB28-3) BjuA.MYB28.2 (BjuMYB28-4) \*\*\* 60.7 56.5 AtMYB29 \*\*\* 66.1 AtMYB76 \*\*\* ![Amino acid sequence alignment of BjuMYB28 proteins. The sequence alignment of the four BjuMYB28 proteins with the known aliphatic glucosinolate-regulating MYB proteins of *A. thaliana* namely, AtMYB28, AtMYB29, and AtMYB76 was performed using Clustal W. Consensus sequences for R2 and R3 domains ([@CIT0015]) are marked as solid lines. The putative nuclear localization signal (LKKRL) is also marked (NLS).](exbotj_ert280_f0001){#F1} The high level of amino acid identity and domain conservation indicated that the four BjuMYB28 proteins are closely related. Their evolutionary relationships vis-à-vis the MYB28 proteins isolated from the species containing the two progenitor genomes of *B. juncea* were compared with publicly reported MYB28-like sequences from other plant genomes. On a maximum likelihood tree, the four BjuMYB28 proteins were grouped together with the AtMYB28 protein into a distinct MYB28 sub-group with high bootstrap support; AtMYB29 along with AtMYB76 was found in a separate subgroup ([Fig. 2](#F2){ref-type="fig"}). All four BjuMYB28 proteins shared a close evolutionary ancestry with MYB28-like sequences from *Brassica* species, particularly those obtained from the species containing the two progenitor genomes. For example, the B-genome-specific MYB28 proteins from *B. juncea* grouped with *B. nigra* proteins with very high bootstrap scores. Similarly, the A-genome specific MYB28 proteins of *B. juncea* grouped nicely with *B. rapa* sequences (including the recently reported sequences, Bra029311 and Bra012961). The phylogentic analysis thus clearly revealed that the four BjuMYB28 proteins are evolutionary conserved and have evolved via duplication (paralogues) and hybridization (homeologues) of two relatively simpler *Brassica* genomes, while retaining a very high level of sequence conservation following allo-polyplodization of A- and B-genomes. ![Evolutionary relationships of BjuMYB28 proteins. Phylogenetic analysis of BjuMYB28 proteins with the MYB proteins involved in aliphatic glucosinolate biosynthesis from *A. thaliana* (At, open diamond), *A. lyrata* (Ala, open triangle), *C. rubella* (Cru, open inverted triangle), *T. halophila* (Tha, open square), *B. rapa* (Bra, closed triangle), *B. nigra* (Bni, closed inverted triangle), *B. oleracea* (Bol, closed circle), and *B. juncea* (Bju, closed diamond) genomes was performed using the MEGA5 program ([@CIT0047]). The evolutionary history was inferred using the maximum likelihood method ([@CIT0040]). The percentage of replicate trees in which the associated proteins clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.](exbotj_ert280_f0002){#F2} *BjuMYB28* genes encode functional MYB28 proteins regulating the accumulation of aliphatic glucosinolates {#s15} --------------------------------------------------------------------------------------------------------- BjuMYB28 proteins share a close evolutionary relationship to the *Arabidopsis* AtMYB28 protein, a member of R2R3-MYB transcription factor family ([@CIT0023]). Although BjuMYB28 proteins lack the typical nuclear localization signal when queried using the PredictProtein software (<http://www.predictprotein.org/>), a conserved SV40-type putative nuclear localization motif (LKKRL; [@CIT0051]) adjacent to the R3 repeat of these proteins was observed ([Fig. 1](#F1){ref-type="fig"}). Thus, to investigate their subcellular localization, a representative BjuMYB28 protein (BjuMYB28-2) was selected and the Pro~35S~:BjuMYB28:YFP construct was introduced into onion epidermal cells by particle bombardment. The transiently transformed cells showed a strong yellow fluorescence signal in the nucleus, thus demonstrating that the BjuMYB28 are predominantly nuclear localized proteins, similar to what was observed for the *Arabidopsis* AtMYB28 protein ([Fig. 3](#F3){ref-type="fig"}). {#F3} In the absence of reverse genetics tools in the allopolyploid *B. juncea*, the functional contribution of each *BjuMYB28* towards controlling the glucosinolate pool and profile was tested in the closest model system, *A. thaliana*. All four *BjuMYB28* genes (under the control of the constitutive CaMV 35S promoter) were over-expressed in two different genetic backgrounds of *A. thaliana*, (i) the homozygous BRC_H161b, a *myb28* knock-down T-DNA insertion mutant, and (ii) the wild-type (Col-0). Two independent homozygous lines of each *BjuMYB28* were analysed for total as well as individual glucosinolate fractions in 4-week-old rosette leaves. The functional complementation of the *BjuMYB28* genes elevated the accumulation of total aliphatic glucosinolates 1.6--2.2-fold compared with the mutant BRC_H161b line ([Fig. 4A](#F4){ref-type="fig"}). Both short- and long-chain aliphatic glucosinolates were found to be elevated in these lines ([Fig. 4B](#F4){ref-type="fig"}, [C](#F4){ref-type="fig"}). The level of 4MSOB, the major glucosinolate present in *Arabidopsis*, was found to be increased by 1.7--2.2-fold. The content of other short-chain aliphatic glucosinolates, like 3MSOP and 5MSOP, was also found to be increased by up to 1.5-fold. Accumulation of the long-chain aliphatic glucosinolate 8MSOO was increased from 1.8--2.7-fold compared with the mutant line ([Fig. 4C](#F4){ref-type="fig"}). The indolic glucosinolates (I3M, 1MOI3M and 4MOI3M) of complemented mutant lines were found to be unaltered in content ([Fig. 4D](#F4){ref-type="fig"}). Thus mutant complementation analysis in *A. thaliana* clearly suggested that all four *BjuMYB28* genes specifically regulate the aliphatic glucosinolate pools without altering the non-aliphatic glucosinolate levels. {#F4} The functional divergence of *BjuMYB28* genes in controlling the levels of aliphatic glucosinolate content and profile was assessed by generating over-expression lines for these homologues in the *A. thaliana* wild-type ecotype Col-0. Total, as well as individual, glucosinolate levels of two over-expression lines of each *BjuMYB28* construct were analysed and compared with the wild-type control. As shown in [Fig. 5A](#F5){ref-type="fig"}, *BjuMYB28* over-expression provided an approximately 1.5--1.9-fold increase in leaf aliphatic glucosinolate accumulation compared with wild-type plants. Levels of 4MSOB, the major glucosinolate present in *Arabidopsis*, were found to be increased by 1.5--1.8-fold ([Fig. 5B](#F5){ref-type="fig"}). Concomitantly, the level of 4MTB (precursor of 4MSOB), showed a significant reduction in over-expression lines for the two A-genome specific *BjuMYB28* genes, thereby reflecting an increased flux of 4C-glucosinolate towards 4MSOB. The contents of short-chain aliphatic glucosinolates like 3MSOP and 5MSOP were also found to be increased up to 2.2- and 1.8-fold, respectively ([Fig. 5C](#F5){ref-type="fig"}). Accumulation of long-chain aliphatic glucosinolates namely 6MSOH, 7MSOH, and 8MSOO, was also increased by 1.3--3.5-fold compared with the wild-type plants ([Fig. 5D](#F5){ref-type="fig"}). Contents of indolic glucosinolates such as I3M, 1MOI3M, and 4MOI3M were found to be unaltered (see [Supplementary Fig. S3](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1) at *JXB* online). {#F5} The increased glucosinolate accumulation in transgenic lines correlated with increased mRNA levels of the glucosinolate pathway genes of *A. thaliana*. As shown in [Supplementary Fig. S4](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1) at *JXB* online, the expression of genes involved in both side-chain elongation (*MAM1, MAM3*) and core biosynthetic steps (*CYP79F1*, *CYP79F2, CYP83A1*, *AtST5b*, *AtST5c*) of the aliphatic glucosinolate biosynthesis pathway were considerably increased in both mutant complementation and over-expression lines compared with their respective backgrounds. The *BjuMYB28* genes tested caused up-regulation of all the aliphatic glucosinolate biosynthetic genes tested, although at variable levels. Over-expression of *BjuMYB28* genes in both wild-type and mutant backgrounds of *Arabidopsis* showed no visible effects on seed germination, plant growth or development. These lines grew normally and did not show any significant differences in response to different abiotic stress conditions (such as salt, heat, dehydration, cold) compared with their corresponding reference backgrounds (data not shown). Thus, both mutant complementation and over-expression studies clearly demonstrated that all four *BjuMYB28* genes encode functional BjuMYB28 proteins regulating aliphatic glucosinolate pools. The four *BjuMYB28* genes exhibit overlapping but distinct expression profiles in *B. juncea* {#s16} --------------------------------------------------------------------------------------------- Genome polyploidy events are often associated with variable expression of the homeologous gene pairs within the genome. The expression profiles of *MYB28* genes were therefore analysed at different developmental stages of *B. juncea* as well as in progenitor species. The efficiency and specificity of gene-specific *BjuMYB28* primer pairs was initially ascertained using a 10-fold serial dilution of the corresponding plasmid DNA. A linear correlation coefficient (*R* ^2^) of 0.99 and above was observed over a 100 000-fold dilution range, which reflected the high efficiency of each primer pair (see [Supplementary Table S4](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1) at *JXB* online). The expression profiles of *MYB28* genes across various developmental stages (or tissue types) of the species representing the three *Brassica* genomes were compared with endogenous control genes. The expression profiles of *MYB28* genes were investigated first in the progenitor species of *B. juncea* namely, *B. rapa* (A-genome) and *B. nigra* (B-genome), each harbouring two *MYB28* paralogues. Both A-genome specific *MYB28* paralogues showed very comparable expression profiles when tested across developing stages of *B. rapa* ([Fig. 6A](#F6){ref-type="fig"}). The two *BraMYB28* paralogues were abundantly expressed in glucosinolate synthesizing tissues such as seedling, leaf, and silique, whereas roots and flowers showed less accumulation of these transcripts. By contrast, when the expression profile of *MYB28* homologues was studied in *B. nigra*, differential expression of the two *BniMYB28* transcripts was observed. The *BniB.MYB28.1* gene was highly expressed across all the developing stages, particularly in seedlings and roots, whereas the *BniB.MYB28.2* transcript showed reduced expression in all the tested tissues ([Fig. 6B](#F6){ref-type="fig"}). {#F6} In order to determine whether there is a bias in transcript levels from one of the two genomes in the polyploid *B. juncea*, qRT-PCR analysis was performed for all four *BjuMYB28* in the same tissue types. All four *BjuMYB28* genes were expressed in most of the tissue types ([Fig. 6C](#F6){ref-type="fig"}). Higher levels of expression were observed in seedling, stem, and siliques compared with root and primary leaf tissues. The expression profile of A-genome-specific *BjuMYB28* genes was almost similar to that observed in *B. rapa* whereas the two B-genome specific homologues showed altered and reduced expression in *B. juncea* in most of the tissue types tested ([Fig. 6](#F6){ref-type="fig"}). In general, the cumulative abundance of the A-genome-specific homologues was higher than the B-genome-specific homologues in most of the tissue types tested. Detailed expression analysis of A- and B-genome-specific *BjuMYB28* genes was further performed at different developmental stages of leaves and siliques in *B. juncea*, the tissues where the biosynthesis of glucosinolates largely occurs. All four *BjuMYB28* genes were expressed throughout leaf development, but had lower transcript abundance during the younger stages. However, the B-genome-specific *BjuMYB28* genes had relatively higher transcript accumulation in the primary and young leaves with the onset of the reproductive phase. A significantly higher accumulation of all four *BjuMYB28* transcripts was observed in the mature and inflorescence leaves ([Fig. 7A](#F7){ref-type="fig"}). During the developing stages of the siliques (5, 10, 15, and 30 d post-anthesis), expression of both A-genome-specific *BjuMYB28* homologues was comparably higher ([Fig. 7B](#F7){ref-type="fig"}). The data clearly reflect the distinct expression of A- and B-genome-specific *BjuMYB28* genes across the developmental stages of allopolyploid *B. juncea*. {#F7} Histochemical analysis of Pro~BjuMYB28~:*uidA* lines in *Arabidopsis* confirms differential regulation of *BjuMYB28* {#s17} -------------------------------------------------------------------------------------------------------------------- In order to confirm the tissue- as well as cell-specific expression of A- and B-genome-specific *BjuMYB28*, Pro~BjuMYB28~:*uidA* lines of all four *BjuMYB28* promoters were developed in the closely related *A. thaliana*. Approximately 1kb upstream of the ATG (translation start codon) of the four *BjuMYB28* genes was isolated from the *B. juncea* genome using a 5′ genome walking protocol. The 5′ upstream regions showed a much lower level of sequence identity compared with the protein-coding regions (see [Supplementary Fig. S5](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1) and [Supplementary Table S5](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1) at *JXB* online). Two independent single-copy transgenic lines of each *BjuMYB28* promoter were used for detailed GUS histochemical analysis in the homozygous T~3~ generation. Reporter gene expression in transgenic *Arabidopsis* lines revealed that all four *BjuMYB28* homologues showed overlapping but distinct expression patterns during the developing stages of *A. thaliana* ([Fig. 8](#F8){ref-type="fig"}). Prominent GUS staining was detected in seedlings, mature rosette leaves, flowers, and siliques of Pro~BjuMYB28~:*uidA* transgenic lines. Among all four promoters, those of *BjuA.MYB28.1* and *BjuA.MYB28.2* exhibited somewhat higher GUS staining than those of the other *MYB28* genes in all floral organs including pistil, anther, and receptacle tissues as well as in the flower stalk ([Fig. 8C](#F8){ref-type="fig"}). Similarly, a stronger reporter gene expression in the developing siliques was also observed for these promoters ([Fig. 8D](#F8){ref-type="fig"}). Thus a higher GUS activity of A-genome versus B-genome-specific *BjuMYB28* promoters was detected in the reproductive tissue. A trace amount of GUS activity was detected in mature roots, senescent leaves, and seeds for all the four *BjuMYB28* promoters tested. {#F8} In the rosette leaves of *Arabidopsis*, the *BjuMYB28* promoters showed cell-specific GUS activity. The *BjuB.MYB28.1* and *BjuB.MYB28.2* promoters showed maximal activity in the edges and lamina of the leaves, respectively, whereas the activity of the *BjuA.MYB28.2* promoter was observed in the mid-vein, primary and secondary veins and also towards the leaf edges ([Fig. 8B](#F8){ref-type="fig"}). The *BjuA.MYB28.1* promoter, however, showed traces of GUS staining in the leaf edges. This non-uniform and cell-specific expression pattern of *BjuMYB28* genes within leaves might have important implications for the regulation of glucosinolate content and profile across different regions of the leaf. Thus, the GUS histochemical data obtained using the Pro~BjuMYB28~:*uidA Arabidopsis* lines confirmed that the four *BjuMYB28* promoters have overlapping but distinct cell and tissue expression patterns. Discussion {#s18} ========== The economically and nutritionally important *Brassica* crops, such as oilseed rape (*B. rapa*, *B. napus*), cabbage (*B. oleracea*), and mustard (*B. juncea*) are rich sources of glucosinolates, particularly the aliphatic glucosinolates. In this study, the isolation, characterization, expression, and functional analysis of *MYB28* gene family homologues, major regulators of aliphatic glucosinolate biosynthesis, is reported in *B. juncea*. The four *BjuMYB28* genes exhibited different, but overlapping tissue- and cell-specific expression patterns, suggesting a co-ordinated role towards controlling aliphatic glucosinolate accumulation in *B. juncea*. Genome origins and phylogeny of *B. juncea MYB28* genes {#s19} ------------------------------------------------------- In this study, four *MYB28* homologues were identified in *B. juncea* and it was possible to determine the genome origin of each homologue based on the sequence identity with the *MYB28* genes from *B. rapa* and *B. nigra*. Two *BjuMYB28* genes (*BjuA.MYB28.1* and *BjuA.MYB28.2*) showed the greatest sequence similarity with *MYB28* sequences from *B. rapa* and, therefore, are believed to originate from the A-genome, whereas the remaining two *BjuMYB28* (*BjuB.MYB28.1* and *BjuB.MYB28.2*) were B-genome-specific. The multiplicity of *BjuMYB28* corresponds well with the allotetraploid genome architecture of *B. juncea*, wherein two genes each are derived from the 'A' (*B. rapa*) and 'B' (*B. nigra*) sub-genomes ([Fig. 1](#F1){ref-type="fig"}; [Table 1](#T1){ref-type="table"}). A recent study also reported the isolation of four functional homologues of a few glucosinolate pathway genes, including *GSL-ELONG* and *GSL-ALK*, which could be equally mapped into 'A'- and 'B'-genome-specific linkage groups ([@CIT0006]). A high level of sequence conservation of the extant *BjuMYB28* was observed with their corresponding *MYB28* homologues from the *B. rapa* and *B. nigra* genomes, as confirmed by their close phylogenetic relationships ([Fig. 2](#F2){ref-type="fig"}). It is presumed that the divergence of the *MYB28* homologues in the *Brassica* lineage might have occurred during the genome triplication events that occurred in the ancestral *Brassica* species around \~13--17 MYA ([@CIT0035]). As a consequence, the diploid *Brassica* species have retained 2--3 divergent copies (paralogues) of most of the genes in their genomes ([@CIT0054]; [@CIT0052]). The recent sequencing of *B. rapa* (<http://Brassicadb.org/>) reported three *MYB28*-like sequences (accession nos. Bra012961, Bra029311, and Bra035929), two of which could be successfully amplified in the current study. Only two MYB28 homologues from the *Brassica* C-genome (*B. oleracea*) are reported in publicly available databases. Using phylogeny, the orthologous MYB28 proteins pairs of the *Brassica* A-, B-, and C-genomes could be determined in this study. Considering the polyploidy level and complex genome architecture of *B. juncea*, the possibility of as yet unidentified *BjuMYB28* sequences that may exist as pseudogenes or functional genes cannot be completely ruled out. Nevertheless, our results provide substantial information on the *MYB28* homologues of *B. juncea*. With the advent of enriched genomic resources, a complete inventory of MYB28 homologues from various *Brassica* species will be possible in the near future, which could explain the variability of glucosinolate content across *Brassica* crops. *BjuMYB28* genes all control aliphatic glucosinolate accumulation {#s20} ----------------------------------------------------------------- Polyploidy is an evolutionary process that plays a key role in generating the diversity of plant species ([@CIT0002]). The evolutionary consequences of duplicated genes after polyploidy include loss or silencing, maintaining ancestral function, and functional divergence. Over time, the function of structurally diverged homologues in allopolyploids can diverge from the ancestral gene, either through subfunctionalization or neo-functionalization ([@CIT0034]; [@CIT0001]). In the current study, over-expression of *BjuMYB28* homologues in two different genetic backgrounds of the phylogenetically close model system *A. thaliana,* demonstrated that the encoded BjuMYB28 proteins are involved in controlling aliphatic (Met-derived) glucosinolate biosynthesis in *B. juncea*, without directly affecting indolic glucosinolate biosynthesis. The *BjuMYB28* genes positively regulate the genes involved in the chain-elongation (*MAM1* and *MAM3*) and the formation of the core structure (*AtSt5b* and *AtST5c*) of aliphatic glucosinolates. The functional data in *A. thaliana* clearly showed that all four BjuMYB28 proteins are positive regulators of the genes involved in aliphatic glucosinolate biosynthesis, controlling the accumulation of both short- and long-chain aliphatic glucosinolates as also reported for the *Arabidopsis* homologue, AtMYB28 ([@CIT0023]). Thus polyploidization of *Brassica* genomes has not altered basic *MYB28* gene function, and all homologues of the *MYB28* gene seem to retain subdivision of gene function in polyploid *Brassica* crops. Amino acid sequence alignment of the four BjuMYB28 proteins showed significant structural variation in their C-terminal half. This variation might be responsible for their differential activation/regulatory control of aliphatic glucosinolate biosynthesis across plant development stages, tissue/cell types or during variable environmental conditions. Variable accumulation of total as well as individual aliphatic glucosinolates in the *Arabidopsis BjuMYB28* over-expression lines was observed in this study ([Figs 4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}). Further experiments in *B. juncea* will gain more insight about the functional divergence of A- and B-genome-specific *MYB28s*. Molecular characterization of more *MYB28*-like sequences from related *Brassica* species will help to understand the significance of variable C-terminal sequences for the regulation of aliphatic glucosinolate biosynthesis. Expression divergence of *MYB28* genes across diploid and allotetraploid *Brassica* species {#s21} ------------------------------------------------------------------------------------------- Studies in allopolyploids have shown that homoeologous genes can be expressed at different levels and can respond differentially to polyploidy in various organs of the plant or in response to various environmental stimuli ([@CIT0001]; [@CIT0033]). Global-wide analysis of gene expression in cotton, wheat, and *B. napus* clearly shows that there are both immediate and long-term alterations in the expression of homoeologous genes arising from polyploidy, such as differential expression, transcriptional bias or gene silencing of homoeologues ([@CIT0011]; [@CIT0003]; [@CIT0027]). The majority of these alterations are known to be caused by *cis*-regulatory divergence between the diploid progenitors, thereby giving rise to transcriptional sub-functionalization. Besides having variation in their coding regions, the gene structures of the *BjuMYB28*s have diverged in several other ways, including the promoter and intronic sequences. For example, when approximately 1kb of upstream sequence of the four *BjuMYB28* genes was scanned in the PLACE (PLant Cis-Acting Regulatory Elements) database (Higo *et al.*, 1991), several *cis*-regulatory elements related to tissue-dependent expression and elements responsive to glucose signalling, abiotic and biotic stress response, and sulphur assimilation were observed (see [Supplementary Table S6](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1) at *JXB* online). The disparity of various *cis*-regulatory elements observed among the four *BjuMYB28* promoters in all probability contribute to the differential expression patterns of *BjuMYB28*s as revealed by qRT-PCR analysis in *B. juncea* and GUS histochemical analysis of Pro~*BjuMYB28*~ *:GUS* transgenic lines developed in *A. thaliana* ([Fig. 8](#F8){ref-type="fig"}). For example, the four *BjuMYB28* homologues have distinct expression patterns ([Fig. 9](#F9){ref-type="fig"}), within leaves and other organs across development. ), and plotted. (B) Graphical representation of the expression profiles of the four *MYB28* homologues in *B. juncea* during the corresponding developmental stages. The colour of the box (data summarized from [Figs 6](#F6){ref-type="fig"} and [7](#F7){ref-type="fig"}) represents the comparative expression score of *BjuMYB28* genes. The two 'A' and 'B' subgenome specific homologues are also marked. (C) The comparative scoring index was constructed from the fold expression values of *BjuMYB28* genes obtained using real-time expression data as indicated.](exbotj_ert280_f0009){#F9} Glucosinolate formation in *B. juncea* leaves seems to be highly localized under the co-ordinated control of the *BjuMYB28* genes ([Fig. 8B](#F8){ref-type="fig"}). Glucosinolate biosynthesis in the mid-vein of a leaf is largely associated with *BjuA.MYB28.2*, whereas all four *BjuMYB28* genes are associated with glucosinolate formation in the outer lamina of a leaf. The non-uniform distribution of glucosinolates in *A. thaliana* leaves has been reported earlier, wherein the major glucosinolates were found to be more abundant in tissues of the mid-vein and the outer lamina of the leaf than the inner lamina ([@CIT0042]). This distribution has been at least partially attributed to the specific spatial expression of the *MYB*s that control aliphatic glucosinolate formation, including *AtMYB28*, *AtMYB29*, and *AtMYB76* ([@CIT0044]). The spatial expression patterns of *BjuMYB28* genes within the leaf also suggest a role in influencing the variable distribution of leaf glucosinolate content in *B. juncea* which, in turn, has important consequences for plant defence. When the feeding pattern of *Helicoverpa armigera* (the cotton bollworm) was studied on *A. thaliana*, the larvae avoided feeding on the mid-vein and periphery of the rosette leaves and fed mainly on the inner lamina. This feeding pattern was a direct consequence of the concentration and distribution of glucosinolates as determined using MALDI-TOF imaging ([@CIT0042]). Interestingly, the two A-genome *MYB28* homologues retained almost similar expression patterns post-polyploidization, whereas the B-genome homologues, particularly *BjuB.MYB28.1* have altered expression patterns in *B. juncea* compared with that in *B. nigra*. When the 5′ upstream region (1kb) of the *BjuMYB28* genes was compared across *B. juncea* and in species harbouring progenitor genomes, the upstream sequences of A-genome-specific homologues were found to be completely identical, whereas the B-genome-specific homologues showed a little divergence (see [Supplementary Fig. S5](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1) and [Supplementary Table S5](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1) at *JXB* online). In addition to the variable 5′ upstream regions, other factors like *trans*-regulatory elements and DNA methylation patterns could also explain the differential expression of homeologous gene pairs in allopolyploid genomes. Comparing the transcripts of the *BjuMYB28* genes from the two genomes, revealed that the both A-genome-specific transcripts in general are overrepresented across *B. juncea* development compared with B-genome transcripts ([Fig. 9](#F9){ref-type="fig"}). This expression bias of A-subgenome-specific *MYB28* homologues potentially suggests its higher transcriptional contribution for controlling the aliphatic glucosinolate biosynthesis in allopolyploid *B. juncea*. In addition to the specific cell- and tissue-level expression of A- and B-genome originating *BjuMYB28s*, all four genes showed higher expression levels with the onset of the reproductive phase. This trend may indicate a greater need for plant defence at the critical times of flowering and seed formation. Aliphatic glucosinolates are found throughout the plant, but the highest accumulation is found in the mature seeds ([@CIT0007]). *BjuMYB28*: a potential candidate for engineering low glucosinolate trait in *B. juncea* {#s22} ---------------------------------------------------------------------------------------- Recent studies on the association of glucosinolate pathway genes with seed-glucosinolate QTLs in *B. juncea* and *B. napus* suggested that *MYB28*, particularly the A-genome-specific orthologues from these two complex allopolyploid genomes, are the major genetic determinants controlling glucosinolate variability ([@CIT0039]; [@CIT0006]; [@CIT0019]; [@CIT0026]). In *B. juncea* out of the six QTLs identified for seed glucosinolate content, two QTLs namely *J3Gsl2* (A2) and *J17Gsl5* (B7) were found to contain the *MYB28* homologues ([@CIT0039]; [@CIT0006]). Recently, [@CIT0026] employing an associative transcriptomics approach to traits in *B. napus*, identified genomic deletions that underlie two quantitative trait loci for the glucosinolate content of seeds. Both the QTLs (occupying linkage groups A9 and C2) were found to contain *B. napus* orthologues of the transcription factor *MYB28* that had been lost from the low-glucosinolate accessions of *B. napus*. Based on previous reports and knowledge generated in this study, an in-depth characterization of *BjuMYB28* genes in native *B. juncea* is currently being performed. Our preliminary data suggested that the A-genome-specific *MYB28* homologues can be exploited for developing low glucosinolate lines in *B. juncea* ([@CIT0004]). The work described in this study has increased our understanding of BjuMYB28 regulatory mechanisms operational in the glucosinolate pathway in allopolyploid *B. juncea*. Our findings provide functional evidence of expression partitioning and subfunctionalization of *MYB28* gene family homologues in regulating aliphatic glucosinolate content in *B. juncea*. The information obtained in the current study should facilitate tissue-specific engineering of aliphatic glucosinolate traits in *Brassica* crops using conventional breeding and/or transgenic approaches in order to reduce anti-nutritive seed glucosinolates to economically acceptable levels while insuring that defence of leaves and other tissues are not compromised. Supplementary data {#s23} ================== Supplementary data can be found at *JXB* online. [Supplementary Table S1](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1). List of primers used in the current study. [Supplementary Table S2](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1). Nucleotide sequence identity (%) of coding DNA sequences (CDS) of *MYB28* homologues isolated from *B. juncea* (Bju), *B. rapa* (Bra), and *B. nigra* (Bni). [Supplementary Table S3](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1). Nucleotide sequence identity (%) of *BjuMYB28* full-length genes. [Supplementary Table S4](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1). Primer amplification efficiency test of *BjuMYB28* genes used in the current study. [Supplementary Table S5](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1). Nucleotide sequence identity (%) of the 5′ upstream region of the *MYB28* homologues isolated from *B. juncea* and its progenitor genomes. [Supplementary Table S6](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1). Summary of various *cis*-regulatory elements present within a 1kb upstream region of the *BjuMYB28* genes, obtained using the PLACE database ([www.dna.affrc.go.jp/PLACE/](www.dna.affrc.go.jp/PLACE/)). [Supplementary Fig. S1](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1). Nucleotide sequence alignment of coding DNA sequences (CDS) of *MYB28* homologues isolated from *Brassica* species. The sequence alignment of CDS of *A. thaliana AtMYB28* (At5g61420), *B. juncea BjuMYB28(1--4*), *B. nigra BniMYB28(1,2*), and *B. rapa BraMYB28(1,2*) was performed using the ClustalW algorithm available in the MegAlign module of DNASTAR software (Lasergene). Dark shading represents conserved residues. [Supplementary Fig. S2](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1). Nucleotide sequence alignment of full-length genomic sequences of four *BjuMYB28* genes. The sequence alignment of *AtMYB28* (At5g61420), and full-length *B. juncea BjuMYB28* genes was performed using the ClustalW algorithm available in the MegAlign module of DNASTAR software (Lasergene). The positions of two introns are marked within the brackets \[\]. Nucleotide in a dark background represents residues differing from the consensus. [Supplementary Fig. S3](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1). Indolic glucosinolate profiles of *BjuMYB28* over-expression (OE) lines in an *Arabidopsis* wt (Col-0) background. Two independent transgenic events for each BjuMYB28 homologue were analysed and the value represent mean ±SE (*n* ≥4). [Supplementary Fig. S4](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1). Transcript levels of glucosinolate pathway genes in rosette leaves of representative (A) *BjuMYB28* mutant complementation (MC) and (B) over-expression (OE) lines in *A. thaliana*. qRT-PCR analysis of aliphatic glucosinolate pathway genes was performed and the transcript accumulation was measured with reference to the *Arabidopsis* wild-type Col-0 and BRC_H161b mutant background, respectively (both set at 1). Values are mean ±SE of three independent biological replicates. Asterisks indicate significant differences in gene expression compared with the respective background (*P* \<0.05, in Fishers LSD test). [Supplementary Fig. S5](http://jxb.oxfordjournals.org/lookup/suppl/doi:10.1093/jxb/ert280/-/DC1). Nucleotide sequence alignment of the 5′ upstream region of four *MYB28* homologues from *B. juncea* and its progenitor genomes. The sequence alignment was performed using the ClustalW algorithm available in the MegAlign module of DNASTAR software (Lasergene). Nucleotide in a dark background represents residues differing from the consensus. ###### Supplementary Data We thank Dr Piero Moranidini for providing the homozygous *myb28* knock-down line (BRC_H161b) and Dr Michael Reichelt for his assistance with the glucosinolate analysis. We thank Dr Arun Jagannath for his critical comments on the manuscript. The central instrumentation and confocal facilities at NIPGR are acknowledged. RA was supported with a Junior Research Fellowship from the Council of Scientific and Industrial Research, India. This work was supported by project schemes (BT/PR271/AGR/36/687/2011 and Rapid Grant for Young Investigators \[RGYI\]) of the Department of Biotechnology, India, and the core research grant was from the National Institute of Plant Genome Research, India.

1. Introduction {#sec1} =============== Around 80% of all acute stroke patients suffer from upper limb paresis hampering daily activities \[[@B1], [@B2]\]. At present, rehabilitation after stroke is mainly based on symptoms of the patients rather than on underlying neurophysiological changes. A better understanding of stroke-induced changes in brain functions is required to develop more individually tailored and more efficient rehabilitation. Integration of somatosensory feedback with motor output is essential for fluent motor performance, and it is tightly coupled with changes in cortical excitability; afferent somatosensory input is known to alter motor-cortex inhibition \[[@B3]--[@B9]\]. Motor-cortex excitability is reflected in the modulation of the 20 Hz rhythm; activation of the motor cortex suppresses this rhythm whereas the subsequent rebound after movement cessation reflects inhibition or deactivation of the motor cortex \[[@B6], [@B10]--[@B13]\]. Both animal and human studies have shown that an acute stroke induces changes in motor-cortex excitability \[[@B14]--[@B18]\]. Our previous MEG studies in stroke patients using tactile \[[@B19]\] and proprioceptive \[[@B20]\] stimulation suggest that alterations in motor-cortex excitability after stroke are probably due to both changes in local excitatory--inhibitory circuits and disturbed afferent input, which lead to impaired sensorimotor integration. To further understand the mechanisms affecting motor-cortex excitability and recovery after stroke, we compared how two different types of afferent input modulate motor-cortex excitability during one-year recovery from stroke. We employed magnetoencephalography (MEG) to compare the effect of tactile and proprioceptive stimulation of the index fingers on the 20 Hz rhythm at one week, one month, and one year after stroke and thereafter correlated the results with clinical recovery of the patients. 2. Methods {#sec2} ========== The data of the control subjects and the passive movement-induced changes in the 20 Hz rhythm in the patients are obtained from our previous two studies \[[@B20], [@B21]\]. Modulation of the 20 Hz rhythm to tactile stimuli, presented here for the first time, was recorded in the same sessions as passive movement data. 2.1. Subjects {#sec2.1} ------------- Thirty patients with first-ever stroke in the territory of the middle cerebral artery and related unilateral upper limb paresis were recruited from the Department of Neurology, Helsinki University Hospital (HUH). Clinical neurological examination was performed at the time of recruitment to include patients with hand weakness or clumsiness. Patients with earlier neurological diseases, mental disorders, prior neurosurgical operations, or unstable cardiovascular/general condition were not included. Seven patients were excluded later during follow-up; two died, four declined the second or third MEG recording, and the data of one patient were contaminated with artifacts preventing reliable analyses. Eventually, 23 patients participated the entire study (10 females, age 45--78 years, mean 65 ± 2 years; [Table 1](#tab1){ref-type="table"}). The control group comprised 22 healthy subjects (11 females, age 42--72 years, mean 59 ± 2.0 years). The Local Ethics Committee of the Helsinki and Uusimaa Hospital District approved the study protocol, and all subjects assigned written informed consent prior to the measurements. 2.2. Clinical Evaluation {#sec2.2} ------------------------ NIHSS (National Institutes of Health Stroke Scale; [Table 1](#tab1){ref-type="table"}) evaluation, hand motor function, tactile sensitivity, and proprioception were assessed in conjunction with the MEG recordings 1--7 days (T~0~), 1 month (T~1~), and 12 months (T~2~) after stroke. According to NIHSS, stroke impairment can be classified as mild (NIHSS \< 8), moderate (NIHSS 8--16), and severe (NIHSS \> 17). An occupational therapist tested the manual dexterity of both the impaired and healthy hands of the patients by using the Box-and-Block test (BB; number of cubes moved from one compartment to another in 60 s; [Table 2](#tab2){ref-type="table"}). The tactile detection threshold was evaluated with von Frey Filaments (20 filaments; 3.22--3.61 normal/reduced light touch; 3.84--4.31 reduced protective sensation; 4.56--6.65 no protective sensation; 6.65 no measurable tactile sense; [Table 2](#tab2){ref-type="table"}). A qualitative test was used for evaluation of proprioception; the impaired hand was placed to different positions, and the patient reproduced the positions without seeing the healthy hand; the ability to mimic the positions with the healthy hand was evaluated to be normal or abnormal. The qualitative test showed that proprioceptive sense of the impaired hand was normal only in 5/23 at T~0~, in 8/23 at T~1~, and in 11/23 at T~2~. 2.3. MEG Recordings and Neuroradiological Evaluations {#sec2.3} ----------------------------------------------------- A whole-scalp MEG system (306 channels; 204 planar gradiometers and 102 magnetometers; Vectorview™; Elekta Oy, Helsinki, Finland) was employed for the recordings. The measurements of 18 control subjects were performed in Aalto University and four controls and all patients with similar devices in the BioMag Laboratory (HUH, Finland). During the recordings, the subjects were either in a sitting or supine (four patients at T~0~) position and instructed not to pay attention to the finger lift or tactile stimulation, to relax, and to avoid excessive blinking. Four indicator coils as well as three anatomical landmarks (right and left preauricular points and nasion) and 50--100 additional points on the head surface were used for coregistration. The MEG and vertical electrooculogram signals were pass-band filtered to 0.03--330 Hz and digitized at 1000 Hz. About 60 averaged trials were accepted for each hand while acquiring continuous data for analysis. In addition, resting state data with eyes open and eyes closed (3 min each) were recorded. To determine the lesion site and size, anatomical magnetic resonance images (MRIs) were taken at T~0~ and T~1~ with a 3T MRI scanner (Philips Achieva 3T, Philips Medical Systems, Best, The Netherlands). The MRIs revealed two patients with cortical, 15 with cortico-subcortical, and six with subcortical infarcts; of which, 16 patients had right and seven left hemispheric lesions. The size of the lesion varied from 0.24 to 218.5 cm^3^ (mean 40 ± 12 cm^3^; [Table 1](#tab1){ref-type="table"}). 2.4. Stimulation {#sec2.4} ---------------- ### 2.4.1. Passive Movement {#sec2.4.1} The index finger was lifted briskly by a laboratory nurse once every 3 s (in the patients, first the healthy and then the impaired side) with a rigid aluminum stick attached with a Velcro strap to the phalanx. Cutaneous tactile stimulation was minimized by covering the middle phalanx with a surgical tape and by ensuring that the fingertip did not touch the device during the movement. A 3-axis accelerometer (ADXL335 iMEMS accelerometer Analog Devices Inc., Norwood, MA, USA) linked to the MEG system was attached on the nail of the index finger to determine the finger kinematics. Reliable accelerometer signals were acquired in 17 controls and 16 patients. The average lag time (time from actual onset of passive finger movement to recorded movement onset) was calculated and used for the subjects with no accelerometer signals. The rhythm and amplitude of the movements were kept constant by monitoring the moving index finger with two optical gates (lower and upper) separated by 30 mm along the direction of the movement; only movements passing through both gates within 500 ms were accepted as valid trials for on-line averaging and later for off-line analysis, \~60 for each hand. In the patients, the peak acceleration of the index finger did not differ significantly between the healthy and the impaired hand at any time point. Neither were differences within one hand observed between T~0~ and T~1~. However, passive movements of both the healthy and impaired hands in the patients were brisker at T~2~ than at T~0~ (*p* \< 0.001) and at T~1~ (*p* \< 0.01). In the controls, the peak acceleration of the passive movement did not differ between the right and left index fingers. At T~2~, the peak acceleration in the patients for both the healthy and impaired hands was brisker than in the controls (*p* \< 0.001) but no significant differences between patients and controls were found at T~0~ or T~1~. The movement duration was significantly shorter (*p* \< 0.01) in the patients versus controls in all measurement sessions. However, the movement duration of either hand of the patients did not differ between T~0~ and T~1~ \[[@B20]\]. ### 2.4.2. Tactile Stimulation {#sec2.4.2} Pneumatic diaphragms driven by compressed air were used to deliver tactile stimuli (duration 140 ms, peak at 50 ms) to the tips of the index fingers alternately with an interstimulus interval (ISI) of 1.5 s (3 s for one side). Around 60 on-line-accepted trials were collected for each finger for later off-line analysis. More detailed description of tactile and proprioceptive stimulation is presented in our previous study in healthy controls \[[@B21]\]. 2.5. Data Analysis {#sec2.5} ------------------ Temporal signal-space separation method (tSSS) \[[@B22]\] was used to suppress environmental magnetic interference from the MEG data. Head movements were compensated with the MaxFilter software (version 2.2.11; Elekta Oy) \[[@B23], [@B24]\]. Only data from the 204 planar gradiometer channels were used for subsequent analysis. To determine the peak amplitudes and frequencies of spontaneous brain activity, the amplitude spectra were estimated from the resting-state data (eyes open) with the Welch method using 2048-sample Hanning-windowed segments. The strongest peaks were found in the 15--25 Hz range in both the controls and the patients; this band was chosen for further analysis in all subjects. The strength of *β*~1~- and *β*~2~-peaks (9--15 and 8--11 fT/cm, resp.) did not differ significantly between the hemispheres, between time points, or between patients and controls. Time-frequency representations (TFR) of passive movement and tactile stimulation responses were calculated over all channels for the 3--40 Hz range with 7-cycle Morlet wavelets, to visually assure the frequency range of the strongest modulation. The temporal spectral evolution method (TSE) \[[@B13]\] was used to quantify the modulation of the 20 Hz rhythm; the continuous data were first filtered to 15--25 Hz, rectified, and averaged (−100--3000 ms) time-locked to stimulus onset. Peak amplitudes of suppression and rebound over the sensorimotor cortex were quantified in both the ipsi- and contralateral hemispheres with respect to the moved/stimulated hand from four channels (two from each hemisphere) showing the strongest suppression/rebound of 20 Hz activity. The relative peak amplitudes were calculated as percentage of amplitude changes with respect to the individual prestimulus baseline (−100--0 ms). 2.6. Statistical Analysis {#sec2.6} ------------------------- The normality of the data was tested with the Kolmogorov--Smirnov (KS) test; with four variables, the null hypothesis of a normal distribution could be rejected at *p* \< 0.05. To ensure that all variables are normally distributed, we converted the original values *x* into new values *y* = ln(*x* + 1) where ln(·) is the natural logarithm. After this transformation, the KS test indicated normal distribution of all variables. These transformed variables were used for statistical analyses. The kinematics of passive movements and clinical test results in the patients between the impaired and healthy hands were compared with a two-way (hand: impaired and healthy; time: T~0~, T~1~, and T~2~) repeated measures ANOVA. The kinematics of passive movements were compared between the patients and the controls (right and left hands pooled) with one-way, six-level (2 × hand; 3 × time) ANOVA \[[@B20]\]. The TSE results from all sessions (T~0~, T~1~, and T~2~) were evaluated in both the affected (AH) and unaffected hemispheres (UH) to both impaired and healthy hand tactile stimulation and passive movement. The variance within factor time, hemispheres (AH/UH), and side of stimulation was analyzed with a two-way within-subject ANOVA. Significant (threshold *p* \< 0.05) main effects (F) were compared with paired sample *t*-tests. Independent sample *t*-tests were used when comparing effects between controls and patients. As rebound amplitudes were clearly larger to passive than to tactile stimuli even in the healthy subjects, direct comparison of amplitudes was not possible. To compare the recovery rates of the rebounds to the two stimulus types, the relative rebounds were normalized with respect to the relative rebound of the healthy hand in the unaffected hemisphere at T~2~. Likewise, to compare the recovery of the hand motor performance (BB test and tactile sense), the clinical scores of the impaired hand were normalized with those of the healthy hand at T~2~. Spearman\'s nonparametric correlation was applied to test for associations between the lesion volumes and clinical variables (scores of BB test, tactile sense) and MEG responses (threshold *p* \< 0.05). 3. Results {#sec3} ========== 3.1. NIHSS {#sec3.1} ---------- According to the NIHSS evaluation, the severity of the impairment caused by stroke varied from mild to moderate; NIHSS \< 17 in all the patients ([Table 1](#tab1){ref-type="table"}). Note that NIHSS was zero in three patients in the acute phase despite of their upper limb paresis. This is due to evaluation of upper arm strength in NIHSS; zero point is obtained if the patient is capable of lifting the arm and holding it up for 10 seconds despite total lack of distal hand movements. 3.2. Tactile Sense {#sec3.2} ------------------ At T~0~, tactile sensitivity of the impaired hand was significantly diminished (4.56 ± 0.22 versus 3.74 ± 0.08; *p* \< 0.01) compared to that of the healthy hand ([Table 2](#tab2){ref-type="table"}). Tactile sensitivity of the impaired hand improved significantly from T~0~ to T~1~ (*p* \< 0.05) but not from T~1~ to T~2~ and remained significantly weaker compared to that of the healthy hand (*p* \< 0.05). In the healthy hand, tactile sensitivity improved significantly from T~0~ to T~2~ (3.74 ± 0.08 versus 3.57 ± 0.04; *p* \< 0.05) but not from T~0~ to T~1~ or T~1~ to T~2~. [Figure 1](#fig1){ref-type="fig"} shows how tactile sensitivity of the impaired and healthy hands recovered during the one-year follow-up. 3.3. Hand Motor Performance {#sec3.3} --------------------------- The results of the BB test of the impaired and healthy hands have been presented in our previous study \[[@B20]\], and they are shown here in [Table 2](#tab2){ref-type="table"}. During the one-year follow-up, BB of the impaired hand improved from T~0~ to T~1~ (*p* \< 0.001) and from T~1~ to T~2~ (*p* \< 0.01). However, at all time points, BB scores of the impaired hand were significantly worse (*p* \< 0.001) than those of the healthy hand. BB improved also for the healthy hand from T~0~ to T~1~ (*p* \< 0.001) but not significantly from T~1~ to T~2~. [Figure 1](#fig1){ref-type="fig"} shows the recovery of BB scores of the impaired and healthy hands (normalized to the scores of the healthy hand at T~2~) during the one-year follow-up. At T~0~ and T~1~, BB scores of the impaired hand were lower than the values of a healthy population (matched for gender, age, and the side of the tested hand) \[[@B25]\] in all patients and at T~2~ in 21/23 patients. The healthy hand BB scores were lower than those of the healthy population in 21 patients at T~0~, 19 at T~1~, and 15 at T~2~. 3.4. Modulation of the \~20 Hz Rhythm {#sec3.4} ------------------------------------- ### 3.4.1. Peak Latencies of Suppression and Rebound {#sec3.4.1} The baseline levels of the 20 Hz rhythm in the patients and between the patients and controls did not differ significantly between the hemispheres or between different time points. In the patients, the suppression of the 20 Hz rhythm peaked at 530 ± 10 ms after passive movement and at 270 ± 10 ms after tactile stimulation; the subsequent rebound peaked at 1370 ± 30 ms and at 690 ± 20 ms, respectively. In the controls, the suppression peaked at 540 ± 10 ms after passive movement and at 300 ± 10 ms after tactile stimulation and the rebound at 1450 ± 30 ms and 790 ± 10, respectively. No differences in peak latencies were detected between the hemispheres or between the patients and control subjects. In all measurements, the peak latencies of suppression and rebound to passive movement were significantly longer (*p* \< 0.001) compared to those to tactile stimulation. No significant differences were detected in the strength of the suppression between the hemispheres of the patients, between different time points, or between the patients and the controls. ### 3.4.2. 20 Hz Rebound Strength to Tactile versus Proprioceptive Stimulation {#sec3.4.2} The maximal 20 Hz rebounds both to passive movement and to tactile stimulation were detected over the same planar gradiometer channels as the strongest beta peaks in the amplitude spectra of the resting-state data; the location of the maximal rebound was found over the rolandic area anterior to that of the maximal suppression. Both tactile and proprioceptive stimulation modulated bilaterally the 20 Hz rhythm, but the effect was much stronger in the contralateral hemisphere to the stimulated hand, in line with earlier findings \[[@B12], [@B13], [@B19]--[@B21]\]. Therefore, in the present study, we compared the rebounds of the hemisphere contralateral to the stimulated hand. The rebound strengths (mean ± SEM) of the patients and controls are presented in [Table 3](#tab3){ref-type="table"}. [Figure 2(a)](#fig2){ref-type="fig"} shows the grand average TSE of the 20 Hz band in the affected and unaffected hemispheres to contralateral tactile stimulation and passive movement; in each patient, the channel showing the maximal rebound was selected, and the TSEs of these channels were then averaged and divided by the mean baseline value. In the controls, no differences between the rebound strengths within one stimulus type between the left and right hemispheres were detected; hence, the rebounds in both hemispheres to contralateral stimuli were pooled \[[@B21]\]. ### 3.4.3. Affected Hemisphere, Impaired Hand Stimulation {#sec3.4.3} [Figure 2(b)](#fig2){ref-type="fig"} shows the relative rebound (% of the baseline) strengths to tactile stimulation and passive movement in the patients during the 12-month follow-up period. Rebounds to tactile stimulation were identified in 13/23 patients at T~0~. At T~1~ and T~2~, all the patients showed reliable rebounds. To passive movement, measurable rebounds were found in 17 patients at T~0~, in 21 at T~1~, and in all 23 patients at T~2~. The rebound strength to tactile stimulation increased significantly from T~0~ to T~1~ and T~2~ (*p* \< 0.001), but no significant increase was detected from T~1~ to T~2~. Accordingly, the rebound to passive movement increased significantly from T~0~ to T~1~ and to T~2~ (*p* \< 0.01 and *p* \< 0.001, resp.) but not from T~1~ to T~2~. The rebound strength to tactile stimulation reached the level of the controls by T~2~ whereas the rebound strength to passive movement remained significantly (*p* \< 0.001) weaker than that of the controls at T~2~ (46% of the rebound of the controls). ### 3.4.4. Unaffected Hemisphere, Healthy Hand Stimulation {#sec3.4.4} The rebounds to both stimuli were identified in all patients at all time points. [Figure 2(b)](#fig2){ref-type="fig"} shows that the rebound strength to tactile stimulation increased significantly from T~0~ to T~1~ and to T~2~ (*p* \< 0.05) but not from T~1~ to T~2~. The rebound strength to passive movement increased significantly from T~0~ to T~2~ (*p* \< 0.01) but not from T~0~ to T~1~ or from T~1~ to T~2~. In the healthy hemisphere, the rebound strengths to tactile stimulation did not differ from those of the controls at any time point whereas the rebound strengths to passive movement were significantly weaker than those of the controls at all time points and remained 67% of the rebound of the controls at T~2~ (*p* \< 0.05). 3.5. Correlation with Clinical Measures {#sec3.5} --------------------------------------- The rebound strength to tactile stimulation or passive movement did not correlate with the lesion volume at any time point. ### 3.5.1. Box-and-Block Test of the Impaired Hand {#sec3.5.1} [Figure 3](#fig3){ref-type="fig"} shows the positive correlation of the AH rebound strength to both stimulus types with BB scores; the stronger the rebound the higher the BB score and the better the motor performance. The Spearman\'s correlation analysis showed that the rebound strengths to both tactile stimulation and passive movement correlated significantly with BB scores at all time points: to tactile stimulation, *r* = 0.63 and *p* \< 0.001 at T~0~, *r* = 0.68 and *p* \< 0.001 at T~1~, and *r* = 0.69 and *p* \< 0.001 at T~2~ ([Figure 3(a)](#fig3){ref-type="fig"}) and to passive movement, *r* = 0.65 and *p* \< 0.001 at T~0~; *r* = 0.78 and *p* \< 0.001 at T~1~, and *r* = 0.59 and *p* \< 0.01 at T~2~ ([Figure 3(b)](#fig3){ref-type="fig"}). Interestingly, the rebound strength at T~0~ correlated significantly with BB scores of the impaired hand at T~2~; the stronger the rebound at T~0~ the better the hand performance at T~2~ ([Figure 3(c)](#fig3){ref-type="fig"}; *r* = 065, *p* \< 0.001 and *r* = 0.57, *p* \< 0.01, to tactile stimulation and passive movement, resp.). ### 3.5.2. Box-and-Block Test of the Healthy Hand {#sec3.5.2} The rebound strength in the unaffected hemisphere to tactile stimulation did not correlate with BB scores of the healthy hand at any time point. The rebound strength in the unaffected hemisphere did not correlate with BB scores of the healthy hand at T~0~ or at T~1~, but a significant correlation was found at T~2~ (*r* = 0.50; *p* \< 0.05). ### 3.5.3. Tactile Sensitivity of the Impaired Hand {#sec3.5.3} The negative correlation of tactile sensitivity with the rebound strength revealed that the better the tactile sensitivity (the thinner the detected von Frey Filament) the stronger the rebound at T~0~ (*r* = −0.57; *p* \< 0.01) and at T~1~ (*r* = −0.56; *p* \< 0.01) but no significant correlation was found at T~2~. Tactile sensitivity of the healthy hand did not correlate with the rebound strength in the UH to tactile stimulation at any time point. 4. Discussion {#sec4} ============= In this study, we compared the effect of two types of afferent input, tactile, and proprioceptive stimulation, on the modulation of the 20 Hz rhythm during one-year stroke recovery. The results showed that the rebound strengths to both stimuli were bilaterally diminished in the acute phase. During the first month of recovery, the rebounds increased but after one month, no significant changes were observed. Temporally similar recovery profiles of the rebounds to both stimuli during the first month suggest that stroke-induced alterations in motor-cortex excitability occur mainly during the first four weeks. This finding confirms and extends the earlier observations indicating a sensitive period for plastic changes during the first weeks after stroke \[[@B26]--[@B31]\]. During this rather short period, changes in gene expression and neurotransmission \[[@B32]--[@B35]\], altered cortical inhibition \[[@B14], [@B36]--[@B42]\], and structural changes \[[@B43]--[@B47]\] enable formation of new networks and reorganization of the sensorimotor cortex. 4.1. Rebound Strength in the Acute Phase after Stroke {#sec4.1} ----------------------------------------------------- In the acute phase, the rebound strengths in the affected and unaffected hemispheres to tactile and proprioceptive stimulation were diminished compared to the controls, indicating increased excitability of the motor cortex ([Figure 2](#fig2){ref-type="fig"}). This is in line with several earlier studies in both animals and humans, showing hyperexcitability both in the affected and unaffected hemispheres after stroke \[[@B14], [@B18]--[@B20], [@B39], [@B48]--[@B53]\]. This hyperexcitability---or disinhibition---is suggested to reflect reduced GABA~A~ergic and increased glutamergic activation in the peri-infarct zone and in the contralesional unaffected hemisphere \[[@B16], [@B17], [@B36], [@B39], [@B40], [@B50]--[@B52], [@B54]\]. As afferent input also affects motor-cortex inhibition, the observed diminished 20 Hz rebound may result both from decreased cortical inhibition and defective afferent input to the motor cortex \[[@B19], [@B20]\]. The behavior of the 20 Hz rhythm followed a similar pattern regardless of the stimulus type, corroborating the assumption that defective afferent input alone is not sufficient to explain the decrease in motor-cortex inhibition. This is further supported by the diminished 20 Hz rebound also in the unaffected hemisphere to healthy hand stimulation in the acute phase after stroke. As the afferent input from the healthy hand to the unaffected hemisphere is likely intact, the diminished 20 Hz rebound of the healthy hemisphere probably indicates decreased intracortical inhibition (ICI), whereas in the affected hemisphere, the weaker rebounds likely are due to both decreased ICI and diminished afferent input to the motor cortex. However, the similar reduction in the rebound strengths to both stimuli indicates that in the acute phase, cortical excitability changes modulate the rebound strongly, and this modulation may itself lead to disturbed sensorimotor integration and hence hampered dexterity. This hypothesis is strengthened by the observation that also the healthy hand function was impaired (as compared to the normative values of a healthy population), although no structural lesions were found in the unaffected hemisphere. 4.2. Recovery of the Rebound during Follow-Up {#sec4.2} --------------------------------------------- The strongest increment of the rebound amplitude occurred from T~0~ to T~1~, whereas no significant increase in the rebound strength was observed from T~1~ to T~2~. Although the changes in the rebound amplitudes to both stimuli followed a rather similar temporal pattern, the rebounds to tactile stimulation reached the level of the controls in both hemispheres during the one-year follow-up, whereas the rebound to passive movement did not. In healthy controls, passive movement has been shown to produce a stronger rebound than electric median nerve stimulation \[[@B9]\] or tactile stimulation \[[@B21]\]. In voluntary movement, the mass of the muscles is known to affect the rebound strength; the greater the mass, the stronger the rebound \[[@B55]\] as a greater mass of moving muscles activate a larger number of sensory afferents. The weaker recovery of the rebound to passive movement compared with tactile stimulation may imply that in our stroke patients, proprioception did not recover as well as tactile sense. However, this remains speculative as we were not able to precisely define the recovery of proprioception in our patients. Furthermore, anticipation and planning of a forthcoming voluntary movement are known to increase the excitability of the motor cortex, which is reflected in the modulation of the 20 Hz activity but also as the Bereitschaftspotential or readiness field \[[@B56], [@B57]\]. Although our stimulation did not involve voluntary movement, the timing of the movements was highly predictable and thus at least the healthy subjects could probably anticipate each stimulus, possibly leading to higher rebounds. In addition, our healthy controls might have actively opposed passive movements more than the patients did, particularly since the muscle strength of the patients was diminished. Yet, the significant increase of the rebound strength from the acute phase by one month after stroke was evident. Future studies should be conducted to explore the relationship between recovery of proprioception, muscle strength, and rebound strength. 4.3. Rebound Strength and Its Association with Clinical Outcome {#sec4.3} --------------------------------------------------------------- In our patients, a stronger rebound (less disinhibition/increased ICI) in the affected hemisphere to both stimulus types was associated with better hand function. Although disinhibition in the acute phase after stroke may be necessary to allow plasticity to a certain extent \[[@B3], [@B5], [@B58]\], it is possible that later on a normalization of excitability is a prerequisite for normal or near-normal (sensorimotor integration) and hand functions. Accordingly, the healthy hand function was impaired in the acute phase, concomitantly with a decreased 20 Hz rebound. Human studies with transcranial magnetic stimulation (TMS) have suggested that reduced short-interval intracortical inhibition (SICI, meaning increased excitability) in an acute stroke enhances afferent input-related long-term potentiation in the motor cortex leading to good motor recovery (measured with modified ranking scale (mRS)) at six months \[[@B59], [@B60]\]. In stroke patients, a H2O15-PET study revealed bilateral hyperexcitability in the acute phase and a reduced excitability at 31 weeks in both hemispheres in association with better recovery in thumb-to-index finger tapping \[[@B61]\]. By using a paired-pulse transcranial magnetic stimulation (TMS) in stroke patients, motor-cortical disinhibition was found in both hemispheres in the acute phase \[[@B39], [@B40]\]; increased ICI in the unaffected hemisphere at three months correlated significantly with good hand motor recovery. Accordingly, in patients with poor motor recovery, ICI in the unaffected hemisphere remained high, in line with other studies showing that prolonged hyperexcitation in the unaffected hemisphere would be harmful for recovery after stroke \[[@B51], [@B52]\]. However, no correlation of increased ICI in the affected hemisphere with clinical recovery was found \[[@B39], [@B40]\]. Another TMS study by Swayne and colleagues (2008) showed that decreased bilateral ICI did not correlate with hand motor performance in the acute phase after stroke (measured weekly until one month with action research arm test (ARAT) and nine-hole peg test (NHPT)). However, in agreement with our findings, increased ICI in the affected hemisphere at three months correlated strongly with hand motor performance suggesting that new intracortical networks probably were already structured and functioning \[[@B47]\]. Similar findings have been observed in animal studies. In rats, autoradiographics revealed a reduction in GABA~A~ receptor expression in the surroundings of acute photothrombic infarcts \[[@B48]\]. Patch-clamp recordings over the primary motor cortex during acute stroke in mice showed that an occlusion in the middle cerebral artery decreased GABAergic tonic inhibition in conjunction with an activation of N-methyl-D-aspartate (NMDA) receptors in the peri-infarct zone \[[@B51]\]. Taken together, both animal and human studies have indicated decreased ICI in the acute phase after stroke and thereafter increased ICI in association with good motor recovery. In the present study, the rebound strength in the affected hemisphere to both stimuli correlated significantly with impaired hand motor recovery at all time points indicating that the observed changes in excitability are closely linked to functional recovery. Furthermore, the stronger the rebound in the affected hemisphere in the acute phase the better the hand motor performance at one year, as measured with the Box-and-Block test. Although, the number of patients in our study does not allow to draw direct conclusions, it is a tempting idea that motor outcome after stroke could be predicted by evaluating the initial inhibitory state of the motor cortex with the 20 Hz rebound. This finding could help to develop tools not only for studying alterations in motor-cortex excitability but also for tailoring rehabilitation according to the observed neurophysiological changes and for predicting motor recovery already at acute stage. 5. Conclusions {#sec5} ============== The temporally similar recovery profiles of the 20 Hz rebounds to both tactile stimulation and passive movement indicate that motor-cortex excitability is increased mainly during the first four weeks after stroke, underlining the importance of early and intensive rehabilitation. Furthermore, we found that the rebound strengths in the affected hemisphere to both stimulus types in the acute phase may reflect functionality of sensorimotor integration and predict motor performance in the long run. Importantly, the close connection of afferent input with excitability changes should be paid attention to when planning novel therapeutic interventions. The rebounds to both tactile stimulation and passive movement appear to be robust neurophysiological markers of stroke-induced cortical excitatory changes. The authors thank Mia Illman for helping with all the MEG recordings, Jyrki Mäkelä for supporting their MEG measurements in BioMag Laboratory (Helsinki University Hospital (HUS)), and Suvi Heikkilä for the assistance in the recordings. The authors thank the HUS occupational therapist for performing hand clinical tests. The study was financially supported by Helsinki University Hospital Research Fund, The Finnish Medical Foundation and Tekes, Finnish Funding Agency for Technology and Innovation, SalWe Research Program for Mind and Body and Seamless Patient Care Grant nos. 1104/10 and 1988/31/2015. Conflicts of Interest ===================== The authors declare that they have no conflicts of interest. {#fig1} {#fig2} {#fig3} ###### Clinical details of the patients. Patient Gender Age NIHSS Lesion --------- -------- ----- ------- -------- --- ---- ---- ------- 1 f 68 0 0 0 rh c 1.78 2 f 59 0 0 0 lh c 0.24 3 f 60 12 6 4 rh cs 24.9 4 m 66 4 3 1 rh cs 71.3 5 m 45 7 2 1 rh cs 84.2 6 f 58 2 0 0 rh cs 31.7 7 f 66 5 2 0 rh cs 4.58 8 m 71 2 1 1 rh cs 26.7 9 m 75 12 6 2 rh cs 35.8 10 m 62 3 1 1 rh cs 21.2 11 m 67 14 10 6 rh cs 218.5 12 m 47 14 7 5 rh cs 149.9 13 f 78 7 4 3 rh cs 55.6 14 m 61 6 4 2 rh cs 124.8 15 m 49 0 0 0 lh cs 3.53 16 m 76 4 3 2 lh cs 2.59 17 f 73 10 4 1 lh cs 2.84 18 m 68 2 1 1 rh s 1.36 19 f 59 4 1 0 rh s 1.95 20 f 75 14 13 4 rh s 13.0 21 m 64 5 2 1 lh s 1.46 22 f 74 15 13 6 lh s 40.0 23 m 74 1 0 0 lh s 0.48 f: female; m: male; rh: right hemisphere; lh: left hemisphere; c: cortical; cs: cortico-subcortical; s: subcortical; NIHSS: National Institutes of Health Stroke Scale; T~0~: 1--7 days; T~1~: 1 month; T~2~: 12 months from stroke. ###### Clinical scores of the patients. Time Box-and-Block (mean ± sem) von Frey (mean ± sem) ------ ---------------------------- ----------------------- ----------------- ------------- T~0~ 22 + 4.7^∗∗∗^ 45 ± 3 4.56 + 0.22^∗∗^ 3.74 ± 0.08 T~1~ 32 ± 4.9^∗∗∗^ 54 ± 2 4.46 ± 0.23^∗∗^ 3.64 ± 0.06 T~2~ 36 ± 5.3^∗∗∗^ 56 ± 2 4.33 ± 0.24^∗^ 3.57 ± 0.04 Box-and-Block: number of blocks replaced in 1 min; tactile sense: von Frey Filaments 1.65--6.65; T~0~: 1--7 days; T~1~: 1 month; T~2~: 12 months from stroke. The significance of the difference between the impaired and healthy hands: ^∗^*p* \< 0.05, ^∗∗^*p* \< 0.01, and ^∗∗∗^*p* \< 0.001. ###### Mean strengths (±SEM) of the 20 Hz rebounds in the patients and the controls. Patients Patients Controls -------------------------------- ---------- ---------- ---------- ---------- ---------- ---------- ---------- Rebound to tactile stimulation 22 ± 6.1 41 ± 6.7 50 ± 7.7 47 ± 4.4 66 ± 9.6 73 + 1.1 57 ± 4.9 Rebound to passive movement 24 ± 4.3 39 ± 6.0 44 ± 5.0 48 ± 4.4 59 ± 6.6 65 ± S.5 97 ± 9.3 AH-impaired: affected hemisphere, impaired hand stimulation; UH-healthy: unaffected hemisphere, healthy hand stimulation; T~0~: 1--7 days; T~1~:1 month; T~2~:12 months from stroke; Contra H: hemisphere contralateral to the stimulation. In the controls, contralateral responses in the left and right hemispheres are pooled. [^1]: Academic Editor: J. Michael Wyss

Related literature {#sec1} ================== For related compounds and further synthetic details, see: Schilf *et al.* (2007[@bb13]). For intra­molecular hydrogen bonds in this type of compound, see: Fernández-G *et al.* (2001[@bb7]); Kabak (2003[@bb8]); Wojciechowski *et al.* (2001[@bb16]); Dey *et al. (*2001); Koşar *et al.* (2004[@bb9]); Lu *et al.* (2008[@bb10]); Qiu & Zhao (2008[@bb12]); Montazerozohori *et al.* (2009[@bb11]); Corden *et al.* (1996[@bb4]); Black *et al.* (2010[@bb1]); Dey *et al.* (2001[@bb5]). Experimental {#sec2} ============ {#sec2.1} ### Crystal data {#sec2.1.1} C~29~H~26~N~2~O~2~*M* *~r~* = 434.52Monoclinic,*a* = 18.1766 (8) Å*b* = 7.9808 (4) Å*c* = 16.0347 (8) Åβ = 92.703 (2)°*V* = 2323.47 (19) Å^3^*Z* = 4Mo *K*α radiationμ = 0.08 mm^−1^*T* = 296 K0.62 × 0.38 × 0.24 mm ### Data collection {#sec2.1.2} Bruker APEXII CCD diffractometerAbsorption correction: integration (*XPREP*; Bruker, 1999[@bb2]) *T* ~min~ = 0.918, *T* ~max~ = 1.00022291 measured reflections3001 independent reflections2724 reflections with *I* \> 2σ(*I*)*R* ~int~ = 0.026 ### Refinement {#sec2.1.3} *R*\[*F* ^2^ \> 2σ(*F* ^2^)\] = 0.034*wR*(*F* ^2^) = 0.087*S* = 1.053001 reflections301 parameters1 restraintH-atom parameters constrainedΔρ~max~ = 0.20 e Å^−3^Δρ~min~ = −0.18 e Å^−3^ {#d5e483} Data collection: *APEX2* (Bruker, 2005[@bb3]); cell refinement: *SAINT-NT* (Bruker, 2005[@bb3]); data reduction: *SAINT-NT*; program(s) used to solve structure: *SHELXTL* (Sheldrick, 2008[@bb14]); program(s) used to refine structure: *SHELXL97* (Sheldrick, 2008[@bb14]); molecular graphics: *PLATON* (Spek, 2009[@bb15]); software used to prepare material for publication: *WinGX* (Farrugia, 1999[@bb6]) and *PLATON*. Supplementary Material ====================== Crystal structure: contains datablocks global, I. DOI: [10.1107/S1600536810015291/hb5392sup1.cif](http://dx.doi.org/10.1107/S1600536810015291/hb5392sup1.cif) Structure factors: contains datablocks I. DOI: [10.1107/S1600536810015291/hb5392Isup2.hkl](http://dx.doi.org/10.1107/S1600536810015291/hb5392Isup2.hkl) Additional supplementary materials: [crystallographic information](http://scripts.iucr.org/cgi-bin/sendsupfiles?hb5392&file=hb5392sup0.html&mime=text/html); [3D view](http://scripts.iucr.org/cgi-bin/sendcif?hb5392sup1&Qmime=cif); [checkCIF report](http://scripts.iucr.org/cgi-bin/paper?hb5392&checkcif=yes) Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: [HB5392](http://scripts.iucr.org/cgi-bin/sendsup?hb5392)). The University of the Witwatersrand and the National Research Foundation (GUN 2069064) are thanked for providing the infrastructure and for the award of a research grant required to carry out this work. Comment ======= The molecular structure of the title compound form two strong intermolecular hydrogen bonds O---H···N involving the hydroxyl and the imine groups, forming S(6) ring motifs which are common to this type of compound (Schilf *et al.*, 2007) and (Fernández-G *et al.*, 2001), and also seen in the work completed by (Kabak *et al.*, 2003), (Wojciechowski *et al.*, 2001), (Dey *et al.*, 2001), (Koşar, *et al.*, 2004), (Lu, *et al.*, 2008) (Qiu & Zhao, 2008), (Montazerozohori *et al.*, 2009), (Corden *et al.*, 1996) and (Black *et al.*, 2010). This causes the dihedral angles between the adjacent phenyl rings and phenyl containing plains to be (C1---C6---C7---C8) 89.8 (2)° and (C17---C22---C23---C24) 87.8 (2)° respectively. These dihedral angles are comparable to (Corden *et al.*, 1996) and (Black *et al.*, 2010). The stereogenic centre on the methyl substituted carbon C15 allows the system to pack in the noncentrosymmetric space group *C2*. The remaining weak interactions in the crystals form unexceptional σ-π interactions. Experimental {#experimental} ============ A mixture of 0.01 mol (2.00 g) of 2-hydroxybenzophenone and 0.005 mole (0.42 ml) of 1,2-propanediamine in 40 ml of methanol was refluxed for 7 h. The excess of solvent (ca. 30 ml) was then evaporated. After cooling to 277 K, a yellow solid was produced. The polycrystalline product was collected by filtration, washed with methanol and dried a yield 54% was obtained. Recrystalization from an ethanol solution yielded yellow blocks of (I). Elemental analysis: *C% 79.67 H% 5.99 N% 6.03*. Refinement {#refinement} ========== The absolute structure of (I) is indeterminate based on the present refinement. All H atoms were refined using a riding model, with a C---H distance of 0.96, for Ar---H a distance of 0.93 Å and for O---H a distance of 0.82 Å, and *U*iso(H) = 1.2*U*eq(C) and 1.5*U*eq(O). The highest residual peak was 0.742 Å from atom C6 with a ρ = 0.20 e Å^-3^. Figures ======= {#Fap1} Crystal data {#tablewrapcrystaldatalong} ============ ------------------------- --------------------------------------- C~29~H~26~N~2~O~2~ *F*(000) = 920 *M~r~* = 434.52 *D*~x~ = 1.242 Mg m^−3^ Monoclinic, *C*2 Mo *K*α radiation, λ = 0.71073 Å Hall symbol: C 2y Cell parameters from 9931 reflections *a* = 18.1766 (8) Å θ = 2.2--28.3° *b* = 7.9808 (4) Å µ = 0.08 mm^−1^ *c* = 16.0347 (8) Å *T* = 296 K β = 92.703 (2)° Block, yellow *V* = 2323.47 (19) Å^3^ 0.62 × 0.38 × 0.24 mm *Z* = 4 ------------------------- --------------------------------------- Data collection {#tablewrapdatacollectionlong} =============== ------------------------------------------------------------ -------------------------------------- Bruker APEXII CCD diffractometer 3001 independent reflections Radiation source: sealed tube 2724 reflections with *I* \> 2σ(*I*) graphite *R*~int~ = 0.026 φ and ω scans θ~max~ = 28°, θ~min~ = 1.3° Absorption correction: integration (*XPREP*; Bruker, 1999) *h* = −23→24 *T*~min~ = 0.918, *T*~max~ = 1.000 *k* = −10→10 22291 measured reflections *l* = −21→20 ------------------------------------------------------------ -------------------------------------- Refinement {#tablewraprefinementdatalong} ========== ------------------------------------- ------------------------------------------------------------------------------------------------- Refinement on *F*^2^ 1 restraint Least-squares matrix: full H-atom parameters constrained *R*\[*F*^2^ \> 2σ(*F*^2^)\] = 0.034 *w* = 1/\[σ^2^(*F*~o~^2^) + (0.0448*P*)^2^ + 0.6417*P*\] where *P* = (*F*~o~^2^ + 2*F*~c~^2^)/3 *wR*(*F*^2^) = 0.087 (Δ/σ)~max~ = 0.001 *S* = 1.05 Δρ~max~ = 0.20 e Å^−3^ 3001 reflections Δρ~min~ = −0.18 e Å^−3^ 301 parameters ------------------------------------- ------------------------------------------------------------------------------------------------- Special details {#specialdetails} =============== ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Experimental. Numerical integration absorption corrections based on indexed crystal faces were applied using the XPREP routine (Bruker, 1999) Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å^2^) {#tablewrapcoords} ================================================================================================== ------ --------------- -------------- -------------- -------------------- -- *x* *y* *z* *U*~iso~\*/*U*~eq~ C1 0.17117 (10) 0.8773 (3) 0.19104 (12) 0.0380 (4) H1 0.1576 0.8561 0.2453 0.046\* C2 0.23795 (11) 0.9528 (3) 0.17780 (14) 0.0452 (5) H2 0.2699 0.9792 0.2229 0.054\* C3 0.25725 (11) 0.9890 (3) 0.09795 (15) 0.0500 (5) H3 0.302 1.0411 0.0891 0.06\* C4 0.21054 (12) 0.9484 (3) 0.03135 (14) 0.0536 (5) H4 0.2237 0.974 −0.0225 0.064\* C5 0.14368 (11) 0.8693 (3) 0.04351 (12) 0.0432 (5) H5 0.1124 0.8412 −0.0019 0.052\* C6 0.12418 (9) 0.8328 (2) 0.12419 (11) 0.0318 (4) C7 0.05455 (9) 0.7387 (2) 0.14143 (10) 0.0308 (4) C8 −0.01204 (10) 0.8365 (2) 0.15614 (11) 0.0317 (4) C9 −0.01477 (11) 1.0091 (2) 0.14096 (13) 0.0398 (4) H9 0.0267 1.0624 0.1219 0.048\* C10 −0.07704 (12) 1.1025 (3) 0.15342 (13) 0.0478 (5) H10 −0.0777 1.2167 0.142 0.057\* C11 −0.13872 (12) 1.0242 (3) 0.18322 (14) 0.0487 (5) H11 −0.1808 1.0867 0.1924 0.058\* C12 −0.13812 (11) 0.8549 (3) 0.19929 (13) 0.0445 (5) H12 −0.1796 0.8042 0.2199 0.053\* C13 −0.07625 (10) 0.7587 (3) 0.18505 (12) 0.0372 (4) C14 0.09851 (12) 0.3771 (3) 0.04854 (12) 0.0445 (5) H14A 0.0548 0.3114 0.0545 0.067\* H14B 0.139 0.3044 0.0374 0.067\* H14C 0.0907 0.4544 0.0031 0.067\* C15 0.11599 (10) 0.4740 (2) 0.12901 (11) 0.0330 (4) H15 0.1592 0.5453 0.1221 0.04\* C16 0.13115 (11) 0.3541 (2) 0.20186 (11) 0.0364 (4) H16A 0.0869 0.2909 0.2124 0.044\* H16B 0.1695 0.2755 0.1882 0.044\* C17 −0.00368 (11) 0.3145 (3) 0.35288 (13) 0.0447 (5) H17 −0.0215 0.4205 0.3389 0.054\* C18 −0.05238 (12) 0.1829 (4) 0.36563 (14) 0.0602 (7) H18 −0.1029 0.2012 0.3604 0.072\* C19 −0.02579 (16) 0.0266 (4) 0.38587 (16) 0.0640 (7) H19 −0.0585 −0.0607 0.3944 0.077\* C20 0.04851 (16) −0.0019 (3) 0.39361 (17) 0.0639 (7) H20 0.066 −0.1085 0.4071 0.077\* C21 0.09762 (13) 0.1270 (3) 0.38147 (14) 0.0477 (5) H21 0.148 0.1073 0.3869 0.057\* C22 0.07149 (10) 0.2860 (2) 0.36114 (11) 0.0331 (4) C23 0.12560 (9) 0.4238 (2) 0.34679 (11) 0.0307 (3) C24 0.14855 (9) 0.5331 (2) 0.41770 (11) 0.0304 (3) C25 0.11331 (10) 0.5252 (2) 0.49331 (12) 0.0368 (4) H25 0.0749 0.4495 0.4988 0.044\* C26 0.13414 (11) 0.6270 (3) 0.56007 (13) 0.0437 (5) H26 0.1102 0.6195 0.6099 0.052\* C27 0.19109 (12) 0.7405 (3) 0.55188 (14) 0.0477 (5) H27 0.2049 0.8105 0.5963 0.057\* C28 0.22735 (11) 0.7505 (3) 0.47865 (13) 0.0461 (5) H28 0.2658 0.8264 0.4741 0.055\* C29 0.20693 (9) 0.6482 (2) 0.41146 (11) 0.0355 (4) N1 0.05252 (8) 0.57835 (19) 0.14636 (9) 0.0338 (3) N2 0.15442 (8) 0.44924 (19) 0.27629 (9) 0.0348 (3) O1 −0.07848 (8) 0.59298 (19) 0.20016 (11) 0.0509 (4) H1A −0.0408 0.5489 0.1844 0.076\* O2 0.24508 (8) 0.6613 (2) 0.34177 (8) 0.0487 (4) H2A 0.2264 0.6006 0.3053 0.073\* ------ --------------- -------------- -------------- -------------------- -- Atomic displacement parameters (Å^2^) {#tablewrapadps} ===================================== ----- ------------- ------------- ------------- -------------- ------------- -------------- *U*^11^ *U*^22^ *U*^33^ *U*^12^ *U*^13^ *U*^23^ C1 0.0384 (10) 0.0346 (9) 0.0407 (9) −0.0049 (8) 0.0003 (8) −0.0001 (8) C2 0.0365 (10) 0.0390 (10) 0.0591 (12) −0.0070 (8) −0.0073 (8) −0.0052 (9) C3 0.0296 (10) 0.0468 (12) 0.0742 (14) −0.0096 (9) 0.0104 (9) −0.0005 (11) C4 0.0464 (11) 0.0669 (14) 0.0487 (11) −0.0111 (11) 0.0144 (9) 0.0084 (11) C5 0.0386 (10) 0.0536 (12) 0.0374 (9) −0.0087 (9) 0.0009 (8) 0.0026 (9) C6 0.0301 (9) 0.0270 (8) 0.0383 (9) −0.0033 (7) 0.0021 (7) 0.0017 (7) C7 0.0299 (8) 0.0341 (9) 0.0280 (8) −0.0069 (7) −0.0017 (6) 0.0013 (6) C8 0.0303 (9) 0.0335 (9) 0.0311 (8) −0.0046 (7) −0.0011 (6) −0.0007 (7) C9 0.0379 (10) 0.0355 (10) 0.0462 (10) −0.0049 (8) 0.0031 (8) −0.0008 (8) C10 0.0544 (12) 0.0365 (10) 0.0526 (12) 0.0058 (9) 0.0034 (10) −0.0034 (9) C11 0.0418 (11) 0.0531 (13) 0.0513 (12) 0.0102 (10) 0.0028 (9) −0.0072 (10) C12 0.0310 (10) 0.0535 (13) 0.0494 (11) −0.0034 (9) 0.0057 (8) 0.0002 (9) C13 0.0315 (9) 0.0409 (10) 0.0389 (9) −0.0042 (8) −0.0020 (7) 0.0016 (8) C14 0.0513 (12) 0.0435 (10) 0.0391 (9) −0.0056 (10) 0.0053 (8) −0.0027 (9) C15 0.0318 (9) 0.0306 (9) 0.0372 (9) −0.0056 (7) 0.0066 (7) −0.0007 (7) C16 0.0406 (10) 0.0294 (8) 0.0391 (9) −0.0029 (7) 0.0018 (7) −0.0018 (7) C17 0.0341 (9) 0.0512 (12) 0.0492 (11) −0.0046 (9) 0.0052 (8) −0.0058 (9) C18 0.0379 (11) 0.089 (2) 0.0546 (13) −0.0233 (12) 0.0085 (9) −0.0164 (13) C19 0.0761 (18) 0.0632 (16) 0.0534 (13) −0.0401 (15) 0.0107 (12) −0.0014 (12) C20 0.0856 (19) 0.0415 (12) 0.0645 (15) −0.0188 (13) 0.0017 (13) 0.0086 (11) C21 0.0495 (12) 0.0368 (10) 0.0564 (12) −0.0059 (9) −0.0017 (9) 0.0050 (9) C22 0.0328 (9) 0.0340 (9) 0.0326 (8) −0.0063 (7) 0.0012 (7) −0.0034 (7) C23 0.0263 (8) 0.0258 (8) 0.0396 (9) 0.0011 (6) −0.0013 (6) 0.0008 (7) C24 0.0260 (8) 0.0266 (8) 0.0382 (8) 0.0013 (6) −0.0028 (6) 0.0002 (7) C25 0.0309 (9) 0.0336 (9) 0.0461 (10) −0.0024 (8) 0.0034 (7) −0.0017 (8) C26 0.0419 (10) 0.0466 (11) 0.0429 (10) −0.0020 (9) 0.0067 (8) −0.0078 (8) C27 0.0472 (11) 0.0480 (12) 0.0473 (11) −0.0063 (10) −0.0032 (9) −0.0140 (9) C28 0.0404 (10) 0.0452 (11) 0.0520 (11) −0.0149 (9) −0.0066 (8) −0.0033 (9) C29 0.0307 (9) 0.0357 (9) 0.0398 (9) −0.0026 (8) −0.0029 (7) 0.0036 (8) N1 0.0307 (7) 0.0322 (8) 0.0385 (8) −0.0061 (6) 0.0031 (6) 0.0001 (6) N2 0.0345 (7) 0.0323 (7) 0.0376 (7) −0.0043 (6) 0.0003 (6) −0.0011 (6) O1 0.0343 (7) 0.0413 (8) 0.0778 (11) −0.0062 (6) 0.0092 (7) 0.0116 (8) O2 0.0440 (8) 0.0613 (10) 0.0410 (7) −0.0221 (7) 0.0020 (6) 0.0007 (7) ----- ------------- ------------- ------------- -------------- ------------- -------------- Geometric parameters (Å, °) {#tablewrapgeomlong} =========================== ----------------------- -------------- ----------------------- -------------- C1---C2 1.380 (3) C15---H15 0.98 C1---C6 1.385 (3) C16---N2 1.460 (2) C1---H1 0.93 C16---H16A 0.97 C2---C3 1.374 (3) C16---H16B 0.97 C2---H2 0.93 C17---C22 1.385 (3) C3---C4 1.371 (3) C17---C18 1.395 (3) C3---H3 0.93 C17---H17 0.93 C4---C5 1.391 (3) C18---C19 1.372 (4) C4---H4 0.93 C18---H18 0.93 C5---C6 1.388 (3) C19---C20 1.369 (4) C5---H5 0.93 C19---H19 0.93 C6---C7 1.508 (2) C20---C21 1.382 (3) C7---N1 1.283 (2) C20---H20 0.93 C7---C8 1.469 (3) C21---C22 1.389 (3) C8---C9 1.399 (3) C21---H21 0.93 C8---C13 1.419 (2) C22---C23 1.500 (2) C9---C10 1.378 (3) C23---N2 1.284 (2) C9---H9 0.93 C23---C24 1.477 (2) C10---C11 1.388 (3) C24---C25 1.399 (2) C10---H10 0.93 C24---C29 1.411 (2) C11---C12 1.376 (3) C25---C26 1.383 (3) C11---H11 0.93 C25---H25 0.93 C12---C13 1.389 (3) C26---C27 1.386 (3) C12---H12 0.93 C26---H26 0.93 C13---O1 1.346 (3) C27---C28 1.376 (3) C14---C15 1.525 (3) C27---H27 0.93 C14---H14A 0.96 C28---C29 1.388 (3) C14---H14B 0.96 C28---H28 0.93 C14---H14C 0.96 C29---O2 1.347 (2) C15---N1 1.460 (2) O1---H1A 0.82 C15---C16 1.525 (3) O2---H2A 0.82 C2---C1---C6 120.49 (18) C16---C15---H15 109.5 C2---C1---H1 119.8 N2---C16---C15 109.55 (15) C6---C1---H1 119.8 N2---C16---H16A 109.8 C3---C2---C1 120.01 (18) C15---C16---H16A 109.8 C3---C2---H2 120 N2---C16---H16B 109.8 C1---C2---H2 120 C15---C16---H16B 109.8 C4---C3---C2 120.00 (18) H16A---C16---H16B 108.2 C4---C3---H3 120 C22---C17---C18 119.5 (2) C2---C3---H3 120 C22---C17---H17 120.2 C3---C4---C5 120.72 (19) C18---C17---H17 120.2 C3---C4---H4 119.6 C19---C18---C17 120.0 (2) C5---C4---H4 119.6 C19---C18---H18 120 C6---C5---C4 119.26 (18) C17---C18---H18 120 C6---C5---H5 120.4 C20---C19---C18 120.5 (2) C4---C5---H5 120.4 C20---C19---H19 119.7 C1---C6---C5 119.48 (17) C18---C19---H19 119.7 C1---C6---C7 118.56 (16) C19---C20---C21 120.3 (2) C5---C6---C7 121.90 (16) C19---C20---H20 119.9 N1---C7---C8 119.60 (16) C21---C20---H20 119.9 N1---C7---C6 122.34 (17) C20---C21---C22 119.8 (2) C8---C7---C6 118.02 (15) C20---C21---H21 120.1 C9---C8---C13 117.66 (17) C22---C21---H21 120.1 C9---C8---C7 121.20 (17) C17---C22---C21 119.81 (18) C13---C8---C7 121.13 (15) C17---C22---C23 121.07 (18) C10---C9---C8 122.01 (19) C21---C22---C23 119.12 (16) C10---C9---H9 119 N2---C23---C24 118.18 (15) C8---C9---H9 119 N2---C23---C22 123.25 (16) C9---C10---C11 119.3 (2) C24---C23---C22 118.55 (15) C9---C10---H10 120.4 C25---C24---C29 117.87 (16) C11---C10---H10 120.4 C25---C24---C23 121.07 (15) C12---C11---C10 120.5 (2) C29---C24---C23 121.06 (15) C12---C11---H11 119.8 C26---C25---C24 121.64 (18) C10---C11---H11 119.8 C26---C25---H25 119.2 C11---C12---C13 120.7 (2) C24---C25---H25 119.2 C11---C12---H12 119.6 C25---C26---C27 119.29 (19) C13---C12---H12 119.6 C25---C26---H26 120.4 O1---C13---C12 118.80 (18) C27---C26---H26 120.4 O1---C13---C8 121.39 (17) C28---C27---C26 120.58 (19) C12---C13---C8 119.81 (18) C28---C27---H27 119.7 C15---C14---H14A 109.5 C26---C27---H27 119.7 C15---C14---H14B 109.5 C27---C28---C29 120.45 (19) H14A---C14---H14B 109.5 C27---C28---H28 119.8 C15---C14---H14C 109.5 C29---C28---H28 119.8 H14A---C14---H14C 109.5 O2---C29---C28 117.95 (17) H14B---C14---H14C 109.5 O2---C29---C24 121.87 (16) N1---C15---C14 108.39 (15) C28---C29---C24 120.17 (17) N1---C15---C16 109.14 (14) C7---N1---C15 122.13 (16) C14---C15---C16 110.66 (15) C23---N2---C16 121.53 (16) N1---C15---H15 109.5 C13---O1---H1A 109.5 C14---C15---H15 109.5 C29---O2---H2A 109.5 C6---C1---C2---C3 2.0 (3) C19---C20---C21---C22 −0.1 (4) C1---C2---C3---C4 −0.7 (3) C18---C17---C22---C21 0.4 (3) C2---C3---C4---C5 −0.5 (4) C18---C17---C22---C23 179.17 (18) C3---C4---C5---C6 0.5 (4) C20---C21---C22---C17 −0.2 (3) C2---C1---C6---C5 −1.9 (3) C20---C21---C22---C23 −179.00 (19) C2---C1---C6---C7 175.34 (18) C17---C22---C23---N2 −93.9 (2) C4---C5---C6---C1 0.7 (3) C21---C22---C23---N2 84.9 (2) C4---C5---C6---C7 −176.5 (2) C17---C22---C23---C24 87.8 (2) C1---C6---C7---N1 −87.8 (2) C21---C22---C23---C24 −93.4 (2) C5---C6---C7---N1 89.5 (2) N2---C23---C24---C25 172.54 (17) C1---C6---C7---C8 89.8 (2) C22---C23---C24---C25 −9.1 (2) C5---C6---C7---C8 −92.9 (2) N2---C23---C24---C29 −7.8 (2) N1---C7---C8---C9 −171.73 (18) C22---C23---C24---C29 170.61 (16) C6---C7---C8---C9 10.6 (2) C29---C24---C25---C26 0.5 (3) N1---C7---C8---C13 7.3 (3) C23---C24---C25---C26 −179.84 (18) C6---C7---C8---C13 −170.35 (16) C24---C25---C26---C27 0.3 (3) C13---C8---C9---C10 0.1 (3) C25---C26---C27---C28 −0.9 (3) C7---C8---C9---C10 179.19 (17) C26---C27---C28---C29 0.7 (3) C8---C9---C10---C11 1.1 (3) C27---C28---C29---O2 −178.9 (2) C9---C10---C11---C12 −0.7 (3) C27---C28---C29---C24 0.2 (3) C10---C11---C12---C13 −0.8 (3) C25---C24---C29---O2 178.35 (17) C11---C12---C13---O1 −178.8 (2) C23---C24---C29---O2 −1.4 (3) C11---C12---C13---C8 2.0 (3) C25---C24---C29---C28 −0.7 (3) C9---C8---C13---O1 179.18 (19) C23---C24---C29---C28 179.59 (17) C7---C8---C13---O1 0.1 (3) C8---C7---N1---C15 177.40 (14) C9---C8---C13---C12 −1.7 (3) C6---C7---N1---C15 −5.0 (3) C7---C8---C13---C12 179.27 (17) C14---C15---N1---C7 −111.3 (2) N1---C15---C16---N2 −66.08 (18) C16---C15---N1---C7 128.14 (18) C14---C15---C16---N2 174.74 (15) C24---C23---N2---C16 −175.93 (15) C22---C17---C18---C19 −0.2 (3) C22---C23---N2---C16 5.8 (3) C17---C18---C19---C20 −0.1 (4) C15---C16---N2---C23 131.47 (17) C18---C19---C20---C21 0.3 (4) ----------------------- -------------- ----------------------- -------------- Hydrogen-bond geometry (Å, °) {#tablewraphbondslong} ============================= --------------- --------- --------- ----------- --------------- *D*---H···*A* *D*---H H···*A* *D*···*A* *D*---H···*A* O1---H1A···N1 0.82 1.84 2.573 (2) 147 O2---H2A···N2 0.82 1.83 2.553 (2) 147 --------------- --------- --------- ----------- --------------- ###### Selected torsion angles (°) ----------------------- ---------- C1---C6---C7---C8 89.8 (2) C17---C22---C23---C24 87.8 (2) ----------------------- ---------- ###### Hydrogen-bond geometry (Å, °) *D*---H⋯*A* *D*---H H⋯*A* *D*⋯*A* *D*---H⋯*A* --------------- --------- ------- ----------- ------------- O1---H1*A*⋯N1 0.82 1.84 2.573 (2) 147 O2---H2*A*⋯N2 0.82 1.83 2.553 (2) 147 [^1]: On leave from: Faculty of Chemistry, Department of General and Coordination Chemistry, Maria Curie-Sklodowska University, Maria Curie-Sklodowska Sq. 2, 20-031 Lublin, Poland.

As the world is witnessing a continuous epidemic of cholera in Haiti in the new millennium, global concerns on this reemerging infection is on the rise.\[[@ref1]\] Although a downward trend in the fatality of this epidemic has been reported recently, the attack rate still remains high at 6.1%.\[[@ref2]\] It is of interest to know that before the recent earthquake, cholera was not seen in Haiti for more than a century and an independent panel convened by the United Nation (UN) concluded that the infection was brought back to Haiti by the United Nation (UN) stabilizing forces.\[[@ref3][@ref4][@ref5]\] Cholera epidemic is usually associated with poor quality of drinking water and other indicators of poor hygiene, which may also provoke epidemics of other infectious agents specially hepatitis A and E.\[[@ref6][@ref7]\] Analyzing previous outbreaks of cholera may help to control these infections in Haiti more effectively and avoid similar tragedies. Continuity of this epidemic for a long period indicates that despite some optimistic views on rapid control, there is still failure in achieving control of this foreign-based epidemic in Haiti.\[[@ref2][@ref8]\] In 2005, Iran experienced an epidemic of cholera with more than 1100 registered cases.\[[@ref3]\] The epidemic started form a local area in central Iran near a vegetable farm. In that farm, a group of illegal immigrants from Pakistan who were suffering from diarrhea were living in tents. The original source of this epidemic was later confirmed with molecular studies.\[[@ref9][@ref10][@ref11]\] The first case was reported in the central city of Qom. The outbreak of watery diarrhea suspected to be cholera was rapidly noticed by the health workers in that village and reported to the local health authorities and then to the ministry. The initial response was local community awareness of the condition, information to health professionals, and strict control on food markets. As the number of cases was increasing and the disease was found to affect many provinces, the ministry announced the epidemics nationally. In addition to the mentioned measures, a national propaganda on hazards of using fresh vegetables was broadcasted on television, radio, internet, magazines, and newspapers. Public awareness had a major impact in the control of this epidemic. In contrary to previous epidemics, which lasted longer,\[[@ref12]\] this epidemic was controlled within weeks and stopped after 4 months. This epidemic once again emphasizes the importance of early detection and monitoring.\[[@ref13]\] The local health workers called "Behvarz" in Iran were the first to suspect the epidemics. The wide network of more than 30,000 of "Behvarzan" in Iran, who worked in more than 13,000 health houses during that time, gave the capacity of early detection of cases and later in monitoring in the progress of the epidemics.\[[@ref14]\] The importance of this capacity for diarrhea surveillance becomes more apparent when one notes that in the recent outbreak of *Escherichia coli* infection in Germany, the possibility of outbreak was only raised after 3 weeks from hospitalization of the first affected case.\[[@ref15][@ref16]\] Medicalization of this public health problem by medical professionals and focusing only on timely diagnosis and treatment of affected cases with antibiotics and hydration was another challenge. Although as a component of national surveillance system, all medical professionals are responsible to report all cases with watery diarrhea to local health authorities, not all of them did this on a timely manner. This was more important in the cities, where initially physicians considered cholera as a disease of rural regions and under estimated the expansible nature of this disease. Considering access of more than 98% of urban and 90% of rural population to sanitary water, it was true that those small communities who were mostly in remote rural areas were the most who were suffering from this outbreak. But the problem was not restricted to them. Even in urban cities with access to sanitary water, food processing at times was associated with use of unsafe water. Examples included ice producing factories, small businesses who prepared fresh vegetables for sale to the restaurants and markets, some of the gardens in vicinity to the cities, which were used for hosting large parties like wedding ceremonies. These places used at times well water, which contributed to the continuation of the epidemic.\[[@ref17]\] Another challenge was those small vegetable farms near the major metropolitans, which were irrigated with waste water or who used natural fertilizers. The control of the outbreak became possible when all of these neglected and hidden routes of transmission of the infection were approached. These interventions were possible though an intersectorial collaboration with special help from ministries of interior, water and energy, and agriculture and even nongovernmental organizations Another issue, which needs consideration, is the rate of consumption of fresh vegetables and fruits in Iran. Before this epidemic, health survey in Iran shows that this rate was around 93%. Just the year after the epidemic, it dropped to 87% and even 4 years after this outbreak it only rose to 88%. Current evidence indicates that low vegetable consumption is associated with obesity, diabetes and higher risk of cardiovascular diseases and increased rate of some cancers. As eating fresh vegetables are among the most effective measures in controlling noncommunicable diseases (NCDs), this event may have a drawback in the current pandemic of NCDs. This means, informing the population about the route of transmission of infectious diarrheal diseases; empowerment to control should also be popularized to avoid this type of misconceptions with resultant increase in NCDs risk factors. In conclusion, control of epidemics like cholera is a multidimensional program. Simplistic approach for these complex problems may even increase the harms and hazards without any benefit. As the epidemic of cholera has not been stopped in Haiti even after 2 years of global effort, health authorities should reevaluate their efforts to find where other facets of this vicious cycle were not approached systematically.

Introduction {#Sec1} ============ Nonalcoholic fatty liver disease (NAFLD) affects a quarter of the adult population and is a leading cause of liver-related morbidity and mortality^[@CR1]^. This condition is defined mainly by its histology and consists of two principal phenotypes which include a fatty liver and steatohepatitis^[@CR2]^. Nonalcoholic steatohepatitis (NASH) is characterized by steatosis, inflammation and hepatocellular ballooning which are predominantly seen in zone III of hepatic lobules^[@CR3]^. NASH has a greater likelihood of progression to cirrhosis than nonalcoholic fatty liver (NAFL)^[@CR4]^. Current disease models predict a two- to three-fold increase in the population burden of cirrhosis and end stage liver disease due to NASH by 2030^[@CR5],[@CR6]^. The development of knowledge in NAFLD has been anchored to the histological assessment of the disease. Two principal concepts in this assessment are disease activity and fibrosis stage. Disease activity represents the factors driving the fibrogenic remodeling of the liver towards cirrhosis and is captured by the NAFLD activity score (NAS), which is the sum of the histological severity scores for steatosis, lobular inflammation, and hepatocellular ballooning^[@CR3]^. On the other hand, fibrosis stage reflects the actual progression towards cirrhosis and is related to clinical outcomes^[@CR7]^. A substantial body of literature has identified a multitude of metabolic, cell stress, death, inflammatory and fibrogenic pathways that underlie these histological manifestations of disease activity and stage^[@CR8]^. These have provided numerous targets for therapeutics which have been translated into over 200 active clinical trials for NASH ([www.clinicaltrials.gov](http://www.clinicaltrials.gov)). There are unfortunately no drugs yet approved for NASH. Several agents have failed altogether and even the drugs that are now in pivotal trials led to resolution of NASH or regression of fibrosis in less than half the individuals who received these agents in phase 2B trials^[@CR9]--[@CR12]^. The reasons for this suboptimal performance are not fully understood. This gap in knowledge is a barrier towards development of more successful therapeutic approaches including the ability to identify which patient may respond best to which therapy, which we begin to address herein. A potential explanation for the limited clinical success of therapeutics is that the biological processes driving the disease phenotype vary with disease severity, even within the relatively limited range of histological severity included in clinical trials. It is also possible that within diseased populations with similar histological patterns and severity of disease, there may be distinct subpopulations with different disease drivers, as seen with several cancers^[@CR13]^. Studies of the human transcriptome in NASH have not investigated these possibilities although specific pathways and genes have been linked to disease severity^[@CR14]--[@CR18]^. To address this gap, we assessed gene expression profiles along the histological spectrum of NAFLD and used them to develop and validate a gene-level score that reflects histological severity. This scoring methodology enabled us to identify patient subpopulations on the basis of their molecular phenotypes. This demonstration is a necessary first step in establishing a foundation for future development of precision medicine approaches for the treatment of NASH. Results {#Sec2} ======= Identification of gene networks that are regulated across disease activity and stage {#Sec3} ------------------------------------------------------------------------------------ We first interrogated the gene expression profile in liver tissue from patients with NAFLD and age- and weight-matched controls. It is important to note that these biopsies were obtained from individuals who were not in a drug treatment trial and were not on any specific NASH drug therapy. The severity of histological features were scored independently by a hepato-pathologist using the NASH CRN scoring system^[@CR3]^. Supplemental Table [1](#MOESM1){ref-type="media"} summarizes histological activity and stage of the samples in this study with their associated clinical profiles and demonstrate that the cohort had the full histological spectrum of the disease. We applied ordinal regression to identify genes whose expression profiles vary with the severity of the NAFLD activity score (NAS) or fibrosis stage. With a false discovery rate (FDR) threshold of 1%, we observed 2970 differentially expressed genes with respect to disease activity and 1656 genes related to fibrosis stage (Supplementary File [1](#MOESM2){ref-type="media"}). The NAS is a composite score that aggregates independent assessments of lobular inflammation, steatosis, and cytological ballooning. Supplementary Fig. [1](#MOESM1){ref-type="media"} shows the distribution of NAS components across the samples. However, very few differentially expressed genes could be associated uniquely with any one of the components, particularly at more stringent FDR thresholds (Supplementary Fig. [2](#MOESM1){ref-type="media"}). We also found no evidence of differential expression with respect to assessments of portal inflammation (all FDR-adjusted p-values \> 0.9). For these reasons, subsequent analyses focus on the full composite NAS score. The gene expression data were integrated with the STRING protein-protein interaction (PPI) network to identify the portion of the network regulated over the spectrum of the disease^[@CR19]^. This procedure generated a PPI network where the edges connecting coordinately expressed genes were preserved. The resulting subnetwork, representing the differentially expressed portion of the transcriptome, was further analyzed to identify "communities" and "hubs". The former are densely connected sets of protein-encoding genes and tend to correspond to biological pathways, while hubs are central to the structure of the network and often represent key regulatory proteins. The resultant networks were differentially regulated with respect to the NAS and fibrosis stage, and are shown in Fig. [1A,B](#Fig1){ref-type="fig"}.Figure 1Integration of differentially expressed genes with a protein-protein interaction network highlights hubs involved in the progression of fatty liver disease. (**A**,**B**) A protein-protein interaction network induced by the differentially expressed genes for both NAS and fibrosis stage, respectively. Each node represents a densely connected community of proteins, whose size represents the number of proteins in the community. The node labels provide a summary of the biological processes enriched in each community, as well as a number which is a community identifier. Edge thickness is proportional to the number of connections between communities. (**C**,**D**) Box plots showing the distribution of eigenvalue centrality in the communities of each network. Communities significantly enriched with hubs (nodes with relatively large centrality) are labeled with their top 5 genes by centrality. Analysis of the NAS PPI network revealed multiple communities, each containing genes that were either up- or down-regulated with respect to increasing NAS (Fig. [1A](#Fig1){ref-type="fig"}). The largest community was related to receptor tyrosine kinase (RTK) activity, Rho GTPase signaling, and immune system activation, followed by communities linked to cell cycle and extracellular matrix (ECM) reorganization (pathway enrichment in each community is provided in Supplementary Files [2](#MOESM3){ref-type="media"} and [3](#MOESM4){ref-type="media"}). Of note, a relatively small community of genes enriched for metabolic functions was linked to severity of the NAS. The increase in expression of genes involved in cell proliferation (community 3) indicates that tissue repair pathways were also progressively activated with increasing disease activity. As a whole, this network captured many processes that are hallmarks of NAFLD. These processes are coordinately regulated over the spectrum of disease activity, which raises the question of what processes, and more specifically, what genes are the key mediators of this coordination--i.e. what are the hubs in this network and what communities are they in? In this study, hubs were defined as nodes in the network with high eigenvector centrality. Communities 1, 3, 8, and 12 were significantly enriched for these hub nodes (Bonferroni adjusted p values: 2e-12, 3e-47, 3e-4, and 3e-6 respectively) (Fig. [1C](#Fig1){ref-type="fig"}), indicating that specific proteins within these communities likely coordinate patterns of regulation across the entire network. For example, community 3 was strongly enriched for hubs related to proliferation (e.g. CDK1), which are regulators of multiple structural proteins involved in regeneration and tissue repair. The epidermal growth factor receptor (EGFR) also formed a hub within this network suggesting a key role for this gene and related downstream signaling in the repair response to increasing disease activity. A detailed list of genes and their centrality values is provided in Supplementary File [4](#MOESM5){ref-type="media"}. The fibrosis stage network, while smaller than the NAS network, had communities enriched for many of the same processes (Fig. [1B](#Fig1){ref-type="fig"}), reflecting the correlation between NAS and fibrosis stage within this predominantly non-cirrhotic population (Supplementary Table [1](#MOESM1){ref-type="media"}). Rho GTPase and cell cycle related signaling were the largest communities that were associated with increasing fibrosis stage. Their linkage with both the NAS and fibrosis stage suggest these to be critical pathways linking disease activity to fibrosis progression. Communities 1--4 and 7 were enriched for hubs (Bonferroni-adjusted p-values: 6e-11, 4e-4, 1e-4, 1e-3, and 2e-12), with the G-protein coupled receptor (GPCR) signaling community (community 7) being the most strongly enriched community. Several chemokine genes, such as CXCR4 and CCR5, are among the most central nodes in this community, reflecting an important role for these genes in fibrosis (Fig. [1D](#Fig1){ref-type="fig"}). However, the corresponding GPCR-associated community in the NAS network (community 11) was not enriched for hubs, suggesting that GPCR pathways are more relevant for fibrosis severity rather than disease activity. Identification of pathways that are differentially expressed with increasing disease activity and fibrosis stage {#Sec4} ---------------------------------------------------------------------------------------------------------------- We next used the Gene Set Variation Analysis (GSVA) which allows the ordinal histological severity score to be regressed against pathway-level abundance values^[@CR20]^. Whereas the PPI network analysis enabled the discovery of communities of genes that are coordinately regulated, the GSVA analysis allowed us to identify specific, established pathways that are differentially regulated with increasing histological severity. Using both approaches allowed us to identify both the biological pathways perturbed across increasing disease severity as well as the relationship between the processes that are perturbed. A total of 586 and 392 Reactome pathways were progressively altered (FDR \< 1%) with increasing severity of NAS and fibrosis stage, respectively. The full set of pathways identified is provided in Supplementary File [5](#MOESM6){ref-type="media"}. The top upregulated pathways, ranked by the strength of their relationship to increasing NAS scores, included mainly those for cell death (intrinsic pathway for apoptosis, programmed cell death), inflammation (Fc epsilon receptor signaling, TNF-receptor 2 noncanonical activation of NFκB, T cell receptor signaling, MHC class II antigen) and cell proliferation (regulation of PTEN, transcriptional regulation of TP53) (Fig. [2A](#Fig2){ref-type="fig"}, Supplementary File [5](#MOESM6){ref-type="media"}). These indicate a role for both innate and adaptive immune systems as drivers of tissue injury while increasing activation of death pathways reflect the primary mechanisms of hepatocyte loss and cell proliferation pathway activation reflects the liver's wound healing response to injury. We next performed a similar analysis to identify pathways progressively repressed with increasing disease activity. Not surprisingly, impaired insulin receptor signaling (IRS) was the top pathway downregulated with increasing NAS. Several neuro-signaling-associated pathways, including acetylcholine nicotinic receptor related pathways, were also amongst the top ten down-regulated pathways. Pathway analysis for fibrosis progression revealed ephrin-signaling to be most tightly related to the severity of fibrosis (Fig. [2B](#Fig2){ref-type="fig"}). Ephrin receptors are the largest subfamily of receptor protein-tyrosine kinases (RTK) and are known to modulate neural migration, angiogenesis, and oncogenesis^[@CR21]^. Ephrins signal via the Ephrin receptors (forward signaling) or by alternate pathways (reverse signaling)^[@CR22]^; specifically, the EphB-mediated forward signaling pathway was activated concordantly with the severity of fibrosis. Rho GTPase signaling pathways, known to modulate oxidative stress, cell migration, phagocytosis, and other cellular processes involving actin reorganization were also closely linked to fibrosis progression^[@CR23]^. Not surprisingly, the expression of cell cycle, extracellular matrix, and inflammatory signaling pathways were also directly related to the severity of fibrosis (Fig. [2B](#Fig2){ref-type="fig"} and Supplementary File [5](#MOESM6){ref-type="media"}). Several inflammation and apoptosis related pathways whose activation level was closely related to the NAFLD activity score, e.g. intrinsic apoptosis pathway and Fc epsilon receptor mediated signaling, were also directly associated with fibrosis stage (Supplementary File [5](#MOESM6){ref-type="media"}). Interestingly, amine-derived hormone pathway expression was progressively and significantly downregulated with increasing fibrosis stage. Further analysis of this pathway indicated suppression of tryptophan hydroxylase-1 and -2 (FDR-adjusted p-value \< 0.01 for both) which are required for serotonin synthesis and dual oxidase-1 (DUOX1) which is a regulator of reactive oxygen species generation^[@CR24]^. Both serotonin and reactive oxygen species can promote fibrosis and the downregulation of their associated pathways likely reflect adaptations to increased fibrogenic drive.Figure 2The top Reactome gene sets that are up- and down-regulated with respect to NAS (**A**) or fibrosis stage (**B**). The y-axes represent the GSVA score, which is a pathway-level quantification of gene abundance, and the x-axes represent the clinical assessment. For disease activity (NAS), pathways related to apoptosis, inflammation (Fc epsilon receptor signaling, TNFR2 signaling, T cell receptor (TCR)), cell proliferation (PTEN, TP53) were top pathways whereas for insulin receptor substrate (IRS) signaling pathway was downregulated. For fibrosis, Ephrin signaling related genes were the top pathway while amine derived hormones and nicotinic acetylcholine receptor pathways were down-regulated. Identifying gene expression changes specific to disease activity or fibrosis {#Sec5} ---------------------------------------------------------------------------- The results from the differential expression analysis indicated that while the expression level of some genes was related to the severity of both activity and fibrosis, others were related to either activity or fibrosis exclusively. To further dissect this, we calculated the posterior probability of each gene being uniquely associated with either the NAS or fibrosis stage **(**Supplementary Fig. [3](#MOESM1){ref-type="media"}). Genes and pathways most uniquely related to NAS were related to metabolism, respiratory chain electron transport, tricarboxylic acid cycle, and lipid metabolism (Supplementary Fig. [3A,C](#MOESM1){ref-type="media"}). This may reflect the upstream metabolic perturbation and fatty acid delivery to mitochondria with increasing disease activity and resultant mitochondrial and electron transport chain activity to generate ATP. On the other hand, it may also reflect the uncoupling of oxidation and phosphorylation and mitochondrial dysfunction that is well known to occur in NASH^[@CR25]^. Genes uniquely related to fibrosis were enriched for protein translation and ribosomal biogenesis (Supplementary Fig. [3B,D](#MOESM1){ref-type="media"}). This likely reflects increased demand for extracellular matrix protein synthesis with increasing fibrosis. Together, these data indicate that while correlated, NAFLD activity and fibrosis stage capture distinct, but overlapping molecular aspects of disease progression. Development of gene-level disease scores {#Sec6} ---------------------------------------- Given that the biological processes that determine disease phenotype and progression are dependent on gene expression, we investigated if individual gene expression levels could predict the histological severity of NAFLD. Specifically, we asked two questions: (1) can the histological severity of the disease be inferred from gene expression, and (2) do individual samples show patterns of pathway regulation that signify distinct regulatory profiles? To address these questions we derived gene-level scores that estimate disease severity as a function of gene expression. The scores correspond to severity with respect to NAS or fibrosis stage, and so we refer to them as gNAS (gene-level NAFLD activity score) and gFib (gene-level fibrosis stage) scores. Ordinal regression models were used to assign a disease progression score for each gene based on its expression for a given patient. The scores were calculated using a 10-fold fitting procedure, where in each fold a set of samples was scored according to models fit to a disjoint set of samples. This procedure simulates a scenario where newly observed samples are scored against a benchmark set of samples. Genes with the highest coefficient of variation of gene-level scores across the dataset convey the greatest information about the relationship between expression and disease severity (see Methods). Thus, we focused on the top 1000 genes based on the coefficient of variation in gNAS and gFib scores. Both gene sets had a 98--99% overlap with differentially expressed genes (FDR 1%). We next ordered the patient samples by mean gNAS and gFib scores from the top 1000 genes and related them to NAS and fibrosis stage respectively (Fig. [3A,C](#Fig3){ref-type="fig"}). These gene-level scores demonstrated a strong correlation with histological grade (Fig. [3B,D](#Fig3){ref-type="fig"}). This implies that given a benchmark transcriptomic dataset (such as the one presented in this study), the histological severity of a newly observed biopsy sample can be approximated from the expression profile of roughly 1000 genes. In this dataset, the discriminating power of this assessment is greatest at the extremes of the disease spectrum.Figure 3Based on the dynamic range of expression and rank order upon ordinal regression of gene expression levels to the NAFLD activity score (NAS) or fibrosis stage, a gene-level score was derived for all genes tested. The distribution of gNAS scores (**A**,**B**) and gFib scores (**C**,**D**). Plots (**A**,**C**) show the distribution of gNAS or gFib scores for the top 1000 genes in each sample. Plots (**B**,**D**) show the relationship between mean gNAS and gFib scores and histological assessments. To determine if severity can be inferred from the expression of a smaller gene set, we used lasso regression to regress mean gNAS or gFib scores against transcript abundance. Lasso regression was chosen for its ability to perform feature selection and to tune the number of features in the model fit. The regularization parameter was tuned such that roughly 20 predictive genes were selected. While cross-validation RMSE values were better with larger gene sets, the differences were modest (Supplementary Fig. [4](#MOESM1){ref-type="media"}). The gNAS and gFib lasso models achieved cross-validated R^[@CR2]^ values of 0.96 and 0.94, respectively (Fig. [4A,B](#Fig4){ref-type="fig"}). These results indicate that the expression levels of a small subset of genes can be used to accurately infer disease severity. The predictor genes span a wide range of biological processes, which includes metabolism, cell-cell interactions, transcription, chromatin dynamics, and transport as well as other processes. The contributions of these specific genes to the model are shown by plotting their variable importance (Fig. [4C,D](#Fig4){ref-type="fig"}) and standardized regression coefficients (Fig. [4E,F](#Fig4){ref-type="fig"}).Figure 4Lasso regression of gene expression values against mean gNAS (**A**,**C**,**E**) or gFib scores (**B**,**D**,**F**). Figures (**A**,**B**) show the results of 5-fold cross-validation for each model, which have 19 and 18 predictors, respectively. The strong performance of the models in cross-validation demonstrates that disease severity can be assessed from the expression levels of a relatively small number of genes. Figures (**C**,**D**) provide the scaled variable importance for model predictors. Figures (**E**,**F**) show the standardized regression coefficients for each model. Identification of distinct gene regulation profiles {#Sec7} --------------------------------------------------- The gene-level resolution and continuous nature of these scores enables many possibilities for making fine distinctions in disease progression and for distinguishing between patients with unique transcriptional profiles. If there are distinct molecular subtypes of NAFLD, driven by distinct biological processes, then this would be reflected as distinct patterns in the distribution gene-level scores within samples. For example, a form of NAFLD driven by lipid metabolism would have relatively high scores for genes associated with lipid metabolism. In such a scenario, lipid metabolism would be referred to as a "leading-edge" disease process. We identified patterns in relative gene-level scores by applying the so-called gene shaving method to the centered and scaled gNAS or gFib scores of the DEGs (1% FDR)^[@CR26]^. This procedure identified distinct clusters of correlated genes with high variance across samples (Fig. [5A,B](#Fig5){ref-type="fig"}). The patterns of gNAS and gFib scores across these genes revealed pronounced sample clusters, which represent groups of patients with distinct, coherent patterns of regulation across the gene clusters (Fig. [5C,D](#Fig5){ref-type="fig"}). Examining the overlap between gNAS- and gFib-based clusters provided further granularity in profile distinction (Fig. [5E](#Fig5){ref-type="fig"}); i.e. patients in any given gNAS cluster were generally distributed over more than one gFib cluster, and vice versa. The overall functional profiles of both the gNAS- and gFib-based gene clusters highlights pathways that are closely linked to the NAFLD disease process, including ECM remodeling, inflammation, metabolism, integrin signaling, compliment, and DNA damage response (Fig. [5F,G](#Fig5){ref-type="fig"}).Figure 5Patterns of gNAS and gFib scores across patient samples reveal distinct molecular profiles. Panels (A,B), respectively, show standardized gNAS and gFib scores across sets of genes that were identified through gene shaving. Sample clusters in these panels show distinct patterns regulation across these genes, and thus represent patients with distinct molecular profiles. Panels (C,D) show the distributions of mean standardized scores for each sample cluster. Within these plots, patterns across gene clusters (x-axis) represent the average molecular profiles of the sample clusters. Panel (E) shows the intersection of the gNAS- and gFib-based sample clusters and provides the number of samples in each cluster pair. Simultaneous consideration of the two partitions provides additional granularity in sample classification. Panels (F,G) show the most strongly represented Reactome pathways in each gene cluster (by Fisher's exact test). The pathways represented are closely linked to NAFLD progression. While the previous analysis provides a rational basis for the classification of patient molecular profiles, it is of limited use in identifying leading-edge processes due to relatively small size of the gene clusters. Furthermore, distinct patterns of regulation across the gene clusters may converge on the same pathways. Therefore, we sought to identify pathway-level summaries of variation in gNAS and gFib scores. For this analysis, we selected the so-called hallmark collection of gene sets from the Molecular Signatures Database (MSigDB), since it concisely summarizes a diverse set of biological processes^[@CR27]^. We computed the mean gNAS and gFib scores of DEGs (1% FDR) in each significantly regulated hallmark gene set (1% FDR). Samples and pathways were then clustered by these values. In the gNAS analysis (Fig. [6A,C](#Fig6){ref-type="fig"}), the result shows at least two distinct sample clusters, and two distinct hallmark clusters. Figure [6C](#Fig6){ref-type="fig"} summarizes the distinct patterns of pathway-level regulation for each gNAS-based sample cluster. Inflammation and apoptosis (hallmark cluster 1) were leading-edge processes for sample cluster 1. This was not the case for sample cluster 2, which instead implicated cell stress, metabolism, and other pathways as leading-edge processes (hallmark cluster 2). Clusters based on gFib scores show similar patterns (Fig. [6B,D](#Fig6){ref-type="fig"}**)**; however, processes associated with morphology and angiogenesis appear as a distinct cluster (hallmark cluster 2).Figure 6Patterns of pathway-level regulation with respect to gNAS and gFib scores. The heatmaps (**A**,**B**) shows the clustering pattern of samples (columns) and MSigDB hallmark pathways (rows) with respect to mean gene-level scores (values represent column-wise Z-scores). Sample clusters show distinct patterns of pathway-level regulation. Panels (C,D) show the mean sample-wise Z-score in each cluster for the gNAS and gFib analyses, respectively. Higher values in both figures are consistent with relatively advanced disease states. Panel E shows the intersection of the gNAS- and gFib-based sample clusters and provides the number of samples in each cluster pair. Discussion {#Sec8} ========== This study provides a snapshot of the pathways that are transcriptionally regulated in NAFLD and the leading-edge pathways associated with increasing disease activity and fibrosis stage. It also provides insights into how these pathways interact and coordinate activation or suppression with increasingly advanced disease. The development of gene-level scores broadly corresponding to histological severity enables inference of disease phenotypes based on transcriptomic profiles and facilitates a procedure for identifying patients with distinct patterns of gene regulation. The methods presented here are general and can be used to distinguish patients based on any high-content molecular profiling technology. Accordingly, these methods have great potential for furthering research into personalized treatment approaches. These data not only provide novel insights in to the specific genes and cellular processes driving the disease phenotype in humans (e.g. Ephrin related signaling), but also enable identification of novel drug targets and hypotheses related to disease drivers. A key finding is that inflammatory pathways, including both the innate and adaptive immune systems, are linked to both histological activity as well as fibrosis; a fact which can be potentially leveraged for therapeutics. If further validated, the methods presented in this study for characterizing molecular heterogeneity may serve as a foundation for precision medicine approaches that identify specific disease drivers in a given patient and therapeutically target the pathways relevant to that individual. The patient clusters identified in this study hint at the presence of distinct molecular subtypes among the patients. This can be inferred from the differences in the leading-edge processes among the patient clusters. A possible biological driver of the clusters is differences in the natural course of the disease. If this is case, the clusters could be interpreted as different patient subtypes with distinct molecular drivers of the disease. A second possibility is that the clusters correspond to snapshots of a dynamic process, in which case the clusters might correspond to various phases of disease progression. Some combination of these two proposals is also possible. However, the lack of association between histological grade and cluster membership suggests that the clusters are driven, at least in part, by something other than disease progression. Future studies will be required to fully elucidate the functional implications of this patient classification strategy. Such studies are important insofar as the establishment of the molecular heterogeneity of NAFLD progression is a priority in the field. Other studies have investigated gene expression changes that accompany disease progression in NAFLD, notably Wruck *et al*. and Moylan *et al*.^[@CR15],[@CR28]^. The former study is a meta-analysis of several transcriptomic datasets from patient liver biopsies that identifies gene expression changes associated with the progression from NAFLD to NASH. The authors identify several functional pathways and gene sets that are significantly regulated over the course of this transition, which are primarily associated with lipid metabolism. The latter study uses a similar approach, comparing mild (fibrosis stage 0--1) to severe (fibrosis stage 3--4) patients. It identifies a somewhat greater diversity of functional process, which includes several core metabolic subsystems as well as proliferation pathways. Our study also identifies several pathways involved broadly in metabolism and proliferation (Fig. [1](#Fig1){ref-type="fig"} and Supplementary Files [2](#MOESM3){ref-type="media"}, [3](#MOESM4){ref-type="media"} and [5](#MOESM6){ref-type="media"}); however, our results also demonstrate widespread regulation of inflammatory processes and the extracellular matrix over the course of the disease. There are several possible reasons for the differences observed across these studies, the most obvious of which include differences in patient cohorts and differences in pathway analysis methods. Perhaps a more subtle difference is the fact that this study identifies disease-associated transcriptional regulation by leveraging all of the information contained in the ordinal histological assessments of the disease, whereas the other studies achieve this by binning patients into "early" and "advanced" disease categories. Thus, our approach is likely more sensitive to gene expression changes that correspond to the phenotypic changes that are summarized by histological scoring. Indeed, Pirhaji *et al*. demonstrated that in the case of Huntington's disease, the use of ordinal regression across a spectrum of disease severity was superior to disease vs. control comparisons in identifying gene expression signatures associated with the disease phenotype^[@CR29]^. In addition to studies that further explore the implications of patient classification on the basis of gene expression, this work invites future longitudinal studies that can be used to validate the progressive changes to gene expression observed in these data. Specifically, tracing changes in gene expression in a set of patients over time would be the best validation of the gene-level scoring system and its ability to infer disease progression. Importantly, the development of gene-level activity and fibrosis scores provides a nuanced and content-rich perspective on disease progression, which enables new ways to evaluate central puzzles in the field, such as the placebo response and spontaneous improvement even in the absence of weight loss. Such investigations are expected to provide insights that can be leveraged to develop novel hypotheses and approaches to reverse the disease process. It is important to note the limitations of this study. We observed relatively few transcriptomic changes uniquely associated with the individual components of the NAS as well as assessments of portal inflammation; however, we observed large effects associated with the composite NAS. There are several potential explanations for this observation. One possibility is that the concomitant progression across all three components of the score may confound analysis of any single component. Additionally, our statistical power for detecting genes associated with the individual NAS components may be limited by the distribution of the scores and/or the sample size. A related limitation of this study is that the patient samples do not uniformly represent the spectrum of the disease. In particular, the majority of the samples represent moderate disease activity (NAS 3--5), with relatively few mild (NAS \< 3) and severe (NAS \> 5) cases. One likely consequence of this sampling bias is a loss of statistical power; i.e., the number of differentially expressed genes we observed is likely an underestimate. We would also expect a moderating effect on the gene-level scores, since model estimates for the extreme ends of the disease spectrum would be associated with relatively high error. Even so, the relationship between expression levels and gene-level scores is monotonic, so the score rankings should be unaffected. In future studies, these issues can likely be addressed through some combination of alternative statistical approaches and a study design structured with the intent of deconvolving the components of the NAS and/or balancing patient samples over the spectrum of the disease. There are also a number of potential confounding factors that were either unavailable for this study, or whose inclusion in this analysis was not possible or straightforward given the study design. For example, some patients were taking concomitant medications which may have altered their gene expression profiles. Also, some known NAFLD-associated genetic variants in genes such as PNPLA3 and TM6SF2 could also influence the patient profiles; however, these data were not available. A broad limitation of this study is that gene expression levels alone may not predict translation into protein or the functionality of the proteins. Ultimately, integrative approaches using larger transcriptomic, proteomic, genomic, and metabolomic data will be needed to build more comprehensive models of disease development and progression. Indeed, some existing studies such as Wruck *et al*. provide high-content data in NAFLD patients that can be used toward this end^[@CR30]^. These limitations notwithstanding, the current study provides a general framework for leveraging the power of high-throughput molecular profiling to develop precise characterizations of NAFLD development and progression. Similar unbiased frameworks will likely serve as a foundation of future precision approaches for the management of NAFLD. Methods {#Sec9} ======= Study population {#Sec10} ---------------- Patients presenting with suspected or known NAFLD who were undergoing a standard of care liver biopsy to diagnose and/or to assess the severity of the disease were enrolled in this study. All subjects were enrolled between 2012 and 2016 at a single tertiary care medical center. The study was approved by the institutional review board of Virginia Commonwealth University, and all subjects provided informed consent. All research was performed in accordance with the guidelines and regulations of the review board and the publisher. The liver biopsy was performed using a percutaneous approach or a transjugular approach in all instances. At the time of the biopsy, 1.5--2 cm core of tissue of 16 gauge diameter was sent for histological assessment and 2--5 mm of tissue was snap-frozen in liquid nitrogen at the bedside within five minutes of obtaining the biopsy. Those with biopsy-proven NAFLD were included for this analysis. Control subjects included those who had normal liver histology and did not have evidence of other common etiologies for liver disease such as hepatitis B and C, hemochromatosis, alcohol-associated liver disease. These subjects were either donors for living donor transplant or had a prior history of ALT fluctuations that was evaluated with a liver biopsy. Assessment of liver histology {#Sec11} ----------------------------- Liver histology was assessed using the NIDDK NASH CRN criteria by two hepato-pathologists^[@CR3]^. NAFLD was diagnosed by the presence of more than five percent steatosis assessed by histological examination. The nonalcoholic nature of the disease was assessed by clinical history and assessment and by exclusion of an alcohol use disorder using the AUDIT questionnaire^[@CR31]^. Steatohepatitis was diagnosed by the presence of steatosis along with hepatocellular ballooning and lobular inflammation with or without fibrosis. Those with borderline or definite steatohepatitis were considered together as steatohepatitis for purposes of this analysis. The severity of individual histological features were scored using NASH CRN criteria and disease activity was determined by computing the NAFLD activity score (NAS) which is a composite of the steatosis, inflammation, and ballooning scores. The fibrosis stage was also scored according to the NIDDK NASH CRN staging system from tissue sections stained with Masson's trichrome stain. Those with stages 1a, 1b, and 1c were considered as stage 1. RNA-seq {#Sec12} ------- RNA was extracted from cells using a Qiagen RNeasy RNA Isolation Kit (Qiagen, Gaithersburg, MD) as per manufacturer's instructions. RNA quantity and quality were assessed using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and Agilent 2100 bioanalyzer (Agilent Technologies; Santa Clara, CA). cDNA libraries were prepared using a TruSeq Stranded mRNA Sample Preparation kit (Illumina, San Diego, CA). RNA-Seq was performed on the Illumina HiSeq2500 next-generation sequencing platform (Illumina, San Diego, CA). Quantification of RNA-seq data {#Sec13} ------------------------------ Transcript expression was quantified using the RNA-seq quasi-mapping tool, Salmon, which was run in GC bias-aware mode^[@CR32]^. Target transcripts were derived from genome assembly GRCh37.75 from Ensembl^[@CR33]^. Transcript-level quantifications from Salmon were transformed into gene-level count estimates using the tximport R package^[@CR34]^ and an Ensembl transcript-to-Entrez gene cross-reference derived from Biomart^[@CR35]^. Genes with low abundance were filtered out of the dataset by applying a minimum expression threshold of greater than 0.5 counts per million (CPM) in at least three samples. Library sizes were adjusted using the TMM normalization method from the edgeR bioconductor package^[@CR36],[@CR37]^. The counts and normalized library sizes were used to transform gene-level counts into log~2~(CPM) values, which were used as the gene-level abundance estimates in subsequent analyses. Differential expression analysis {#Sec14} -------------------------------- Genes that were differentially expressed across the ordinal spectrum of fibrosis stage or NAS were identified using ordinal regression, which is an approach similar to the one used by Pirhaji *et al*. in Huntington's disease^[@CR29]^. Specifically, using the 'ordinal' R package, we fit a cumulative link logit model to each gene j:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathrm{log}\,{\rm{it}}({\rm{P}}(Y\le i|{\hat{x}}_{j}))={\alpha }_{ij}-{\hat{\beta }}_{j}^{T}{\hat{x}}_{j}$$\end{document}$$where i is an ordinal value (i.e. $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$i\in \{0,1,2,3,4,5,6\}$$\end{document}$ for NAS and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$i\in \{0,1,2,3,4\}$$\end{document}$ for fibrosis stage), Y is the sample score (i.e. the clinical call), and x is a vector of predictors, which in this case is gene expression and the sex of the patient. The two-tailed z-test was used to test the null hypothesis that the gene abundance regression coefficient is equal to zero. The resulting p-values were adjusted across all genes using the Benjamini-Hochberg method. For each gene, we calculated the Bayesian posterior probability of the null hypothesis being false using the method described by Allison *et al*.^[@CR38]^. This value can be interpreted as the posterior probability that a gene is differentially expressed. Fold changes reported throughout this report correspond to the difference in the mean log~2~ CPM between the top two and bottom two levels of the ordinal range. Protein-protein interaction network analysis {#Sec15} -------------------------------------------- The differential expression results were integrated with the human STRING v10 protein-protein interaction network^[@CR19]^. The network was obtained using the 'STRINGdb' R package from Bioconductor^[@CR39]^. It was pruned to include only high-confidence interactions--i.e. interactions with combined scores of 700 or greater. For each differential expression analysis, the posterior probabilities of differential expression were assigned to their corresponding nodes. Edge weights were calculated as the product of the posterior probabilities of their incident nodes. Thus, edge weights represent the joint posterior probability of differential expression of the interacting proteins. The networks were further pruned to include only edges with weight 0.98 or greater. The giant component of the resulting network is the differentially regulated portion of the protein-protein interaction network. Communities in these networks were identified using the Louvain algorithm implemented in the 'igraph' R package^[@CR40]^. Gene set enrichment of the network communities was calculated using Fisher's exact test and human Reactome gene sets^[@CR41]^. Analysis of regulation with respect to NAS vs fibrosis {#Sec16} ------------------------------------------------------ Identification of differential expression that is exclusive to NAS or fibrosis stage was based on the posterior probability of differential expression with respect to each measure. We calculated the posterior probability that a gene i is exclusively regulated with respect to NAS as$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${P}_{i}^{exNAS}={P}_{i}^{NAS}\times (1-{P}_{i}^{Fib})$$\end{document}$$and exclusively regulated with respect to fibrosis stage as$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${P}_{i}^{exFib}={P}_{i}^{Fib}\times (1-{P}_{i}^{NAS})$$\end{document}$$where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${P}_{i}^{NAS}$$\end{document}$ is the posterior probability of differential expression with respect to NAS and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${P}_{i}^{Fib}$$\end{document}$ the posterior probability of differential expression with respect to fibrosis stage. Gene sets enriched for exclusively regulated genes were identified using the 'geneSetTest' function from the 'limma' Bioconductor package, which performs a rank-based competitive test^[@CR42]^. Pathway analysis {#Sec17} ---------------- Additional pathway analyses were performed using the gene set variation method (GSVA) followed by ordinal regression^[@CR20]^. GSVA generates pathway-level quantifications from gene-level quantifications. To identify differentially expressed pathways, we used the pathway-level quantifications from GSVA in conjunction with the same ordinal regression strategy that was used for the gene-level analysis. Calculation of gene-level NAS and fibrosis scores {#Sec18} ------------------------------------------------- The ordinal regression models, once fit, can be used to predict the probability of assignment to an ordinal level given the expression value of a gene. We used this feature of the model to assign a score for each gene in every sample--i.e. the gNAS and gFib scores. Specifically, the gene-level scores are the weighted mean of the possible ordinal scores, where the predicted probabilities serve as weights:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${v}_{ij}({x}_{ij},{m}_{i})=\sum _{s\in \sigma }(s\times {\rm{P}}({S}_{j}=s|{x}_{ij},{m}_{i}))$$\end{document}$$ Here, v~ij~ is the gene-level score for a gene (i) in a sample (j); *σ* is the set of all possible ordinal scores; x is the gene expression value; and m is a model fit. To reduce bias in the scores we used a 10-fold fitting procedure, where samples in each holdout set were scored using models fit to the samples in the complementary set. Sample-level scores were derived from gene-level scores by computing the mean of the gene-level scores of the 1000 genes with the greatest coefficient of variation (CV). Genes with the highest CV have the greatest information content since $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\rm{P}}(S=s|{x}_{ij})={\rm{P}}(S=s)$$\end{document}$ when gene expression and sample score are independent (i.e. when expression is uninformative), in which case $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathrm{var}({\hat{v}}_{i})$$\end{document}$ is minimized. Prediction of sample-level scores from clinical measurements and gene expression {#Sec19} -------------------------------------------------------------------------------- Regularized regression models were fit using the 'glmnet' and 'caret' packages in R^[@CR43],[@CR44]^. Regularization and, when applicable, mixing parameters were selected using a 10-fold 50-repeat cross-validation on a parameter tuning grid. In the case of lasso regression of sample-level scores against gene abundance, we selected somewhat suboptimal (in terms of resampling statistics) regularization parameters in order to achieve a small predictor set of approximately 20 genes. To assess model performance and generalizability, we performed a 5-fold cross-validation using the regularization parameters found in the previous step. Final estimates of coefficients (and variable importance) were derived from models that were fit to the entire dataset. Identification of gene and pathway clusters based on gNAS and gFib scores {#Sec20} ------------------------------------------------------------------------- To identify gene clusters, the gNAS and gFib scores were standardized for each sample across DEGs (1% FDR). The resulting Z-scores were used as input to the gene shaving algorithm using a 10% shaving rate and an *a priori* selection of four clusters^[@CR26]^. This yielded two sets of gene clusters: one based on gNAS scores, and the other based on gFib scores. Sample clusters were identified by performing hierarchical clustering on the samples using Euclidean distance and Ward's linkage method. The resulting dendrogram was cut such that the resulting partition maximized the median silhouette width, and the number of clusters that was produced was greater than 2, but less than 20. Pathway clusters were identified by first determining which hallmark gene sets^[@CR27]^ were differentially regulated at a 1% FDR. This was achieved using the previously described GSVA method. Each differentially regulated pathway received a score for each sample that was equal to the mean gNAS or gFib score of the corresponding DEGs (1% FDR). These values were standardized for each sample, and the resulting values were clustered using the same hierarchical clustering method described above. Supplementary information ========================= {#Sec21} Supplementary figures and tables Supplementary file 1 Supplementary file 2 Supplementary file 3 Supplementary file 4 Supplementary file 5 **Publisher's note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary information ========================= **Supplementary information** accompanies this paper at 10.1038/s41598-019-48746-5. The work was supported by the following grants: NIDDK RO1 DK105961 (Principal Investigator: Arun Sanyal). NIDDK T32 DK 07150 (Principal Investigator: Arun Sanyal). NCATS CTSA. RR REDCAP. A.O., R.V., A.A., M.S., F.M., M.C., M.I. and A.S. designed, facilitated, and executed sample collection strategy. S.H., R.F., R.V., A.A., M.S., F.M. and A.S. developed the concept for the study. S.H., M.L., N.D. and J.T. performed data analysis, prepared the figures, and developed the computational approach. S.H., R.F., B.K.C., M.L., N.D., J.T, B.W., F.M., M.C., M.I. and A.S. provided interpretation of the analyzed data. The manuscript was primarily written by S.H. and A.S. with secondary contributions, input, and feedback from all other authors. All raw data and relevant metadata are available through the Gene Expression Omnibus, Accession Number GSE130970. Arun Sanyal: Dr. Sanyal is President of Sanyal Biotechnology and has stock options in Genfit, Akarna, Tiziana, Indalo, Durect. He has served as a consultant to Hemoshear, Echosens, AbbVie, Astra Zeneca, Nitto Denko, Ardelyx, Conatus, Nimbus, Amarin, Salix, Tobira, Takeda, Novo Nordisk, Fibrogen, Jannsen, Gilead, Boehringer, Lilly, Zafgen, Novartis, Pfizer, Immuron, Exhalenz and Genfit. He has been an unpaid consultant to Intercept, Immuron, Galectin, Fractyl, Syntlogic, Affimune, Chemomab, Nordic Bioscience and Bristol Myers Squibb. His institution has received grant support from Gilead, Salix, Tobira, Bristol Myers, Shire, Echosens, Intercept, Merck, Astra Zeneca, Malinckrodt, Cumberland and Novartis. He receives royalties from Elsevier and UptoDate. Stephen Hoang, Ryan Feaver, Banumathi Cole, Mark Lawson, Nathan Day, Justin Taylor, and Brian Wamhoff are Employees of HemoShear Therapeutics.

1. Introduction {#sec1} =============== Providing appropriate treatment to all stroke patients remains a genuine challenge. Sixteen million strokes occur every year \[[@B1]\], one every four minutes. Of these, 25% of patients die, 40% remain disabled, and 25% develop dementia. Stroke represents a considerable economic burden, with costs reaching 8.3 billion euros per year in France (Regional Health Agency report, 2013). Diabetes is a known independent risk factor for stroke, increasing its incidence threefold and worsening both the severity of the event and stroke-related mortality \[[@B2]\]. These observations are all the more alarming considering the anticipated doubling of the prevalence of diabetes by 2030 (World Health Organization, 2012). Early recanalization following proximal cerebral artery occlusion is critical; stent retriever thrombectomy combined with recombinant tissue plasminogen activator (rtPA \[Alteplase\]) reduces disability at 90 days and improves functional independence when performed within 8 hours poststroke \[[@B3]\]. Nevertheless, the limited therapeutic time window and the scarcity of interventional neuroradiology centers mean that very few patients actually receive effective treatment. The search for strategies to extend the limited therapeutic window is challenging, particularly those focusing on restoring neurological damage. Cell therapy-based approaches hold considerable promise. Translational research favors transplantation of autologous cells from various tissues and organs, in a more or less differentiated state. Expansion of mesenchymal stem cells requires several weeks of culture processing. In contrast, bone marrow-derived mononuclear cells (BM-MNC) can be obtained simply by density gradient centrifugation rendering them immediately ready for use. BM-MNC transplantation improves poststroke neurological deficit in rodents \[[@B4]\], and clinical trials have shown the feasibility of local \[[@B5]\], intra-arterial \[[@B6], [@B7]\], or intravenous \[[@B8], [@B9]\] administration. The underlying mechanisms are not fully understood: the transplanted cells show rare differentiation into endothelial or neural cells \[[@B10]\], but they do contribute to brain repair processes by stimulating endogenous angiogenesis \[[@B11]\], which in turn induces neurogenesis \[[@B12]\] and modulates the inflammatory response \[[@B13]\], thus counteracting the inflammatory cascade that creates a hostile environment compromising the survival of new cells. Peripheral blood-derived MNC (PB-MNC) transplantation may be an interesting alternative because unlike BM-MNC, PB-MNC can be easily collected at the bedside. Furthermore, in a mouse model of hind limb ischemia, we previously showed that pretreating PB-MNC with ephrin-B2 considerably improves their therapeutic potential, especially when isolated from diabetic patients \[[@B14]\]. Ephrin-B2 belongs to the Eph/ephrin family of membrane-bound cell signaling molecules that are essential for embryonic development of the nervous system and vasculature and for neurogenesis and angiogenesis in adults \[[@B15], [@B16]\]. In addition, both ephrin-B2 and its receptor EphB4 are expressed in mature monocytes and lymphocytes and regulate their motility, adhesion, transmigration, and the secretion of inflammatory mediators \[[@B17]\]. These data suggest that activating the ephrin-B2/EphB4 pathway of mononuclear cells may provide notable therapeutic benefits in stroke. We therefore set out to evaluate the therapeutic potential of PB-MNC isolated from diabetic patients (a population at risk for stroke) and stimulated (PB-MNC+) or not by ephrin-B2, administered intravenously into healthy, nondiabetic, adult male C57Bl6J mice subjected to focal cerebral ischemia. Our primary objective was to assess infarct volume and neurological deficits in mice poststroke; the secondary objective was to assess whether peripheral blood transplanted cells (PB-MNC or PB-MNC+) also affected poststroke angiogenesis, inflammatory response, and tissue repair, as described for BM-MNC. Lastly, we investigated the in vitro properties of PB-MNC+. 2. Materials and Methods {#sec2} ======================== 2.1. PB-MNC Cells and Ephrin-B2/Fc Binding {#sec2.1} ------------------------------------------ Blood samples from type-2 diabetic patients from the CUDC (Centre Universitaire du Diabète et de ses Complications) Department of the Lariboisière Hospital (Paris) were collected in heparinized tubes, during the patients\' routine annual check-up. All patients gave written informed consent. Mean age of the cell donors was 62 ± 8 years old and 61% were males with known diabetes for a duration of 14 ± 8 years; 73% had hypertension, 51% had dyslipidemia, 24% were current smokers, and 21% were former smokers. In addition to oral antidiabetic drugs (gliclazide, glibenclamide, metformin, glimepiride, exenatide), donors were on hypocholesterolemic drugs (rosuvastatin, atorvastatin, ezetimibe), antiplatelet therapy, and antihypertensive drugs (calcium channel blockers, angiotensin II receptor blockers, beta-blockers, angiotensin-converting enzyme inhibitors, and thiazide diuretics). PB-MNC were isolated by density gradient centrifugation using Pancoll (PAN Biotech, Aidenbach, Germany). Cells were washed with PBS and incubated in M199 medium with or without 15 *μ*g/mL of recombinant ephrin-B2/Fc (R&D systems, Minneapolis, MN) at 37°C for 30 minutes. Cell viability was assessed using the Muse Count & Viability Kit and a Muse Cell Analyzer (Millipore Corporation, Hayward, CA) and was determined as \>96%. Optimal stimulation conditions have been described previously \[[@B14]\]. The subcellular composition of PB-MNC transplants and the PB-MNC cellular ephrin-B2/Fc-binding subsets were determined using flow cytometry analysis as previously described \[[@B14]\]. Briefly, to identify cellular subpopulations, we incubated the cells (30 min at 4°C) with a mixture of directly conjugated antibodies specific for the following surface antigens: V450-antihuman CD3, PerCP-Cy5.5-antihuman CD4, FITC-antihuman CD8, APC-antihuman CD19, APC-H7-antihuman CD14, V500-antihuman-CD45, and a PE-Cy7-antihuman CD34 (all from BD Biosciences). The cells were then washed twice with PBS and analyzed using a LSRII flow cytometer (BD Biosciences). The percentage of positive cells was determined using the FlowJo software (Tree Star, Ashland, OR). In a series of similar experiments, PB-MNC preincubated with biotinylated ephrin-B2/Fc were incubated with the above mixture of surface antigen-specific antibodies supplemented with PE-streptavidin (BD Biosciences) to determine the percentage of ephrin-B2-binding cells within each PB-MNC cellular subset. 2.2. Animal and Group Distribution {#sec2.2} ---------------------------------- All animal experiments and surgical procedures were performed in accordance with the European Community Directive (2010/63/EU), the ARRIVE (Animal Research Reporting In Vivo Experiments) guidelines, and the French national guidelines for the care and use of laboratory animals. The study was approved by the Local Ethics Committee in Animal Experimentation (protocol number CEEALV/2012-11-02). Experiments were performed on male adult C57/BL6 mice (age, 10--12 weeks; 20--25 g) (Janvier Labs, Le Genest-Saint-Isle, France). Focal cerebral ischemia was induced at D0 and intravenous injection of PBS, PB-MNC, or PB-MNC+ performed on D1. Poststroke neurological deficit was first assessed on D1 before randomization to cell transplantation and then at D3 and D14 to assess differential outcome between groups. Infarct volume and blood-brain barrier (BBB) permeability were evaluated at D3, and angiogenesis, neurogenesis, and inflammation were evaluated at D3 and D14 ([Figure 1](#fig1){ref-type="fig"}). Microarray analyses were performed on PB-MNC and PB-MNC+, and in vitro properties of these cells were assessed on adhesion assays and capillary-like tube formation assays on Matrigel. The examiner was blinded to all scoring and data analyses to prevent sample identification (phosphate-buffered saline- (PBS-) treated control mice, PB-MNC, or PB-MNC+ transplantation). Codes were attributed to each mouse by an independent team member. The sample size calculation was not possible a priori (new feasibility and mechanistic study), but based upon the experience of the team, given the surgery procedure and the outcome assessments. 2.3. Focal Cerebral Ischemia and Intravenous PB-MNC Administration {#sec2.3} ------------------------------------------------------------------ Focal cerebral ischemia was induced by electrocoagulation of the left middle cerebral artery leading to permanent occlusion (pMCAo), as previously described \[[@B18]\]. Between 18 and 24 hours after pMCAo, animals were randomized to intravenous PBS, PB-MNC, or PB-MNC+. Stratified randomization was achieved by assigning each mouse to a group according to the degree of neurological impairment. Cells were then injected via the retro orbital vein (5 × 10^5^ cells suspended in 200 *μ*L of PBS per mouse, equivalent to 25 million cells/kg). The dose of cells injected was determined on the basis of the dose-effect relationships established previously in a mouse model of hind limb ischemia \[[@B14]\]. A mix of different patients\' blood samples was administered to the mice. For the surgical procedure, mice were anesthetized using isoflurane (initially 2%, followed by 1.5 to 1.8% in O~2~). Intraperitoneal buprenorphine (0.1 mg/kg) was administered 30 minutes before starting anesthesia and repeated every 12 hours for 48 hours. No immunosuppressants were used. 2.4. Neurological Deficit Assessment {#sec2.4} ------------------------------------ Neurological deficit was assessed at D1 and pMCAo mice were compared to nonoperated mice, before randomization into the three groups. Following randomization to PBS, PB-MNC, or PB-MNC+, neurological deficit was assessed at D3 and D14 by calculating a global neurological score (/38): the lower the score, the more severe the deficit, as based on six neurological tests (neurological score, circle test, grip and string tests, beam walking, and pole test) \[[@B18]\]. In addition, at the same time points, the two hind paws were immersed in a water-soluble, nontoxic liquid ink. Mice were allowed to walk freely toward a bright box, along a dark 8/3/100 cm corridor lined with paper. Each mouse performed the test three times, and the best track was recorded. Results are expressed as the toe spread, determined by calculating the distance between the first and fifth toes, the smallest value indicating the greatest deficit \[[@B19]\]. 2.5. Infarct Volume and Brain Atrophy Assessment {#sec2.5} ------------------------------------------------ Infarct volume was assessed three days after pMCAo, on coronal 30 *μ*m sections stained with cresyl violet. The cortical infarct area was measured on every eighth section (30 *μ*m thick) and volume (mm^3^) calculated using ImageJ software (National Institutes of Health, Bethesda, MD) \[[@B18]\]. Brain atrophy was assessed at day 14, as follows: the total brain volume (left hemisphere + right hemisphere, excluding bulbus olfactorius, cerebellum, and brainstem) and the size of the lateral ventricles (left ventricle + right ventricle) were measured by manually drawing respective regions of interest on every 8th coronal section integrating the distance between sections and the thickness of each section using NIH ImageJ (v 1.33, National Institute of Health, USA) analysis software. Brain matter was then calculated by subtraction of ventricle volume from total brain volume. Data are expressed as arbitrary units (a.u.). 2.6. Immunohistochemistry and Morphological Analysis {#sec2.6} ---------------------------------------------------- Coronal 30 *μ*m free-floating sections were incubated with primary antibody overnight at 4°C. Anti-Ki67 (1 : 200, Abcam, UK), antidoublecortin (DCX) (1 : 100, Santa Cruz, USA), anticollagen IV (1 : 200, Abcam, UK), anti-CD31 (1 : 200, BD Biosciences, USA), anti-Iba1 (1 : 200, Wako, Japan), and anti-GFAP (1 : 400, Millipore, Burlington, USA) antibodies were used to detect proliferative cells, neuroblasts, basal lamina of endothelial cells, endothelial cells, microglia/macrophage cells, and astrocytes, respectively. Appropriate Alexa Fluor 594- or 488-labeled secondary antibodies (1 : 400, Molecular Probes, Eugene, OR) were applied for one hour at room temperature. Specificity was checked by omitting the primary antibody. Cell counts and staining density measurements were performed at three coronal brain levels (+0.80, −0.80, and −1.20 mm relative to bregma), all consistent within the infarct area. Cell proliferation was evaluated in the peri-infarct area and in the subventricular zone (SVZ) and expressed as the mean number of Ki67+ cells in each area. Angiogenesis was assessed in three regions of interest (0.06 mm^2^ each) located in the peri-infarct area and expressed as the average number of Ki67+/CD31+ cells per region of interest. Microvessel density was evaluated after collagen IV immunolabeling by calculating the integrated pixel density of collagen IV immunolabeling per total microscopic field (20x), using NIH ImageJ software. Microglia/macrophage density was evaluated in the peri-infarct area as described above after Iba-1 immunolabeling. Glial scar was evaluated in the peri-infarct area after astrocyte immunolabeling and quantification performed as described above for angiogenesis. Neurogenesis was evaluated in the whole section by assessing neuroblast density after DCX immunolabeling. Neuroblast (DCX+) migration was calculated by measuring the distance from the SVZ to the cell cluster nearest to the ischemic area. The maximal distance that SVZ cells covered (*μ*m) was measured and the mean value calculated between the three sections. 2.7. Evaluation of Blood-Brain Barrier Permeability {#sec2.7} --------------------------------------------------- BBB permeability was assessed three days poststroke, as previously described \[[@B12]\]. 2.8. Quantitative Real-Time Analysis of mRNAs (qRT-PCR) for BBB Components, Angiogenesis, and Inflammatory Cytokines {#sec2.8} -------------------------------------------------------------------------------------------------------------------- Total RNA was isolated from ipsilateral hemispheres of ischemic brains using the RNeasy Lipid Mini Kit (Qiagen, Hilden, Germany) according to standard procedures. Reverse transcription was performed with Ready-to-Go RT-PCR Beads (GE Healthcare, Uppsala, Sweden) and real-time PCR was performed using a Mesa Green qPCR kit (Eurogentec, Angers, France) on a Mastercycler Realplex \[[@B18]\] (Eppendorf AG, Hamburg, Germany). We used custom-designed primers for all genes of interest ([Table 1](#tab1){ref-type="table"}) and a ready-to-use primer for the housekeeping peptidylpropyl isomerase A (cyclophilin A) (Qiagen, Courtaboeuf, France). We ran all assays in triplicate. We normalized the results for each individual gene to the level of the housekeeping gene encoding cyclophilin A. Results are expressed as arbitrary units (a.u.). 2.9. Plasma Cytokine Measurements {#sec2.9} --------------------------------- Fourteen days of poststroke, blood samples were collected in heparinized microtubes, centrifuged at 4°C, and plasma samples were collected. Vascular endothelial growth factor-A (VEGF-A) and brain-derived neurotrophic factor (BDNF) (R&D systems, France) levels were detected by ELISA according to manufacturer\'s protocols. 2.10. Microarray {#sec2.10} ---------------- PB-MNC isolated from seven type-2 diabetic patients and stimulated or not with ephrin-B2 were incubated in M199 medium for one hour or four hours and then processed to determine differential gene expression. At the end of incubation, cells were collected and total RNA was isolated using NucleoSpin® RNA (Macherey-Nagel GmbH & Co. KG, Düren, Germany). Gene expression analysis of PB-MNC was performed by the Genomics Services Unit of MACSmolecular (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) using the Agilent Whole Human Genome Oligo Microarrays, dual color (Agilent Technologies, Santa Clara, CA, USA). RNA from unstimulated and stimulated cells from the same patient was, respectively, Cy3- and Cy5-labeled using the Agilent Low Input Quick Amp Labeling Kit (Agilent Technologies). The corresponding cRNA were combined and hybridized overnight to Agilent Whole Human Genome Oligo Microarrays 4×44K using the Agilent Gene Expression Hybridization Kit, according to the Agilent 60-mer oligo microarray processing protocol (Agilent Technologies). Fluorescence signals of the hybridized Agilent Oligo Microarrays were detected using the Agilent DNA microarray scanner. The Agilent Feature Extraction Software (FES) was used to read and process the microarray image files, including determination of feature intensities and ratios with background subtraction and normalization, rejection of outliers, and calculation of statistical confidence intervals (*P* values). The FES-derived output data files were further analyzed using the Rosetta Resolver gene expression data analysis system (Rosetta Biosoftware, Seattle, WA, USA) to determine the differential gene expression. 2.11. In Vitro Adhesion Assay {#sec2.11} ----------------------------- Human umbilical vein endothelial cells (HUVEC) were isolated and cultured as previously described \[[@B20]\]. Two days before isolating PB-MNC, HUVEC were seeded in a 0.2% gelatin-coated 96-well plate at 10^4^ cells/well. PB-MNC were obtained from ten patients and labeled with a Vybrant CFDA-SE Cell Tracer (Molecular Probes, Eugene, OR, USA) according to manufacturer\'s recommendations; PB-MNC were stimulated or not with ephrin-B2/Fc and added to a HUVEC monolayer at 7.5 × 10^4^ cells/well. After incubation for one hour at 37°C, the percentage of adherent PB-MNC was determined using a Victor3 spectrofluorimeter (PerkinElmer, Turku, Finland). 2.12. Capillary-Like Tube Formation Assay on Matrigel {#sec2.12} ----------------------------------------------------- Cells were obtained from seven patients and cocultured with HUVEC on Matrigel to allow the formation of a capillary-like network. HUVEC and PB-MNC were seeded on the preformed Matrigel gel in a *μ*-Slide Angiogenesis ibiTreat Microscopy Chamber (Ibidi, Martinsried, Germany) at 7 × 10^3^ cells/well. After culture at 37°C for 24 hours in a humidified atmosphere containing 5% CO~2~, capillary-like structures were observed with an Axio Observer.Z1 fluorescence microscope (Carl Zeiss MicroImaging GmbH, Göttingen, Germany). Images were taken using a Baumer TXD14 digital monochrome progressive scan camera (Baumer Optronic GmbH, Radeberg, Germany) and Archimed 4.7.0 software (Microvision Instruments, France). The number of branch points of the capillary tube formation per microscopic field was quantified using HistoLab software (Microvision Instruments). 2.13. Cell Tracking {#sec2.13} ------------------- At 1 hour, 1 day, 3 days, and 14 days after cell transplantation, ipsilateral and contralateral hemispheres, lungs, spleen, liver, and kidneys were removed and snap frozen in liquid nitrogen, after flushing the brain vasculature. The presence of human cells was detected using human genomic DNA polymerase chain reaction (PCR). DNA samples (100 ng per reaction) were subjected to PCR to detect a 1171 bp fragment of the human chromosome 17-specific satellite region \[[@B12]\]. P17H8 sequence was amplified using the Light Cycler 1.5 Instrument (Roche Diagnostics), the Light Cycler FastStart DNA Master SYBR Green I, and the following primers: forward, 5′-ACACTCTTTTTGCAGGATCTA-3′ and reverse, 5′-AGCAATGTGAAACTCTGGGA-3′. As a positive control, 0.5 pg of human genomic DNA isolated from PB-MNC from the patients (0.0005% of mouse DNA, low detection limit) was added to the fourth replicate of each sample. As a negative control, the template was replaced by PCR-grade water. For quantification, standard curves were generated by serial dilution of a human genomic DNA in the presence of 100 ng of mouse genomic DNA isolated from the liver or from the tibialis anterior muscle. Specificity of the amplified PCR product was assessed by performing a melting curve analysis (the specific p17H8 product melts at 84°C). PCR products were also analyzed by 1.5% agarose gel electrophoresis to determine the presence of P17H8-specific band of 1171 bp. 2.14. Statistical Analysis {#sec2.14} -------------------------- Statistical analyses were performed with Prism 5 Software (Prism v5.03; GraphPad, San Diego, CA). Data are expressed as mean ± SD. A paired *t*-test was used to compare HUVEC properties when PB-MNC or PB-MNC+ were added to the HUVEC monolayer. Data comparisons between the three groups were performed using the Kruskal-Wallis test with a post hoc Dunn\'s test. Comparisons of neurogenesis, angiogenesis, and inflammation between D3 and D14 in each group were made with an unpaired *t*-test. The sample size was based on the low operative mortality; if secondary hemorrhage occurred during procedure, mice were excluded from the analysis. Statistical significance was set at *P* \< 0.05. 3. Results {#sec3} ========== 3.1. Survival and Neurological Deficit {#sec3.1} -------------------------------------- No mice died during the two-week follow-up, regardless of the transplant group. At D1, we observed a significant reduction in the neurological score in operated (pMCAo) compared to nonoperated mice (33.1 ± 3.0 versus 36 ± 1.0, resp., *P* \< 0.001). Operated mice were then randomized to PB-MNC+, PB-MNC, or PBS. At D3, neurological scores increased in all three groups compared to D1, though the differences between PB-MNC+, PB-MNC, and PBS were not significant (35.86 ± 0.26, 36.29 ± 0.36, and 35.00 ± 0.77, resp.). At D14, the neurological score of the PB-MNC+ group was significantly higher than that of the PBS group (35.8 ± 1.8 versus 35.1 ± 1.4; *P* \< 0.05); the difference between the PB-MNC+ group and the PB-MNC group was however not significant (35.1 ± 1.4) (*N* = 12--13) ([Figure 2(a)](#fig2){ref-type="fig"}). No significant intergroup differences were observed on the ink test assessment, regardless of the time point (data not shown). 3.2. Infarct Volume, Brain Atrophy, and Glial Scar {#sec3.2} -------------------------------------------------- At D3, we observed a significant reduction in infarct volume in mice treated with PB-MNC+ (4.3 ± 1.5 mm^3^) compared to those receiving PB-MNC (8.4 ± 4.4 mm^3^, *P* \< 0.05) or PBS (11.6 ± 4.2 mm^3^, *P* \< 0.01) (*P* = 0.0017) (*N* = 9--11) ([Figure 2(b)](#fig2){ref-type="fig"}). There was no hemorrhagic transformation. At D14, infarct volume in all groups was barely detectable and was therefore not measured. Brain atrophy calculation was not statistically significantly different between PB-MNC+ (124.7 ± 2.9 a.u.), PB-MNC (135.4 ± 8.8 a.u.), and PBS groups (128.6 ± 9.4 a.u.) (*N* = 4). Glial cell density was not different between PB-MNC+ (39.1 ± 5.8 cells), PB-MNC (40.7 ± 4.7 cells), or PBS (43.6 ± 6.8 cells) (*N* = 4--7). 3.3. Cell Proliferation {#sec3.3} ----------------------- At D3, Ki67+ cell levels in the peri-infarct area were significantly increased in the PB-MNC+ group compared to the PBS group (132.9 ± 41.3 cells versus 66.6 ± 24.5 cells, resp.; *P* \< 0.05), but there were no significant differences between the number of Ki67+ cells in the PB-MNC group (98.8 ± 7.0 cells) and the two other groups (*N* = 4--6) ([Figure 3(a)](#fig3){ref-type="fig"}). In the subventricular zone (SVZ), there was also a significant increase in Ki67+ cells in mice treated by PB-MNC+ compared to PBS-treated mice (187.6 ± 20.2 cells versus 110.4 ± 32.6 cells; *P* \< 0.01), but no significant difference between Ki67+ cells in PB-MNC-treated mice (153.0 ± 36.2) and PBS-treated mice (*N* = 5--6) ([Figure 3(b)](#fig3){ref-type="fig"}). At D14, very few Ki67+ cells were present in the peri-infarct area of mice in all three groups. In the SVZ, although Ki67+ cells continued to proliferate, differences between the PB-MNC+, PB-MNC, and PBS groups were no longer significant (125.7 ± 42.0, 153.8 ± 61.7, and 162.3 ± 70.5 cells, resp.) (*N* = 4--6) ([Figure 3(b)](#fig3){ref-type="fig"}). By D14, there were no significant differences in Ki67+ cell numbers in the SVZ compared to D3 in the PBS and PB-MNC groups, whereas this parameter was significantly decreased in the PB-MNC+ group (*P* = 0.017) ([Figure 3(b)](#fig3){ref-type="fig"}). 3.4. Angiogenesis {#sec3.4} ----------------- Angiogenesis was assessed by double immunolabeled Ki67+/CD31+ cell counts, microvessel density after collagen IV immunostaining, and levels of mRNA encoding proangiogenic proteins. At D3, the number of Ki67+/CD31+ cells in the peri-infarct area was not significantly different between PB-MNC+, PB-MNC, or PBS-treated mice (76.4 ± 15.3, 55.3 ± 22.3 and 56.9 ± 15.9 cells, resp.) (*N* = 4) nor was microvessel density (14.4 ± 1.5 a.u., 15.0 ± 0.8 a.u., and 13.9 ± 0.4 a.u., resp.) (*N* = 4--5) ([Figure 4](#fig4){ref-type="fig"}). The levels of mRNA encoding for main proangiogenic factors were not significantly different between the PB-MNC+, PB-MNC, and PBS groups. Interestingly, we observed a significant increase in transforming growth factor (TGF)-*β* mRNA expression in PB-MNC+ compared to PBS-treated mice (*P* \< 0.05) but no difference between PB-MNC, PBS, or PB-MNC+ groups. TGF-*β* receptor type-2 (TGFR-2) mRNA expression was not significantly different between the three groups (*N* = 5--8) ([Table 2](#tab2){ref-type="table"}). Ki67+/CD31+ cell quantification in the peri-infarct area was not performed on D14 because of the scarcity of Ki67+ cells in all three groups. We did however calculate microvessel density, which was significantly increased in the PB-MNC+ group compared to the PBS group (17.7 ± 2.0 a.u. versus 14.0 ± 0.9 a.u., *P* \< 0.05) (*N* = 5--7); microvessel density in the PB-MNC group (15.8 ± 2.6 a.u.) was not significantly different to that of the other two groups ([Figure 4](#fig4){ref-type="fig"}). Levels of mRNA encoding Ang-1, Ang-2, Tie-2, eNOS, VEGF-A, VEGFR-2, PDGF-B, PDGFR-1, TGF-*β*, and TGFR-2 were not significantly different between the PB-MNC+, PB-MNC, or PBS groups (*N* = 5--7) (data not shown). Between D3 and D14, microvessel density increased significantly in the PB-MNC+ group (*P* = 0.032) but was not significantly modified in the PB-MNC or PBS groups. Plasma VEGF levels measured at D14 were significantly greater in mice treated with PB-MNC+ compared to the PBS group (73.5 ± 6.7 versus 41.6 ± 5.9 ng/L) (*P* \< 0.001). There were however no significant differences between plasma VEGF levels in the PB-MNC group (55.7 ± 10.3 ng/L) and the other two groups (*N* = 6--7). 3.5. BBB Permeability {#sec3.5} --------------------- BBB permeability was assessed at D3 using Evans blue extravasation and by determining the expression levels of mRNA encoding BBB proteins. PB-MNC, stimulated or not with ephrin-B2, did not modify Evans blue extravasation (*N* = 6--7) or occludin, claudin-5, ZO-1, or VE-cadherin mRNA expression compared to PBS (*N* = 5--7) ([Figure 5](#fig5){ref-type="fig"}). 3.6. Inflammation {#sec3.6} ----------------- Inflammatory response was assessed at D3 and D14 by measuring microglia/macrophage cell density using Iba-1 immunostaining and by quantification of mRNA encoding proinflammatory cytokines interleukin-1*β* (IL-1*β*), IL-6, interferon-*γ* (IFN-*γ*), tumor necrosis factor-*α* (TNF-*α*), macrophage chemoattractant protein-1 (MCP-1), and the anti-inflammatory cytokine TGF-*β* and its receptor, TGFR-1. At D3, there was an overall significant difference in microglia/macrophage density between the three groups (*P* = 0.043; [Figure 6](#fig6){ref-type="fig"}). In mice treated with PB-MNC+, Iba1+ cell density was significantly lower than in PBS-treated mice (27.4 ± 7.3 versus 47.4 ± 22.3) (*P* \< 0.05); we observed no significant difference in Iba1+ cell density between the PB-MNC+ group compared to PB-MNC-treated mice (34.2 ± 7.6) (*N* = 7--10). We found no significant differences between PB-MNC+, PB-MNC, and PBS groups in terms of proinflammatory cytokines ([Table 3](#tab3){ref-type="table"}). TGF-*β* mRNA expression was significantly greater in mice treated with PB-MNC+ than in PBS-treated mice *P* \< 0.05 ([Table 2](#tab2){ref-type="table"}). At D14, microglia/macrophage density was not significantly different between the PB-MNC+, PB-MNC, and PBS groups (5.3 ± 1.7, 3.3 ± 1.2, and 6.8 ± 3.5 a.u., resp.; [Figure 6](#fig6){ref-type="fig"}) (*N* = 5--6), but had decreased dramatically compared to D3: PB-MNC+ (*P* = 0.0025), PB-MNC (*P* = 0.0012), and PBS (*P* = 0.0007). No intergroup differences in proinflammatory and anti-inflammatory cytokines were observed at D14, except for TNF-*α* mRNA expression, which was significantly higher in the PB-MNC+ group (1.4 ± 0.8 a.u.) than the PBS group (0.005 ± 0.006 a.u.) (*P* \< 0.01), whereas TNF-*α* mRNA expression in the PB-MNC group (0.9 ± 0.6 a.u.) was not significantly different compared to the other two groups (*N* = 5--7). 3.7. Neurogenesis and Neuroblast Migration to the Infarct Area {#sec3.7} -------------------------------------------------------------- Neurogenesis was assessed by calculating DCX-positive neuroblast density and by measuring the distance covered by these cells as they migrated from the SVZ to the peri-infarct area. At D3, there were no significant differences in neuroblast density between PB-MNC+, PB-MNC, and PBS groups (52,262 ± 14,825 *μ*m^2^; 47,579 ± 14,099 *μ*m^2^; and 57,225 ± 21,634 *μ*m^2^, resp.) or in the migration distance of DCX+ cells toward the ischemic area (825.6 ± 177.4 *μ*m; 618.8 ± 184.5 *μ*m; and 765 ± 135.5 *μ*m, resp.) (*N* = 4--5) ([Figure 7](#fig7){ref-type="fig"}). Likewise, at D14, there were no significant differences in neuroblast density between the three groups (18,287 ± 5037 *μ*m^2^; 18,528 ± 9851 *μ*m^2^; and 12,476 ± 5223 *μ*m^2^, resp.) nor in the migration distance of DCX+ cells toward the peri-infarct area (500.5 ± 145.3 *μ*m; 592.3 ± 135.9 *μ*m; and 526.1 ± 212.5 *μ*m, resp.) (*N* = 4--5). Between D3 and D14, we observed a significant reduction in neuroblast density in the PB-MNC+ (*P* = 0.008), PB-MNC (*P* = 0.016), and PBS (*P* = 0.029) groups and a significant reduction in the neuroblast migration distance in the PB-MNC+ group (*P* = 0.032); no significant difference was observed between the same two time points in the PB-MNC and PBS groups. BDNF, a major mediator of neuroplasticity, was also assessed; plasma BDNF levels measured at D14 were significantly greater in the PB-MNC+ groups (35.8 ± 3.7 ng/L) than the PB-MNC group (21.5 ± 1.4 ng/L) (*P* \< 0.05) and the PBS group (20.5 ± 1.2 ng/L) (*P* \< 0.01) (*N* = 6--7). 3.8. Cell Tracking {#sec3.8} ------------------ PCR analysis revealed human genomic DNA in both the ipsilateral and contralateral hemispheres of mice injected with PB-MNC or PB-MNC+, as early as one hour postinjection and persisting one day later. One hour after injection, human DNA levels were lower in the contralateral hemispheres and (with the exception of the lungs) in the peripheral organs (the spleen, liver, and kidneys) than in the ischemic hemispheres; the signal increased however after one day, indicating that the engrafted cells had reached and were retained in the mouse brain ([Figure 8](#fig8){ref-type="fig"}). At D3, the signal was faint in the ischemic hemisphere, but was stronger in the contralateral hemisphere of mice treated with PB-MNC+ and was not detected in peripheral organs. At D14, no signal was detectable in either the brain or the peripheral organs. 3.9. The Subcellular Composition of PB-MNC Transplants and the PB-MNC Cellular Ephrin-B2/Fc-Binding Subsets {#sec3.9} ----------------------------------------------------------------------------------------------------------- PB-MNC isolated from diabetic patients (*N* = 8) were mainly composed of CD45+ cells (leucocytes) (97.5 ± 1.4%): CD3+ cells (T-lymphocytes) (64.6 ± 2.8%), CD3+/CD4+ cells (CD4+ T-lymphocytes) (43.1 ± 9.0%), CD3+/CD8+ cells (CD8+ T-lymphocytes) (19.2 ± 3.7%), CD19+ cells (B-lymphocytes) (6.6 ± 4.1%), CD14+ cells (monocytes) (15.8 ± 4.5%), and also for a small fraction of CD34+ cells (endothelial progenitor cells) (0.5 ± 0.3%). We have previously shown that only a fraction of 11.6 ± 11.6% cells within diabetic PB-MNC was capable to bind ephrin-B2/Fc \[[@B14]\]. Using multicolor flow cytometry analysis, we determined that the percentage of ephrin-B2/Fc-binding cells varied considerably, depending on PB-MNC subpopulation. The highest capacity to bind ephrin-B2/Fc was detected for CD34+ progenitors (73.3 ± 41.5%) and monocytes (66.6 ± 34.3%), while only a small proportion of T-lymphocytes, the largest fraction of PB-MNC, bound ephrin-B2/Fc (3.0 ± 2.4%). B-lymphocyte demonstrated an intermediate binding capacity (24.0 ± 24.5%) (*N* = 5--6) ([Figure 9](#fig9){ref-type="fig"}). 3.10. PB-MNC Response to Ephrin-B2 Stimulation by Microarray Analysis {#sec3.10} --------------------------------------------------------------------- To determine the mechanisms underlying the potentiating effect of ephrin-B2 on PB-MNC, we compared gene expression profiles of stimulated versus unstimulated cells, at 1 and 4 hours poststimulation, using Agilent Whole Human Genome Oligo Microarrays. At 1 hour, of the approximately 41,000 genes analyzed, the expression of 312 ± 101 genes was significantly changed, of which 90 ± 25 genes were downregulated and 223 ± 79 were upregulated more than twofold compared to unstimulated cells (*N* = 5) ([Figure 10(a)](#fig10){ref-type="fig"}). At 4 hours, expression of an approximately 2.8-fold larger set of genes was modified, with expression profiles only partially overlapping with those obtained at 1 hour (total modulated 861 ± 496; downregulated 350 ± 208; upregulated 511 ± 307, *N* = 5). Of these genes, several were significantly upregulated or downregulated in three or more of the five patients (*P* \< 0.0001), at one or both time points ([Figure 10(a)](#fig10){ref-type="fig"}). Among them, we found the principal components of major regulatory clusters, involving inflammatory responses (*IFNγ*, *IL6*, several members of the interleukin 1 pathway, including *IL1A*, *IL1B*, *IL1RN*, and the TNF pathway members *TNF*, *TNFAIP6*, *TNFSF15*, *TNFRSF9*), angiogenesis (*CXCL10*, *PTGS2*), immune regulation (*CD80*, *CD274*, *CSF3*, *IL23R*), leukocyte chemotaxis (*CCL2*, *CCL3*, *CCL4*, *CCL20*, *CCL23*, *CCL3L3*, *CXCL1*, *CXCL2*, *CXCL5*, *CXCL9*), transcriptional regulation (*EGR1*, *GATA6*, *HEY1*), proliferation (*CDKN2B*, *FGFR1*), adhesion (*ITGB8*), extracellular matrix remodeling (*ADAMTS4*), cell-cell communication (*GJB2*), cell signaling (*EDNRB*, *IRS1*, *RIN2*), ion transmembrane transport (*KCNJ2*, *CLIC4*), and response to oxidative stress (*SOD2*). *IL23R* was upregulated in all tested patients one hour postephrin-B2 stimulation, and eight genes (*IL1A*, *IL1B*, *CCL2*, *CDKN2B*, *TNFAIP6*, *HEY1*, *ITGB8*, and *GJB2*) were upregulated in all patients four hours poststimulation. These may therefore be considered as potential gene candidates for activating the ephrin-B2 mechanism. 3.11. Adhesion of Ephrin-B2-Stimulated PB-MNC to HUVEC Monolayer {#sec3.11} ---------------------------------------------------------------- To assess the ability of PB-MNC+ to adhere to endothelial cells, we performed an *in vitro* adhesion assay. There was a 3.5-fold increase in the number of adherent cells with PB-MNC+ compared to PB-MNC (34.7 ± 7.8 versus 10.8 ± 5.3%, resp., *P* \< 0.0001) (*N* = 10) ([Figure 10(b)](#fig10){ref-type="fig"}). 3.12. Capillary-Like Tube Formation Assay on Matrigel {#sec3.12} ----------------------------------------------------- To investigate the ability of human PB-MNC to stimulate angiogenesis *in vitro*, we performed an *in vitro* 3D Matrigel assay. Marked phenotypic differences in the capillary-like tubular patterns were observed between PB-MNC+/HUVEC and PB-MNC/HUVEC cocultures. Branch point density was significantly increased in the presence of PB-MNC+ compared to PB-MNC (62.2 ± 7.9 versus 52.2 ± 9.0, resp., *P* = 0.02) (*N* = 7), suggesting the formation of a more complex capillary-like network with greater covering capacity ([Figure 10(c)](#fig10){ref-type="fig"}). 4. Discussion {#sec4} ============= We speculated that PB-MNC from type-2 diabetic patients, stimulated by ephrin-B2 (PB-MNC+) and transplanted into mice 18--24 hours after induction of experimental cerebral ischemia, would be more effective than PBS or unstimulated PB-MNC in promoting stroke recovery. Our results show that IV transplantation of PB-MNC+ significantly reduces cerebral infarct volume and increases plasma BDNF levels compared to mice receiving PB-MNC or PBS. Compared to IV PBS, PB-MNC+ accelerated functional recovery and was associated with early cell proliferation in the SVZ and peri-infarct area, decreased microglia/macrophage cell density and TGF-*β* upregulation, and late angiogenesis. *In vitro* studies confirmed that ephrin-B2 modified the gene profile of PB-MNC, which acquired enhanced proangiogenic properties. Our data suggest that IV transplantation of ephrin-B2-stimulated PB-MNC from diabetic donors enhances brain repair. Although over 400 trials ([http://ClinicalTrials.gov](http://clinicaltrials.gov/) web service) are currently investigating novel repair strategies for stroke patients, very little is known about ideal cell sources, total doses required, optimal administration routes, therapeutic windows, or even the mechanisms involved. We opted to adhere as closely as possible to the latest translational research, by exploring a strategy that favors PB-MNC transplantation, thus excluding cell culture expansion and any risk of graft rejection since patients receive autologous cell transplantation. The simplicity and rapidity of the ephrin-B2 treatment procedure (30 minutes) makes bedside cell injection feasible at any time. We also assessed the consequences of diabetes on the ability of cells to maintain their tissue repairing properties. The current pandemic of diabetes, a known cardiovascular risk factor associated with stroke, made this disease a particularly relevant context for research \[[@B21]\]. PB-MNC from diabetic patients differ (from healthy donor cells) in their phenotype and may therefore have exhausted their inherent aptitude to instigate neurogenesis and angiogenesis. It has been shown previously that diabetic endothelial progenitor cells exhibit impaired proliferation, adhesion, and incorporation in adhesion and matrigel assays \[[@B22]\] or, in vivo, cannot repair retina vasculature \[[@B23]\]. Actually, we did compare the effects induced by ephrin-B2 in PB-MNC isolated from healthy donors and from diabetic patients in our previous study \[14 and unpublished data\]. First, using the in vitro adhesion assay, we showed that PB-MNC from diabetic patients present a roughly 35% decreased ability to adhere to HUVEC, compared to healthy PB-MNC. Stimulation with ephrin-B2 resulted in restoration of impaired adhesion of diabetic PB-MNC so that the maximal response obtained at optimal time and concentration is comparable for diabetic PB-MNC and healthy PB-MNC. Secondly, using an in vivo hind limb ischemia model, we showed that the pretreatment with ephrin-B2 increased the therapeutic potential of PB-MNC isolated from healthy donors and from diabetic patients to a similar extent. Finally, we showed in our present study that mononuclear cells from peripheral blood of diabetic patients, and stimulated by ephrin-B2, are capable to enhance neurorepair in stroke. Very few studies have assessed the efficacy of PB-MNC in the treatment of ischemic diseases. Experimental data have shown that cellular functions are reinforced through hypoxic preconditioning of human PB-MNC from healthy donors transplanted into a mouse hind limb model of ischemia \[[@B24]\]. Only one experimental model of cerebral hypoxia-ischemia has shown that transplantation of nonpreconditioned PB-MNC from healthy human donors reduced infarct volume and improved neurological deficit; brain recovery mechanisms were not specifically investigated \[[@B25]\]. Results from clinical studies using intracoronary transplantation of nonpreconditioned PB-MNC following acute myocardial infarction are however conflicting \[[@B26]\]. To the best of our knowledge, Broquères-You et al. \[[@B14]\] are the only group to have studied PB-MNC from diabetic donors and shown more extensive postischemic neovascularization when PB-MNC were stimulated by ephrin-B2. Our results also suggest that ephrin-B2 potentiated the neurorepair properties of PB-MNC from diabetic donors. Using microarray analysis, we showed that PB-MNC+ upregulate the genes that are commonly involved in proliferation, angiogenesis, and inflammation, processes all known to modulate neural plasticity \[[@B27]\]. *In vitro* analysis of PB-MNC adhesion to HUVEC monolayers, and also of capillary-like tube formation in coculture with HUVEC on Matrigel, confirmed the enhanced proangiogenic properties of PB-MNC+ compared to unstimulated PB-MNC. In our cerebral ischemia model, significantly more proliferative cells were observed in the peri-infarct area of the PB-MNC+ group as early as D3, followed by a significant increase in microvessel density and plasma VEGF-levels at D14, suggesting that angiogenesis was stimulated between D3 and D14 in that group. Angiogenesis is one of the mechanisms accounting for the neurorepair properties of BM-MNC \[[@B11]\], by promoting postischemic neurogenesis \[[@B12]\]. In our study, we found that early cell proliferation was induced by PB-MNC+ in the SVZ, a niche from which neuroblast progenitors are predominantly generated after stroke \[[@B12]\]. Moreover, plasma BDNF levels were increased in the PB-MNC+ group at D14. Because BDNF is known to have pleiotropic properties involved in neurorepair (angiogenesis, prevention of neuronal death, modulation of local inflammatory factors) in addition to neurogenesis and neural plasticity \[[@B27]\], the repair processes were likely stimulated by PB-MNC+ transplantation and persisted at D14. However, intergroup DCX+ cell density differences were not significant, on D3 or on D14. An earlier neuron progenitor marker such as glial fibrillary acidic protein (GFAP) or Mash1 and an intermediate time point analysis may have been more appropriate to detect neurogenesis in the mice treated with PB-MNC+ \[[@B28]\]. Furthermore, we could not show either that transplantation with PB-MNC+ increased the distance covered by neuroblasts to the infarct area at D14, on the contrary. We can speculate that PB-MNC+ may have accelerated neuroblast differentiation into neurons, mature neurons do not expressing anymore DCX. Evans blue extravasation and BBB gene expression assessments showed that BBB permeability was not modified by treatment. TGF-*β* mRNA however, which has a role in vessel maturation and stability \[[@B29]\], was significantly increased in mice treated with PB-MNC+. Moreover, the results of our microarray analysis revealed the upregulation of several gene products linked to TGF-*β* signaling, including integrin *β*8 that has been implicated in both TGF-*β*-mediated inflammation suppression \[[@B30]\] and stabilization of nascent brain vessels \[[@B31], [@B32]\], as well as IL-1*β* \[[@B32]\] and Hey1 \[[@B33]\]. Thus, the increased expression of TGF-*β* mRNA at D3 may also be a marker of an anti-inflammatory response. Indeed, microglia/macrophage cell density was lower in the PB-MNC+ than the PBS group, suggesting that PB-MNC+ may limit the early excitotoxic cascade triggered by ischemia and exert a cerebroprotective effect, as was previously shown following intranasal administration of TGF-*β* in mice poststroke \[[@B34]\], and therefore have contributed to the significant reduction in infarct volume that we observed. Although we did not specifically investigate potential sources of TGF-*β*, astrocytes are known to have an anti-inflammatory effect in the subacute poststroke period and are a major source of TGF-*β* \[[@B35]\] so may well be good candidates. At D14, TNF-*α* was significantly increased in the PB-MNC+ group. The role of TNF-*α* in stroke is controversial and either deleterious or beneficial in stroke, depending on the local source of production (microglia, lymphocytes, astrocytes, or neurons), the timing poststroke, and the specific receptor subtype that is activated \[[@B36]\]. A dedicated study to elucidate these points is warranted. The decreased microglia/macrophage density following PB-MNC+ treatment, and the absence of upregulation of proinflammatory cytokines, suggests that human cell transplantation did not induce rejection in our immunocompetent recipients. One hour after injection, we detected human DNA in the ischemic brain hemisphere and the lungs; 24 hours later, it had increased in the contralateral hemisphere and in all peripheral organs collected, including the lungs. The signal persisted in the contralateral hemisphere of PB-MNC+ transplanted mice on D3 and to a lesser extent in the ipsilateral hemisphere in both PB-MNC and PB-MNC+ groups, but had totally disappeared by D14. The absence of notable differences between PB-MNC and PB-MNC+ suggests that PB-MNC recruitment was not significantly modified by ephrin-B2 stimulation, except at D3 in the contralateral hemisphere; this requires confirmation in a larger mouse population. Taking into account the higher ephrin-B2-binding efficiency of monocytes and CD34+ progenitors, we hypothesize that these cellular subpopulations represent the principal ephrin-B2 targets within PB-MNC and contribute for a major part to the therapeutic effects of ephrin-B2-treated PB-MNC. The recent experimental findings on the role of monocytes in therapeutic efficacy of MNC isolated from umbilical cord blood (UCB-MNC) \[[@B37]\] and the contribution of CD34+ cells to the neuroprotective effect of UCB-MNC \[[@B38]\] support our hypothesis. Our study has several limitations. First, we evaluated one single-cell dose, though it was the highest tolerated dose determined in our previous experiments as inducing the maximal proangiogenic effect \[[@B14]\]. Second, defining optimal timing of the injection is a genuine challenge. Our study design included a single injection at what is currently the most frequently studied injection time point, that is, not immediately poststroke, because of the deleterious excitotoxic cascade, but not too late, particularly if both neuroprotection and neurorepair are targeted. Third, our assessment time points may have been too far apart to identify a specific mechanism. This might account for our inability to demonstrate neurogenesis or to show significant differences between PB-MNC+ and PB-MNC-treated mice for some objectives. Fourth, the known rapid recovery of C57Bl6J mice subjected to pMCAo, particularly when the infarct is small, prevented us from showing any significant differences between PB-MNC+ and PB-MNC-treated mice on the neurological score assessment or the ink test. Fifth, we did not show any difference in glial scar or prevention of brain atrophy at day 14; as it might occur later in the time course, it could be an interesting point to look at as well as prevention of cognitive decline, but this would deserve a specific study. Lastly, the use of animals with no vascular risk factors may have overestimated the potential of the therapeutic strategies we tested \[[@B39]\]. There is a paucity of data on the host environment in the context of cell transplantation. We do know that diabetic microangiopathy has the potential to interfere with repair mechanisms \[[@B29]\] and the cerebral "diabetic state" may generate inflammation that affects the global response to cell transplantation. BM-MNC have been tested in older mice and found to be beneficial in this population \[[@B40]\], though not in spontaneously hypertensive rats \[[@B4], [@B41]\]. Transplantation in diabetic mice should be the next step in the development of such cell therapy strategies. Moreover, although this was not our objective, the question of the ideal therapeutic window remains open. In a comparable study using intravenous administration of human umbilical cord blood mononuclear cells at 4, 24, and 72 hours and 14 or 120 days after permanent middle cerebral artery occlusion in spontaneously hypertensive rats, it was shown that the administration was effective up to 72 hours after stroke, which enlarges the window compared to recanalization strategies \[[@B42]\]. Whether this is also the case in mice transplanted with human PB-MNC demands a specific study. We have shown for the first time that IV transplantation of PB-MNC+ from diabetic donors, 18--24 hours after mouse cerebral ischemia, significantly reduces infarct volume, enhances cell proliferation and angiogenesis, and modulates inflammation. We have also shown that unstimulated PB-MNC were not significantly more effective on these endpoints than PBS, despite a trend towards improvement. Finally, we have shown that diabetic human PB-MNC, when stimulated by ephrin-B2, retain their neurorepair properties when transplanted poststroke. This strategy allowed us to assess the beneficial effect of MNC stimulated by ephrin-B2 in a nondiabetic ischemic brain. In line with current translational research, future studies should explore (i) whether the diabetic state of the recipient influences the response to cell transplantation and (ii) whether PB-MNC collected from diabetic patients in the acute poststroke period maintain the same aptitude since stroke is known to influence the systemic inflammatory response and bone marrow activation. Focus should be given to the gene candidates identified in our study since triggering the TGF-*β* pathway may underlie the ephrin-B2-mediated therapeutic poststroke effects. BM-MNC and PB-MNC should also be compared to assess the efficacy of the source. This work was supported by INSERM, the French National Institute for Health and Medical Research; Rose Hilal received a grant from the French Ministry of High Education and Research. Disclosure ========== Results have been partly published since first submission in the form on an abstract (Keio J Med 2017;66(3):55. Doi: [10.2302/kjm.66-005-ABST](http://10.2302/kjm.66-005-ABST)). BIL holds a patent (WO2011117559 \[Angiostim\]) on proangiogenic compositions, a method for preparing same, and uses thereof (PB-MNC & Ephrin B2). Conflicts of Interest ===================== The authors declare no conflicts of interest. Authors\' Contributions ======================= Tatyana Merkulova-Rainon and Nathalie Kubis contributed equally to this work. {#fig1} {#fig2} {#fig3} {#fig4} {#fig5} {#fig6} {#fig7} {#fig8} {#fig9} {#fig10} ###### Sense and antisense primer sequences used for RT-PCR. Name Sense Antisense --------------------------- -------------------------- ------------------------- Occludin CCTCCAATGGCAAAGTGAAT CCCCACCTGTCGTGTAGTCT Claudin 5 GCTCTCAGAGTCCGTTGACC ATCTAGTGCCCCCAGGATCT TJP-1 gene (ZO-1 protein) ACCCAGCAAAGGTGTACAGG CCGTAGGCGATGGTCATAGT VE-cadherin CAATGACAACTTCCCCGTCT CGTTTGGGGTCTGTCTCAAT Angiopoietin 1 GGTCAACAGAATCGCCACTT CCTGTTCCCATTTGCTGTTT Angiopoietin 2 AATGTTCCGTGGGAGTTCAG AACCTGTGCCCACCACTTAG Tie-2 (or TEK) TCTGGGTGGCCACTACCTAC TGAAAGGCTTTTCCACCATC eNOS AAGCTGCAGGTATTTGATGC TATAGCCCGCATAGC VEGF-A CCTTAATCCAGAAAGCCTGACATG AAAGTGCTCCTCGAAGAGTCTCC VEGFR-2 GGCGGTGGTGACAGTATCTT GTCACTGACAGAGGCGATGA PDGF-B GGCCACACACCTTCTCTGAT GTGGAGGAGCAGACTGAAGG PDGFR-1 CACCTTCTCCAGTGTGCTGA GGAGTCCATAGGGAGGAAGC TGF-*β* (1) TTGCTTCAGCTCCACAGAGA TGGTTGTAGAGGGCAAGGAC TGFR-2 GCAAGTTTTGCGATGTGAGA GGCATCTTCCAGAGTGAAGC Interleukin-1*β* ACCTTCCAGGATGAGGACATGA AACGTCACACACCAGCAGGTTA Interleukin-6 AGTTGCCTTCTTGGGACTGA TCCACGATTTCCCAGAGAAC Interferon-*γ* GCTTTGCAGCTCTTCCTCAT GTCACCATCCTTTTGCCAGT TNF-*α* TGGCCTCCCTCTCATCAGTTC TTGGTGGTTTGCTACGACGTG MCP-1 (or CCL2) AGGTCCCTGTCATGCTTCTG TCTGGACCCATTCCTTCTTG TJP-1: tight junction protein 1; ZO-1: zona occludens-1; VE-cadherin: vascular endothelial-cadherin; eNOS: endothelial nitric oxide synthase; VEGF-a: vascular endothelial growth factor-a; VEGF-R2: vascular endothelial growth factor receptor 2; PDGF-B: platelet-derived growth factor-B; PDGFR-1: platelet-derived growth factor receptor-1; TGF-*β*: transforming growth factor-*β*; TGFR-2: transforming growth factor-*β* receptor-2; TNF-*α*: tumor necrosis factor-*α*; MCP-1: monocyte chemoattractant protein-1. ###### Relative proangiogenic factor transcript levels in treated and untreated mice at D3 (*N* = 5--8). Transcript PBS PB-MNC PB-MNC+ *P* values ------------ ----------- ----------- ----------- ------------- Ang-1 0.7 ± 0.4 0.9 ± 0.3 1.0 ± 0.3 0.42 Ang-2 0.8 ± 0.4 0.9 ± 0.5 0.9 ± 0.6 0.99 Tie 2 1.2 ± 0.4 1.2 ± 0.6 1.8 ± 1.4 0.84 eNOS 0.5 ± 1.6 0.9 ± 0.6 1.0 ± 0.6 0.40 VEGF-A 0.6 ± 1.2 0.9 ± 0.6 0.9 ± 0.8 0.79 VEGFR-2 0.7 ± 0.3 0.9 ± 0.5 1.0 ± 0.4 0.55 PDGF-B 2.5 ± 1.3 2.6 ± 0.6 2.4 ± 0.6 0.75 PDGFR-1 0.6 ± 0.2 1.0 ± 0.3 1.0 ± 0.6 0.07 TGF-*β* 0.7 ± 0.3 0.9 ± 0.3 1.2 ± 0.4 *P* \< 0.05 TGFR-2 3.7 ± 0.4 4.3 ± 1.6 3.2 ± 1.3 0.62 Ang-1: angiopoietin-1; Ang-2: angiopoietin-2; Tie-2: angiopoietin tyrosine kinase receptor; eNOS: endothelial nitric oxide synthase; VEGF-A: vascular endothelial factor-A; VEGFR-2: VEGF receptor-2; PDGF-B: platelet-derived growth factor-B; PDGFR-1: PDGF receptor-1; TGF-*β*: transforming growth factor-*β*; TGFR-2: transforming growth factor-*β* receptor-2. Data are presented as mean (arbitrary units) ± SD. ###### Relative proinflammatory cytokines transcript levels in treated and untreated mice at D3 (*N* = 5--7). Transcript PBS PB-MNC PB-MNC+ *P* values ------------ ----------- ----------- ----------- ------------ IL-1 *β* 1.9 ± 1.0 5.4 ± 5.2 4.0 ± 3.6 0.37 IL-6 1.0 ± 0.4 1.4 ± 0.3 2.1 ± 1.4 0.15 IFN-*γ* 2.5 ± 2.5 2.0 ± 1.5 2.1 ± 1.1 0.89 TNF-*α* 0.4 ± 0.2 0.6 ± 0.3 0.8 ± 0.8 0.60 MCP-1 0.7 ± 0.5 1.0 ± 0.5 1.4 ± 1.0 0.39 IL-1*β*: interleukin-1*β*; IL-6: interleukin-6; IFN-*γ*: interferon-*γ*; TNF-*α*: tumor necrosis factor-*α*; MCP-1: macrophage chemoattractant protein-1. Data are presented as mean (arbitrary units) ± SD. [^1]: Academic Editor: Eva Mezey

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INTRODUCTION ============ Nutrient excess and nutrient deficiency in the diets of preschool children can lead to permanent modification of metabolic pathways and increased risk of diet-dependent diseases in adults. Children are most susceptible to the adverse consequences of bad eating habits. An unbalanced diet can lead to delayed physical, cognitive and emotional development. Excessive supply of saturated fats and simple sugars combined with a deficiency of vitamins, minerals and highly available protein contributes to weight gain and glucose metabolism disorders. The results of numerous studies indicate that obesity in preschool children poses serious health risks and increases the prevalence of obese adolescents. Abdominal obesity increases the risk of insulin resistance 3-fold. A BMI greater than the 85 percentile increases the risk of unbalanced cholesterol levels 2.4-fold, unbalanced LDL cholesterol levels -- 3-fold, unbalanced HDL cholesterol levels -- 3.4-fold, unbalanced triglyceride levels -- 7.1-fold, and hypertension -- 4.5-fold.^[@B1]^ The objective of this study was to evaluate the eating habits and the diets of preschool children as risk factors for excessive weight, obesity, insulin resistance and the metabolic syndrome. METHODS ======= The study was conducted on 350 randomly selected preschool children attending kindergartens in south-eastern Poland. It was carried out in three stages between October 2013 and February 2014. In the first stage, children were subjected to anthropometric measurements, which involved the determination of height with the use of a stadiometer exact to 0.1 cm and body weight with the use of the Tanita BC 545N scales exact to 10 g. The measurements were used to calculate the body mass index (BMI) adjusted for age and gender in a centile chart (WHO). The Cole index (CI), also known as the relative body mass index (RBMI), was calculated for every subject to determine the participants\' weight relative to the average BMI at the 50^th^ percentile, with the use of the following formula: Patient\'s BMI RBMI = -------------------------------- x 100 \[%\] BMI at the 50th percentile CI values are expressed as percentages ([Table-I](#T1){ref-type="table"}), and they are used to assess the nutritional status of children and adolescents.^[@B2]^ In the second stage of the study, parents were asked to fill out a questionnaire designed by the author to provide information about the family\'s eating habits, meal preparation methods, frequency of consuming various foods and knowledge about the nutritional requirements of preschool children. The parents participated in a 3-day dietary recall covering one day of the weekend. The children\'s preferences regarding food products and ready-made meals was evaluated in the third stage of the study. The subjects were presented with a picture-based questionnaire and were asked to mark different products with \"like\", \"don\'t like\" and \"don\'t care\" emoticons. The survey was carried out with the involvement of trained assistants who were students of the University of Life Sciences in Lublin graduating in nutritional sciences. {#F1} Three-day dietary recalls were processed and evaluated in the Dieta 5 application developed by the National Food and Nutrition Institute in Warsaw based on Polish food composition and nutrition tables.^[@B3]-[@B5]^ The average nutritional value and composition of the evaluated diets was determined in the Dieta 5 application. RESULTS ======= The surveyed children were aged 4-6 years (average age 4.97 ± 0.84 years) and they attended public kindergartens. 58% of the surveyed subjects were girls. 5-year-olds had the highest share of the analyzed population. According to the parents\' declarations, the children\'s material and family situation was evaluated as satisfactory in 67% of cases, highly satisfactory in 21% of cases, unsatisfactory and unstable in 12% of cases. The results of anthropometric measurements revealed that 14.6% of all children (n=51) were overweight ([Table-II](#T2){ref-type="table"}). The majority of overweight subjects were girls (34), mostly 5-year-olds. The average RBMI was determined at 107.6% for girls and 103.3% for boys, where it did not exceed normal levels in any age group. Despite the above, not all boys were characterized by healthy body weight. The group of 4-year-olds included 3 boys with general obesity and RBMI values in the range of 123.5 -- 131.2%. The results of dietary recall interviews revealed that most children ate regular meals on weekdays, including 4 meals in the kindergarten and 2 meals at home. On weekends, 28% of the polled subjects ate only 3 meals daily, 8.8% children skipped breakfast and their first meal of the day was lunch served around noon. 95.4% children ate dinner, but the last meal of the day was served very late at 9 to 10.30 p.m. The results of dietary recall interviews indicate that nutrient deficiencies (calcium, phosphorus, iron, vitamins E and D, unsaturated fatty acids and plant-based proteins) as well as nutrient excess (sodium, vitamin A, saturated fatty acids, simple and added sugars) were common in the analyzed diets ([Table-III](#T3){ref-type="table"}). The average energy value of the analyzed diets was 1473.2 kcal ± 234.7, which was insignificantly above the recommended level. The evaluated diets were rich in high-calorie foods and added sugar. All children snacked between meals and had the highest preference for sweets and sweetened products. Sweetened fruit juice was consumed by 66% of the subjects, and sweetened soft drinks -- by 44.6% of the children at least once a week. All children declared to have a preference for sweets: 203 subjects (58%) ate at least one serving of sweets per day, 118 children (33.7%) ate sweets several times a week, and only 8.3% of children ate sweets less frequently. The favorite products indicated by children were: candy, chocolate, biscuits, salty snacks, crisps, jelly sweets, nuts, crackers and fruit. DISCUSSION ========== The number of overweight children continues to increase and it poses a serious global problem.^[@B6]-[@B9]^Undesirable social behavior is the second most important contributor to excessive body weight after genetic factors. Parents and caretakers have very little knowledge about the impact of healthy nutrition on children\'s health and development. Overweight children are much more likely to become overweight and obese adults. Research results indicate that children aged 3 to 9 years with BMI above the 80^th^ percentile are three times more likely to suffer from obesity between the ages of 24 and 39, and the risk is four-times higher in overweight adolescents.^[@B10]^ According to the International Obesity Task Force,^[@B11]^ every fifth child in Europe is overweight. A Health Behavior in School-age Children (HBSC) study conducted in Poland in 2010 revealed that 18.3% of Polish adolescents aged 11-12 years were overweight and 3.4% were obese.^[@B12]^ ###### Classification of excessive body weight and obesity in children and adolescents based on the Cole index (CI). **RBMI value** **Nutritional status** ---------------- ------------------------ \< 75% Wasting 75--89% Malnutrition 90--109% Normal 110--119% Overweight \>120% Obesity ###### Age structure and average RBMI values of the analyzed population **Gender** **Age** ------------------------------ --------- --------- --------- Girls \[number of children\] 4 years 5 years 6 years 65 86 52 Average RBMI 106% 111.3% 105.5% Number of overweight girls 7 23 4 Boys \[number of children\] 4 years 5 years 6 years 37 67 43 Average RBMI 108.4% 99% 102.5% Number of overweight boys 8 2 7 ###### Average concentrations of selected nutrients in the analyzed diets ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ **Nutritional requirement** **Average content in the analyzed diets ±**\ **Average content in the analyzed diets expressed in \[%\] of nutritional requirements** ***p*** **standard deviation** ------------------------- ----------------------------- ---------------------------------------------- ------------------------------------------------------------------------------------------ ---------- Protein 45g 37.09 g ± 8.8 84.22 \< 0.001 Carbohydrates 220g 194.7 g ± 87.9 88.5 \< 0.001 including simple sugars 20-25g 140.3 g ± 63.8 561 -701 \< 0.001 Total fat 45g 47.3 g ± 19.7 105.1 \< 0.001 Saturated fat 15g 29.47 g ± 10.1 196.5 0.009 Monounsaturated fat 22.5g 13.78 g ± 7.4 61.24 \< 0.001 Polyunsaturated fat 7.5g 4.05 g ± 2.2 54 0.004 Fiber 15g 11.9 g ± 4.7 79.3 \< 0.001 Calcium 800 mg 553.25 mg ± 232.7 69.15 \< 0.001 Magnesium 150 mg 166.5 mg ± 84.3 111 0.004 Phosphorus 1000 mg 641.6 mg ± 248.2 64.16 \< 0.001 Iron 8 mg 6.64 mg ± 2.28 83 \< 0.001 Sodium 1000 mg 2554.7 mg ± 358.4 255.47 \< 0.001 Potassium 2100 mg 1552.7 mg ± 179.5 73.9 \< 0.001 Vitamin A 2000 IU 2670 IU ± 291.1 133.5 \< 0.001 Vitamin E 10 mg 4.02 mg ± 0.8 40.2 0.004 Vitamin C 50 mg 90.24 mg ± 47.2 180.48 \< 0.001 Vitamin D 500 IU 234 IU ± 67.9 46.8 0.009 Folates 0.2 mg 0.06 mg ± 0.1 30 \< 0.001 ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ In the past decade, the incidence of obesity and the metabolic syndrome has increased at an alarming rate in South Asia, in particular among women and children. The main causes of obesity are economic development, changes in the traditional diet, lower levels of physical activity and genetic factors. In South Asia, the risk of metabolic and cardiovascular diseases has increased due to higher consumption of fat and abdominal fat deposition.^[@B13],[@B14]^ Excessive weight gain during childhood and adolescence and abdominal fat deposition could be the first factors that increase the risk of the metabolic syndrome.^[@B15]^ In this study, 51 children were overweight, and most of them accumulated fat in the abdominal region or were characterized by general obesity. Snacking between meals and a preference for sweet-tasting foods can contribute to metabolic diseases such as hypertension, hyperglycemia, higher triacylglycerol (TG).^[@B16]^ Obesity prevention starting as early as in infancy is the most effective method of lowering the risk of the metabolic syndrome. Products rich in omega-3 and omega-6 fatty acids should be incorporated in the children\'s diet. In the surveyed population, the average consumption of unsaturated fatty acids covered nutritional requirements in 54% due to low consumption of fish. 45.1% of the polled children did not like and did not eat fish, whereas 17.1% of the subjects ate fish once a week, mostly in the form of fried bread-coated products. Body weight is also determined by the content of saturated fatty acids in the diet. In the analyzed population, the supply of saturated fatty acids accounted for 196% of nutritional requirements due to the consumption of highly processed fried foods. 211 children ate a hamburger at least once a week, 262 children -- a slice of pizza, and 180 subjects -- French fries as a serving of vegetables. 94.8% of the evaluated population consumed at least 15 g of hard cheese daily, which is a rich source of protein, but also fat. Other authors have demonstrated that high fast food intake leads to a three-fold increase in the risk of being overweight in children aged 6 to 11 years.^[@B17]^ Fast food is characterized by low nutritional value and a very high content of fat and saturated fatty acids.^[@B18]^ Those factors contribute to excessive body weight during childhood. The consumption of milk and dairy products that meet daily calcium requirements can inhibit the development of abdominal obesity in children aged 6 to 11 years. Several observational studies have demonstrated correlations between the consumption of dairy products and obesity in children, which indicates that milk protein plays an important role in body weight control.^[@B19]^ High and regular consumption of calcium, in particular from wholesome dairy products, can prevent excessive body weight and obesity.^[@B20]^ Milk and dairy product consumption was low in the studied population. 91 children (26%) did not like milk and did not consume milk in pure form or in milk soup. 180 children consumed 1 serving of milk or yogurt daily, and only 79 of subjects drank 500 ml of milk daily and ate other dairy products. The average calcium content of the analyzed diets covered only 69.19% of nutritional demand for this element.Similar results were reported by Uush, in whose study calcium intake in all age groups (1-3, 4-7, 8-12 years) was significantly below (39%, 30.9%, 24.4%) the recommended levels.^[@B21]^ Distribution of meals and regularity in eating are also important factors in body weight control. Breakfast avoidance can lead to excessive hunger, overeating,^[@B22]^ eating larger portions and excessive calorie intake in successive meals.^[@B23]^ In the analyzed population, children tended to skip regular meals during the weekend. A late or skipped breakfast can disrupt appetite regulation. The consumption of sweet snacks at all times of the day further contributes to meal skipping and irregular meal patterns. Preschool children should eat 4-5 meals at regular times of the day. Regular meal patterns aid digestion and the utilization of nutrients and energy by the body. The consumption of added sugars increases the energy value and lowers the nutritional value of a child\'s diet. Higher intake of simple sugars increases fasting blood glucose levels and impairs insulin secretion. Excessive supply of dietary glucose, fructose and saccharose can lead to blood sugar imbalance and insulin resistance.^[@B24],[@B25]^ Simple sugar intake was very high in the analyzed diets, which contained 5-7 times more sugar on average than recommended for children aged 4-6 years. The above resulted from excessive consumption of sweets, juice, sweet soft drinks, sweetened tea and the children\'s general preference for sweet-tasting foods. Regular breakfasts consumption, higher intake of milk, oils rich in unsaturated fatty acids, fresh vegetables and fruit minimize the risk of excessive body weight, insulin resistance and the metabolic syndrome.^[@B26]^ Researchers and campaign authors havenoted a significant decrease in obesity, in particular among children aged 2 to 5 years (from 13.9% to 8.4%, p=0.03).^[@B27]^ In South Asia, Europe and the United States, which have the highest childhood obesity rates, the main objective of obesity prevention campaigns should be to increase awareness about diet-dependent diseases. There is a great need for community programs promoting physical activity and healthy eating habits among children. CONCLUSIONS =========== Irregular meal patterns and an unbalanced diet can impair physical and cognitive development in children. Diets characterized by excessive energy value and nutritional deficiency can lead to health problems. In most cases, excessive weight gain in children can be blamed on parents and caretakers who are not aware of the health consequences of high-calorie foods rich in fats and sugar. There is a dire need for social campaigns that promote healthy eating habits and prevent uncontrolled weight gain. In recent years, several healthy lifestyle campaigns in the US has given highly promising results. ***Conflict of interest*** ***:*** The work was financed by public funds for science and higher education. There is no conflict of interest.

Introduction {#sec1} ============ Fluorescence-based techniques are the working horses in life sciences, especially when it comes to optical microscopy. Here, technical developments complemented with optimized fluorescence probes have paved the road for the investigation of many biological processes on a (sub)cellular level, e.g., using single-molecule spectroscopy or STED microscopy.^[@ref1],[@ref2]^ In order to adapt fluorescence techniques on new topics and/or to optimize them, novel fluorescence dyes with tailored optical properties are needed. Here, \[1,3\]dioxolo\[4,5-*f*\]\[1,3\]benzodioxoles (DBD dyes) have shown many promising properties. The photophysical and chemical properties of the DBD dyes can be tuned (decay time, quantum yield, spectral position of emission maximum, as well as Stokes shift and their functional groups for bioconjugation) and can be excited by two-photon absorption, and they show a high photochemical stability.^[@ref3]−[@ref5]^ Förster resonance energy transfer (FRET) is a well-established concept to monitor the binding interaction between antigen and antibody with a very high sensitivity and is successfully applied in clinical diagnostics.^[@ref6]−[@ref9]^ DBD dyes in combination with coumarins were also characterized with respect to FRET parameters, with special emphasis on the orientation factor.^[@ref10],[@ref11]^ Recently, DBD compounds have been also characterized in the context of membrane probes.^[@ref10],[@ref12]^ Hydrophobic motifs like oligospirothioketal groups^[@ref10]^ or alkyl chains^[@ref12]^ were used as an anchor to the probes in membranes and vesicles. The length of the anchor was also directing the DBD compound to laterally ordered membrane domains. Due to the presence of a maleimide moiety, further biofunctionalization, e.g., to proteins, was successfully carried out.^[@ref12]^ For studying and understanding the interactions and signal pathways on cell membranes (e.g., of receptor--ligand interactions), liposomes are frequently used as model systems.^[@ref9],[@ref13]^ Liposomes can also be seen as a class nanomaterial with a surface that can be designed to be biocompatible. They are adjustable in size, and more importantly for the investigation of ligand--receptor interactions their surface can be tailored (e.g., by the insertion of proteins), mimicking certain properties of biomembranes.^[@ref14]−[@ref16]^ Liposomes form small compartments (μm to subμm scale) consisting of a hydrophobic bilayer with polar surfaces on the in- and outside of the compartment. Because of these unique properties, they are used in many different analytical methodologies.^[@ref17],[@ref18]^ In the hydrophobic bilayer, nonpolar molecules can be trapped. In contrast, inside the liposome, hydrophilic compounds can be located. The combination of hydrophilic and hydrophobic regions in a well-defined volume can be used for labeling (parts) of the liposome and for the development of assays.^[@ref19],[@ref20]^ A major feature of liposome-based assay formats was the potential for signal amplification because the number of markers "hosted" in the vesicle or on the surface of vesicles can be much higher than the number directly conjugated to a biomolecule (e.g., antibody). Enzyme-linked immunoadsorbent assays are reported with hundreds of enzyme molecules per vesicle yielding a significant signal amplification.^[@ref21]−[@ref24]^ Homogeneous as well as heterogeneous assay formats using liposomes are on hand, and often the signal enhancement is based on a liposome lysis (complement-mediated or melittin-mediated) upon biorecognition.^[@ref25],[@ref26]^ In our studies we used the liposomes in a 2-fold way: as a mediator phase to directly interface conjugation of hydrophobic biomolecules with hydrophobic probes and as a potential scheme for signal amplification. Here, we present the first results with respect to (i) the direct labeling of biomolecules to hydrophobic dye molecules and (ii) the proof-of-principle for a binding interaction on the surface of the liposomes. The hydrophobic part of the liposomes was used to host a hydrophobic fluorophore (DBD mm239), which subsequently was attached to an antibody. In our experiments, an antibody directed against rhodamine (DC7-Ab) was used. An advantage of using DC7-Ab is that rhodamine derivatives for labeling purposes of biomolecules are commercially available, and it has therefore a broad spectrum of possible "modified" antigens which could be disseminated in future applications. The binding interaction of rhodamine and DC7-Ab was characterized in detail before using fluorescence techniques with special emphasis on Förster resonance energy transfer (FRET) to evaluate the success of the binding.^[@ref27]^ Results and Discussion {#sec2} ====================== Some spectroscopic properties of the mm239-DTT in DMSO as well as immobilized in liposomes are shown in [Table [1](#tbl1){ref-type="other"}](#tbl1){ref-type="other"} (for mm239 only and in other solvents see ref ([@ref12])). The DBD derivative is characterized by a large Stokes shift (Δλ ∼ 90 ± 5 nm) and long fluorescence lifetime (τ ∼ 15 ns) in both samples. ###### Spectroscopic Properties of mm239-DTT in Liposomes probe solvent λ~abs~ (nm) λ~em~ (nm) Δλ (nm) τ (ns) --------------------- --------- ------------- ------------ --------- -------- mm239-DTT DMSO 370 456 86 14.3 mm239-Liposome- DTT PBS 369 464 95 15.4 According to [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"} (left), before the reaction with DTT, the fluorescence intensity of mm239 dye (in liposomes) was very weak due to the quenching effect of the maleimide group, which is a result of a photoinduced electron transfer.^[@ref12]^ After reaction with DTT, the fluorescence intensity increased significantly by a factor of about eight. {#fig1} Like the fluorescence intensity, the fluorescence decay kinetics was also changed. While for mm239 in DMSO and also in liposomes a distinct quenching (biexponential decay, see [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}, right) was observed, upon modification with DTT the fluorescence decay became monoexponential, showing a fluorescence decay time of 15 ns in liposomes (see [Table [1](#tbl1){ref-type="other"}](#tbl1){ref-type="other"}). The long fluorescence decay time and the large Stokes shift of about Δλ = 90 nm in combination with the DTT for further functionalization made mm239-DTT very intriguing as an optical probe in biosensing. However, the successful incorporation of mm239-DTT into the liposome phase could not be concluded directly from the data. Incorporation of DBD Dye in Liposomes {#sec2.1} ------------------------------------- Previously, the incorporation of mm239 into GUV membranes has been shown indirectly by fluorescence microscopy.^[@ref12]^ Here, additional fluorescence depolarization experiments were performed to monitor the incorporation of dyes (and the conjugation of antibodies to liposomes, vide infra) directly. A full depolarization of the fluorescence was found in DMSO, and subsequently the steady-state fluorescence anisotropy *r* was calculated as zero. On the other hand, in the presence of liposomes (in PBS) the fluorescence anisotropy *r* increased up to *r* = 0.16 for mm239-DTT ([Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}, right). {#fig2} Complementary results from time-resolved fluorescence anisotropy data analysis yielded, in the case of mm239-DTT in DMSO, a fast, single-exponential decay kinetics ([Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}, left) with a short rotational correlation time Φ = 0.3 ns, which is typical for small organic molecules in neat solvents.^[@ref28]^ In the presence of liposomes, the rotation of mm239-DTT was distinctly slower ([Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}, left). In the data analysis the "wobble-in-a-cone" model was used.^[@ref29]−[@ref31]^ Based on this model the rotational correlation time calculated for the anisotropy decay kinetics increased to Φ = 1.42 ns, and a semicone angle of θ~*k*~ = 48° was found. The increase of the steady-state anisotropy as well as the rotational correlation time of the dye in the presence of liposomes showed once more that the movement of the dye was restricted and underlined its incorporation into the lipid phase of the liposome. Binding of R6G (Antigen) to Liposome-Immobilized Ab {#sec2.2} --------------------------------------------------- In the next step, the binding of an antibody (Ab) to the liposome surface was investigated. In principle the maleimide group in DBD derivatives could react directly with thiol groups which are frequently present in proteins as cysteine residue under reducing conditions. However, in the case of Ab, thiols are often found in oxidized form as disulfide bridges which play an important role in keeping the tertiary structure and activity of antibodies. Direct labeling would require cleavage of disulfide bonds by using reducing agents which in some cases cause the loss of Ab activity. Instead, in this study an indirect labeling using dual functional cross-linker SMPH was applied, which also aimed to increase the efficiency of the conjugation of the Ab to the liposome because the distance to the liposome surface was enlarged this way (see [Figure [7](#fig7){ref-type="fig"}](#fig7){ref-type="fig"}). The successful binding of the DC7-Ab to the liposome loaded with mm239-DTT was tested in fluorescence depolarization experiments (see [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}). For liposomes after conjugation with DC7-Ab the fluorescence anisotropy *r* of mm239-DTT increased further up to *r* = 0.19, and the rotational correlation time was increased to Φ = 2.42 ns. The increase of both parameters is a very strong indication of DC7-Ab conjugation to the liposome surface. Only the semicone angle of θ~*k*~ = 52° (calculated based on the wobble-in-a-cone model) did not change significantly. The almost unchanged cone angle indicates that the location of the dye in the lipid phase of the liposome is not altered by the attachment of the antibody, which could be an effect of the increased distance due to the cross-linker. To show that the labeling as such and the presence of the liposome did not interfere with the function of the antibody, the binding of the respective antigen was investigated. The DC7-Ab is directed against R6G. Because of the spectral overlap between the emission spectrum of mm239-DTT and the absorption spectrum of R6G (see [Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}), a potential FRET pair is on hand. The calculated Förster radius of *R*~0~ = 5.5 nm is large enough to monitor the binding of R6G to DC-7-Ab bound to mm239-DTT in liposomes but on the other hand small enough to show a cross sensitivity between different liposomes of different mm239 dyes in one liposome at low to medium loading. Consequently, upon binding between the partners, FRET is expected to yield a decrease in the donor emission (quantum yield as well as fluorescence decay time) and a simultaneous increase of the acceptor emission, if the distance *R* between mm239 and R6G in the complex is in the range of 0.5*R*~0~ \< *R* \< 2*R*~0~ (with *R*~0~ being the Förster radius), which should be the case for the liposomal system investigated. {#fig3} Therefore, in order to monitor the binding between the mm239-DTT-DC7 and R6G, the fluorescence (intensity as well as decay kinetics) of both fluorophores was measured. The binding experiments were performed by preparing samples, which had the same concentration of mm239-DTT-DC7-labeled liposomes but different amounts of R6G. According to the intensity spectra shown in [Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"} (left), the intensity of the donor part (mm239 moiety) decreased when the concentration of acceptor R6G was increased, and at the same time the acceptor-related fluorescence increased. Both observations point to a FRET which proved the successful binding of DC7-Ab to the liposome surface. {#fig4} Complementary to the steady-state emission spectra the fluorescence decay kinetics of the donor (mm239) and acceptor (R6G) of DC7-Ab-conjugated mm239-labeled liposomes were measured and analyzed. In [Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"} (right) the decay kinetics of the donor and acceptor emissions are shown at different donor--acceptor ratios. The acceptor fluorescence was measured under donor-specific excitation conditions in order to obtain the energy transfer related decays. The donor fluorescence decay time decreased from about 16 ns (for DC7-Ab-conjugated mm239-labeled liposomes) to ∼5 ns for the highest R6G concentration investigated (see [Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}). At the same time the fluorescence decay kinetics of R6G became biexponential, showing a steady increase of the fluorescence decay. Because R6G was also excited directly to a small extent at the donor excitation wavelength of λ~ex~ = 375 nm, a combination of direct excited and via FRET excited emission kinetics was detected. As a trend from the analysis of the decay kinetics an increasing fraction of FRET-related R6G emission was found. For that fraction the determined decay time was 13 ± 1 ns, which was in agreement with the nonquenched donor fluorescence decay time and further stressed the active FRET in the liposome-based immune complex. Single-molecule FRET experiments (PIE-FRET) were also carried out in order to avoid the averaging out effect, contribution from free donor emission, and the influence of directly excited acceptor emission, which often obscure ensemble FRET measurements yielding the wrong FRET parameters (see [Figure [5](#fig5){ref-type="fig"}](#fig5){ref-type="fig"}). The distance between the donor and acceptor was determined to be *R* ∼ 5.1 nm (0.94*R*~0~). {#fig5} In [Table [2](#tbl2){ref-type="other"}](#tbl2){ref-type="other"} the results of the ensemble-based (intensity as well as decay time related) and PIE-FRET results of the calculated FRET efficiencies are compared. For the calculation of the ensemble-related FRET efficiencies the direct excitation of the acceptor and its contribution to the signal had to be taken into account. On the other hand the PIE-FRET experiments were performed at a very low liposome concentration. Measurements were carried out at several different dilutions of the liposomes but yielded always the same distance distribution, which was expected since the distance between donor and acceptor in the complex is always the same. Due to the high affinity of the Ab for its antigen, the R6G binding was high, and therefore basically all the donor sites became involved in the FRET. Consequently, no signal of "free" donor was found. ###### Comparison of FRET Efficiency Based on Different Techniques   FRET efficiency (%) -------------- --------------------- by intensity 64 by lifetime 68 by PIE-FRET 59 Conclusion {#sec3} ========== The labeling of biomolecules with optical probes, that are often highly hydrophobic molecules, requires a compromise in the solvent polarity. However, many biomolecules are very sensitive to organic solvents and may be irreversibly altered (or even destroyed) in their function in organic solvents (mixtures). DBD dyes but also other classes of dyes can be used in the proposed labeling scheme without the necessity of adding additional (often) charged groups to the dyes in order to improve their water solubility. Using liposome as a mediator phase such labeling can be nicely accomplished. Here, the liposomes (or their surface) can be seen as "small" reaction vessels offering the hydrophobic environment for the probes and their hydrophilic surface for the attachment of the biomolecules. The full reaction scheme (see [Figure [7](#fig7){ref-type="fig"}](#fig7){ref-type="fig"}) could be demonstrated step by step based on the change in the fluorescence parameters of mm239 in liposomes. First, the modification with DTT was indicated by a significant increase in the fluorescence intensity as well as the fluorescence decay time. The successful incorporation and the subsequent labeling to an Ab were demonstrated by the distinct changes of the fluorescence depolarization of the DBD dye mm239. The connection of the Ab to the surface of the liposome was also stressed by the anisotropy data. Finally, the binding activity of the Ab (when connected to the liposome) was demonstrated in FRET experiments. Here, R6G was used as antigen. Depending on the specific needs it can be envisaged that after the successful conjugation the liposomes may be destroyed, releasing the labeled biomolecules, or can be developed into a signal amplification scheme as has been shown before for different liposome-based assays. Labeling of biomolecules often leads to the conjugation of more than one dye molecule per antibody (unless there is only one specific functional group such as R--SH per biomolecule), which then can lead to unwanted effects like self-quenching or loss of binding activity.^[@ref32]^ With the proposed labeling scheme using liposomes as a mediator phase, the possibilities of multiple labelings of the biomolecule are distinctly reduced due to steric considerations, and at the same time an efficient, relatively simple strategy for signal amplification (on a single liposome level) is on hand. The assay response can be easily scaled with respect to binding sites per liposome via the loading of the vesicle with DBD dye derivative. This can be controlled, even on a single liposome level using laser fluorescence microscopy techniques. Since liposomes are "nanocontainers" the consumption of analytes/chemical can be minimized. In addition, even a multianalyte detection on one liposome can be envisaged, if different Abs are attached to the liposome surface using tailored DBD dyes, which allow an excellent tuning of the absorption/emission parameters. Work is in progress to evaluate the sensitivity of the liposome-based assay relative to enzyme-based conventional ELISA assays. Although the experiments shown here were carried out in PBS buffer, additional experiments and fine adjustments are needed to transfer this test scheme to real-world samples. Materials and Methods {#sec4} ===================== DBD derivative (mm239) was synthesized by the group of Prof. P. Wessig (Institute of Chemistry, University of Potsdam).^[@ref12]^ The chemical structure of the compound is depicted in [Figure [6](#fig6){ref-type="fig"}](#fig6){ref-type="fig"}. {#fig6} Succinimidyl-6-(*b*-maleimidopropionamido)hexanoate (SMPH), dimethyl sulfoxide (DMSO), dithiothreitol (DTT), chloroform (CHCl~3~), and phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich Chemie GmbH, Munich, Germany. The GC71-DC7 anti-TAMRA-antibody (DC7-Ab) was synthesized by Hybrotec GmbH, Am Mühlenberg 11, Potsdam, Germany. 1-Palmitoyl-2-oleoyl-*sn*-glycero-3-phosphocholine (POPC) and rhodamine 6G were purchased from Avanti Polar Lipids, Alabama, and Sirah Lasertechnik GmbH, Grevenbroich, Germany, respectively The fluorescence spectra were measured in DMSO and PBS. The experiments with samples containing antibodies were carried out in PBS. All chemicals and solvents were of analytical grade. Liposome Preparation {#sec4.1} -------------------- The incorporation of mm239 into the phospholipid vesicles (liposomes) was carried out based on a rehydration technique (see [Figure [7](#fig7){ref-type="fig"}](#fig7){ref-type="fig"}). The mixture of phospholipid and mm239 was first dissolved in CHCl~3~; subsequently, CHCl~3~ was evaporated by applying a gentle stream of nitrogen gas, followed by using vacuum overnight to fully remove the remaining organic solvent residues. The PBS solution was added to dry lipid film and incubated at 50 °C for 1 h. Freeze-and-thaw cycles (×5) were performed by immersion in liquid nitrogen followed by thawing in a water bath (50 °C). Finally extrusion through the 100 nm pore size polycarbonate membrane (Whatman, United Kingdom) was carried out, and homogeneous mm239-labeled large unilamellar vesicles (LUV) were obtained. The size of the liposomes was determined using dynamic light scattering (Zetasizer NanoZS, Malvern Panalytical) to 70 ± 5 nm (with a PDI of 0.13). {#fig7} In order to couple DC7-Ab to liposomes, DTT was added to the mm239-labeled liposome sample solution with the molar ratio of 10:1. The mixture was then incubated for 1 h at RT, followed by removing the excess amount of DTT by using Amicon Ultra 0.5 mL centrifugal filters with a molecular weight cut off (MWCO) of 3 KDa (Merck, Germany) (centrifugation for 20 min twice). DC7-Ab was treated with the hetero cross-linker SMPH (reaction molar ratio between DC7-Ab and SMPH = 1:20). The reaction mixture was incubated overnight at 4 °C, the NHS ester group of the SMPH cross-linkers reacted with primary amines in DC7-Ab to yield stable amide bonds. The mixture was then put in Amicon Ultra 0.5 mL centrifugal filters with MWCO of 10 kDa and centrifuged to remove excess amount of SMPH (twice for 20 min at 4 °C). The DTT-mm239-labeled liposome solution was mixed with SMPH-labeled DC7-Ab (molar ratio 1:1) and incubated at RT for 1 h. The maleimide functional group in SMPH-labeled DC7-Ab reacted with the free thiol group on the DTT-mm239-labeled vesicle to form a thioether bond. Steady-State Fluorescence Spectroscopy {#sec4.2} -------------------------------------- The steady-state fluorescence measurements were performed using a Fluoromax 4 spectrofluorometer (Horiba Jobin Yvon, Japan) operated in the single photon counting mode. Excitation wavelengths of λ~ex~ = 370 nm were applied in the fluorescence experiments to record the emission spectra of the mm239 dye. The fluorescence anisotropy excitation spectra were measured by exciting the mm239 with vertical polarized light, and the emission was detected with the polarizer set to vertical (*I*~vv~) and horizontal (*I*~vh~) orientation, respectively. The polarized emission was recorded at λ~em~ = 470 nm. The anisotropy *r* was calculated according to equation [eq [1](#eq1){ref-type="disp-formula"}](#eq1){ref-type="disp-formula"} Here, *G* is the correction factor to take into account the different efficiencies of the optical components for vertical and horizontal polarized light. The *G* factor value was calculated according to [eq [2](#eq2){ref-type="disp-formula"}](#eq2){ref-type="disp-formula"}. Time-Resolved Fluorescence Depolarization {#sec4.3} ----------------------------------------- To measure the fluorescence decay kinetics, a FL920 spectrometer (Edinburgh Instruments, UK) equipped with a multichannel plate (Europhoton, Germany) operated in the time-correlated single photon counting (TCSPC) mode was used. A laser diode (EPL-375, Edinburgh Instruments, λ~ex~ = 375 nm) with a pulse width of 55 ps and a repetition rate of 10 MHz was used as the excitation source. A supercontinuum source SC450-AOTF (Fianium, UK, λ~ex~ = 520 nm, pulse width of 50 ps, 20 MHz repetition rate) was applied for the investigation of the R6G fluorescence decay kinetics. The fluorescence decays were recorded with an emission polarizer (Glan Thompson prism, Thorlabs, Germany) set to magic angle conditions at λ~em~ = 470 nm for mm239 and at λ~em~ = 580 nm in the case of R6G. The time-resolved fluorescence anisotropy *r*(*t*) was calculated based on [eq [3](#eq3){ref-type="disp-formula"}](#eq3){ref-type="disp-formula"}. In the depolarization measurements photons were collected for 900 s.The *G* factor was determined according to [eq [4](#eq4){ref-type="disp-formula"}](#eq4){ref-type="disp-formula"}. Single-Molecule Spectroscopy (SMS) {#sec4.4} ---------------------------------- The pulsed interleaved excitation FRET (PIE-FRET) SMS experiments were performed using a MicroTime 200 instrument (PicoQuant, Germany). R6G was excited at λ~ex~ = 532 nm using a supercontinuum laser source (SC-400-2, Fianium, England) in combination with an AOTF filter. The laser beam was focused to a 50 μL droplet of sample on the cover slide or to a 200 μL sample in chambered cover glass (Nunc, Lab-Tek, Thermo Scientific, Germany) by an oil immersion objective (100×, NA 1.4, Zeiss PlanApo, Germany). The emission light was collected by the same objective and separated from excitation light by a dual band dichroic mirror (ZT 405/532rpc-UF1, AHF Analysentechnik AG, Tübingen, Germany). After passing a 50 μm pinhole, the emission light was detected by two avalanche photodiodes operated in the single photon counting mode. As the second laser source a picosecond pulsed diode laser (λ~ex~ = 405 nm) was used to excite mm239. For the pulsed interleaved excitation the 532 nm laser pulse was electronically delayed by 23.5 ns with respect to the 405 nm laser pulse. The donor (mm239) and acceptor (R6G) emissions were separated using a beam splitter (FF552-Di02--25 × 36, Semrock, USA). Suitable bandpass filters (SP492 nm and LP560 nm in the donor and in the acceptor detection path, respectively) were inserted to further eliminate the excitation wavelength and reduce spectral crosstalk. All experiments were performed at room temperature (RT, *T* = 25 ± 1 °C). The data acquisition and the calculations of the autocorrelation curves were performed using the SymPhotime software (PicoQuant, Germany). All other data sets were further analyzed using Origin (OriginLab, Corp., USA). The calibration was carried out by measuring Rhodamine 6G in PBS with a diffusion coefficient of (4.14 ± 0.1) × 10^--6^ cm^2^/s at 25 °C to determine the focal volume (described by an optical plane *w*~o~ and an optical axis *z*~o~). The authors declare no competing financial interest. Ms. Hoa T. Hoang is grateful for funding through Excellence Initiative of the German Research Foundation (DFG) (School of Analytical Sciences Adlershof (SALSA)) (project number 8711110399).

1 Introduction ============== Sequencing distant relatives is an established approach to identify causal variants in Mendelian disorders, but is also increasingly applied to identify genetic risk variants in complex disorders. When familial aggregation of a phenotype is observed at a rate much higher than the prevalence in the general population, one possible explanation can be the segregation of a rare and highly penetrant disease variant. We recently devised a statistical framework for such a setting, based on the notion that sequencing DNA in extended multiplex families can help to identify such high penetrance disease variants too rare in the population to be detected through tests of association in population-based studies ([@bty976-B1]). Specifically, when several affected subjects per family are sequenced, evidence that a rare variant may be causal can be quantified from the probability of sharing alleles by all affected relatives given it was seen in any one family member under the null hypothesis of complete absence of linkage and association. We presented a general framework for calculating such sharing probabilities when two or more affected subjects per family are sequenced, and show how information from multiple families can be combined by calculating a *P*-value as the sum of the probabilities of sharing events at least as extreme ([@bty976-B1]). Sequencing three affected second cousins from a family with multiple oral cleft cases, we successfully employed this approach to identify a causal nonsense mutation in the gene CDH1 ([@bty976-B2]). Recently, we extended the methodology to allow for gene- or region-based tests, and partial sharing of variants ([@bty976-B3]). We also investigated sharing of rare copy number variations, and implemented a global test for an enrichment of variant sharing ([@bty976-B4]). The novel Rare Variant Sharing software package RVS is an open source implementation of these methods. The software builds on existing infrastructure for family-based genetic studies and probability propagation in graphical networks (probabilistic expert systems) to calculate sharing probabilities in pedigrees. RVS differs from other methods commonly employed for sequence data analyses in pedigrees as no parental or founder data is required (which can be of great advantage, as it can be difficult to obtain DNA from past generations), only affected subjects are sequenced, and estimates of population variant frequencies are not required ([Supplementary Material](#sup1){ref-type="supplementary-material"}). 2 Features ========== 2.1 Calculating sharing probabilities and *P*-values ---------------------------------------------------- The primary function `RVsharing` of the RVS package is to calculate exact variant sharing probabilities in extended multiplex pedigrees. The genetic variant can be a single nucleotide variant (e.g. [@bty976-B2]) or a copy number variant (e.g. [@bty976-B4]). Given the variant has been observed in the family, what is the probability that it is shared identical-by-descent by all sequenced affected relatives? For a single family, this probability can be interpreted directly as a *P*-value for testing the null hypothesis of absence of linkage and association. For example, the sharing probability of the CDH1 nonsense mutation among the three second cousins with oral clefts is 1/745 ([Fig. 1](#bty976-F1){ref-type="fig"} top, and [@bty976-B2]). For variants seen in more than one family, the *P*-value can be obtained as the sum of the probability of events at least as extreme as the observed sharing, and can be calculated using the function `multipleFamilyPValue`. The function `multipleVariantPValue` generalizes `multipleFamilyPValue` across multiple variants. This function also provides a filtering option based on the 'potential *P*-values', which is the significance a variant could achieve if it was shared by all affected, and usually only variants that could pass a multiple comparisons correction are retained in an analysis ([@bty976-B1]). Of note, even if no individual variant might achieve a significant *P*-value, it is possible that all variants considered jointly exhibit more sharing than can be explained by random chance ([@bty976-B4]). The `enrichmentPValue` function can compute a single *P-*value to test for such an enrichment of variant sharing. For the analysis of a genomic region such as a gene, the function `RVgene` implements the *P*-value computation described in [@bty976-B3], and also offers a partial sharing test which considers probabilities of sharing among a given subset of the affected subjects, calculated using the `carriers` argument in the `RVsharing` function. Both `multipleFamilyPValue` and `RVgene` take as input objects of class `snpMatrix` storing single nucleotide variant genotypes extracted from ped or Variant Call Format (vcf) files by functions from the `VariantAnnotation` and `snpStats` Bioconductor packages. ![**Top:** Pedigree with three affected second cousins sharing a rare nonsense variant in gene CDH1. Affected subjects in this family are indicated by filled left parts of the symbols. Individuals 402, 404 and 405 were sequenced, indicated by shaded right parts of the symbols. **Bottom:** The exact sharing probabilities (in percent, y-axis) as a function of variant frequency (in percent, x-axis) for the three second cousins. The RVS sharing probability calculated under the assumption of 'no IBS without IBD' is 1/745. The dotted gray line indicates significance after a Bonferroni correction for 16 potentially causal variants analyzed ([@bty976-B2]). A population allele frequency of up to 2.05% (vertical line) would have still yield significant variant sharing](bty976f1){#bty976-F1} 2.2 Follow-up and sensitivity analyses -------------------------------------- The two key assumptions in the variant sharing methodology are that the damaging variant is sufficiently rare in the population that only one founder introduces it into the pedigree (i.e. identical-by-state implies identical-by-descent) and that the founders are unrelated. Violations of either of these assumptions inflate the actual sharing probability, and thus can lead to false positive identifications. The first assumption is critical when the allele frequency of the damaging variant is unknown, which is commonly the case. Using the argument `alleleFreq` in the `RVsharing` function allows for a fast and convenient sensitivity analysis of the rare variant assumption. Exact sharing probabilities in the pedigree can be calculated for any assumed population rare variant allele frequency by making extensive use of the underlying probabilistic expert system, propagation the genotype probabilities from the founders to the sequenced subjects. For the CDH1 nonsense mutation ([@bty976-B2]) a population allele frequency of up to 2.05% would still have yielded a significant sharing probability under a Bonferroni correction, where a total of 16 potentially causal variants were assessed ([Fig. 1](#bty976-F1){ref-type="fig"}, bottom). When founders of the pedigree are related, computing the variant sharing probabilities becomes even trickier. The correction described in [@bty976-B1]) uses the mean kinship coefficient among founders passed as argument to the RVsharing function using the `kinshipCoeff` parameter. When estimates of kinship coefficients are only available for descendants, the `ComputeKinshipPropCoef` function can be used to infer the mean kinship coefficient of the founders. Any complex relationships among founders can be specified using the argument `founderDist`. In the RVS implementation, sharing probabilities in pedigrees with inbreeding can be calculated directly and no longer require the gene-dropping approach described in [@bty976-B1]). Gene dropping remains available by specifying the `nSim` argument in `RVsharing`. 3 Conclusion ============ Rare genetic variant sharing analysis is a powerful approach to identify causal variants underlying complex disease risk. The open source RVS Bioconductor package provides convenient functionality to carry out such analyses with excellent scalability (Table 3 in [@bty976-B3], and [Supplementary Material](#sup1){ref-type="supplementary-material"}). To facilitate reproducible research, a software vignette with an example of a workflow is provided at the Bioconductor website. Supplementary Material ====================== ###### Click here for additional data file. The RVS package includes some basic code written by Samuel G. Younkin, which we gratefully acknowledge. The previous RVsharing CRAN package has been deprecated. Funding ======= This work was supported by NIDCR \[grant R03-DE-02579 to I.R.\]. *Conflict of Interest*: none declared.

BR : brassinosteroid CPD : constitutive photomorphogenesis and dwarfism EBL : 24-epibrassinolide IEF : isoelectric focusing

RNA serves important and diverse functions inside the cell ========================================================== In 1981, Thomas Cech and colleagues observed self-splicing RNA in a 26 S rRNA precursor[@b1][@b2]. In 1983, Sidney Altman found that ribonuclease P could cleave tRNA in the absence of protein[@b3]. In 2002, it was discovered that even mRNAs could bind small metabolites and regulate protein expression[@b4][@b5][@b6]. Today, RNA is recognized as extensively active, with roles in regulating genes, preparatory cleavage, metabolite sensing, and immune response. RNAs achieve this diverse activity through intricately regulated structure, with catalytic RNAs such as riboswitches maintaining highly conserved functional regions[@b7][@b8]. RNA chemistry and the need for accurate structures ================================================== RNA structure is a challenge to determine experimentally because it can fold into many different structures. For example, during RNA transcription, synthesized RNA regions fold locally[@b9], sampling hairpins and short-range motifs. After transcription completes, RNA molecules are able to fold completely and sample long-range interactions[@b10]. With the numerous structures available for RNA to fold into, long-lived misfolded RNA intermediates often occur[@b11][@b12][@b13]. In addition, heterogeneous folding pathways exist for the same RNA sequence[@b14][@b15][@b16][@b17][@b18][@b19][@b20]. As a result, RNA has a highly dynamic folding landscape, which is challenging to capture using techniques such as x-ray crystallography and NMR spectroscopy[@b21][@b22]. Further, due to only recent interest in the diversity of RNA function in biology, there is a deficiency in available RNA experimental structures. However, RNA structure is key to understanding its function and for development of RNA-based applications. Due to the lack of available experimental structures of RNA, computational models of RNA are vital to predict RNA structures. Secondary structure methods for RNA =================================== Currently, there are a variety of structure prediction methods available to elucidate RNA structure. Secondary structure prediction methods predict base pairing contacts for a given RNA sequence[@b23]. If homologous sequences exist, comparative sequence analysis[@b24][@b25][@b26][@b27] remains the most accurate secondary structure technique. One of the most popular secondary structure prediction methods is dynamic programming. Using nearest neighbor energies[@b28] and the sequence of the RNA, dynamic programming methods, such as Mfold[@b29][@b30] or ViennaRNA[@b31][@b32][@b33], exhaustively compare and build secondary structures to achieve the minimum free energy structure. However, dynamic programming schemes face certain limitations[@b34], such as difficulty predicting pseudoknot structures. Various secondary structure programs[@b35][@b36][@b37] have been developed to predict the folding of these structures. Recently, it has been shown that incorporating results from the experimental method SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension)[@b38] can moderately increase accuracy of secondary structure prediction[@b39][@b40][@b41][@b42][@b43][@b44]. Despite its utility, secondary structure prediction is ultimately limited to 2-D base paired RNA structures. For RNA based therapeutics and *de novo* design, 3-D RNA structure must be determined. 3-D structure prediction models =============================== Tertiary or 3-D structure prediction methods use template, graph theory, and physics based modeling to sample and predict relevant 3-D RNA structures[@b45][@b46]. Template based modeling uses predefined, small motifs to assemble RNA structures from their sequence. Template based models include the MC-Fold/MC-Sym pipeline[@b47], BARNACLE[@b48], RSIM[@b49], 3dRNA[@b50], RNAComposer[@b51], Vfold[@b52][@b53][@b54][@b55], RNA-MoIP[@b56] and FARNA/FARFAR[@b57][@b58][@b59] available in the Rosetta package[@b60]. Similar to template based modeling, ASSEMBLE[@b61] and RNA2D3D[@b62] use homologous RNA structures to predict the new RNA structure (with manual refinement available). In graph theory techniques, RNA is depicted topologically to build RNA structures; this improves sampling and even allows for creation of novel RNA motifs. Graph theory techniques[@b63] are utilized by RAG/RAGTOP[@b64][@b65][@b66][@b67] and others[@b68][@b69][@b70][@b71]. In physics based methods, the RNA is built from sequence into a 3D structure, and these 3D RNA structures are sampled using Monte Carlo or Molecular Dynamics (MD) protocols. Due to the high charge density of RNA and the associated large computational cost to sample structures, many tertiary structure models use coarse-grained representations of RNA[@b72]. In coarse-grained (CG) models, atomic sites are grouped together and represented as a "bead" or pseudoatom. Typical coarse-grained models depict a few pseudoatoms per nucleotide. This results in a reduction in the degrees of freedom and lowers the simulation cost of the model, as compared with simulating the all-atom structure. Physics based coarse-grained models with one pseudoatom per nucleotide include YAMMP/YUP[@b73][@b74], an adaptable user input required model, and NAST[@b75][@b76], which assumes ideal helices from secondary structure and uses MD and clustering to build loops. iFoldRNA[@b77][@b78], Denesyuk *et al*.[@b79][@b80], and TOPRNA[@b81][@b82][@b83] use three pseudoatoms per nucleotide to depict phosphate, sugar, and nucleobase groups. iFoldRNA uses discrete Molecular Dynamics and replica exchange Molecular Dynamics to sample structures, with non-bonded parameters decomposed from nearest neighbor energies. Similarly, the model by Denesyuk *et al*.[@b79][@b80] derives its parameters from nearest neighbor energies and experimentally determined structures. TOPRNA captures effects of secondary structure constraints on loop conformations and free energies. HiRE-RNA[@b84][@b85][@b86] depicts six-seven pseudoatoms per nucleotide with five pseudoatoms along the backbone. SimRNA[@b87][@b88], Bernauer *et al*.[@b89], as well as the previous generation and current RACER model studied[@b90][@b91][@b92], all represent RNA with five pseudoatoms per nucleotide. SimRNA uses a Monte Carlo sampling algorithm with parameters from statistical potentials. The model by Bernauer *et al*. similarly uses statistics from high-resolution crystal structures for parameterization yet also derives all-atom potentials for structure refinement. The RACER RNA Model =================== The CG RNA model RACER (RnA CoarsE-gRained) developed and applied in this work is a physics-based model, derived from RNA structural statistics, refined using RNA thermodynamics, and applied in molecular dynamics simulations of folding and complexation of RNAs. In the results section, we first introduce the potential energy functions used in the RACER model, with a focus on the newly implemented effective vdW potential. Second, we demonstrate how RACER parameters were optimized using statistical potentials derived from PDB statistics. Additionally, we provide motivation for modeling RNA as a modeling RNA as a 1D molecule and the associated 1D correction we made to the non-bonded PMFs. Third, we show how we validated RACER using simulated annealing simulations for RACER structure prediction capability and generation of funnel free energy landscapes. Fourth, we apply RACER to generate folding free energy predictions for a testing set of RNA hairpins and duplexes, and we compare our results to experiments. In the discussion section, we summarize the changes made to the RACER model and emphasize RACER's ability to capture folding free energies and to predict structures. In the methods section, we show (1) the ability of RACER to map between all-atom and coarse-grained representations for use in multiscale simulations, (2) details on the folding free energy calculations, and (3) implementation instructions for those wishing to use RACER. Results ======= Model ----- ### Potential energy functions The total potential energy function of the RACER model includes bond stretching, angle bending, torsion, effective vdW, hydrogen bonding, and electrostatics, labeled as *E*~*bond*~, *E*~*angle*~, *E*~*torsion*~, *E*~*vdW_eff*~, *E*~*hb*~, and *E*~*ele*~ respectively (see [Eq. 1](#eq1){ref-type="disp-formula"}). The RACER model is currently implemented in TINKER[@b93]. In RACER, RNA nucleotides consists of 5 pseudoatoms per nucleotide, with a total of 9 pseudoatom types (shown in [Fig. 1](#f1){ref-type="fig"}). The RACER model used here differs from previous publications[@b90][@b91] in that we employ a novel effective vdW potential to better capture the short-range non-bonded interactions among the pseudoatoms, which we found to be essential for correctly capturing the folded state. As a result, we had to re-parameterize the other non-bonded contributors including the electrostatics and hydrogen bonding potential. ### Bonded Potential Energies The potential energy functions which retain the same functional form between the previous model and RACER are the bonded potential energy functions. Bond and angle potentials are represented by harmonic terms: and . The torsion potential of [Eq. 2](#eq4){ref-type="disp-formula"} uses the first 3 terms of a Fourier series expansion for the torsion potential, where *ϕ* is the torsion angle, and *k*~*n*~ and *δ*~*n*~ are the spring constant and phase angle of expansion term *n*. ### Improved Effective vdW Potential The RACER model includes a newly implemented effective potential (vdW~eff~) that significantly improves the fit of RACER to non-bonded statistical potentials. In the previous model[@b92] the vdW-like non-bonded potential was modeled using a Buckingham function. However, this was found to significantly overestimate repulsion at short distances when compared with statistical potentials. The new effective vdW~eff~ potential ([Eq. 5](#eq12){ref-type="disp-formula"}) allows for tuning the repulsion at short distances through a third parameter γ, enabling a closer fit to the statistical non-bonded potential of mean force (PMF) ([Fig. 2](#f2){ref-type="fig"}). The vdW~eff~ does not represent the true vdW interaction, but rather the potential of mean force between a pair of pseudoatoms. However, based on statistical potentials, the non-bonded interactions between most pairs of pseudoatoms we sampled exhibited vdW potential-like behavior. The new functional form for vdW~eff~ potential taken from ref. [@b94] is shown in [Eq. 3](#eq8){ref-type="disp-formula"}, where ε is the minimum well depth and σ is the distance of minimum energy, and γ is a parameter allowing for fine-tuning of the slope of the short-range interaction. [Figure 2b](#f2){ref-type="fig"} presents a comparison between the vdW~eff~, Lennard Jones, and Buckingham potentials while [Fig. 2c--e](#f2){ref-type="fig"} show the effects of the three parameters σ, ε, and γ on the vdW~eff~ potential. The combining rules for unlike pseudoatom types *i* and *j* in the vdW~eff~ potential are: , , and . ### Hydrogen Bond and Electrostatics Energies The hydrogen bond ([Eq. 4](#eq11){ref-type="disp-formula"}) and Debye-Huckel electrostatics ([Eq. 5](#eq12){ref-type="disp-formula"}) potential energy terms are of the same form as used previously. However, we reparametrized the hydrogen bond and Debye-Huckel potentials with the introduction of the new vdW~eff~ term. In the hydrogen bond potential *ε*~*hb*,*max*~ is the maximum potential found at the hydrogen bond equilibrium distance *σ*~*hb*,*eq*~. is the magnitude of the vector from atom j to atom i, while is a directional component with *θ*~*i*~ and *θ*~*j*~ defined in [Fig. S1](#S1){ref-type="supplementary-material"}. For hydrogen bond parameterization, the maximum potential *ε*~*hb*,*max*~, was increased from 0.5 kcal/mol to 2.0 kcal/mol. Other hydrogen bond parameters including equilibrium distance *σ*~*hb*,*eq*~ of 2.9 Å and cutoff of 6 Å (base edge) remain the same as the previous model. Hydrogen bond potential energy is computed for both canonical (GC, AU) and noncanonical base pairs. For Debye-Huckel [Eq. 5](#eq12){ref-type="disp-formula"}, *q*~*i*~ is the charge of atom i, *r*~*ij*~ is the distance between atom i and atom j, *D* is the dielectric constant, and *ξ* is the Debye length. A dielectric constant *D* of 25 was determined to be optimal under the new model potential, compared to 78 from the previous model. In depth discussion of Debye-Huckel and hydrogen bond optimization can be found in the SI. Model improvement and Parameterization -------------------------------------- ### Statistical potentials The premise of our parameter optimization was to fit to both RNA structure and experimental free energies. First, we updated model statistical potentials from experimentally determined crystal structures. We downloaded all available Protein Data Bank (PDB, <http://www.rcsb.org/>) RNA structures as of RNA structures as of Feb. 10, 2015, (excluding RNA-protein and RNA-DNA combination structures) totaling \~1100 entries. Our previous model fit to statistical potentials used approximately 668 structures. For RACER, our updated parameterization includes an additional \~400 structures, which led to various modifications in the potentials. The method of statistical potentials involves fitting energy functions to statistically derived potential of mean force (PMF) curves. The PMFs are determined by taking the probability distribution *P*(*r*) of occurrences from the PDB structure set and then extracting the free energy *G*(*r*), with the reference distribution *ref* setting the minimum interaction at 0 kcal/mol. One of the major improvements in the current model is to adopt a new nonbonded effective potential form to capture the intricate short range behavior observed in nonbonded statistical potentials that standard vdW potential forms (including our model's previously used Buckingham potential) cannot capture. The Buckingham and other common vdW functions are too stiff at short range with a steep slope, whereas the nonbonded statistical potentials reveal much softer behavior. We have identified a more "flexible" vdW~eff~ potential that better captures this short range behavior, which is critical for local packing of RNA molecules. When we implemented the new potential, it was also necessary to re-parameterize the torsion, electrostatics, and hydrogen bond interactions for consistency. ### 1-D PMF for RNA In this work, we determined that modeling RNA as one-dimensional rather than a three-dimensional, isotropic molecule is more appropriate when extracting the statistical potentials from PDB structures. This choice is justified as there is an abundance of short, linear helices found in PDB structures of RNA. Additionally, folded RNA typically forms prolate ellipsoids[@b95]. Similarly, in the PDB structure of 16S rRNA more than half of the nucleotides are base paired[@b24]. Therefore, treating RNA as a one-dimensional molecule for capture of local interactions is not unreasonable. Additionally, 3D PMFs are more appropriate for systems with isotropic distance distributions, such as molecular liquids[@b96][@b97][@b98] and proteins[@b99][@b100][@b101]. Our motivation for modeling RNA as a 1D molecule came from the observation of divergence of 3D radial distribution functions (RDF) at distances greater than 10 Å, and as a result the potential of mean force (PMF) that was derived from the RDF did not converge to zero at large separation (see [Fig. 3](#f3){ref-type="fig"}). The cause of this divergence at long distances is the inherent volumetric effect of the 3D RDF, while the PDB structures we sample are mostly small and linear. The statistical potentials do include some larger ribosomal structures, but these are too few to cause the observed divergence. Contrary to 3D RDFs, when 1D radial distribution functions were used the PMF asymptotically approached zero for long distances (see [Fig. 3](#f3){ref-type="fig"}), reinforcing the discussion that the set of RNAs used here in statistical potentials can be adequately sampled as linear 1D, rather than 3D RDFs. The main difference between 3D and 1D RDFs is the normalization factor. For 3D RDFs, normalization is done over a volumetric shell *4πr*^*2*^*dr*, whereas 1D RDFs normalizes over an incremental distance, *dr*. Specifically, the non-bonded PMF is evaluated via Boltzmann inversion as where *g*(*r*) is the radial distribution function, normalized probability function discussed above. When treating RNA as a 3D isotropic molecule, the 3D RDF, as was done previously[@b90][@b91], is given by , where *n*~*ij*~(*r*) is the number of atom type *j* at distance *r* from atom type *i, N*~*i*~ and *N*~*j*~ are the total number of *i* and *j* atoms respectively, and *V* is the volume of the system. Now we treat RNA as a "1D", linear molecule to more adequately parameterize the vdW~eff~ potential, and the RDF becomes . Structure Prediction -------------------- ### Folding RNA by simulated annealing We tested RACER with simulated annealing simulations to (1) validate that RACER can accurately fold experimentally determined RNA structures and to (2) ensure the native structure has the lowest energy on its energy landscape. We ran simulated annealing simulations on a testing set of 14 RNAs, duplexes and hairpins, that have known experimentally determined structures[@b90]. This test set of 14 RNAs was included as part of the 1100 structures used to compute our statistical potentials; however, the contributions of the 14 RNA test set (\~1% of training set) to the statistical potentials and thus parameterization is negligible. From annealing simulations on this set of 14 RNAs, RACER is able to predict 13 out of 14 RNA molecules with RMSD \< 5 Å, and 6 RNA molecules with RMSD \< 2.5 Å. The average RMSD between the predicted lowest-energy structures and native structures is 2.93 Å. This average RMSD is improved from our previously published average RMSD of 3.31 Å; additionally, our model now has the capability to predict free energy landscapes of RNA in addition to structure prediction. The simulated annealing protocol involved running MD sequentially for 5 ns at temperatures in order of 298(K), 400, 1000, 900, 800, 700, 600, 500, 400, 298 K, for a total simulation time of 50 ns, with structures saved every 10 ps. Given the high temperatures used, we used a 1fs time step for annealing simulations. Results for structure prediction using simulated annealing are given in [Table 1](#t1){ref-type="table"}. These predicted RMSD values are calculated between PDB structures and the minimum potential energy structures found by RACER. ### Energy landscapes Analyzing the energy landscapes of the 14 RNAs in our training set was an important part of our optimization. RNAs are complex molecules that may adopt stable and long lived misfolded structures. However, it is assumed the final native structures, at least *in vitro*, should have the lowest free energy for the given environment[@b102]. Here, annealing simulations are used to generate a large number of unfolded structures for each RNA. Each of these structures is then energy minimized to 0 K. The energy and RMSD (with respect to the native structure) of each structure are used to characterize the energy landscape. The energy-RMSD landscapes for all 14 RNAs are given in SI, [Table S1](#S1){ref-type="supplementary-material"}. The energy vs RMSD landscapes for all 14 RNAs show clear "funnel" shapes skewed toward the native structure. As examples, we present the energy landscapes for two favorably predicted structures (157D and 1AL5, 1.45 Å and 1.26 Å RMSD repectively) in [Fig. 4a,b](#f4){ref-type="fig"}, and the energy landscapes for the two most unfavorably predicted structures (1F5G and 1I9X, 8.91 Å and 4.56 Å RMSD respectively), where the lowest energy structures have large RMSD in [Fig. 4c,d](#f4){ref-type="fig"}. RACER predicted structures for PDB ID: 157D, 1AL5, 1F5G, and 1I9X are shown in [Fig. 4](#f4){ref-type="fig"}. RACER predicted structures 157D and 1AL5 agree well with experiment (inset in [Fig. 4a,b](#f4){ref-type="fig"}). The RACER predicted structure for 1F5G (8.91 Å RMSD) has collapsed into a torus-like structure, with very little backbone twist (inset in [Fig. 4c,d](#f4){ref-type="fig"}). A possible explanation for this observed behavior is the non-canonical base pairing present in 1F5G. While RACER can capture non-canonical base pairing through the hydrogen bond potential, these hydrogen bonds need further calibration relative to canonical interactions. The RACER predicted structure for 1I9X (4.56 Å RMSD) forms an extended helix compared to the crystal structure. This is likely due to two bases flipped out of the helix in the crystal structure, while RACER incorporates these bases back into the helix. In the crystal structure for 1I9X, several water molecules stabilize these bases. In the RACER model, this stabilization is challenging to capture due to the implicit treatment of solvent via the Debye-Huckel potential. Additionally, energy landscapes allow us to identify possible meta-stable intermediates, which are high-RMSD (\~8 Å) "local" funnels observed in plots for 1DQF and 1QCU. The meta-stable structure of 1DQF at the local minimum, shown in [Fig. S2](#S1){ref-type="supplementary-material"} resembles the toroidal structure observed for 1F5G, but for 1QCU an extended, base stacking meta-stable structure is observed. For 1DQF, the local funnel structure has increased torsional potential energy (\~30 kcal/mol) over the global-minimum structure, although both have similar vdW~eff~ and hydrogen bond potentials. For 1QCU, the local funnel structure has a more stabilizing hydrogen bond potential (\~−15 kcal/mol) than the global-minimum structure; however, in the global-minimum structure, the Deby-Huckel electrostatics and vdW~eff~ potentials compensate hydrogen bonds to result in an overall more stabilizing intermolecular energy than the local-funnel structure. It is important to note that for both of these RNAs, the RACER global-minimum structure is very close to the experimental structures. In the process of validating and optimizing our model by energy landscape analysis, we noticed the importance of a dedicated hydrogen bond potential for base paring, as the vdW~eff~ potential is not well suited for distinguishing between base stacking and base pairing interactions[@b103]. The hydrogen bond potential allows for directional base pairing and helps in separating the base stacking and base pairing interactions effectively. Equilibrium Pulling Simulations ------------------------------- ### Experimental free energies To test RACER, we focused on capturing experimental melting free energies of canonical helices[@b104] and hairpins[@b105]. We used RACER to perform equilibrium pulling simulations, and we compared free energy differences to two sets of experimental thermodynamic data: RNA melting free energies from Turner and coworkers[@b28] and folding free energies from single molecule force experiments. Five hairpins of size 10, 10, 12, 14, and 18 nt and five duplexes of size 6, 6, 8, 8, and 10 base pairs were selected from melting free energy experiments, and the TAR RNA hairpin was chosen to compare RACER to single molecule force experiments. Hairpin sequences 30, 11, 33, 47, and 19 from the [Supplementary Information](#S1){ref-type="supplementary-material"} of[@b105] are referred to here as h1, h2, h3, h4, and h5, and duplex sequences 35, 48, 71, 78, and 90 of[@b104] are referred to here as d1, d2, d3, d4, and d5. TAR is a 52 nt, 21 bp hairpin with two internal loops. In melting free energy experiments, a solution of RNAs of known sequence are heated while measuring UV absorption. As helical and single stranded RNAs absorb light at different wavelengths, the absorption will change over heating as the RNA denatures. By fitting a curve to absorption vs temperature the melting free energy can be determined[@b106][@b107][@b108]. Turner and co-workers have published a compendium of melting free energies for small RNA motifs and structures using nearest neighbor energy parameters and RNA secondary structure prediction[@b28][@b104][@b109]. Additionally, we compared our model to RNA single molecule force experiments. In single molecule force experiments, folded RNA molecules are unfolded by mechanical force using techniques such as optical tweezers or atomic force microscopy. Using the end-to-end extension as a reaction coordinate, the free energy of unfolding can be determined from position vs. time data. A recent single molecule research study of the trans activation response (TAR) element of HIV extracted the free energy of folding at zero force under the assumption of the worm-like chain model[@b110]. Here we study the same TAR RNA as used in the single molecule force experiments. Melting and pulling experiments for all RNAs were simulated by umbrella sampling simulations pulling the RNAs apart from their ends (see [Fig. S3](#S1){ref-type="supplementary-material"} for example simulation setup showing end-to-end reaction coordinate). Free energy values were then computed using the Weighted Histogram Analysis Method (WHAM) software distributed by Alan Grossfield[@b111]. Details of these simulations are included in the Methods section. Although exact energy landscapes at equilibrium for both TAR and melting free energy helices are unknown, folding free energies can be computed according to [Eq. 6](#eq17){ref-type="disp-formula"}. The folded free energy, *ΔG*, is found by integrating over all folded conformations at end-to-end extension *r* with free energy *Δω*. Folded free energy is then normalized to volumetric entropy, with standard state volume *V*~*ref*~ of 1660 Å^3^. *kT* is the Boltzmann constant multiplied by temperature (298 K). ### Unfolding free energies from RACER MD simulations The free energies computed from equilibrium pulling MD simulations (WHAM) using RACER are in excellent agreement with experimental measurements, with a correlation coefficient (R^2^) of 0.93 for 11 RNAs tested ([Table 2](#t2){ref-type="table"} and [Fig. 5](#f5){ref-type="fig"}). For additional comparison, we also included the melting free energies from Mfold, a widely-used secondary structure prediction program that has been parameterized using the experimental melting thermodynamic data (Mfold predicted structures are shown in [Fig. S4](#S1){ref-type="supplementary-material"}). The unfolding free energies evaluated by RACER and Mfold[@b30] are presented in [Table 2](#t2){ref-type="table"} along experimental values and the length of each MD simulation. The correlation plots for RACER and Mfold show both models have close R^2^ correlation coefficients of 0.93 and 0.96 respectively. However, Mfold's linear fit has a slightly higher slope (1.5) than RACER (1.2) as Mfold over predicts the stability of the duplexes. Note that RACER is a 3D particle based physical model developed for molecular dynamics simulations, whereas Mfold predicts secondary structures from sequences based on nearest neighbor energy parameters. In RACER we explicitly compute the entropy contributions to the free energy through molecular dynamics sampling. ### Pulling generated RNA structures Ensemble model structures for folded states are shown in [Figs S5 and S6](#S1){ref-type="supplementary-material"}. In the folded states, TAR, h4 and h5 are observed to form helices while h1--h3 form base pairs and stacking interactions but without regular helical structure. For duplexes, the two RNA strands form canonical base pairs resulting in proper helices. The terminal nucleotides of d5 are observed to break base pairing with one nucleotide rotating out of the helix while the other remains stacked, but this is also observed in experiment[@b112]. In pulling experiments, free energy vs end-to-end extension plots show two distinct energy minima corresponding to folded and unfolded states[@b113][@b114][@b115]. In the RACER model unfolded (extended) states remain stabilized by vdW~eff~ base stacking interactions, so the location of unfolded free energy is difficult to determine directly from free energy landscapes of RNAs. While the free energy landscapes predicted by RACER show an energy well around the folded state, there is a flat to monotonically increasing curve observed at large extensions ([Figs 6](#f6){ref-type="fig"} and [7](#f7){ref-type="fig"}, blue curve, also see [Fig. S7](#S1){ref-type="supplementary-material"}). The location of the unfolded state is paramount to computing the folded free energy ΔG using [Eq. 6](#eq17){ref-type="disp-formula"}. To determine unfolded state location, we plotted the gradient of the free energy, the 'force' as a function of extension ([Figs 6](#f6){ref-type="fig"} and [7](#f7){ref-type="fig"}, black curve). From these force vs. extension plots, the predicted free energy of the unfolded state was taken to be the free energy value where the force is very low (\~0.1 kcal/mol/Å), i.e. before the RNA reaches the over-stretched regime ([Figs 6](#f6){ref-type="fig"} and [7](#f7){ref-type="fig"}, red lines). A 4 Å running average of 'force' over extension was used to eliminate noise ([Figs 6](#f6){ref-type="fig"} and [7](#f7){ref-type="fig"}). Histogram figures showing equal sampling of the pulling windows are included in [Figs S8 and S11](#S1){ref-type="supplementary-material"}. Additionally, the uncertainty of the free energy landscape as computed by a Monte Carlo bootstrap error analysis in the WHAM program by Alan Grossfield[@b111] is shown as a range in [Figs S12 and S14](#S1){ref-type="supplementary-material"}. Discussion ========== Statistical potential summary ----------------------------- RACER, a coarse-grained RNA model, can accurately predict native structures and capture RNA folding free energy. The functional forms and parameters in RACER were determined by systematic optimization against native structures and melting free energies for a number of RNA molecules. We found that the statistical potentials[@b92] used in the previous model were over stabilizing and the 3D PMFs diverged at long distances. As a result, we treat RNA as a one-dimensional rather than three-dimensional molecule, and use a 1D RDF when fitting to PMFs. Our optimization procedure led us to incorporate a more general effective van der Waals potential energy function (vdW~eff~) to describe the interactions among pseudoatoms. As a result of implementing a new non-bonded potential energy, we have also reparametrized both electrostatic and hydrogen bond potential energy functions. As the RNA backbone is highly charged, a Debye-Huckel electrostatics term is included for each phosphate pseudoatom; a dielectric of 25 was chosen in order to capture both folded and unfolded RNA structures. A directional hydrogen bond potential was reparametrized in order to accurately distinguish base pairing (hydrogen bond, some vdW~eff~) and base stacking (vdW~eff~) interactions. We found that the hydrogen bond potential was pivotal to accurate folding free energies as both folded and unfolded RNA have base stacking interactions, while only folded RNA have base pairing (hydrogen bond) interactions. Thermodynamic summary --------------------- For a structure prediction model, thermodynamic accuracy is important to ensure that the energy landscape correctly represents RNAs with varying size and sequence. Our energy landscape analysis suggests that even relatively small RNAs may have complex energy landscapes, and there are many RNA structures at low potential energy. Therefore, explicit consideration of entropy through techniques such as MD is crucial to capture the free energy landscapes of RNA structures. Folding free energy values for six RNA hairpins of size 10--52 nts and five duplexes of size 6--10 bp were determined by umbrella sampling simulations with WHAM-computed free energy. For hairpins, we determined that umbrella sampling simulations with a reaction coordinate of end-to-end extension is appropriate for capturing folding free energy. For duplexes, the same protocol is found to be appropriate, with the addition of a restraint preventing the single strands from long-lasting intra-strand interactions (e.g. hairpin-like structures). Pulling free energy landscapes of hairpins and duplexes clearly revealed the folded state and we used the gradient (force) of pulling free energy to define the location of the unfolded state. Given the low computational cost of RACER, over 0.8 ms of umbrella sampling and simulated annealing simulations are presented. Overall, the MD-calculated free energy results using the RNA model are in excellent agreement (R^2^ = 0.93) with experimental folding free energy values while preserving accurate structure prediction. In this work, we present RACER, a novel RNA coarse-grained model that captures both RNA structure and thermodynamics for increased utility to RNA folding investigations. Methods ======= Mapping from all-atom to coarse-grained structures -------------------------------------------------- A notable feature of our model is the ability to map to and from all-atom experimental crystal structures. Each of our model's pseudoatoms represents an atomic site in nucleotides; for example, the sugar pseudoatom is assigned the C4' atom position on ribose. Moreover, our model captures the planarity of the nucleobase with three pseudoatoms. Given a novel (structure undetermined) RNA sequence, our model can first predict the three-dimensional structure in coarse-grained coordinates and then map to all-atom coordinates with further minimization, producing an equivalent to an all-atom experimentally determined structure. As a result, our RNA model is well suited to perform multiscale simulations in the future. Pulling methods --------------- Melting and pulling experiments are modeled by using umbrella simulations pulling the RNA molecule apart from its terminal ends. A harmonic potential of 1 kcal/mol/Å^2^ spring constant is used to restrain the RNA ends at the sugar pseudoatoms (C4' sugar atomic site). Simulation extensions ran from 5.5 Å up to fully extended lengths (59.5, 76.5, 86.5, 106.5, and 307.5 Å for 10, 12, 14, 18, and 52 nt hairpins assuming 5.9 Å per nt contour length) with a spacing of 1 Å between windows. Duplexes are similarly pulled apart from the sugar pseudoatoms at one terminal end with a 1 kcal/mol/Å^2^ spring constant; the other terminal end is restrained between two terminal sugar pseudoatoms with a 1 kcal/mol/Å^2^ spring constant. Duplex extensions ranged from 5.5 Angstroms up to fully extended lengths (80.5, 100.5, and 124.5 Angstroms for 6, 8, and 10 base pair duplexes respectively) with umbrella window spacing of 1 Å. For the duplexes and shorter hairpins of size 10 and 18 nt, 1 μs of Molecular Dynamics was run for each window. For the TAR hairpin, 100 ns was found to be sufficient given the longer end-to-end extension (more windows) needed. We used a 4 fs time step for pulling simulations. From the umbrella simulations, the free energy landscapes were computed by the Weighted Histogram Analysis Method[@b116] (WHAM) using the program distributed by Alan Grossfield[@b111]. Computational efficiency of the RACER Model ------------------------------------------- All annealing and pulling simulations (total of 0.86 ms) were computed on a local computer cluster. For all simulations discussed below a 4 fs time step was used, and the CPUs used are an early generation Intel Xeon E5345 2.33 GHz CPU. Using one CPU core for each simulation, 1 μs of simulation of the 10 nt hairpin h1 took 22 hours, 1 μs of simulation of the 18 nt hairpin h3 took \~60 hours, and 100 ns of simulation of the 52 nt hairpin TAR took \~48 hours. Additionally, 1 μs simulation of duplex d35 required 30 hours, while 1 μs for duplex d90 required 74 hours. Recently, RACER has been implemented with OpenMP allowing parallelization to multiple cores. In the future, we will implement our model on GPUs, using the software package OpenMM[@b117]. Implementation of RACER on GPUs will allow for even better efficiency. As a result of the improved computational efficiency offered by the coarse-graining, it will be possible to simulate RNAs at physiologically relevant timescales. Implementation and parameters ----------------------------- The TINKERMD implemented RACER model is available free of charge at <http://biomol.bme.utexas.edu/tinker-openmm/index.php/TINKER-OPENMM:Development-rna>. The parameters and conversion programs are included in the distribution. Conversion tutorials are posted online at <http://biomol.bme.utexas.edu/tinker-openmm/index.php/TINKER-OPENMM:Tutorials-rna>. Additional Information ====================== **How to cite this article:** Bell, D. R. *et al*. Capturing RNA Folding Free Energy with Coarse-Grained Molecular Dynamics Simulations. *Sci. Rep.* **7**, 45812; doi: 10.1038/srep45812 (2017). **Publisher\'s note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Material {#S1} ====================== ###### Supplementary Information We are grateful for support from the Robert A. Welch Foundation (Grant F-1691 to P.R.) and the National Institutes of Health (Grants GM106137 and GM114237 to P.R.). The authors declare no competing financial interests. **Author Contributions** P.R. designed and supervised the research. D.R.B., S.Y.C., and P.R. prepared the manuscript. D.R.B., H.S., and P.R. parameterized the model. D.R.B., and S.Y.C. conducted the pulling and annealing simulations. D.R.B., S.Y.C., and P.R. analyzed the simulations. {#f1} {#f2} {#f3} {#f4} ![Correlation plot between predicted free energy from RACER and experimental free energy in kcal/mol.\ RACER simulation predicted free energy is compared with Mfold minimum free energy as well as unity slope (dashed line). RACER and Mfold have the same correlation free energy predictive capability (R^2^ = 0.93 to experimental free energies, experiment), but RACER has a slope closer to unity (slope = 1.2), while Mfold over-stabilizes the free energy of larger RNAs (slope = 1.5). Error bars present on RACER data come from a Monte Carlo bootstrap error analysis as implemented in WHAM by Alan Grossfield[@b111] (most errors are within the data point).](srep45812-f5){#f5} {#f6} {ref-type="supplementary-material"}).\ Umbrella sampling pulling simulations were run for 1 μs for each window, with a 1 Å window separation. The unfolded state is determined as the state right before the force (derivative of the free energy, curves shown in black) sharply increases from low (\<0.1 kcal/mol/Å) to high due to overstretching. 0.1 kcal/mol/Å and the location of the unfolded state are denoted by the red lines. The folding free energy (ΔG~f~ kcal/mol) is included for each RNA. A 4 Å running average of force (black curves) is shown to eliminate noise.](srep45812-f7){#f7} ###### Predicted (minimum energy) RMSD values compared to Protein Data Bank (PDB) structures from simulated annealing. PDB ID 157D 1AL5 1DQF 1F5G 1I9X 1KD5 1LNT 1QCU 1ZIH 2AO5 2JXQ 2K7E 353D 472D Avg. ---------- ------ ------ ------ ------ ------ ------ ------ ------ ------ ------ ------ ------ ------ ------ ------------- RMSD (Å) 1.45 1.26 3.04 8.91 4.56 2.88 2.65 0.96 3.11 1.83 1.13 3.58 2.24 3.47 2.93 ± 0.54 ###### Unfolding free energy values for RNAs from experiment (Expt.), Mfold predicted, and RACER predicted. Hairpin Length (nt) Expt. ΔG (kcal/mol) Mfold ΔG (kcal/mol) RACER ΔG Length per window (kcal/mol) ------------ ----------------- ---------------------------- --------------------- -------------- ------------------------------ h1 10 −3.5 ± 0.3[@b105][@b118] −5.3 −2.8 ± 0.12 1 μs h2 10 −0.3 ± 0.1[@b105][@b119] +0.9 −1.4 ± 0.12 1 μs h3 12 −4.4 ± 0.2[@b105][@b120] −3.4 −4.8 ± 0.14 1 μs h4 14 −2.2 ± 0.08[@b105][@b121] −2.2 −5.7 ± 0.15 1 μs h5 18 −8.2 ± 0.2[@b105][@b122] −8.4 −7.9 ± 0.22 1 μs TAR 52 ≈−21.5 ± 4.3[@b110] −31.3 −29.7 ± 0.36 0.1 μs **Duplex** **Length (bp)**         d1 6 −7.56 ± 0.3[@b104] −11.4 −7.8 ± 0.15 1 μs d2 6 −4.95 ± 0.2[@b104] −9.8 −7.5 ± 0.14 1 μs d3 8 −12.32 ± 1.2[@b104] −17.0 −12.8 ± 0.17 1 μs d4 8 −10.11 ± 0.2[@b104][@b106] −14.7 −11.1 ± 0.18 1 μs d5 10 −12.69 ± 0.5[@b104] −18.1 −14.0 ± 0.19 1 μs           Total: 860 μs Molecule length in nucleotides or basepairs and the simulation length per window are also shown. Error is take from a Monte Carlo bootstrap error analysis as implemented in the WHAM program by Grossfield[@b111].

Prebiotics were first defined as "nondigestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacterial species already resident in the colon, and thus attempt to improve host health".^[@ref1]^ This concept has been updated and adapted many times,^[@ref2]−[@ref4]^ but there is no doubt that galactooligosaccharides (GOS) make up an important category of prebiotics. GOS are a mixture of oligosaccharides produced from lactose by β-galactosidase enzymes, comprising a number of galactose units, with a terminal glucose or galactose.^[@ref5]−[@ref7]^ They are produced via the double-displacement reaction catalyzed by β-galactosidase enzymes. In the first step, the glycosidic linkage of lactose is cleaved, and the galactosyl unit covalently binds to the enzyme forming a galactosyl--enzyme intermediate while releasing the glucose. In the second step, an acceptor substrate attacks the intermediate, resulting in formation of a product with the galactosyl moiety. β-Galactosidase enzymes perform two reactions depending on the acceptor substrate. With water serving as the acceptor substrate, galactose is released via hydrolysis; with lactose or other carbohydrates serving as acceptor substrates, GOS products are formed via transgalactosylation.^[@ref8]−[@ref11]^ β-Galactosidase enzymes belong to glycoside hydrolase (GH) families 1, 2, 35, and 42.^[@ref12]^ The β-galactosidase from *Bacillus circulans* ATCC 31382 (BgaD, GH2) is a 189 kDa enzyme, and its commercial preparation, Biolacta N5, is widely used in the food industry.^[@ref13]−[@ref15]^ BgaD has an activity toward lactose higher than those of and a thermal stability better than those of other β-galactosidase enzymes.^[@ref16]^ A study investigated the influence of organic solvents on the regioselectivity of BgaD transglycosylation.^[@ref17]^ The product specificity of BgaD with lactose as a substrate was studied using methylation analysis, mass spectrometry (MS), and nuclear magnetic resonance spectroscopy; in this way, 11 GOS structures were identified.^[@ref18]^ A detailed analysis of the commercial product Vivinal GOS, synthesized by the *B. circulans* enzyme, resulted in identification of a total of 43 GOS structures.^[@ref6],[@ref7]^ In *B. circulans*, BgaD is present in different isoforms resulting from C-terminal cleavage by an endogenous protease.^[@ref19]^ Three isoforms were reported by Vetere and Paoletti;^[@ref20]^ four isoforms (BgaD-A, BgaD-B, BgaD-C, and BgaD-D) were reported in the commercial enzyme preparation of β-galactosidase from *B. circulans*.^[@ref19]^ In recent years, these BgaD isoforms have been characterized in more detail, facilitated by their cloning and recombinant overexpression in *Escherichia coli*.^[@ref21]^ All four isoforms have similar transgalactosylation activity at high lactose concentrations.^[@ref9],[@ref22]^ The products synthesized by recombinant BgaD-D were also studied in detail, showing a product profile similar to that of Vivinal GOS.^[@ref6],[@ref7],[@ref10]^ Mutagenesis studies have shown that the C-terminal discoidin domain of BgaD is essential for the hydrolytic activity of the enzyme.^[@ref23]^ A few BgaD-D mutants have been biochemically characterized in our previous work, resulting in identification of residues Glu532 and Glu447 as the nucleophile and acid/base catalysts, respectively.^[@ref9]^ Mutagenesis of the Arg484 changed the GOS linkage specificity.^[@ref11]^ The high-resolution crystal structure of the shortest isoform of β-galactosidase from *B. circulans* \[BgaD-D, Protein Data Bank (PDB) code 4YPJ\] was reported recently.^[@ref24]^ Details of the structure--function relationships of this enzyme determining its GOS product linkage specificity have remained unknown. Here we report the identification and site-directed mutagenesis of further residues in the active site of BgaD-D. Their functional roles were characterized by analyzing the biochemical properties of the mutant enzymes and the structures of the GOS produced. The data provide insight into how these residues contribute to determining enzyme activity and GOS linkage specificity. Experimental Procedures {#sec2} ======================= Bacterial Strains {#sec2.1} ----------------- *E. coli* DH5α (Phabagen) was used for DNA manipulation, and *E. coli* BL21 (DE3) was used for protein expression. Sequence and Structure Alignment {#sec2.2} -------------------------------- T-Coffee and Jalview were used for the alignment of the amino acid sequences of β-galactosidases from GH2 ([Table [1](#tbl1){ref-type="other"}](#tbl1){ref-type="other"} and [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}): BgaD-D (E5RWQ2) from *B. circulans* ATCC 31382, BgaA (A0A0H2UP19) from *Streptococcus pneumoniae* serotype 4, BIF3 (Q9F4D5) from *Bifidobacterium bifidum* DSM 20215, BbgIII (A4K5H9) from *Bi. bifidum* NCIMB 41171, and BbgIII (D4QAP3) from *Bi. bifidum*. PyMOL (The PyMOL Molecular Graphics System, version 1.2, Schrödinger, LLC) was used for the structural alignment ([Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}) of BgaD-D from *B. circulans* (PDB code 4YPJ) and the nucleophile mutant (E645Q) of BgaA in complex with *N*-acetyl-lactosamine (LacNAc) from *S. pneumoniae* (PDB code 4CUC). ###### Comparison of the Amino Acid Sequences of β-Galactosidase BgaD-D from *B. circulans* ATCC 31382 and Other β-Galactosidases in the GH2 Family enzyme name enzyme source identity (%) UniProt code PDB code ------------- ---------------------------- -------------- -------------- ---------- BgaD-D *B. circulans* ATCC 31382 100 E5RWQ2 4YPJ BgaA *S. pneumoniae* serotype 4 49 A0A0H2UP19 4CU6 BIF3 *Bi. bifidum* DSM 20215 44 Q9F4D5   BbgIII *Bi. bifidum* NCIMB 41171 43 A4K5H9   BbgIII *Bi. bifidum* 26 D4QAP3 5DMY {ref-type="other"}](#tbl1){ref-type="other"}. The amino acid residue color coding used is in Clustal X format. For details, see <http://www.jalview.org/help/html/colourSchemes/clustal.html>; gaps in the alignments are denoted with hyphens.](bi-2017-00207h_0001){#fig1} {#fig2} Site-Directed Mutagenesis {#sec2.3} ------------------------- Plasmid pET-15b-LIC containing the rBgaD-D-encoding gene from *B. circulans* was obtained from a previous study and used as a template for site-directed mutagenesis.^[@ref10]^ On the basis of the results of the sequence and structure alignments, mutations of residues Arg185, Asp481, Lys487, Tyr511, Trp570, Trp593, Glu601, and Phe616 were introduced by various primers ([Table S1](http://pubs.acs.org/doi/suppl/10.1021/acs.biochem.7b00207/suppl_file/bi7b00207_si_001.pdf)) using the QuikChange site-directed mutagenesis kit (Stratagene). The polymerase chain reaction (PCR) products were cleaned up with a PCR purification kit after digestion by DpnI (Thermo Fisher). Then the PCR products were transformed into *E. coli* DH5α competent cells (Phabagen) via heat shock for overnight growth on LB agar plates (containing 100 μg/mL ampicillin). For every mutant, colonies were randomly chosen and inoculated into 5 mL of LB medium (containing 100 μg/mL ampicillin) for DNA amplification overnight. The plasmid DNA of the overnight cultures was purified using a miniprep kit (Sigma-Aldrich) and sequenced (GATC Biotech). Recombinant Protein Expression and Purification {#sec2.4} ----------------------------------------------- The sequence-verified plasmids were transformed into *E. coli* BL21 (DE3) (Invitrogen) competent cells for protein expression as described previously.^[@ref10]^ Briefly, after growth on LB agar plates (containing 100 μg/mL ampicillin), colonies were inoculated into 10 mL of LB medium (containing 100 μg/mL ampicillin) for overnight preculture. The overnight cultures were inoculated into 1 L of LB medium (containing 100 μg/mL ampicillin) and incubated at 37 °C until the cell density reached 0.6 at 600 nm; then 1 mM isopropyl β-[d]{.smallcaps}-thiogalactopyranoside was used for the induction of recombinant protein expression. The cells were cultured at 30 °C overnight and harvested by centrifugation at 10876*g* for 15 min. The cell pellets were washed with 20 mM Tris-HCl buffer (pH 8.0) and centrifuged again. The cell pellets were lysed with B-PER protein extract reagent (Thermo Scientific) at room temperature for 1 h. The cell debris was removed by centrifugation; the remaining supernatants were mixed with HIS-Select Nickel Affinity Gel (Sigma) and incubated at 4 °C overnight. Unbound proteins were washed away with 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl. The recombinant proteins were eluted with 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl containing 100 mM imidazole. Subsequently, the imidazole was removed by ultrafiltration (30 kDa cutoff, Amicon, Merck) Enzyme Activity Assay {#sec2.5} --------------------- For the activity assay of the wild-type and mutant enzymes, 0.5--1 mg/mL (protein concentration determined by a Nanodrop 2000 UV--vis spectrophotometer) amounts were incubated with 10% (w/w) lactose in 100 mM sodium phosphate buffer (pH 6.0) for 5 min at 40 °C. The incubation mixtures were withdrawn and immediately inactivated with 50 μL of 1.5 M NaOH. The reaction mixtures were neutralized with 50 μL of 1.5 M HCl after 10 min. The total activity (units) toward lactose was defined as the amount of enzyme required to release 1 μmol of Glc/min. The released glucose was measured using a GOPOD kit ([d]{.smallcaps}-glucose Assay Kit, Megazyme). The activity of the wild-type enzyme was regarded as 100%; activities of all mutant enzymes were relative to that of the wild-type enzyme. The kinetic parameters (*K*~m~ and *k*~cat~) of mutant enzymes were determined using 10 different lactose concentrations ranging from 10 to 500 mM. GOS Production and Analysis {#sec2.6} --------------------------- GOS were produced by using 3.75 units/g lactose β-galactosidase wild-type and mutant enzymes with 50% (w/w) lactose \[in 100 mM sodium phosphate buffer (pH 6.0)\] incubated at 60 °C for 20 h to reach the highest GOS yield. We stopped the reactions by heating the mixtures at 100 °C for 10 min. The reaction mixtures were diluted 1000-fold with Milli-Q water and analyzed by high-pH anion exchange chromatography (HPAEC) coupled with a pulsed amperometric detector (PAD) on an ICS3000 chromatography workstation (ThermoScientific). The analysis was performed by injecting 5 μL on a CarboPac PA1 analytical column (2 mm × 250 mm) with the following elution buffers: (A) 100 mM sodium hydroxide, (B) 600 mM sodium acetate in 100 mM sodium hydroxide, (C) Milli-Q water, and (D) 50 mM sodium acetate. The separation conditions were the same as used in our previous study.^[@ref10]^ The quantity of β-[d]{.smallcaps}-Gal*p*-(1→3)-β-[d]{.smallcaps}-Gal*p*-(1→4)-[d]{.smallcaps}-Glc*p* was determined by using a calibration curve of this compound (Sigma) ranging from 4 to 200 μg/mL. The comparisons of other GOS fractions were based on the peak intensities of HPAEC--PAD profiles of the wild-type and mutant enzymes. The quantification of the GOS yield {GOS yield (grams) = initial lactose (grams) -- \[remaining lactose (grams) + galactose (grams) + glucose (grams)\] after 20 h} was based on a calibration curve of galactose, glucose, and lactose ranging from 10 to 1000 μM. Results {#sec3} ======= Structural Alignment {#sec3.1} -------------------- The β-galactosidase proteins BgaD-D of *B. circulans* and BgaA of *S. pneumonia* share 49% sequence identity. A structural alignment of BgaD-D (PDB code 4YPJ) with the catalytic region of BgaA (PDB code 4CU6) and with the inactive BgaA mutant E645Q in complex with LacNAc (PDB code 4CUC) ([Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}) guided our selection of active site residues to be mutated. The residues targeted for mutagenesis were grouped in four sets, mainly based on their location in the active site. Site-Directed Mutagenesis and Enzyme Activity {#sec3.2} --------------------------------------------- Mutations were introduced by site-directed random mutagenesis as described in [Experimental Procedures](#sec2){ref-type="other"}. The mutants were obtained via two rounds of mutagenesis. Universal primer pairs ([Table S1](http://pubs.acs.org/doi/suppl/10.1021/acs.biochem.7b00207/suppl_file/bi7b00207_si_001.pdf)) were used for the first round, and 20 colonies were selected randomly for sequencing. Specific primers ([Table S1](http://pubs.acs.org/doi/suppl/10.1021/acs.biochem.7b00207/suppl_file/bi7b00207_si_001.pdf)) were used in the second round of mutagenesis for a full coverage of amino acid (side chain) classes, or to obtain a specific amino acid residue. The mutants obtained in the two mutagenesis rounds and their relative enzyme activities (release of Glc from lactose) are listed in [Table [2](#tbl2){ref-type="other"}](#tbl2){ref-type="other"}. For residues Arg185, Glu601, and Tyr511, a total of 6, 11, and 5 different mutants were obtained after two rounds, respectively; all mutants were completely inactive ([Table [2](#tbl2){ref-type="other"}](#tbl2){ref-type="other"}). Four Trp570 mutants were found in the first round of mutagenesis; another six mutants were selected in the second round. Of these mutants, Trp570Tyr had the highest activity, 37.4 ± 0.4% compared to that of the wild-type enzyme, followed by Trp570Phe (16.1 ± 1.0%) and Trp570Leu (14.6 ± 0.8%). The activity of the other Trp570 mutants was rather low ([Table [2](#tbl2){ref-type="other"}](#tbl2){ref-type="other"}). One round of Trp593 mutagenesis yielded a total of 11 mutants. Among them, only Trp593Tyr and Trp593Phe retained activity, 3.3 ± 0.2 and 70.7 ± 0.7% of the wild-type enzyme activity, respectively ([Table [2](#tbl2){ref-type="other"}](#tbl2){ref-type="other"}). For Phe616, a total of eight mutants were obtained in the first round. With the specific primers, the other 11 mutants were also obtained. Phe616Trp (37.2 ± 1.5%) and Phe616Tyr (73.2 ± 0.6%) had the highest activity among all these mutants ([Table [2](#tbl2){ref-type="other"}](#tbl2){ref-type="other"}). In the first round, six mutants of Asp481 were acquired, and another seven mutants were obtained in the second round. Only Asp481Glu, Asp481His, Asp481Ser, Asp481Asn, and Asp481Gln retained very little activity ([Table [2](#tbl2){ref-type="other"}](#tbl2){ref-type="other"}). For Lys487, the universal primers resulted in eight mutants; most of them had relatively high activity (65.5 ± 0.8 to 105.5 ± 1.2%), except for Lys487Cys (3.5 ± 0.4%) ([Table [2](#tbl2){ref-type="other"}](#tbl2){ref-type="other"}). ###### Relative Total Activities (release of Glc from lactose) of the Various *B. circulans* ATCC 31382 β-Galactosidase BgaD-D Mutant Proteins site round mutant relative activity[a](#t2fn1){ref-type="table-fn"} site round mutant relative activity -------- ------- -------- --------------------------------------------------- -------- ------- -------- ----------------------------------     WT 100 Phe616 1 Val 1.9 ± 0.01 Arg185 1 Glu [b](#t2fn2){ref-type="table-fn"}     Glu 1.4 ± 0.01     Gly [b](#t2fn2){ref-type="table-fn"}     Gly 3 ± 0.1     Leu [b](#t2fn2){ref-type="table-fn"}     Lys 0.8 ± 0.01     Pro [b](#t2fn2){ref-type="table-fn"}     Gln 3.2 ± 0.08     Ser [b](#t2fn2){ref-type="table-fn"}     Arg 1.3 ± 0.01     Lys [b](#t2fn2){ref-type="table-fn"}     Asp 4.6 ± 0.1 Glu601 1 Pro [b](#t2fn2){ref-type="table-fn"}     Leu 22.9 ± 1.6     Phe [b](#t2fn2){ref-type="table-fn"}   2 Trp 37.2 ± 1.5     Gln [b](#t2fn2){ref-type="table-fn"}     His 13.4 ± 0.2     His [b](#t2fn2){ref-type="table-fn"}     Ser 5.3 ± 1.1     Ala [b](#t2fn2){ref-type="table-fn"}     Thr 4.9 ± 0.03     Arg [b](#t2fn2){ref-type="table-fn"}     Asn 11.6 ± 0.1     Tyr [b](#t2fn2){ref-type="table-fn"}     Cys 3.9 ± 0.08     Cys [b](#t2fn2){ref-type="table-fn"}     Pro 3.8 ± 0.7     Gly [b](#t2fn2){ref-type="table-fn"}     Ala 3.4 ± 0.1     Thr [b](#t2fn2){ref-type="table-fn"}     Ile 2.8 ± 0.04   2 Asp [b](#t2fn2){ref-type="table-fn"}     Met 1.4 ± 0.05 Tyr511 1 Cys [b](#t2fn2){ref-type="table-fn"}     Tyr 73.2 ± 0.6     Pro [b](#t2fn2){ref-type="table-fn"} Asp481 1 Tyr [b](#t2fn2){ref-type="table-fn"}     Ser [b](#t2fn2){ref-type="table-fn"}     Phe [b](#t2fn2){ref-type="table-fn"}   2 Trp [b](#t2fn2){ref-type="table-fn"}     Glu 7.3 ± 0.5     Phe [b](#t2fn2){ref-type="table-fn"}     His 2.1 ± 0.6 Trp570 1 Gly 5.1 ± 0.08     Ala [b](#t2fn2){ref-type="table-fn"}     Thr 6.5 ± 0.2     Ser 6.1 ± 0.2     Arg 3.6 ± 0.1   2 Lys [b](#t2fn2){ref-type="table-fn"}     Glu 5.2 ± 0.3     Arg [b](#t2fn2){ref-type="table-fn"}   2 Tyr 37.4 ± 0.4     Asn 6.7 ± 0.3     Phe 16.1 ± 1.0     Gln 3.3 ± 0.3     Ala 4.2 ± 0.05     Leu [b](#t2fn2){ref-type="table-fn"}     Val 3.0 ± 0.1     Trp [b](#t2fn2){ref-type="table-fn"}     Cys 6.1 ± 0.1     Gly [b](#t2fn2){ref-type="table-fn"}     Leu 14.6 ± 0.8 Lys487 1 Met 105.5 ± 1.2 Trp593 1 Val [b](#t2fn2){ref-type="table-fn"}     Phe 73.2 ± 0.7     Leu [b](#t2fn2){ref-type="table-fn"}     Leu 93.7 ± 0.4     Ser [b](#t2fn2){ref-type="table-fn"}     Gln 83.1 ± 0.6     Ala [b](#t2fn2){ref-type="table-fn"}     Ser 76.3 ± 0.9     Gly [b](#t2fn2){ref-type="table-fn"}     Gly 65.5 ± 0.9     Thr [b](#t2fn2){ref-type="table-fn"}     Asn 65.5 ± 0.8     Pro [b](#t2fn2){ref-type="table-fn"}     Cys 3.5 ± 0.4     Gln [b](#t2fn2){ref-type="table-fn"}             Tyr 3.3 ± 0.2             Phe 70.7 ± 0.7             His [b](#t2fn2){ref-type="table-fn"}         Total activity. Activities of all mutant enzymes are relative to that of the wild-type (WT) enzyme (100%, 103.4 μmol min^--1^ mg^--1^). Enzyme activity was measured in triplicate experiments with 10% (w/w) lactose at 40 °C. Activity not detectable (detection limit of 0.1 mg/mL glucose). Catalytic Properties {#sec3.3} -------------------- On the basis of the activity compared to that of the wild-type (WT) enzyme, and interesting product profiles (see the next section), the catalytic properties of a selection of enzymes were determined ([Table [3](#tbl3){ref-type="other"}](#tbl3){ref-type="other"}). For all enzymes, the *k*~cat~ was lower (12.3 ± 1.1 to 184.7 ± 3.4 s^--1^) than that of the WT enzyme (199.8 ± 5.3 s^--1^), while the substrate affinity (*K*~m~) ranged from 21.9 ± 4.7 to 246.5 ± 66.0 mM; the WT enzyme has a *K*~m~ of 112.9 ± 12.7 mM.^[@ref11]^ The catalytic efficiency (*k*~cat~/*K*~m~) of the WT enzyme is 1780.7 ± 152.0 s^--1^ M^--1^; in almost all mutants, the efficiency was lower (178.4 ± 16.5 to 1546.3 ± 50.0 s^--1^ M^--1^), except for Trp593Phe that has a much higher *k*~cat~/*K*~m~ of 2990.2 ± 184.2 s^--1^ M^--1^. ###### Kinetic Properties of the *B. circulans* ATCC 31382 Wild-Type β-Galactosidase BgaD-D and Mutant Proteins enzyme *K*~m~[a](#t3fn1){ref-type="table-fn"} (mM) *k*~cat~[a](#t3fn1){ref-type="table-fn"} (s^--1^) *k*~cat~/*K*~m~ (s^--1^ M^--1^) ------------------------------------ --------------------------------------------- --------------------------------------------------- --------------------------------- WT[b](#t3fn2){ref-type="table-fn"} 112.9 ± 12.7 199.8 ± 5.3 1780.7 ± 152.0 Trp570Tyr 75.6 ± 11.0 27.2 ± 0.9 398.6 ± 20.0 Trp570Phe 246.5 ± 66.0 44.6 ± 7.1 178.4 ± 16.5 Trp593Phe 21.9 ± 4.7 63.6 ± 0.6 2990.2 ± 184.2 Phe616Trp 180.0 ± 8.0 114.7 ± 3.5 587.7 ± 5.2 Phe616His 156.1 ± 9.8 48.9 ± 1.8 321.3 ± 27.2 Phe616Tyr 112.6 ± 3.9 183.9 ± 4.0 1504.6 ± 57.2 Asp481Gln 57.4 ± 13.8 12.3 ± 1.1 217.9 ± 37.9 Lys487Ser 119.5 ± 5.6 184.7 ± 3.4 1546.3 ± 50.0 Lys487Gly 176.8 ± 12.6 177.7 ± 7.1 1001.5 ± 37.2 Kinetic parameters (*K*~m~ and *k*~cat~) were determined with 10 different lactose concentrations ranging from 10 to 500 mM. Data from our previous study.^[@ref11]^ Mutant GOS Profiles {#sec3.4} ------------------- For the wild type (WT) and a selection of mutant enzymes showing sufficient activity, incubations using 3.75 units/g of lactose were performed for 20 h, and the HPAEC--PAD GOS product profiles were compared ([Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}). {#fig3} Because galactose is released by hydrolysis, the hydrolysis activity is reflected by the intensity of peak **1** (galactose). For all three Trp570 mutants, the hydrolysis activity increased, as evidenced by the increased intensity of peak **1** in the HPAEC--PAD profile ([Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}A), whereas the hydrolytic activity of all Trp593 and Phe616 mutant enzymes was comparable with that of the WT enzyme \[peak **1** ([Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}B,C)\]. Most notable changes in relative product intensities were observed in structures **8**, **11**, **13**, and **17** for the mutant enzymes at Trp570, Trp593, and Phe616 ([Table [4](#tbl4){ref-type="other"}](#tbl4){ref-type="other"}). In the case of Trp570Tyr and Trp570Phe, structures **8a** \[β-[d]{.smallcaps}-Gal*p*-(1→2)-[d]{.smallcaps}-Glc*p*\] and **8b** \[β-[d]{.smallcaps}-Gal*p*-(1→3)-[d]{.smallcaps}-Glc*p*\] were similar in level to that of the WT enzyme, whereas the levels of the (β1→4) elongations (peaks **13a** and **13b**) significantly increased. Levels of structures **11** \[β-[d]{.smallcaps}-Gal*p*-(1→4)-β-[d]{.smallcaps}-Gal*p*-(1→4)-[d]{.smallcaps}-Glc*p*\] and its (β1→4) elongation (peak **17**) were significantly decreased. In the case of Trp593Tyr and Trp593Phe, a similar observation was made, except that levels of structures **8a** and **8b** also were increased compared to the WT product profile. Mutant Trp570Leu showed a completely different profile, with a hydrolytic activity much higher than those of any of the other mutants. The HPAEC--PAD profile showed two major product peaks for **11** and **17** and only minor peaks for other products. However, quantitation of the peaks ([Table [4](#tbl4){ref-type="other"}](#tbl4){ref-type="other"}) showed that peak **11** is comparable with that of the WT enzyme, while all other peaks showed a reduced product yield. ###### Total GOS Yields and Yields of the Major GOS Structures **8**, **11**, **13**, and **17** Obtained for the *B. circulans* ATCC 31382 BgaD-D Trp570, Trp593, and Phe616 Mutants, Compared to the Wild-Type Enzyme[a](#t4fn1){ref-type="table-fn"} enzyme GOS yield[b](#t4fn2){ref-type="table-fn"} structure **8** yield[c](#t4fn3){ref-type="table-fn"} structure **11** yield[c](#t4fn3){ref-type="table-fn"} structure **13** yield[c](#t4fn3){ref-type="table-fn"} structure **17** yield[c](#t4fn3){ref-type="table-fn"} ------------------------------------ ------------------------------------------- ------------------------------------------------------- -------------------------------------------------------- -------------------------------------------------------- -------------------------------------------------------- WT[d](#t4fn4){ref-type="table-fn"} 63.5 ± 0.8 100 100 100 100 Trp570Tyr 65.8 ± 1.7 133.5 ± 0.9 52.7 ± 1.0 103.8 ± 1.6 41.3 ± 1.0 Trp570Phe 65.4 ± 0.9 110.1 ± 0.7 44.1 ± 0.5 91.6 ± 2.0 31.0 ± 0.6 Trp570Leu 43.8 ± 0.7 21.8 ± 0.5 93.9 ± 3.1 [e](#t4fn5){ref-type="table-fn"} 67.9 ± 2.7 Trp593Phe 66.0 ± 1.0 122.5 ± 5.1 65.7 ± 1.7 126.2 ± 5.1 53.7 ± 1.6 Trp593Tyr 65.2 ± 2.1 146.6 ± 7.2 49.3 ± 2.1 128.4 ± 1.9 35.4 ± 1.9 Phe616Trp 50.8 ± 2.4 80.8 ± 1.7 132.1 ± 7.2 75.7 ± 0.8 119.8 ± 6.0 Phe616Leu 61.0 ± 1.4 114.4 ± 1.6 91.3 ± 3.1 120.2 ± 2.5 82.7 ± 3.3 Phe616His 59.6 ± 1.2 99.8 ± 3.3 93.4 ± 1.5 110.6 ± 0.8 80.4 ± 2.4 Phe616Asn 57.3 ± 1.4 82.0 ± 2.6 106.5 ± 8.0 93.6 ± 4.7 97.3 ± 9.6 Phe616Tyr 58.3 ± 0.6 105.9 ± 4.3 108.0 ± 0.1 114.9 ± 2.0 108.3 ± 0.5 GOS was produced using 3.75 units/mL β-galactosidase wild-type and mutant enzymes with 50% (w/w) lactose \[in 100 mM sodium phosphate buffer (pH 6.0)\] incubated at 60 °C for 20 h. Yields are calculated as grams of GOS produced from 100 g of initial lactose. Calibration curves for lactose, galactose, and glucose ranging from 10 to 1000 μM were used for quantification. The yields are relative to that of the wild-type enzyme (100%), estimated by comparing the peak intensities in the HPAEC--PAD profiles. Data from our previous study.^[@ref11]^ Unable to quantify. Enzymes with a mutation at Phe616 showed HPAEC--PAD profiles very similar to that of the WT enzyme. Quantitation of peaks **8**, **11**, **13**, and **17** showed some differences in intensity compared with the WT enzyme, but the changes were mostly minor. In case of Phe616 Trp, -Asn, and -Tyr mutants, levels of structures **11** and **17** were slightly increased, while levels of **8** and **13** were either slightly decreased or the same compared to that of the WT. For the other Phe616 mutants, the intensities showed the opposite response ([Table [4](#tbl4){ref-type="other"}](#tbl4){ref-type="other"}). Mutations at Asp481 and Lys487 showed different effects on the product profiles ([Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}D,E). Mutation of Asp481 to Glu, Ser, or Asn reduced the intensity of peaks **8**, **11**, and **13** ([Table [5](#tbl5){ref-type="other"}](#tbl5){ref-type="other"}), whereas the intensity of peak **12** \[β-[d]{.smallcaps}-Gal*p*-(1→3)-β-[d]{.smallcaps}-Gal*p*-(1→4)-[d]{.smallcaps}-Glc*p*\] increased significantly. Whereas the WT enzyme produces only minor amounts of **12** (0.2 ± 0.05 g/100 g of lactose), all three mutants produced a significant amount of **12**. Notably, in case of Asp481Asn, the intensity of peak **4** \[β-[d]{.smallcaps}-Gal*p*-(1→4)-[d]{.smallcaps}-Glc*p*\] also was increased ([Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}D). ###### Total GOS Yields and Yields of the Major GOS Structures **8** and **11--13** Obtained for the *B. circulans* ATCC 31382 BgaD-D Asp481 and Lys487 Mutants, Compared to the Wild-Type Enzyme[a](#t5fn1){ref-type="table-fn"} enzyme GOS yield[b](#t5fn2){ref-type="table-fn"} structure **8** yield[c](#t5fn3){ref-type="table-fn"} structure **11** yield[c](#t5fn3){ref-type="table-fn"} structure **12** yield[d](#t5fn4){ref-type="table-fn"} structure **13** yield[c](#t5fn3){ref-type="table-fn"} ------------------------------------ ------------------------------------------- ------------------------------------------------------- -------------------------------------------------------- -------------------------------------------------------- -------------------------------------------------------- WT[e](#t5fn5){ref-type="table-fn"} 63.5 ± 0.8 100 100 0.2 ± 0.05 100 Asp481Glu 56.4 ± 2.2 51.5 ± 2.0 47.5 ± 1.5 4.8 ± 0.8 17.1 ± 3.2 Asp481Ser 55.3 ± 3.6 59.3 ± 1.9 70.1 ± 4.1 2.6 ± 0.4 30.3 ± 3.1 Asp481Asn 68.8 ± 1.7 68.5 ± 2.9 16.6 ± 0.8 4.9 ± 0.8 20.2 ± 4.7 Lys487Met 59.0 ± 1.9 99.8 ± 1.5 97.3 ± 3.2 [f](#t5fn6){ref-type="table-fn"} 103.2 ± 3.4 Lys487Phe 58.3 ± 1.5 74.7 ± 0.2 105.6 ± 1.3 1.6 ± 0.2 68.7 ± 1.0 Lys487Leu 59.1 ± 1.1 101.3 ± 0.7 101.9 ± 2.4 [f](#t5fn6){ref-type="table-fn"} 97.7 ± 4.1 Lys487Gln 58.6 ± 2.9 98.9 ± 1.2 98.8 ± 1.7 [f](#t5fn6){ref-type="table-fn"} 98.1 ± 2.9 Lys487Ser 63.6 ± 1.0 77.5 ± 3.1 68.2 ± 1.3 5.0 ± 0.4 57.5 ± 3.7 Lys487Gly 62.0 ± 2.7 63.1 ± 0.2 66.0 ± 2.7 9.7 ± 1.1 36.9 ± 3.7 Lys487Asn 59.8 ± 1.3 68.5 ± 1.8 104.4 ± 5.0 1.7 ± 0.2 58.5 ± 2.0 GOS was produced using 3.75 units/mL β-galactosidase wild-type and mutant enzymes with 50% (w/w) lactose \[in 100 mM sodium phosphate buffer (pH 6.0)\] incubated at 60 °C for 20 h. Yields are calculated as grams of GOS produced from 100 g of initial lactose. Calibration curves for lactose, galactose, and glucose ranging from 10 to 1000 μM were used for quantification. The yields are relative to that of the wild-type enzyme (100%), estimated by comparing the peak intensities in the HPAEC--PAD profiles. Yields are expressed as grams of product obtained from 100 g of initial lactose. A calibration curve of structure **12** \[β-[d]{.smallcaps}-Gal*p*-(1→3)-β-[d]{.smallcaps}-Gal*p*-(1→4)-[d]{.smallcaps}-Glc*p*\] ranging from 4 to 200 μg/mL was used for its quantification. Data from our previous study.^[@ref11]^ Not detectable. Changing Lys487 to a Met, Leu, or Gln had no significant effect on the product profile, also evidenced by quantitation of peaks **8** and **11--13** ([Table [5](#tbl5){ref-type="other"}](#tbl5){ref-type="other"}). Mutation of this residue to Phe or Asn resulted in a reduced yield for **8** and **13**, while the yields of **11** and **12** both increased. The most remarkable changes were observed for mutants Ly487Ser and Lys487Gly, showing a significant increase in **12**, even up to 9.7 ± 1.1 g/100 g of lactose. Discussion {#sec4} ========== Prebiotic GOS produced from lactose by β-galactosidase enzymes are drawing a great deal of attention; they have been added to infant formula because their molecular size is similar to that of human milk oligosaccharides and because of their beneficial functions and positive effects on intestinal health.^[@ref25]−[@ref28]^ The beneficial functions of GOS were further shown by studies regarding calcium absorption, metabolic activities, and protection against colorectal cancer.^[@ref29]−[@ref32]^ To understand how different β-galactosidases synthesize such a range of GOS structures, and how they may be tailored to produce more specific GOS mixtures, it is essential to obtain detailed (three-dimensional) structural information about both the enzymes and their GOS products. Although several crystal structures of β-galactosidase enzymes have been determined and served to improve our understanding of the enzyme reaction mechanism,^[@ref33]−[@ref36]^ their detailed structure--function relationships are still largely unexplored, especially regarding their GOS product linkage specificity. In this study, we have characterized the functional roles of eight amino acid residues in the active site of BgaD-D from *B. circulans* ATCC 31382, close to the substrate binding site, constituting four groups. Arg185 and Glu601 Are Essential for Activity and Substrate Binding {#sec4.1} ------------------------------------------------------------------ Arg185 and Glu601 are located near the nonreducing end hydroxyl groups of the lactosyl moiety of LacNAc in subsite −1 ([Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}A). All six mutants of Arg185, and all 11 mutants of Glu601, had lost detectable enzyme activity. As shown in [Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}A, Arg185 and Glu601 in 4YPJ correspond to Arg288 and Glu716 in *S. pneumoniae* BgaA, respectively; they are located at the −1 subsite of BgaA and are hydrogen-bonded to the 4-OH group of the galactosyl unit of LacNAc ([Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}A). Given the conservation of these residues, it is likely that Arg185 and Glu601 in BgaD-D have a similar essential function, namely assisting in the binding and positioning of the substrate. ![Superposition of the *B. circulans* ATCC 31382 BgaD-D residues (PDB code 4YPJ) subjected to mutagenesis \[(A) Arg185, Glu601, and Tyr511, (B) Trp570, Trp593, and Phe616, and (C) Asp481 and Lys487\] with the nucleophile mutant (E645Q) of BgaA of *S. pneumoniae* in complex with LacNAc (PDB code 4CUC). The color coding and labeling are the same as in [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}.](bi-2017-00207h_0004){#fig4} Tyr511 Is Essential for Activity {#sec4.2} -------------------------------- Residue Tyr511 is conserved among the GH2 family β-galactosidase enzymes ([Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}); both in BgaA and in BgaD, its OH group makes a hydrogen bond interaction (2.6 Å) to the nucleophilic Glu residue. The nearby nucleophilic residue Glu532 attacks the substrate while it is productively bound; a previous study proposed that Tyr511 assists in the catalytic mechanism by donating its proton to Glu532, before it attacks the substrate to form the covalent galactosyl--enzyme intermediate.^[@ref24]^ None of the mutants obtained were active. Our experimental results confirm that the hydroxyl group of Tyr511 is essential for enzymatic activity. Residues Trp570, Trp593, and Phe616 Form an Aromatic Pocket Shaping the Substrate Binding Site {#sec4.3} ---------------------------------------------------------------------------------------------- Trp570 is located near the +1 subsite, while Trp593 and Phe616 are located near the −1 subsite ([Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}B). From the 10 mutants of Trp570, Trp570Tyr retained 37.4 ± 0.4% of the WT activity ([Table [2](#tbl2){ref-type="other"}](#tbl2){ref-type="other"}). The *k*~cat~ of Trp570Tyr decreased 7.3-fold compared to that of the wild-type enzyme, while the *K*~m~ decreased 1.5-fold ([Table [3](#tbl3){ref-type="other"}](#tbl3){ref-type="other"}). Trp570Phe retained 16.1 ± 1.0% of the WT activity; for the other substitutions, the activity was lower ([Table [2](#tbl2){ref-type="other"}](#tbl2){ref-type="other"}). The *k*~cat~ of Trp570Phe decreased 4.5-fold, while the *K*~m~ increased 2.2-fold ([Table [3](#tbl3){ref-type="other"}](#tbl3){ref-type="other"}). The kinetic parameters of Trp570Tyr and Trp570Phe show that the mutations at this site have a stronger influence on the turnover rate (*k*~cat~) than on *K*~m~ and may affect acceptor binding. The GOS yields of the Trp570Tyr and Trp570Phe mutants were comparable with that of the WT enzyme (63.5 ± 0.8 g of GOS from 100 g of initial lactose), while Trp570Leu can produce only 43.8 ± 0.7 g of GOS from 100 g of initial lactose ([Table [4](#tbl4){ref-type="other"}](#tbl4){ref-type="other"}). Mutants Trp570Tyr and Trp570Phe produced more of structure **8** and less of structures **11** and **17**, showing their preference for (β1→2) and (β1→3) over (β1→4) linkages. In contrast, Trp570Leu produced only 21.8 ± 0.5% of structure **8** compared to the wild type (100%), and the yield of structures further elongated from this (structure **13**) was too low to be quantified. The Trp570Leu yield of structure **11** was comparable to that of the wild-type enzyme. These results show the strong preference of this mutant to synthesize GOS with (β1→4) linkages. Notably, these three mutants also have a higher hydrolytic activity, as evidenced by a much higher yield of galactose (structure **1**) compared to that of the wild-type enzyme ([Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}A), i.e., 3.0, 5.9, and 10.1 times that of the wild type for Trp570Tyr, Trp570Phe, and Trp570Leu, respectively. Among the mutants of Trp593, activity was observed for only Trp593Tyr and Trp593Phe (3.3 ± 0.2 and 70.7 ± 0.7%, respectively) ([Table [2](#tbl2){ref-type="other"}](#tbl2){ref-type="other"}). Both the *K*~m~ and the *k*~cat~ of Trp593Phe decreased ([Table [3](#tbl3){ref-type="other"}](#tbl3){ref-type="other"}), resulting in a relatively low activity (70.7 ± 0.7%) compared to that of the wild-type enzyme ([Table [2](#tbl2){ref-type="other"}](#tbl2){ref-type="other"}). As shown in panels B and F of [Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"} and [Table [4](#tbl4){ref-type="other"}](#tbl4){ref-type="other"}, the GOS yields of mutants Trp593Tyr and Trp593Phe were comparable to that of the wild-type enzyme. These two mutants synthesized more of structures **8** and **13** and less of structures **11** and **17**, showing their preference for (β1→2) and (β1→3) over (β1→4) linkages. In the case of Phe616, the Phe616Tyr mutant had the highest activity, with 73.2 ± 0.6% of WT activity remaining, followed by Phe616Trp, which has 37.2 ± 1.5% of the WT activity ([Table [2](#tbl2){ref-type="other"}](#tbl2){ref-type="other"}). The kinetic parameters (*K*~m~ and *k*~cat~) of Phe616Tyr changed slightly compared to those of the wild-type enzyme ([Table [3](#tbl3){ref-type="other"}](#tbl3){ref-type="other"}). For Phe616Trp, the *K*~m~ increased while the *k*~cat~ decreased ([Table [3](#tbl3){ref-type="other"}](#tbl3){ref-type="other"}), resulting in a much lower activity. Phe616Trp produced more of structures **11** and **17** and less of structures **8** and **13** ([Table [4](#tbl4){ref-type="other"}](#tbl4){ref-type="other"}), showing its preference for synthesizing (β1→4) linkages. In contrast, Phe616Leu has a preference for synthesizing (β1→2) and (β1→3) linkages ([Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}C and [Table [4](#tbl4){ref-type="other"}](#tbl4){ref-type="other"}). The aromatic pocket formed by residues Trp570, Trp593, and Phe616 in BgaD shapes its active site. We found that mutations at these positions negatively affected enzyme activity, especially when the mutations were non-aromatic. Moreover, they changed the linkage preference and size of the products. For example, Trp570Leu produced only 21.8 ± 0.5% of structure **8** compared to the wild-type enzyme, and its elongation product, structure **13**, was not detectable. Apparently, the mutation enhanced the percentage of small oligosaccharides produced, achieving similar results as reported in a previous study.^[@ref37]^ Our results also show that Trp570 is essential for the transgalactosylation and hydrolysis activities, suggesting that it is involved in selection of the acceptor substrate, either water or carbohydrates. Together, the observed effects of mutations of residues in the aromatic pocket suggest that the geometry of this pocket is important to recognize and orient donor and acceptor substrates. Asp481 and Lys487 Are Involved in Determining Linkage Specificity {#sec4.4} ----------------------------------------------------------------- Asp481, Arg484, and Lys487 are located near subsite +1 ([Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}C). Mutations of the first residue (Asp481) dramatically reduced the enzyme activity. Among all mutants, only five (Glu, His, Ser, Asn, and Gln) retained some activity ([Table [2](#tbl2){ref-type="other"}](#tbl2){ref-type="other"}). Because Asp481 is relatively close to the acid/base catalyst Glu447 (3.9 Å), mutations at this position may affect the orientation and/or acidity of Glu447 and thus influence catalysis. Mutation Asp481Gln increased the substrate binding affinity (*K*~m~ decreased 2-fold) but decreased the turnover rate (*k*~cat~ decreased 16-fold) ([Table [3](#tbl3){ref-type="other"}](#tbl3){ref-type="other"}). The GOS profiles of the Asp481 mutants changed strongly compared to that of the wild-type enzyme ([Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}D). The amounts of all major GOS structures produced by the wild-type enzyme (structures **8**, **11**, and **13**) decreased, while that of structure **12** increased significantly ([Table [5](#tbl5){ref-type="other"}](#tbl5){ref-type="other"}), similar to results found previously for mutations of Arg484.^[@ref11]^ It is also notable that with mutant Asp481Asn, the yield of allolactose (structure **4**) increased 2.3-fold compared to that of the wild-type enzyme ([Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}D). The results show that the mutations at this site favor synthesis of GOS with (β1→3) and (β1→6) linkages. In BgaA of *S. pneumoniae*, the corresponding Asp599 (Asp481 in BgaD) forms a hydrogen bond with the 6-OH group of the GlcNAc moiety at the +1 subsite ([Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}C); thus, considering its position and interaction, Asp481 may play a role in acceptor substrate binding. The third residue in this group, Lys487, is relatively far from the +1 subsite ([Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}C). The activity of its mutants is relatively high compared to those of the other mutant enzymes except for Lys487Cys, retaining only 3.5 ± 0.4% of WT activity ([Table [2](#tbl2){ref-type="other"}](#tbl2){ref-type="other"}). Compared to those of the wild-type enzyme, the *K*~m~ and *k*~cat~ values of Lys487Ser showed only minor changes. In comparison, the *K*~m~ value of Lys487Gly increased by 57% and resulted in a lower catalytic efficiency (*k*~cat~/*K*~m~ = 1010). Compared with those of the wild-type enzyme, the GOS profiles and the relative amounts of the major structures did not change significantly for Lys487Met, Lys487Leu, or Lys487Gln. Mutants Lys487Phe, Lys487Ser, Lys487Gly, and Lys487Asn all have increased yields of structure **12** ([Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}E and [Table [5](#tbl5){ref-type="other"}](#tbl5){ref-type="other"}). Especially for Lys487Gly, the yield of structure **12** is comparable to that of Arg484Ser and Arg484His.^[@ref11]^ The latter mutations thus favor the synthesis of GOS with (β1→3) linkages. Considering the slightly larger distance from the +1 site, mutations of Lys487 may change the microenvironment of the +1 subsite, therefore affecting the linkage specificity indirectly. Conclusions {#sec5} =========== In conclusion, we pinpointed several residues in the active site of BgaD that are important for the β-galactosidase reaction activity and specificity, by affecting substrate binding or transglycosylation specificity. Residues Arg185 and Glu601 at subsite −1 are essential for the β-galactosidase reaction because they affect substrate binding. A tyrosine residue near the catalytic residues (Tyr511) is also essential for the enzyme activity. An aromatic triplet (Trp570, Trp593, and Phe616) shaping the active site is important for correct substrate binding and determining the linkage distribution. Residues in or near subsite +1 (Asp481, Asp484, and Lys487) may affect the acceptor substrate orientation. Mutants derived produced large amounts of the trisaccharide β-[d]{.smallcaps}-Gal*p*-(1→3)-β-[d]{.smallcaps}-Gal*p*-(1→4)-[d]{.smallcaps}-Glc*p* (structure **12**) in the product mixture, thus leading to more diverse GOS mixtures with potential industrial applications. Our study thus provides important insights into the understanding of the structure--function relationships of β-galactosidase enzymes, especially regarding their GOS product linkage specificity, and provides guidance for rational protein engineering of these enzymes with the aim of producing tailor-made prebiotic GOS mixtures. The Supporting Information is available free of charge on the [ACS Publications website](http://pubs.acs.org) at DOI: [10.1021/acs.biochem.7b00207](http://pubs.acs.org/doi/abs/10.1021/acs.biochem.7b00207).Primers used in this study and additional figures ([PDF](http://pubs.acs.org/doi/suppl/10.1021/acs.biochem.7b00207/suppl_file/bi7b00207_si_001.pdf)) Supplementary Material ====================== ###### bi7b00207_si_001.pdf The authors declare no competing financial interest. This work was funded by the China Scholarship Council (to H.Y.) and by the University of Groningen (to T.P., X.M., S.S.v.L., and L.D.). We also thank Prof. Johannis P. Kamerling for stimulating discussions.

1. Introduction {#sec1} =============== Stillbirth remains a major unresolved public health issue and is identified globally as a priority area for targeting prevention strategies in perinatal health. Many deaths remain unexplained despite investigation, and there is an urgent need to identify and target appropriate areas for research and prevention of stillbirth. In developed country settings with low neonatal mortality rates, stillbirths now comprise the majority of perinatal deaths with no significant change in rates over the past 20 years \[[@B1]\]. There is even evidence of a recent increase in stillbirth rates in Australia, UK, and USA which has been attributed to an increase in population risk factors such as advanced maternal age, nulliparity, and obesity \[[@B2], [@B3]\]. In Australia, stillbirths account for around two thirds of all perinatal deaths with a reported rate of 7.4 per 1000 births in 2007 \[[@B1]\]. Many of these deaths remain unexplained with proportions documented as 41% of stillbirths in NSW and 28% nationally \[[@B4]\]. Stillbirths nearer to term are more likely to be classified as unexplained than very preterm stillbirths \[[@B5]\]. We recently reported that in NSW a striking 60% of term stillbirths were unexplained \[[@B4]\]. A number of these "unexplained" deaths equate to "unexplored" with the proportion of unexplained deaths shown to be lower in settings with an extensive test protocol and higher autopsy rates \[[@B6], [@B7]\]. Infection has been documented as particularly important in early stillbirths with a strong association between intrauterine infection and births before 28 weeks \[[@B8]\]. Intrauterine infection is caused by ascending infection from the lower genital tract \[[@B9]\], is usually asymptomatic until labour onset, and the gold standard diagnosis is most commonly retrospective after histopathologic examination of the placenta \[[@B10]\]. Ascending intrauterine infection elicits both maternal (chorioamnionitis) and fetal inflammatory responses (chorionic and umbilical vasculitis, funisitis) \[[@B8]\]. Recent data from a hospital cohort of 459 stillbirths has shown a rise in incidence of chorioamnionitis in later gestation stillbirths suggesting that some late unexplained stillbirths may be due to infection \[[@B10]\]. Intrauterine infection is a recognized cause of stillbirth, and it is postulated that undiagnosed infection may account for a proportion of unexplained deaths \[[@B10], [@B11]\]. Assessing the relationship between intrauterine infection, inflammation, and stillbirth on a comparative basis is difficult, as there are different methods of reporting on the pathological findings and no international standardised criteria. There are also widely differing gestational ages after which an intrauterine death is defined as a stillbirth with countries such as Sweden until recently defining stillbirth as death beyond 28 weeks gestation compared with the 20 week gestation definition of Australia. Both these differences likely explain why the incidence of chorioamnionitis in the literature is variably reported from \<10 to \>90% \[[@B11]--[@B23]\]. There are considerably fewer studies that report on the incidence of a fetal inflammatory response despite accumulating evidence indicating that this is the histopathology most correlated with outcomes related to intrauterine infection \[[@B24]--[@B28]\]. The majority of earlier published studies of stillbirth and chorioamnionitis are small and comprise case series, case-control studies, or hospital-based cohorts \[[@B12], [@B13], [@B14]--[@B23]\]. There are no large population-based studies utilising record linkage of histological chorioamnionitis and stillbirth. We have shown recently, in the largest published hospital cohort of placentae of stillborn infants, an overall incidence of chorioamnionitis of 36% and demonstrated the novel finding of increased incidence at extremes of gestational age, with \>60% of placentae showing chorioamnionitis at both 22 and 41 weeks. \[[@B10]\]. We also found that a fetal inflammatory response was strongly associated with spontaneous onset of labour and its absence with unexplained antepartum death. These findings support the "failure to rescue by birth" hypothesis. This hypothesis suggests that the fetal immune system responds to intrauterine infection by a sequence which leads to early labour and that a fetus who dies in utero in the presence of histological chorioamnionitis may have been unable to mount an immune response large enough to trigger the onset of labour \[[@B11], [@B29]\]. The purpose of this study was to determine whether our novel findings could be replicated in a large population based cohort with placental pathology performed in different centres. 2. Methods {#sec2} ========== This was a Statewide population-based cohort study using deidentified linked data from two NSW data sets: the New South Wales Midwives Data Collection and the Perinatal Death Data from the NSW Maternal and Perinatal Committee. Placental pathology was performed by each area referral pathology service, and reports were scanned and linked to the dataset. Ethical approval was obtained from the NSW Department of Health Ethics Committee Ref No DoHEC 2005-06-11. 2.1. Data Sources {#sec2.1} ----------------- The New South Wales Midwives Data Collection (MDC) is a population-based surveillance system covering all births in NSW public and private hospitals, as well as home births \[[@B30]\]. It encompasses all live births and stillbirths of at least 20 weeks gestation or at least 400 grams birth weight. The MDC requires the attending midwife or doctor to complete a notification form when a birth occurs, and collects demographic, maternal health, pregnancy, labour, delivery, and perinatal outcome data (see Appendix S1 in Supplementary Material available online at doi:10.1155/2011/456728). The NSW Maternal and Perinatal Committee is a quality assurance committee established under the NSW Health Administration Act 1982 and is privileged under this Act to carry out confidential reviews of both maternal and perinatal deaths \[[@B30]\]. Members are appointed by the Minister for Health. A subgroup called the Perinatal Outcomes Working Party (POWP) reviews and classifies perinatal deaths using the Perinatal Society of Australia and New Zealand (PSANZ)---Perinatal Mortality Classification System which has been documented to have a high interobserver reliability with a kappa value of 0.83--0.95 \[[@B32], [@B31]\]. Information available to the POWP at review is forwarded by hospitals and includes a confidential report on perinatal death (Appendix S2), postmortem, and placental pathology reports as well as any other information considered relevant by the local hospital perinatal death review committee. Information considered by the Committee is confidential. 2.2. Placental Examination {#sec2.2} -------------------------- Guidelines for placental reporting are included in the PSANZ Guidelines on Perinatal Mortality Audit \[[@B31]\]. Placental examinations were performed by an Anatomical Pathologist at the local pathology service where the stillbirth occurred and reports forwarded to the Perinatal Outcomes Working Party. For this study, all the placental reports were then further reviewed by one researcher (AG) for the presence of chorioamnionitis and a fetal inflammatory response. Chorioamnionitis was defined as linear aggregation along tissue planes of neutrophil polymorphs of maternal origin in the subchorionic fibrin, chorion or amnion of the peripheral membranes, or the fetal plate of the placenta. Fetal Inflammatory response was defined as the presence of umbilical vasculitis and/or funisitis or inflammation of the chorionic plate. Umbilical vasculitis was defined as migration of fetal neutrophil polymorphs into or through the media of the umbilical arteries or vein, usually in the direction of the amniotic surface of the cord. Funisitis was defined as further migration of fetal neutrophil polymorphs into the Wharton\'s jelly of the umbilical cord. 2.3. Study Population {#sec2.3} --------------------- Records for babies from the Midwives Data Collection and Perinatal Deaths data compiled by the NSW Maternal and Perinatal Committee subgroup POWP were linked using probabilistic record linkage methods in Automatch software (MatchWare Technologies Inc, Silver Spring Md, USA). Both datasets covered the years 2002--2004. Babies were included if they had no congenital abnormality and had placental histopathology performed. Cause of death classification was extracted from the perinatal death database on all stillborn infants that had been reviewed by the Perinatal Outcomes Working Party. Demographic data were collected from both datasets. 2.4. Definitions {#sec2.4} ---------------- Gestational age was determined by certain dates confirmed by ultrasound before 20 weeks gestation or if dates uncertain by ultrasound alone prior to 20 weeks or if unavailable by examination of the newborn infant. Stillbirth was defined as a baby born of at least 20 weeks gestation or 400 g birth weight who did not, at any time after delivery, breathe or show any evidence of life such as a heartbeat. Spontaneous labour was defined by the initiation of regular painful uterine contractions without medical or surgical intervention. Unexplained antepartum death was defined as the death of a normally formed fetus prior to the onset of labour where no predisposing factors are considered likely to have caused the death. 2.5. Statistical Analysis {#sec2.5} ------------------------- Statistical analysis was performed using SPSS (Statistical Package for Social Sciences) version 14.0. Independent proportions were compared using the chi-squared test or Fishers exact where appropriate. Multivariate logistic regression was performed to assess the relationship of fetal inflammatory response with labour and unexplained death adjusting for confounders. Curve fit analysis using least squares regression fitting was performed on the distribution of incidence of chorioamnionitis using Igor Pro software (version 5.02; Wavemetrics, Inc, Lake Oswego, OR, USA). Best fit model was ascertained by the minimisation of the chi-square of the residuals. 3. Results {#sec3} ========== There were a total of 258 045 births in NSW over the three-year study period. The total number of perinatal deaths reviewed and classified by the Perinatal Outcomes Working Party was 1877, and of these, there were 1264 stillbirths and 613 neonatal deaths. Of the 1264 stillbirths, 1096 stillbirths (87%) had no congenital abnormality, and 952 (87%) had placental pathology performed and were included in the cohort. For 95 % of the cohort (901/952), there was additional descriptive data available from the Midwives Data Collection. Demographic data is shown in [Table 1](#tab1){ref-type="table"}. 3.1. Incidence of Histological Chorioamnionitis {#sec3.1} ----------------------------------------------- Histological chorioamnionitis without a fetal response was present in 12.5% (119/952), and histological chorioamnionitis with a fetal response was present in 10.1% (96/952). No babies had a fetal response without histological evidence of chorioamnionitis. Overall, the incidence of histological chorioamnionitis was 22.6% (215/952). The distribution of chorioamnionitis by completed week of gestation is shown in [Figure 1](#fig1){ref-type="fig"}. The incidence was increased at the extremes of gestation, and curve fit analysis by the least squares method showed that residual values were lowest for a bimodal Gaussian distribution. This distribution remained the best fit when a sensitivity analysis was performed without the 3 babies born at 42 weeks. 3.2. Relationship of Fetal Response to Labour Onset {#sec3.2} --------------------------------------------------- There was a significant association between spontaneous onset of labour and the presence of a fetal inflammatory response. The proportion of stillborn babies who had a fetal inflammatory response whose mothers had spontaneous onset of labour was 67% (65/96) significantly higher than the 27% (238/856) with no fetal inflammatory response. Adjusted odds ratios are shown in [Table 2](#tab2){ref-type="table"}. 3.3. Absence of Fetal Response and Unexplained Death {#sec3.3} ---------------------------------------------------- The absence of a fetal inflammatory response was strongly associated with the classification of unexplained antepartum death. The proportion of stillborn babies without a fetal inflammatory response who were classified as unexplained deaths was 53% (454/856) compared with only 16% classified as unexplained with a fetal inflammatory response. Adjusted odds ratios are shown in [Table 2](#tab2){ref-type="table"}. The association of a fetal inflammatory response with the other PSANZ classifications for the cause of death is documented in [Table 3](#tab3){ref-type="table"}. 4. Discussion {#sec4} ============= This represents the largest study of histological chorioamnionitis in a stillborn cohort to date and is truly population based. We have confirmed our previously published findings of a bimodal distribution of chorioamnionitis in stillbirth with increased incidence in both early and late gestation. We have also reproduced the striking correlation between the presence of a fetal inflammatory response and the spontaneous onset of labour, and the absence of that fetal inflammatory response and the classification of unexplained antepartum death using a standardised classification system. The strengths of this study are that it is population based and, therefore, includes a large number of stillbirths from a birth cohort of more than 250,000 women reducing the likelihood that our findings may have occurred by chance. With a larger dataset, we were able to perform multivariate analysis to test the associations of labour and unexplained death with fetal inflammatory response and adjust for potential confounders. This represents further contribution to the area as the majority of previous studies have provided univariate data only \[[@B12], [@B13], [@B14]--[@B23]\]. The classification of cause of death uses the PSANZ Perinatal Death Classification system which has been endorsed nationally and has now also been shown to perform well against other classification systems used internationally \[[@B33]\]. The members of the committee who attribute the classification all have clinical content knowledge, relevant investigations, and confidential reports forwarded from the hospital of delivery and are experienced in classifying perinatal deaths. There are, however, inherent weaknesses in using large population-based datasets. The detail that can be obtained is limited to what routine information is collected and there is often a significant time lag between when the information is collected to when it is published or available to researchers. Placental pathology was performed in different laboratories and by different pathologists; however, previous literature has shown that even with different pathologists intra- and interobserver agreement of the presence of intrauterine inflammation remains consistently high as does the clustering of findings that relate to ascending infection \[[@B34]\]. Furthermore, all reports were reviewed and coded by one clinical researcher. A further issue as in many studies that assess stillbirth is the unavailability within the same cohort of placental pathology on live born infants. It is well documented that there is a high incidence of chorioamnionitis in preterm live born babies that decreases with gestation \[[@B35]\]. There is minimal data in term live born babies but what is published suggests this incidence is low. Previous studies that have assessed chorioamnionitis in later gestation in live born babies have shown a slight increase in healthy term infants of 3.6% at term and 5.1 % after term \[[@B36]\]. A more recent study that assessed fetal inflammatory response at term showed an incidence of 4% and associated this with both microbial invasion of the amniotic cavity and intra-amniotic inflammation \[[@B37]\]. Both these indicate a significantly lower incidence at term than that reported in stillbirths in both this cohort and in our previous hospital cohort. In summary, we confirm our previous findings of a bimodal distribution of histological chorioamnionitis in stillbirth. This consistent finding of an increased incidence of histological chorioamnionitis in term stillborn infants is important and requires further exploration. On average, 20--70% of stillbirths are unexplained and the unexplained group increases with gestation with 60% of NSW stillbirths remaining unexplained at 37 weeks or greater \[[@B4]\]. Our findings suggest that undiagnosed infection may be implicated in a proportion of these unexplained deaths. We have also been able to demonstrate clearly, adjusting for confounders, the significant relationships between spontaneous onset of labour and fetal response and the absence of that fetal response and the classification of unexplained antepartum death. This contributes to the "failure to rescue by birth" hypothesis that a fetus who is unable to mount a sufficient immune response to trigger labour may be more likely to die in utero \[[@B11], [@B29]\]. Future research in this area requires elucidation of the underlying biological mechanisms of intrauterine inflammation and its consequences, further study of histological chorioamnionitis in term infants, and standardisation of placental reporting. Priority areas related to underlying mechanisms include molecular and genetic studies assessing fetal and maternal immune function. The incidence and significance of histological chorioamnionitis needs to be determined for term live born infants within large cohorts where antenatal, delivery, and outcome data are known. Finally, the development of an international standardized approach to placental pathological examination and reporting is long overdue and will enable comparison of these findings in different settings. Supplementary Material {#supplementary-material-sec} ====================== ###### Appendix S1: The New South Wales Midwives Data Collection (MDC) is a population-based surveillance system covering all births in NSW public and private hospitals, as well as home births. It encompasses all live births and stillbirths of at least 20 weeks gestation or at least 400 grams birth weight. Appendix S2: The NSW Maternal and Perinatal Committee is a quality assurance committee established under the NSW Health Administration Act 1982 and is privileged under this Act to carry out confidential reviews of both maternal and perinatal deaths. Information available to the POWP at review is forwarded by hospitals and includes a confidential report on perinatal death (Appendix S2), postmortem, and placental pathology reports as well as any other information considered relevant by the local hospital perinatal death review committee. ###### Click here for additional data file. The authors have no conflict of interests to declare. Adrienne Gordon designed the study, linked the datasets, extracted data, and reviewed all placental reports. Monica Lahra and Adrienne Gordon interpreted data and performed statistical analysis. Camille Raynes-Greenow provided assistance with presentation of results and the final manuscript. Heather Jeffery provided overall supervision and advice. All authors reviewed the study findings and read and approved the final paper. Ethical approval was obtained from the NSW Department of Health Ethics Committee ref. no. DoHEC 2005-06-11. The authors are grateful to Lee Taylor and the Centre for Epidemiology and Research at the NSW Department of Health for record linkage and providing the MDC and perinatal death data. They would also like to thank Dr. Patrick Groenestein, F.R.A.C.P., Ph.D., The George Institute, Sydney, Australia, for his assistance with the curve fit statistical analysis and Professor Sandy Raeburn for comments on the final paper. M. Lahra and A. Gordon were supported by the National Health and Medical Research Council Public Health Scholarships. {#fig1} ###### Demographic details of stillborn cohort. Variable *n* = number Percentage (*n/N*) ------------------------------- -------------- -------------------- \*Maternal age group (year s)  \< 20 44 4.9  20--24 143 15.9  25--29 246 27.3  30--34 275 30.5  35--39 148 16.4  ≥40 45 4.8 \*Primigravid  Yes 423 46.9  No 478 53.1 \*Preexisting diabetes  Yes 16 1.8  No 885 98.2 \*Gestational diabetes  Yes 27 3  No 874 97 Chronic hypertension  Yes 29 3  No 923 97 Preeclampsia  Yes 59 6.2  No 893 93.8 Labour onset  Spontaneous 303 31.8  Induced 534 56.1   No labour 105 11  Not stated 10 1.1 Delivery type  Vaginal 577 60.6  Forceps 16 1.7  Ventouse 15 1.6  Vaginal breech 181 19  Caesarean 151 15.9  Other 8 0.8  Not stated 4 0.4 Antepartum stillbirth  Yes 864 90.8  No 88 9.2 Baby gender  Male 465 48.8  Female 476 50  Indeterminate 4 0.4  Not stated 7 0.7 Unexplained stillbirth  Yes 469 49.3  No 483 50.7 Spontaneous onset of labour  Yes 303 31.8  No 649 68.2 Weight \< 10th Percentile\*\*  Yes 217 22.8  No 735 77.2 Post mortem performed  Yes 393 41.3  No 559 58.7 Gestational age (weeks)  20--24 228 24  25--29 178 18.7  30--36 259 27.2   ≥37 287 30.1 \*Data for these variables collected from MDC available on 901/952 stillborn babies. \*\*Calculated using Australian National Birth weight Percentiles \[[@B32]\]. ###### Relationship of fetal response to spontaneous onset of labour and unexplained fetal death. Fetal response *n* = 96 (%) Absence of fetal response *n* = 856 (%) Unadjusted odds ratio (95% CI) Adjusted odds ratio\* (95% CI) *P* value --------------------------------------------- ----------------------------- ----------------------------------------- -------------------------------- -------------------------------- ----------- Spontaneous onset of labour (*n* = 303/952) 65 (67%) 238 (27%) 5.4 (3.5--8.6) 4.4 (2.7--7.2) \< 0.0001 Unexplained death (*n* = 469/952) 15 (16%) 454 (53%) 6.1 (3.5--10.7) 4.5 (2.4 -- 8.4) \<0.0001 \*Adjusted for: fetal growth restriction \< 10th percentile, bleeding during pregnancy, maternal medical conditions, maternal hypertension, fetal or maternal proven infection, and gestational age. ###### Relationship of fetal response to cause of death. Fetal response ---------------------------------------- --------------------- ---------------- --------- ------- Absent Present Total PSANZ classification of cause of death Perinatal infection 20 18 38 52.6% 47.4% 100.0% Hypertension 58 0 58 100.0% .0% 100.0% Antepartum haemorrhage 84 3 87 96.6% 3.4% 100.0% Maternal disease 45 1 46 97.8% 2.2% 100.0% Perinatal conditions 76 5 81 93.8% 6.2% 100.0% Hypoxic peripartum death 18 5 23 78.3% 21.7% 100.0% Fetal growth restriction 31 3 34 91.2% 8.8% 100.0% Spontaneous preterm 69 46 115 60.0% 40.0% 100.0% Unexplained antepartum death 454 15 469 96.8% 3.2% 100.0% No obstetric antecedent 1 0 1 100.0% .0% 100.0% Total 856 96 952 89.9% 10.1% 100.0% [^1]: Academic Editor: Francesco DeSeta

Case Report {#sec1-1} =========== A 20-year-old boy reported to us with a history of right eye (RE) uneventful mitomycin-C (MMC)-assisted trabeculectomy done 21 days before for medically uncontrolled secondary glaucoma. He had underwent vitrectomy with silicone oil tamponade for traumatic retinal detachment 4 months back. His chief complaint was gradual blurring of RE vision since last 1 week. Left eye (LE) ophthalmic history was unremarkable. On examination his best corrected visual acuity (BCVA) in RE was 6/36 and 6/6 in LE. Intraocular pressure (IOP) in RE was 36 mm Hg (with maximum tolerated topical and oral antiglaucoma therapy) and 12 mm Hg in LE. Anterior segment examination of RE revealed a superior filtering bleb with a 2 mm area of yellowish infiltration suspicious of blebitis \[[Fig. 1](#F1){ref-type="fig"}\]. Anterior chamber was quiet. Small silicone oil bubble and subluxated traumatic cataract were the other significant findings. Posterior segment examination revealed optic disc cupping of 0.7, attached retina with old laser scars, and silicone oil-filled vitreous cavity without any signs of vitreous inflammation. LE ocular examination was unremarkable. {#F1} Anterior segment ocular coherence tomography (AS-OCT) of RE revealed low reflective inner cavity with a thick hyperreflective bleb wall with irregular bumpy surface suggestive of failing filtering bleb \[[Fig. 2](#F2){ref-type="fig"}\]. {#F2} He was on topical moxifloxacin eye drops three times a day and cyclopentolate eye drops once at night. With a provisional diagnosis of blebitis, we planned RE bleb revision and scleral patch grafting with retinal re-intervention backup. After opening up the conjunctival flap over the bleb area, we found three leftover MMC sponges in the area of yellowish infiltration \[[Fig. 3](#F3){ref-type="fig"}\]. The MMC sponges were sent for pathological assessment and the subconjunctival space was thoroughly washed with balance salt solution. The trabeculectomy fistula was found to be patent and functional intraoperatively. Lastly the conjunctiva was closed with watertight sutures. Since we could not see posterior segment inflammation intraoperatively, retinal re-intervention was not done. {#F3} Postoperatively, the RE BCVA improved to 6/18 at 2 weeks. The IOP was 10 mm Hg with a diffuse filtering bleb \[[Fig. 4](#F4){ref-type="fig"}\]. The MMC sponge did not yield any positive growth on culture. He maintained BCVA of 6/18 and stable IOP in normal range at 3 and 6 months follow-up visits on topical anti-glaucoma medications without any recurrences of inflammation. One year later he underwent combined cataract surgery, Ahmed glaucoma valve shunt with silicone oil removal and is presently maintaining BCVA of 6/12 and stable IOP. {#F4} Discussion {#sec1-2} ========== Trabeculectomy with the use of antimetabolite agents is the most successful and popular treatment for the glaucoma.\[[@ref1]\] However, its high success rate is being confounded by various antimetabolite-related complications such as hypotonus maculopathy, cataract, and various bleb-related complications specially bleb related endophthalmitis.\[[@ref2][@ref3]\] The incidence of bleb-related infections has been reported to 0.55% for blebitis and 0.45%--1.3% for bleb-associated endophthalmitis.\[[@ref4]\] Various devices like scleral shields, methyl cellulose sponges, gel foam discs, and cellulose sponges are available to deliver antimetabolite agents. When used in surgical procedures, very fine particles of this lint frequently become dislodged. It stays at the site as foreign particles which may potentially initiate inflammation and contribute to early postoperative failure of the filtering bleb. Choudhary S, *et al.* has reported a case of granulomatous inflammation 3 weeks postoperatively after combined phacoemulsification and trabeculectomy surgery with MMC due to retained microfragments of methyl cellulose sponge.\[[@ref5]\] Similarly Shin, *et al.* has reported a case of a retained cellulose sponge in the subconjunctival space 3 weeks after the primary surgery.\[[@ref6]\] In our case, yellowish infiltration was seen exactly on the filtering bleb caused due to retained sponges presented after 3 weeks. We suspected it to be a case of blebitis because of its location and appearance. Anterior chamber was quiet and there was no bleb leak. We believe that the leftover MMC sponge acted as a mechanical barrier preventing migration of cellular debris and inflammatory cells into the anterior chamber. Furthermore, AS-OCT helped us in suspecting that it is not only a failing bleb but also something else. Functioning filtering bleb is described as having multiform wall reflectivity with the pattern of multiple internal layers and microcysts on AS-OCT.\[[@ref7]\] Here, AS-OCT showed low reflective inner cavity with a thick hyperreflective bleb wall with irregular bumpy surface \[[Fig. 2](#F2){ref-type="fig"}\]. Needless to say, it is very important to differentiate blebitis and endophthalmitis to plan the treatment protocols. Commonly accepted definition of blebitis is anterior segment inflammation with mucopurulent material in or around the bleb, with anterior chamber cells but without hypopyon. Thus anterior chamber and vitreous examination for hypopyon and inflammatory cells can aid to differentiate the above two entities.\[[@ref8]\] Various methods have been described in the literature to avoid retained antimetabolite sponges. Shin *et al.* has suggested thorough irrigation and inspection of the subconjunctival space and counting the sponge.\[[@ref6]\] Poole *et al.* suggested use of polyvinyl alcohol (PVA) microsurgical sponge for antiproliferative agent delivery as its cut pieces do not leave behind any microfragments, when soaked with antimetabolite.\[[@ref9]\] Intraoperative use of MMC injection has also been studied showing equal efficacy as MMC sponges.\[[@ref10]\] This can also be considered as a useful procedure in view to avoid retained MMC sponges. We usually count the MMC sponges while placing under the conjunctiva and match the number during the removal and wash the area thoroughly using continuous irrigation after removing the sponges. Regular follow-ups and detailed clinical examination help in titrating treatment protocols. We believe early surgical reintervention coupled with frequent postoperative follow-ups and good patient compliance favored good visual outcome in our case scenario. Declaration of patient consent {#sec2-1} ------------------------------ The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship {#sec2-2} --------------------------------- Nil. Conflicts of interest {#sec2-3} --------------------- There are no conflicts of interest.

Introduction {#Sec1} ============ The wild strawberry, *Fragaria chiloensis* L. Duch., is a native species from Chile, which is distributed from the arctic circle in the west of North America to the southernmost point of Chile and Argentina^[@CR1],[@CR2]^. The species produces fruits, which follow a non-climacteric pattern, and are characterized by an attractive appearance and quality attributes such as taste and aroma, in addition to its high nutritional value imparted by elevated concentrations of minerals, vitamins and antioxidants^[@CR3]^. Several attributes make the fruit of *Fragaria chiloensis* an attractive agronomic resource, but the accelerated fruit softening and a short flowering reproductive period are negative aspects affecting production^[@CR4],[@CR5]^. The fruit ripening process comprises a highly coordinated, irreversible and genetically programmed event which involves many biochemical, physiological and organoleptic modifications that lead to the development of a soft and edible ripe fruit with ideal quality features. This is concomitant with various changes such as chlorophyll degradation, carotenoid or anthocyanin biosynthesis, increased respiration, essential oils, flavor and aroma components, increased activity of cell wall-degrading enzymes, and transient increases in hormonal production that take place during fruit ripening^[@CR6]^. During ripening of fleshy fruit important modifications in the cell wall structure take place related with wall components whose solubility percentage increases, polymer length decrease, and linkages between many kinds of polymers are modified to produce a decrease in fruit firmness^[@CR7]--[@CR9]^. NAC genes are a transcription factor family (TF), which have been found to play important roles in plant development and environmental responses. Members of this family have been reported to be involved in the ripening and softening of fleshy fruits such as citrus^[@CR10]^, banana^[@CR11]^, tomato^[@CR12]^, and peach^[@CR13]^. The acronym of NAC comes from the first three genes described, which contain the domain: NAM (No Apical Meristem) from petunia^[@CR14]^, ATAF1 and ATAF2 (GenBank accession numbers X74755 and X74756) and CUC (Cup-Shape Cotyledon) from *Arabidopsis thaliana*^[@CR15]^. NAM has been associated in determining positions of meristems and primordia in petunia^[@CR14]^. ATAF1 and ATAF2 are negative regulators of defense response against pathogens in Arabidopsis^[@CR16],[@CR17]^, while CUC genes are involved in shoot apical meristem (SAM) formation and cotyledon separation in the embryogenesis^[@CR18]^. These TFs have been identified as large multi gene families^[@CR19]^: 117 genes have been described in Arabidopsis, 151 in rice, 79 in grape, 26 in citrus, 163 in poplar, 152 in soybean and 152 in tobacco^[@CR20]--[@CR23]^. These TFs share a NAC domain consisting of 130--150 amino acids at their N-terminus that allows for their interaction with DNA, which is divided into five subdomains, A--E, from N-terminus to C-terminus respectively^[@CR24]^. The protein also contains a transcriptional regulatory (TR) domain at the C-terminal region, and some NAC TFs have a transmembrane domain within the TR domain^[@CR25]^. Phylogenetic analyses of NACs from nine plant species revealed that these TFs are divided into 21 subfamilies, some of which are specific to either monocots or eudicots^[@CR26]^. To date there are some reports involving members of the NAC family in processes related to ripening of fleshy fruit. For example, in tomato (*Solanum lycopersicum*) SlNAC4 shows high transcript accumulation in sepals and the early stages of ripening. RNAi assays of SlNAC4 results in delays to the ripening process of tomato fruit, by reducing transcript levels of genes related to the ethylene pathway and a decrease in carotenoid content^[@CR12]^. Other reports have shown that the overexpression of *SlNAC1* results in a reduction of carotenoids by altering carotenoid pathway flux and decreasing ethylene synthesis mediated mainly by reduced expression of ethylene biosynthetic genes, thus leading to yellow or orange mature fruits^[@CR27]^. In banana (*Musa acuminata*) six NAC genes (*MaNAC1*-*MaNAC6*) have been described to show a differential expression profile between skin and pulp, in which MaNAC1 and MaNAC2 play a direct role in fruit ripening^[@CR11]^. More recently, a NAC gene from kiwifruit has been associated with monoterpene production, and it has been proposed to be crucial in promoting the synthesis of aroma during ripening^[@CR28]^. Information about NAC transcription factors related to cell wall remodeling in fleshy fruits has not been widely described. The genes encoding the suite of enzymes required promoting cell wall remodeling needs to be coordinately regulated in a temporal and spatial manner. In this sense, the activation of secondary wall biosynthetic genes is modulated by a transcriptional network, including secondary wall NAC (SWN) master switches and their downstream transcription factors^[@CR29]^. The role of NAC TFs in the ripening and softening process of Chilean strawberry is still unknown. A partial fragment of a NAC gene was identified as differentially expressed in a suppressive substractive hybridization (SSH) library prepared from Chilean strawberry fruit^[@CR30]^. Using rapid amplification of cDNA ends (RACE) the full-length coding sequence was obtained. This gene, termed *FcNAC1* was characterized to elucidate its participation during the ripening of the Chilean strawberry fruit. This characterization included the analysis of its sequence and the confirmation of a functional DNA binding domain, its nuclear localization, and attempts to explain its transcriptional regulatory effect on genes related to cell wall remodeling associated with fruit softening. To complete the study the role of hormones that participate in ripening of strawberry fruit on the transcriptional regulation of FcNAC1 was analyzed. Results {#Sec2} ======= Cloning the Full-length of FcNAC1 and Sequence Analysis {#Sec3} ------------------------------------------------------- Starting from a partial fragment (939 bp) of a putative NAC gene^[@CR30]^ the full-length sequence of FcNAC1 was cloned from ripe strawberry fruit RNA samples using 3′ RACE method, employing FcNAC1-RACE1 and FcNAC1-RACE2 primers (Table [1](#Tab1){ref-type="table"}). After performing the two RACE reactions, two fragments of 373 bp for FcNAC1-RACE1 and 461 bp for FcNAC1-RACE2 were obtained. The combined *FcNAC1* sequence (GenBank accession number AKC96459.1) contained an ORF of 999 bp and codes for a deduced polypeptide sequence of 332 amino acid residues with a theoretical molecular mass of 37.3 kDa and an isoelectric point of 7.0. FcNAC1 shares a 99.4% amino acid identity with FvNAC and 62.6% with SND2 (Supplementary Table [1](#MOESM1){ref-type="media"}). FcNAC1 protein sequence contains a highly conserved region towards the N-terminal, corresponding to the NAC domain which is divided into five sub-domains, A to E (Fig. [1A](#Fig1){ref-type="fig"}). The sub-domain C contains the nuclear localization signal (Supplementary Fig. [1](#MOESM1){ref-type="media"}). The protein contains an extension at the N-terminal (NTE). The transcriptional regulatory domain identified at the C-terminal displays low similarity among the sequences. A transmembrane domain was not predicted in the FcNAC1 protein.Table 1Nucleotide sequence of the primers (5′ → 3′) used in this study.NameForward primer sequenceReverse primer sequenceFcNAC1-RACE1AGGAGGCCAGAATAGGGAAAFcNAC1-RACE1TTCCTCCAAAGAAGGCTGGTFcNAC1-RealtimeTGGTATGAGCGGCCTCAGGCTGCCCTCTCTTCTTCCTCFcGAPDH1TCCATCACTGCCACCCAGAAGACTGAGCAGGCAGAACCTTTCCGACAGFcNAC1-Full-lengthCACCATGACATGGCACTCAGATGAGGGTTTCCTTTGCTTGTATCTGCAFcNAC1-GW1CCTCTCTTCTTCCTCTTCCTCATCTGFcNAC1-GW2GCAGAGGAGGGAGTTATTGACTGAACFcNAC1-GW3CTATAGGGTGGCCACAAGACGFcNAC1-GW4GAGTGGATGAAGCTTTCTGGTATCFigure 1Schematic representation of FcNAC1 protein and phylogenetic analysis with NAC sequences. (**A**) Schematic representation of the primary structure of FcNAC1 showing the NTE (N terminal extension), the DNA binding domain (NAC domain) divided into five subdomains (A to E), and the transcriptional regulatory domain, as described in Puranik *et al*.^[@CR24]^. (**B**) Phylogenetic analysis where FcNAC1 groups with members of the NAC family related to wall formation, close to AtSND2. We used amino acid sequences of NAC members with described function: AtNAC2 (AB049071.1), AtNAP (NM_105616.4), AtCUC1 (EU550396.1), AtCUC2 (AB002560), HvNAC6 (AM500854.1), AtNAC1 (NP_175997), TdNAM-B1 (KF541318), AtNST1 (NM_130243), AtSND1 (EF101892), AtSND2 (NC_003075.7), AtNST2 (NP_191750), OsNAC6 (AB028185.1), PeNAM (X92205), PvNAP (XP_007158644), SlNAC1 (NM_001247553.3), OsSNAC1 (KM265360.1), and AtTIP (NP_197847). The transcription factor OsMYB (CAA72218) was used as outlier. The phylogenetic tree was performed with MEGA5 software employing the neighbor joining method, with 1000 replicates. Phylogenetic analysis of FcNAC1 protein {#Sec4} --------------------------------------- A phylogenetic analysis was performed to reproduce the different groups with known function that have been previously reported^[@CR31]^. The analysis included 21 NAC protein sequences including representatives from Arabidopsis, tomato, barley, rice, citrus, petunia and bean with known function, and one homologous sequence from woodland strawberry (*Fragaria vesca*). FcNAC1 protein was grouped into the cluster related to cell wall related NACs, near to FvNAC and SND2 (Fig. [1B](#Fig1){ref-type="fig"}). Additionally, a phylogenetic tree including 33 other recently reported proteins^[@CR32]^ was built, and FcNAC1 grouped in the Vascular-related NAC Domain (VND) cluster (Fig. [2](#Fig2){ref-type="fig"}). FcNAC1 was clustered in the same branch with SND2, a member of the NAC family related to cell wall formation.Figure 2Phylogenetic analysis of FcNAC1 with VNS (*V*ND, *N*ST and *S*MB) proteins as Master Regulators of cell wall formation. In the analysis, members of the NAC family with function related to cell wall remodeling were included. Representative genes of NST, SMB, VND and ancestral groups were considered for the analysis^[@CR32]^. The phylogenetic tree was performed with MEGA5 software employing the neighbor joining method, with 1000 replicates. Subcellular localization of FcNAC1 protein {#Sec5} ------------------------------------------ As a transcription factor, FcNAC1 should be localized in the nucleus. Its nuclear localization was predicted using the web server NLS Mapper (<http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi>) (Supplementary Fig. [2](#MOESM1){ref-type="media"}). In order to demonstrate its *in vivo* subcellular localization the coding sequence was fused to GFP protein sequence and cloned into pK7FWG2.0 vector under the control of 35 S promoter. The plasmid was transfected into Agrobacterium (LBA4404) and then tobacco leaves were agro-infiltrated. After 3 days the fluorescence from FcNAC-GFP fusion protein was measured. A strong fluorescence signal was detected in the nucleus (Fig. [3](#Fig3){ref-type="fig"}), which co-localized with the fluorescence of the nuclear stain SyTo^®^ 84, confirming the nuclear localization of FcNAC1.Figure 3*FcNAC1* subcellular localization of the transcription factor by transient transformation of tobacco leaves. Co-localization assay of FcNAC1 protein fused to GFP in vector pK7WGF2.0. *Agrobacterium tumefaciens* LV300 strains were used to transiently transform young leaves from tobacco plants (6-week-old). Syto84 staining was used to locate the nucleus. GFP: Green Fluorescent Protein, Syto84: nuclear staining, MERGE: superposition of GFP and Syto84 images. The white bar indicates 10 µm. FcNAC1 transcript levels in developing strawberry fruit and other tissues {#Sec6} ------------------------------------------------------------------------- *FcNAC1* displays a differential expression pattern during fruit development in *F*. *chiloensis* (Fig. [4](#Fig4){ref-type="fig"}). *FcNAC1* showed low transcripts level at C1 and C2 stages, which correspond to unripe fruit stages. An increment in the transcriptional level occurs at the C3 stage, maintaining a high transcript level at C4 stage (Fig. [4A](#Fig4){ref-type="fig"}). Other tissues were tested to identify the specific expression of *FcNAC1*. Gene expression analysis showed that *FcNAC1* transcript abundance was high in flowers when compared with the other vegetative tissues. Similar low transcript levels were detected in roots, runners and leaves (Fig. [4B](#Fig4){ref-type="fig"}).Figure 4Levels of *FcNAC1* transcripts in different *F*. *chiloensis* tissues. Quantification of transcripts was performed by qRT-PCR employing specific primers: (**A**) during development and ripening of *Fragaria chiloensis* fruit (C1, C2, C3 and C4); (**B**) different plant tissues (leaves, flowers, roots, stem and runners). RNA extractions from the samples were performed using the CTAB method. *FcGAPDH* was used as internal calibrator gene. Different letters indicate statistical difference between fruit stages (**A**) or plant tissues (**B**). Significant differences were determined at *p* ≤ 0.05 (LSD Fisher test). In order to facilitate the comparison of transcripts level, C1 fruit stage in A, and leaf in B were normalized to 1. All pictures were taken by one of the authors. Changes in the expression of FcNAC1 by hormones {#Sec7} ----------------------------------------------- In order to test the hormonal effect on the transcription of *FcNAC1*, fruit at the C2 stage were treated with ABA or synthetic auxin (NAA) (Fig. [5A](#Fig5){ref-type="fig"}). The relative expression level of *FcNAC1* in untreated C2 fruit (control) displayed a decreasing expression pattern during the observation period (12 h at room temperature). In response to ABA, a rapid accumulation of *FcNAC1* transcripts was observed after 1 h of treatment, and then a significant reduction was observed after 2 and 12 h. In contrast, a reduction in the expression level of *FcNAC1* transcripts was observed after 1 h of auxins treatment, and maintained during the following 12 h. These results indicate that FcNAC1 displays a different expression response to ABA and auxins.Figure 5Effect of hormones on the transcription of *FcNAC1*. (**A**) Expression changes of *FcNAC1* in response to ABA and auxins. *F*. *chiloensis* fruit at the C2 stage was subjected to hormonal treatments (1 mM ABA and 1 mM ANA) or immersed in buffer (control). Samples were collected just after treatment (10 min), and after 1, 2 and 12 h, and stored at −80 for subsequent RNA extractions by the CTAB method. Expression analyses were performed through qRT-PCR. For the same treatment, different letters indicate significant differences during the observation period. Significant differences were determined at *p* ≤ 0.05 (LSD Fisher test). (**B**) Analysis of *FcNAC1* promoter. By means of the genome Walker technique, a sequence of 1488 bp corresponding to the promoter sequence of FcNAC1 was obtained and *in silico* analyzed with PlantCARE database (<http://bioinformatics.psb.ugent.be/webtools/plantcare/html/>). Different putative cis-elements related to several hormonal responses were identified along the sequence. Analysis of FcNAC1 promoter sequence {#Sec8} ------------------------------------ In order to understand the transcriptional regulation of *FcNAC1*, its promoter sequence was isolated. A 1488 bp sequence was obtained from genomic *F*. *chiloensis* DNA. An *in silico* analysis was carried out to search for putative *cis*-regulatory elements related to hormonal response. The promoter of FcNAC1 contains five ABA-related sequences, in addition to sequences related to auxins, gibberellins, methyl jasmonate and salicylic acid responses (Fig. [5B](#Fig5){ref-type="fig"}). The *cis*-regulatory elements identified in the promoter of FcNAC1 might explain their differential hormonal response. Putative *cis*-regulatory elements for NAC binding were also identified in the promoter (Supplementary Fig. [3](#MOESM1){ref-type="media"}). Functional analysis of FcNAC1 gene and promoter sequences in transiently transformed tobacco {#Sec9} -------------------------------------------------------------------------------------------- For functional analysis independent genomic constructs were built for *FcNAC1* (35 S:*FcNAC1-GFP*) and for the promoters of two cell wall degrading genes (*FcExp2* and *FcPL)* (promoter:*LUC*) which have been reported to be important for *F*. *chiloensis* softening^[@CR33]^. These constructs were cloned and transfected into Agrobacterium (GV3101), then agro-infiltrated into *N*. *benthamiana* leaves for transient expression^[@CR34]^. The expression of FcNAC1 protein did not change the Luc/Ren activity ratio in the case of the promoter of *FcExp2*, however a significant increase was observed in the activity of the promoter of *FcPL* (Fig. [6](#Fig6){ref-type="fig"}). This indicated that the FcNAC1 protein is able to activate the promoter of *FcPL*. As a positive control FvMYB10 and FvbHLH, increased the activity of the FvDFR promoter, a gene described to be transcriptionally regulated by these two transcription factors^[@CR34]^. There were no significant differences in the transactivation activity of *FvDFR* promoter sequence by FcNAC1.Figure 6Functional assay of *FcNAC1* gene by transient transformation of tobacco leaves. The ratio of LUC/REN activities obtained after Dual Luciferase interaction assay of *FcNAC1* with the promoter sequences of *FvDFR* (Dihydroflavonol reductase, 1560 bp) (XP_004308377), *FcEXP2* (Expansin 2, GenBank KC527027, 847 bp) and *FcPL* (Pectate lyase, GenBank KC527025, 1038 bp) in transiently transfected *N*. *benthamiana* leaves are shown. *GUS* represents the plasmid 35SPro-*GUS* (negative control). *FvMYB10* (GenBank EU155163.1) and *FvBHLH* (Basic helix loop helix XP_004308377; 1932 bp) were used as positive controls of the technique. Bars represent the bioluminescent measure of 4 replicates ± standard deviation. Different letters indicate statistical differences (*p* ≤ 0.05) compared to respective *GUS* control. Structural modeling of FcNAC1's NAC domain {#Sec10} ------------------------------------------ The 3D monomer structure of FcNAC1 NAC domain was built using the chain A of crystallographic structure of ANAC019 from *Arabidopsis thaliana* (PDB code: 3SWM) as template. The pairwise alignment of the NAC domain from FcNAC1 (from residue 56 to 222) predicted with SMART software (<http://smart.embl-heidelberg.de/>) (167 residues in total) against chain A of the template shares 28.5% identity (Fig. [7A](#Fig7){ref-type="fig"}). In terms of geometric stability, 2 nanoseconds of Molecular Dynamics Simulation (MDS) were performed and the protein model showed a RMSD of \~1.5 Å (Supplementary Fig. [4](#MOESM1){ref-type="media"}). The structural model was validated through PROCHECK, showing that 82.4% of the residues were located in the most favoured regions with no poorly modeled residues identified (Table [2](#Tab2){ref-type="table"}). In addition, ProSA analysis confirmed the good quality of the model (Z-score: −4.64) (Supplementary Fig. [5](#MOESM1){ref-type="media"}). The final structural conformation of FcNAC1 NAC domain forms a twisted anti-parallel β-sheet, which is composed of one α-helix, one α-helix 3~10~, five β sheets and eight loops. It can be observed that this β-sheet is packed between an N-terminal α- helix and a short α-helix 3~10~ on the other side (Fig. [7B](#Fig7){ref-type="fig"}).Figure 7Homology modeling of the NAC domain present in FcNAC1 protein. (**A**) The alignment between the template (3SWM chain A) and FcNAC1 NAC domain, including structural information. Spirals and arrows at the top of the sequences represent the secondary structure of the template, while those at the botton correspond to predicted secondary structure of FcNAC1. Triangles indicate the important residues for the dimerization process and asterisk indicate conserved residues for DNA interaction. (**B**) Homology modeling of FcNAC1 NAC domain using MODELLER software. Fifty models were built and the best was subjected to energy minimization and molecular dynamics with the purpose of relaxing the structure and minimizing steric impediments. It is possible to appreciate that the cluster of β sheets is flanked on both sides by the α-helix and the α-helix 3~10~. α-helices were labeled as α, and β-sheets as β. (**C**) Homodimer structure of FcNAC1 NAC domain showing the potential residues involved in the salt/bridge interaction. (**D**) Protein-DNA positioning between the homodimer of FcNAC1 and the DNA sequence co- crystallized with the template 3SWM (5′- GTCTTGCGTGTTGGAACACGCAACAG -3′). (**E**) Visualization of the homodimer structure of FcNAC1 NAC domain highlighting the positively charged residues involved in the protein-DNA interaction.Table 2Validation of FcNAC1 NAC domain structure by PROCHECK program.Core (%)^**a**^Allow (%)^**b**^Gener (%)^**c**^Disall (%)^**d**^FcNAC1-NAC domain82.416.80.80^a^Most favorable regions.^b^Additional allowed regions.^c^Generously allowed regions.^d^Disallowed regions. The FcNAC1 homodimer structure using both chains of the template, A and B, was built (Fig. [7C](#Fig7){ref-type="fig"}). The physical interaction of both monomers involved the conserved N-terminal sequence. The dimerization interaction involved two prominent salt bridges between Lys67 and Glu74 from adjacent chains. On the other hand, the interaction of the homodimer with DNA was also tested, showing that β-sheets β3 and β4 are involved in protein-DNA interaction, allowing the recognition of major DNA grooves (Fig. [7D](#Fig7){ref-type="fig"}). Additionally, the presence of positively charged residues (Lys189 and Lys192) in this domain, allows the interaction of the DNA sequence and FcNAC1 transcription factor (Fig. [7E](#Fig7){ref-type="fig"}). Discussion {#Sec11} ========== The characterization of the *F*. *chiloensis* NAC transcription factor, named *FcNAC1*, revealed a protein sharing a highly conserved NAC domain and a variable C-terminal region that can act as a domain of transcriptional activation. The domains identified in FcNAC1 were initially described in *A*. *thaliana* and *Oryza sativa*^[@CR35]^. Multiple alignments showed that FcNAC1 shares high similarity with sub-domains B, C and D from FvNAC and SND2 proteins. A nuclear localization signal was predicted which was confirmed by the transient transformation into tobacco cells of FcNAC1 fused to a reporter gene. Other members of this NAC family also contain the same nuclear localization: a NAC from *S*. *lycopersicum* was shown to be located in the nucleus of onion epidermal cells^[@CR36]^, as was also the case for AtNAC2^[@CR36]^. MtNST1, described as a secondary cell wall master switch, was also identified in the nucleus of epidermal tobacco cells^[@CR37]^. The tomato SlNAC3 has been localized in the nucleus of onion epidermal cells and reported to respond to environmental and endogenous stimuli and is likely to function during the response of plants to salt and drought stresses^[@CR38]^. The different NAC protein sequences characterized to date can be grouped into six functional sub-groups through phylogenetic analysis, which confirms the groups described previously^[@CR31],[@CR32]^. In this case, FcNAC1 grouped with SND2, which has been related to cell wall formation^[@CR39]^. The overexpression of *SND2* in *Arabidopsis* up-regulates the biosynthesis pathways of genes associated to cellulose, xylan, mannan, pectins and lignin polymerization, playing a crucial role in the secondary cell wall transcriptional network^[@CR40]^. Recently a new classification has grouped NAC genes into three groups^[@CR32]^. *FcNAC1* grouped in the VND cluster, as well as SND2, which is involved in cell wall biosynthesis. *FcNAC1* could be involved in similar functions, contributing to the fruit softening process. The ripening of *F*. *chiloensis* fruit, and in particular its softening process, has been studied and several enzymes related to cell wall assembly/disassembly have been shown to increase during ripening. The transcript abundance of *FcEXP2* (*Expansin 2*), *FcPL* (*Pectate lyase*), *FcPG* (*Polygalacturonase*)^[@CR5]^ and *FcXTH1* (*Xyloglucan transglycosylase/hydrolase*) all increased during ripening development of *F*. *chiloensis* fruit, as well as their activity. In addition, it has been reported that fruit firmness dramatically falls during the transition from C2 to C3 stage^[@CR41]^. In accordance with this profile, *FcNAC1* transcripts level increase in the same developmental period, remaining at a high level until the end of fruit ripening. Kang *et al*.^[@CR42]^ reported transcriptomic analysis during development of *Fragaria vesca* fruit, starting from flowering. Interestingly, *FvNAC* gene, with close homology to *FcNAC1*, showed a high accumulation of transcripts after nine days post anthesis. This indicated that the expression pattern is similar in *F*. *chiloensis* and *F*. *vesca*. Coincidently, a peak of transcript accumulation was observed in flowers of both species. In *F*. *vesca*, transcripts coding for *EG*, *Exp2*, *PG*, *PL* and *XTH* were also identified as differentially expressed during fruit development^[@CR41]^. Moreover, the analysis of the promoter sequences of these genes in *F*. *vesca* contains cis-elements that are recognized by NAC members. Therefore, the involvement of *FcNAC1* in the regulation of genes related to cell wall remodeling might be expected. In the commercial strawberry *Fragaria x ananassa*, the concentration of auxins decays during development of the fruit, while the concentration of abscisic acid increases^[@CR43]^. The literature describes that abscisic acid (ABA) plays a crucial role in the regulation of strawberry fruit ripening and exogenous applications of this hormone promotes the ripening of this berry^[@CR44]^. There is information about members of the NAC family and its relationship with different types of stresses, where it has been possible to show the different responses by NAC members from various species such as *S*. *lycopersicum*, *A*. *thaliana*, *Oryza sativa*, *S*. *tuberosum*, *Triticum aestivum*, *Brassica napus*, *Zea mays*, *sugarcane*, *Petunia hybrida and Citrus sinensis* to diverse kinds of stimuli (heat, cold, salt, water, wounding, insect/pathogen, methyl jasmonate and abscisic acid) producing variations in the transcription profiles of these NACs members^[@CR45]^. Information is available on the specific modulation of diverse NAC transcription factor members by ABA; *ANAC019*, *ANAC055*, *ANAC072*, *RD26*, *RD20*, *ZmSNAC1*, *ANAC2*, *SNAC2*, *OsNAC5*, *OsNAC10*, *DgNAC1* and *CarNAC3* respond to ABA and, in some cases, ABA signaling^[@CR46]^. In agreement with this, cis-elements related to hormonal response were identified in the promoter of *FcNAC1* suggesting the participation of hormones in its transcriptional regulation. ABA and auxins were able to modulate the accumulation of *FcNAC1* transcripts as it has been previously reported in *S*. *lycopersicum*^[@CR45]^. An increase in the accumulation of transcript of *FcNAC1* by ABA and a decrease in the transcripts level after treatment with auxins were observed in *F*. *chiloensis* fruit. The subgroup of NAC TFs related to cell wall biosynthesis (SWN, Secondary wall NAC domain) are able to promote the expression of diverse transcription factors, such as *SND2*, *SND3*, *MYB46*, *MYB103*, *MYB85*, *MYB52*, *MYB54*, *MYB69*, *MYB43*, *MYB20*, and *KNAT7*, which led to the proposal that a transcriptional network including SWNs and their downstream targets is involved in the regulation of secondary wall biosynthesis^[@CR46]^. This activation can be carried out by binding an imperfect palindromic 19-bp consensus present in the promoter sequences and designated as a secondary wall NAC binding element (SNBE) (T/A)NN(C/T) (T/C/G)TNNNNNNNA(A/C)GN(A/C/T) (A/T). Studies using the reporter gene *GUS* showed that SWNs bind to SNBE sequences in the promoters of their direct target genes, and thereby activate their expression in the secondary wall-forming cells^[@CR47]^. The same SNBE elements have been identified in the promoter sequence of *FcNAC1* containing the conserved CGT \[A/G\] core sequence that is present in the SNBEs sequences. The interaction between NAC TFs and these motifs have been reported using EMSA experiments, demonstrating that NAC proteins bind DNA as dimers^[@CR48]^. The dual luciferase experiment performed in this work shows the transactivation of a cell wall remodeling gene by a NAC TF of strawberry. NAC TFs are able to modulate the expression of genes acting during fruit ripening, such as those involved in color development^[@CR13]^ or aroma formation^[@CR28]^. FcNAC1 interacts with the promoter sequence of *FcPL*, increasing the expression of genes controlled by this promoter. However, the response was not observed with the promoter of *FcEXP2* or the control promoter, *FvDFR*. This evidence allowed us to hypothesize a possible role of *FcNAC1* in the transcriptional regulation of genes related to pectin metabolism, and in this way, contributing to the process of cell wall remodeling during development and softening of the *F*. *chiloensis* fruits. The 3D structure of the NAC domain present in FcNAC1 was obtained through homology modeling. The homodimeric structure is stabilized by salt-bridges between Lys67 and Glu74 from adjacent FcNAC1 chains. In the literature it has been described that dimerization is supported by Arg25 and Glu32 in the case of ANAC019^[@CR49]^. We hypothesized that Lys could be fulfilling the role of Arg in the dimerization process. On the other hand, NAC, WRKY and GCM TF families share a central β-sheet domain with similar topology, which is responsible for mediating the interaction with DNA^[@CR49]^. The FcNAC1 model showed a motif between β-sheets β3 and β4, which may participate in protein-DNA interaction, and in agreement, the presence of two positively charged residues identified in this motif is congruent with its interaction with DNA. In summary, *FcNAC1* is implicated as a transcriptional regulator during the softening process of the Chilean strawberry fruit. The evidence provided will contribute to understanding the regulatory network that takes place during development and ripening of *F*. *chiloensis* fruit. Future work to identify or generate mutants of *FcNAC1* will aim to confirm the level of this involvement. Materials and Methods {#Sec12} ===================== Plant material {#Sec13} -------------- Chilean strawberry fruit at different stages of development (C1, C2, C3 and C4) and vegetative tissues (flowers, roots, leaves, runners and stem) were collected from plants grown in a commercial field at Contulmo city, Biobío Region, Chile (latitude 38°04′8.6′′S, longitude 73°14′2.96′′W). The fruit was classified into four different developmental stages according to weight and color of the receptacle and achenes as previously reported: C1, small fruit with green receptacle and green achenes; C2, large fruit with green receptacle and red achenes; C3 turning stage, fruit of white receptacle and red achenes; and C4, ripe fruit with pink receptacle and red achenes^[@CR5]^. After harvest, the peduncle and calyx of each fruit were removed, and the fruit cut longitudinally into two halves, frozen in liquid nitrogen and stored at −80 °C for later use. Isolation and cloning of strawberry FcNAC1 cDNA {#Sec14} ----------------------------------------------- From an SSH (suppressive substractive hybridization) library prepared form *F*. *chiloensis* fruit at different developmental stages^[@CR30]^ an EST contig was identified as differentially expressed, and by means of BLAST analysis (<https://blast.ncbi.nlm.nih.gov/Blast.cgi>) its tentative homology to plant NAC sequences was revealed. With the aim to isolate the full-length cDNA sequence of FcNAC1, RACE (Rapid Amplification of cDNA Ends) reactions using RNA from ripe fruit stage as template were performed using the BD Smart RACE cDNA Amplification kit (Clontech, USA). Two 3′-RACE PCR reactions were performed using the Universal Primer A Mix (Clontech) and primers FcNAC1-RACE1 and FcNAC1-RACE2 (Table [1](#Tab1){ref-type="table"}). The expected amplicons obtained were cloned into pGEM-T Easy Vector (Promega, USA) and sequenced at Macrogen, Inc. (Korea). Then a new set of primers was designed to obtain the full-length cDNA of FcNAC1: FcNAC1-Full-length-F and FcNAC1-Full-length-R (Table [1](#Tab1){ref-type="table"}); the [CACC]{.ul} sequence was incorporated to the forward primer for directional cloning. This amplified sequence was cloned into the pENTER^TM^/SD/D-TOPO^®^ vector included in the pENTER^TM^ Directional TOPO^®^ Cloning kit (Invitrogen). PCR reactions were employed to confirm the presence and directionality of FcNAC1 sequence by using the primers mentioned before. Then plasmids were purified employing GeneJET Plasmid Miniprep kit (Thermo Scientific^TM^), and sequenced at Macrogen, Inc. (Korea). The amino acid sequence of FcNAC1 has been submitted to NCBI under the accession number: KP966107.1. Transient expression of FcNAC1 in Nicotiana benthamiana leaves and sub-cellular localization {#Sec15} -------------------------------------------------------------------------------------------- Gateway^®^ LR Clonase^TM^ II Enzyme Mix kit (Invitrogen) was used to perform a 35S:*FcNAC1*-*GFP* construction. The manufacturer's instructions were used for recombination using equivalent amounts of entry vector (160 ng/ul) and destination vector (pK7WGF2.0). The *35 S:FcNAC1-GFP* construct cloned in pK7FWG2.0 vector was introduced into *Agrobacterium tumefaciens* strain LBA4404 ElectroMAX^TM^ (Invitrogen) by thermic shock in liquid nitrogen. Transformed bacteria were plated on a selective medium yeast mold agar containing streptomycin (PhytoTechnology Laboratories) and spectinomycin (PhytoTechnol. Lab.) at a final concentration of 100 µg/ml each. Resistant colonies were analyzed by PCR for the presence of full-length *FcNAC1* gene using the primers mentioned before. A positive colony was cultured in selective YM (100 ml) and incubated at 28 °C until an O.D.~600~ between 0.6 and 0.8. Agroinfiltration suspension was used to inject the abaxial face of young tobacco leaves (two weeks old) and samples were analyzed after three days of infiltration. Syto^®^ 84 Orange Fluorescent Nucleic Acid stain (Thermo Scientific^TM^) was used to label the nucleus. Subcellular localization of FcNAC1 in transient transformed leaves samples was analyzed through visualization of the tissue under a confocal fluorescence microscope (Carl Zeiss Confocal microscopy LMS 700) employing phase contrast image. RNA extraction and Expression analysis by Real Time PCR (RT-qPCR) {#Sec16} ----------------------------------------------------------------- Samples of RNA were extracted from 2 g of *F*. *chiloensis* fruit bulk from each developmental stage (C1, C2, C3 and C4) and other vegetative tissues (flowers, roots, stem, runners and leaves) using the CTAB method with modifications^[@CR50]^. RNA samples were treated with DNase I amplification grade (Invitrogen), and then cleaned using an RNeasy Plant Mini Kit (Qiagen). cDNA synthesis using First Strand cDNA Synthesis Kit (Fermentas) was performed following the manufacturer's instructions. Three biological sample replicates from each fruit stage or tissue were considered. Specific primers designed for 5′-UTR region of *FcNAC1* and 3′-UTR region of glyceraldehyde 3-phosphate dehydrogenase (*FcGAPDH1*; as internal control)^[@CR51]^ were used (Table [1](#Tab1){ref-type="table"}). Primers for 5′-UTR region of *FcNAC1* were designed by Primer3 (<http://frodo.wi.mit.edu/primer3/>). Primers were tested by RT-PCR. For RT-qPCR analysis Maxima SYBR Green/ROX qPCR Master Mix (2×) (Fermentas) was used following manual instructions, in a DNA engine Opticon 2 Real-Time PCR System (MJ Research, Watertown, MA). The amplification efficiency was determined through a dilution series of a cDNA mix prepared from different fruit samples. All qPCR reactions were carried on in duplicates, and water was used as negative controls in each run. Relative expression levels correspond to means of three biological replicates ± standard deviation normalized against the gene expression level of *FcGAPDH1* (constitutive gene), employing C1 stage as the calibrator of fruit samples and leaves as the calibrator of vegetative tissue samples. Different letters of significance indicate significant differences in expression level (*p* ≤ 0.05). Genome walker and analysis of FcNAC1 promoter sequence {#Sec17} ------------------------------------------------------ The *FcNAC1* promoter sequence was obtained by the Genome Walker technique using GenomeWalker^TM^ Universal kit (Clontech) according to the manufacturer's instructions using specific primers designed (Table [1](#Tab1){ref-type="table"}). DNA samples were extracted from 2 g of *F*. *chiloensis* leaves bulk using the CTAB method with modifications^[@CR50]^. DNA samples were subjected to enzymatic digestions with different restriction enzymes: EcoRI, XbaI, HindIII and XhoI, following the protocol described in the kit. After cloning, sequencing and assembling these sequences, the TATA box and cis-elements were predicted using the online sequence analysis program PlantCARE (<http://bioinformatics.psb.ugent.be/webtools/plantcare/html/>)^[@CR52]^. Phylogenetic analysis {#Sec18} --------------------- The deduced amino acid sequences were analyzed using the translate tool of ExPASy (<http://ca.expasu.org>). The similarity search was performed using the local alignment tool (BLAST, National Center for Biotechnology Information, USA). The multiple alignments of amino acid sequences were performed using ESPript 3.0 (<http://espript.ibcp.fr/ESPript/ESPript/>)^[@CR53]^ incorporating secondary structural information. The phylogenetic tree was built using MEGA software (Version 5.2, <http://megasoftware.net>)^[@CR54]^, using neighbor joining method and 1000 bootstrap replicates. Hormonal treatments {#Sec19} ------------------- *Fragaria chiloensis* fruit at the C2 stage were treated with 1 mM ABA (Abscisic acid) and 1 mM NAA (1-Naphthaleneacetic acid) according to Opazo *et al*.^[@CR51]^ with some modifications. For these assays, C2 maturation stage was considered as it has been reported that the cross-talk between AUX and ABA occurs at this stage of fruit development in *Fragaria*^[@CR34]^. Fruit lots of 24 fruits were incubated during 10 min with ABA, ANA or remained untreated. Samples of 6 fruits were collected just after treatment (10 min), and after 1 h, 2 h and 12 h of treatment; the fruit was immediately frozen in liquid nitrogen and stored at −80 °C until use. RNA extractions and RT-qPCR reactions were performed as indicated above. Dual luciferase assay of transiently transformed Nicotiana benthamiana leaves {#Sec20} ----------------------------------------------------------------------------- Two DNA fragments corresponding to promoter sequences of genes related to cell wall remodeling, *PL* (*Pectate lyase*, GenBank KC527025; 1038 bp) and *EXP2* (*Expansin 2*, GenBank KC527027; 847 bp), which contain cis*-*elements in to response NAC transcription factors, were isolated by the introduction of restriction sites: 5′ NotI and 3′ NcoI for *PL*, and 5′ BamHI and 3′ NotI for *EXP2*. Then these sequences were inserted into the cloning site of pGreenII 0800-*LUC* vector^[@CR33]^. The promoter sequence of *FvDFR* (Dihydroflavonol reductase (AB211139); 1560 bp) was used as a positive control of the technique. The internal control was *Renilla* (*REN*) *LUC* driven by 35 S promoter. The full-length coding sequence of *FcNAC1* (GenBank AKC96459.1) was inserted into the pHEX2 vector under the 35 S promoter by Gateway, according to the manufacturer's instructions. *FvMYB10* (GenBank EU155163.1) and *FvBHLH* (Basic helix loop helix, XP_004308377; 1932 bp) fused to pHEX2 vector were used as positive control of the technique. Since *GUS* gene (*uidA*) has not been related to cell wall remodeling pathways, it was fused to pHEX2 vector to function as a negative control. All the constructs were transformed into *A*. *tumefaciens* GV3101, and these cultures were incubated at 28 °C for 2 days. Leaf disks were punched 3 days after infiltration, and then subjected to luminescence assay. Luc/Ren ratio activity was measured using the Orion Microplate Luminometer (Berthold Detection Systems, <http://www.titertek-berthold.com/>) as previously reported Espley *et al*.^[@CR55]^ and reported as mean of four technical replicates ± standard deviation. Homology modeling of NAC domain of FcNAC1 {#Sec21} ----------------------------------------- The search for an appropriate crystal structure available in the Protein Data Bank (PDB)^[@CR56]^ with the closest homology to FcNAC1 was performed through BLAST. The structure of ANAC019 (PDB: 3SWM chain A) was selected as template to build the NAC domain present in FcNAC1. To build the structure the pipeline described by Morales-Quintana *et al*.^[@CR57]^ was used. The molecular dynamics was performed following the strategy reported by Morales-Quintana *et al*.^[@CR58]^. Statistical analysis {#Sec22} -------------------- For gene expression analysis a random design with three biological replicates and two technical replicates were used. Statistical analyses were performed using the SPSS v.14 package. Analysis of variance (ANOVA) and significant differences were determined at *p* ≤ 0.05 (LSD Fisher test). Electronic supplementary material ================================= {#Sec23} Supplementary figures and tables **Electronic supplementary material** **Supplementary information** accompanies this paper at 10.1038/s41598-018-28226-y. **Publisher\'s note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This study was primarily funded by CONICYT grants Anillo Ciencia y Tecnología ACT-1110 and FONDECYT 1171530. C. C.-O. acknowledges UTalca and CONICYT for his doctoral scholarship. Thanks to Jocelyn Guajardo and Macarena Gonzalez for their technical assistant, and Patricio Ramos for helping with the use of the confocal microscope. The funders had no part in the design of the study or collection, analysis, and interpretation of data, neither in writing the manuscript. The study was conceived and designed by C.C.-O., R.H., A.C.A., M.A.M.-L. The experimental work was carried out by C.C.-O. Bioinformatics analyses were designed by Y.S. The hormonal treatment was carried by C.C.-O. and A.M.-Y. The dual luciferase analysis was performed by C.C.-O. with the critical supervision of A.C.A., R.E. and B.P. The manuscript was written by C.C.-O. and R.H., and critically reviewed by M.A.M.-L. and A.C.A., R.E. Each author participated sufficiently in the work to take public responsibility for appropriate portions of the content. All authors read, edited and approved the final manuscript. Competing Interests {#FPar1} =================== The authors declare no competing interests.

###### Summary box - Universal health coverage (UHC) is at the forefront of the discussions on how to achieve the health-related Sustainable Development Goals (SDG). - A prominent part of the UHC agenda is to ensure that people are not impoverished due to high healthcare expenditures. While this is crucial, it is not sufficient to protect people from hardship in times of ill health, as illustrated in the ongoing Covid-19 pandemic where lack of income security creates barriers for people to adhere to infection control measures. - Social protection systems ensuring income security when unable to work due to sickness are as important as schemes designed to reduce out-of-pocket healthcare expenditure. Yet, this is not part of the UHC framework and not sufficiently visible in the SDG Target on social protection. - This contrasts sharply with the high prioritisation of income security in times of ill health when universal social protection systems were built in the last century in many of today's high-income countries. Introduction {#s1} ============ Poor health can trap individuals, families and communities in a vicious disease-poverty cycle. While ensuring universal access to affordable healthcare in times of need is essential to break this cycle, income security in time of sickness or injury for all is equally important. Recent evidence indicates that people who cannot work or are not allowed to work due to illness face high indirect costs linked to income loss, which can be compounded by the opportunity cost of time spent seeking and staying in care. For example, the ongoing Covid-19 pandemic illustrates that lack of income security leads to economic hardship for individuals and creates barriers for adhering to infection control measures,[@R1] and similar challenges have previously been well-documented concerning tuberculosis.[@R3] Both access to healthcare services and income security in case of illness are enshrined in the human rights to health and social security and in international standards on social protection.[@R4] Income security acts on both the social determinants and the adverse consequences of ill health. The provision of sickness benefits is the primary responsibility of the State, usually implemented by social protection institutions under the joint stewardship of the health, social and labour sectors. Yet, while access to healthcare services is at the forefront of the 2030 Agenda through a dedicated Target on Universal Health Coverage (UHC), income security in case of ill health has limited visibility within the Sustainable Development Goals (SDGs) and is under-researched, especially in low-income and middle-income countries (LMICs).[@R6] Unpacking SDG targets and indicators {#s2} ==================================== The SDGs constitute an unprecedented opportunity to accelerate synergistic actions on health and social protection. Achieving the health Targets under Goal 3 will contribute to social well-being. Moreover, the UHC Target (3.8) has a specific indicator for financial protection (3.8.2), which measures occurrence of catastrophic out-of-pocket healthcare expenses. The focus is on direct medical costs while income security in times of ill health is not included. This was a conscious choice as the indicator measures what UHC intends to achieve: access to needed healthcare without financial hardship from paying for these services.[@R7] SDG Target 1.3 on social protection aims to implement nationally appropriate social protection systems and measures for all, including floors. In principle, this scope includes income security in case of ill health. Still, this dimension is currently missing in the related monitoring indicator 1.3.1 ('Percentage of the population covered by social protection floors/systems') which reports social protection coverage for children, unemployment, old age, disability and work injury benefits, but not for sickness benefits.[@R8] This exclusion directly correlates to a shortage of comparable data across countries. Indicators cannot capture all that is important of course, but they are an opportunity to set an accountability framework fostering the collection and publication of more and better data. In this respect, progress needs to be made. In line with the nine branches of social security defined under the International Labour Organization (ILO) Convention No. 102, the World Social Protection Database provides information on whether the legal framework includes entitlements to income support in case of sickness and collects national-level data on effective coverage for this contingency. Yet, more reporting is necessary to allow for the elaboration of global estimates.[@R9] These challenges relate to the setup of such guarantees. Indeed, many countries chose to cover this contingency through an employer's liability (ie, there is no social protection scheme as such, each employer is responsible to continue to pay the worker's salary during sick leave). This model has two effects. First, this form of protection is often limited to those covered under national labour legislation while those in informal employment remain unprotected. Second, it can create a disincentive for employers to hire and retain workers from groups prone to sickness, as the full cost of sick leave falls on them. This is a concern for small and medium enterprises where resources can be limited.[@R10] The labour force in LMICs is still largely informal. More efforts are necessary to extend social protection coverage, including income security in case of sickness, to those in informal employment and facilitate their transition to the formal economy, which also contributes to fostering decent work under Goal 8 and the broader SDG agenda. Learning from history {#s3} ===================== Income security in times of ill health has been part of social protection systems in many high-income countries (HIC) for over half a century, often longer than universal access to healthcare. After a long period of heterogeneous and small-scale union-based or guild-based mutual funds, the first national legislations on social insurance came into force around the turn from the 18th to the 19th century in countries that are today classified as HIC, but at the time had fiscal space that was no larger than today's poor countries. The 20th century saw scale-up in fits and starts towards universalism through periods of devastating wars and economic depression.[@R11] The Bismarck and Beveridge models did not only concern health coverage as defined today under the UHC framework. They were models for comprehensive social health protection, including both access to healthcare without hardship and income security in times of sickness.[@R13] One underpinning argument was that income security coupled with rehabilitation would help prevent permanent incapacity to work due to chronic conditions and hence reduce the burden on disability pension and poverty relief schemes. Another was that income security would facilitate implementation of infectious disease control measures. None of the early schemes had only healthcare benefits. In many countries, including Germany, UK and Sweden, sickness benefits came first, followed by gradual introduction of healthcare benefits. Lord Beveridge stated upfront in his 1942 report that UK had by then already made progress on social insurance, and argued that it was now time to include also healthcare coverage since 'a plan for social security assumes a concerted social policy in many fields'.[@R14] The human rights framework and international labour standards followed this approach, considering income security in case of sickness an integral part of social health protection.[@R5] As early as 1927, the ILO adopted the first convention on sickness benefits, which was subsequently included in the Social Security (Minimum Standards) Convention, 1952 (No. 102), the Medical Care and Sickness Benefits Convention, 1969 (No. 130) and Recommendation, 1969 (No. 134). Those instruments call on member states to set up systems ensuring protection in case of 'incapacity for work resulting from a morbid condition and involving suspension of earnings'. However, their global implementation was hampered by various factors, and the available data suggests effective coverage remains very low.[@R15] In spite of the inclusion of income security during sickness in the Social Protection Floors Recommendation, 2012 (No. 202), the recent United Nations resolutions do not elaborate on it[@R16] and more needs to be done to better reflect it in the SDG framework. Moving forward {#s4} ============== Despite the Universal Declaration of Human Rights including 'the right to security in the event of sickness' and WHO's definition of health as including 'social well-being', attention to income security in times of sickness remains limited in the global health field. A reason is perhaps that UHC and scientific advances are expected to solve the problem through swift cures for most conditions. Medical and allied science have advanced tremendously. Healthcare services can cure more diseases and reduce risk of long-term disability. Still, UHC will not eliminate the risk of income insecurity in case of sickness. The global tuberculosis and HIV/AIDS strategies are ahead of the game.[@R17] They include policy commitments on social protection and monitoring tools. Heavily subsidised healthcare services have been scaled up globally for those diseases, which is probably why the limitations of affordable healthcare alone to prevent poverty effects of diseases have become obvious. Evidence indicates that patients who pay little out of pocket for quality healthcare still face high indirect costs.[@R19] The national tuberculosis patients cost surveys coordinated by WHO show that patients experience variable levels of direct medical costs depending on the country context, but also high direct non-medical costs (mostly transport and nutrition) and income loss, creating additional incentives to forgo care.[@R3] There is good reason to believe that income security is an equally important global challenge for people with both communicable and non-communicable diseases, including diabetes, cancer, cardiovascular diseases and mental health problems. The long-term solution should not be disease-specific social protection schemes but universal systems that provide better ways to extend income security protection in case of sickness for all. Let's not wait until 2030 to put this issue firmly on the global health agenda and in the discussions on the future of social protection.[@R20] All authors are members of the Health and Social Protection Action Research & Knowledge Sharing network (SPARKS), an international interdisciplinary research network. SPARKS' multi-sectoral team characterizes and evaluates the direct and indirect effects of social protection strategies on health, economic, and wider outcomes. **Handling editor:** Seye Abimbola **Contributors:** All authors conceptualised the paper, wrote it jointly and approved the final version. **Funding:** Funding was received from the Swedish Research Council (2018-05174). **Competing interests:** None declared. **Patient consent for publication:** Not required. **Provenance and peer review:** Not commissioned; externally peer reviewed. **Data availability statement:** No additional data are available.

As of April 2020, more than 2 million people worldwide had tested positive for COVID‐19, and more than 200,000 deaths are attributed to this virus. It is estimated that around 15% of patients diagnosed with COVID‐19 will develop severe health complications, and around 5%--10% will require intensive level care due to the seriousness of the symptoms and the high mortality risk (3%--5%) (Baud et al., [2020](#jocn15314-bib-0003){ref-type="ref"}; Murthy, Gomersall, & Fowler, [2020](#jocn15314-bib-0013){ref-type="ref"}). At the time of writing, COVID‐19 has caused the need for hospitalisation of thousands of people due to the serious pneumonia type symptoms that result in extreme breathing difficulty. Critical care units in hospitals around the world are treating people experiencing potentially life‐threatening COVID‐19 symptoms. In some of these settings, the pressure on staff is compounded by a lack of adequate personal protection equipment (PPE) and staff shortages, as well as shortages of beds and mechanical ventilators. Despite the challenges, nurses who work in critical and intensive care units deliver the care required and we have witnessed their courage in recent media reports, with nursing and medical personnel describing the difficulties they face on a daily basis in providing care to these very ill and infectious patients. The current situation has generated a range of stressors that could negatively impact nurses and other health workers (Jackson et al., [2020](#jocn15314-bib-0009){ref-type="ref"}; Usher, Durkin, & Bhullar, [2020](#jocn15314-bib-0015){ref-type="ref"}). Critical care nurses may be particularly affected by severe emotional distress which has been associated with the development of compassion fatigue (CF) and/or burnout (Alharbi, Jackson, & Usher, [2020](#jocn15314-bib-0002){ref-type="ref"}). Indeed, Li et al. ([2020](#jocn15314-bib-0011){ref-type="ref"}), caution against ignoring vicarious traumatisation caused by the COVID‐19 pandemic. There is a known emotional impact for nurses' witnessing prolonged suffering of patients in environments such as intensive and emergency care units (Alharbi, Jackson, & Usher, [2019](#jocn15314-bib-0001){ref-type="ref"}). This impact is particularly related to their perceived inability to alleviate the suffering of those in their care. Research evidence shows that health professionals can experience various psychological problems when working in high‐pressure and high‐risk scenarios, such as in times of disaster and pandemic. Kang et al. ([2015](#jocn15314-bib-0010){ref-type="ref"}) found an increased risk for the onset of post‐traumatic stress disorder symptoms among rescuers following the 2010 Yushu earthquake in China. The contextual factors surrounding COVID‐19; such as the ease of transmission, lack of immunity among global populations, delayed testing, limited medical equipment, uncertainty of the pandemic trajectory and the general level of anxiety within the community all combine to place increasing pressure on health and welfare systems (Centers for Disease Control & Prevention, [2020](#jocn15314-bib-0004){ref-type="ref"}). CF and its related symptoms are a particular issue for critical care nurses in disaster contexts because the expectation to confront and cope with the need for care can exceed the ability to provide it, potentially (indirectly) leading to emotional distress in staff (Mathieu, [2014](#jocn15314-bib-0012){ref-type="ref"}). In addition to witnessing/experiencing patient suffering and death more frequently; having the responsibility for decisions related to resource rationing and utilisation means critical care nurses are at heightened risk of developing CF and moral injury during pandemics (Doherty & Hauser, [2019](#jocn15314-bib-0006){ref-type="ref"}). Moreover, nurses working under COVID‐19 conditions (like so many other healthcare workers) are vulnerable to exposure to risk of infection, and have the added concern of potentially contracting the virus themselves or unknowingly exposing family members and friends to heightened risk. The concern about being infectious can lead to a reluctance to seek out assistance from family or friends and may reduce the capacity to be compassionate in the workplace (Wallace, Wladkowski, Gibson, & White, [2020](#jocn15314-bib-0017){ref-type="ref"}). Craigie et al. ([2016](#jocn15314-bib-0005){ref-type="ref"}) refer to the "cost of caring" or the occupational hazard of working in critical care settings. The literature has clearly established that burnout and CF are high among all health professionals but especially so for those who work in environments where they are confronted daily with large numbers of people for whom the outcome is dire; such as the case for those diagnosed with COVID‐19 and requiring admission to emergency or intensive care units (Wallace et al., [2020](#jocn15314-bib-0017){ref-type="ref"}). Burnout is not just a term for being overworked; rather, it is a measurable condition that takes a heavy personal toll on health care providers, leads to lower quality care and increased errors (Alharbi et al., [2020](#jocn15314-bib-0002){ref-type="ref"}). Similar to burnout, CF carries a heavy personal toll, including isolation from others, excessive drinking and over‐eating, drug use and other detrimental coping measures. CF also increases absenteeism and turnover, and lowers morale (Alharbi et al., [2020](#jocn15314-bib-0002){ref-type="ref"}). Importantly, it is known to be linked to situations where nurses believe their actions will not make a difference (Portnoy, [2011](#jocn15314-bib-0014){ref-type="ref"}). This is unfortunately potentially the case for many patients with COVID‐19 as once they are admitted to critical care units, events have shown us that many will not survive. Evidence to gain empirical insights into the impact of COVID‐19 on nurses is only just beginning to emerge, with some unexpected findings. Wu et al. ([2020](#jocn15314-bib-0018){ref-type="ref"}) recently conducted a study of 220 health professionals (physicians and nurses) to compare the frequency of burnout between those professionals working on the COVID‐19 front line in Wuhan province (*n* = 110) and those working in their usual hospital wards in hospitals (*n* = 110). Notably, the authors reported that burnout frequency was in fact lower among health professionals working on the COVID‐19 front line compared to those working their usual wards (13% versus 39%, respectively (Wu et al., [2020](#jocn15314-bib-0018){ref-type="ref"}). Although these results were somewhat unexpected, the conclusion drawn by the researchers provides an interesting insight into the nature of burnout and CF. They theorise the lower‐level of burnout among the front‐line workers may be the result of these health professionals having to place all of their focus on achieving positive outcomes for patients (Wu et al., [2020](#jocn15314-bib-0018){ref-type="ref"}). This explanation, however, arguably implies that the nurses' focus is not on their own emotional well‐being. Moreover, as discussed previously a leading risk factor for the development of CF among intensive care nurses is their tendency to put the care needs of the patient above their own needs. According to Wu et al. ([2020](#jocn15314-bib-0018){ref-type="ref"}), the focus of front‐line carers on what they are trying to achieve rather than the personal impact of what they are trying to achieve may explain the more favourable outcomes for this group. For the nurses working in critical care environments, such as intensive care units, there is no escaping the daily parade of seriously ill patients with predicted poor outcomes in times such as those we are currently witnessing. We have seen and read of nurses describing situations where all patients have died on a unit during the course of an evening. It is hard to imagine the effect this has on the nurses working that shift. For many of the nurses in this situation, there may be little support. Hendin et al. ([2020](#jocn15314-bib-0007){ref-type="ref"}) have developed a framework for the provision of end of life care by nurses to patients faced with immediate death from COVID‐19 or similarly highly transmissible acute respiratory infections. At the centre of the framework is naturally a focus on minimising risks of transmission to nurse professionals. However, the authors also recommend the framework be underpinned by the "imperative for workplace colleagues to support each other and to perform frequent debriefs" (p. 3). This, they conclude, is vital to reduce the risk of front‐line nurses developing psychological problems including CF, burnout and vicarious trauma (Hendin et al., [2020](#jocn15314-bib-0007){ref-type="ref"}). Furthermore, as Horesh and Brown ([2020](#jocn15314-bib-0008){ref-type="ref"}) argue, the most concerning aspect of the health care sector (as opposed to healthcare worker) response to COVID‐19 for critical care nurses is the lack of a clear set of guidelines on how best to manage self‐care and well‐being. Current recommendations to front‐line healthcare workers are to ensure work‐life balance, practice deep breathing, facilitate mindfulness and support other when possible are modes of therapy or coping that independently or in combination can provide a positive effect (Van Zyl & Noonan, [2018](#jocn15314-bib-0016){ref-type="ref"}). However, these fall short of a formal and set of guidelines (supported with resources) that critical care nurses can refer to specifically to direct their self‐care efforts to manage their well‐being (Horesh & Brown, [2020](#jocn15314-bib-0008){ref-type="ref"}). At the very least, improved self‐care, both in and out of the hospital environment, is necessary to help critical care nurses to reduce the risk of developing CF. In conclusion, large‐scale public health events such as the COVID‐19 pandemic require a dedicated and highly demanding response from critical care nurses. To support these nurses, the broader response to COVID‐19 must include multiple stakeholders including, but not limited to, senior nursing staff, government policymakers, technology designers, hospital administrations, as well as members of the broader community. The decision and actions of stakeholders can play a central role in assisting nurses to manage the competing care demands caused by increased acuity, increased patient numbers, clinical uncertainty and limited access to necessary equipment. Hence, in addition to critical care nurses doing all they can to protect their own and their colleagues' well‐being, they need to work with other stakeholders to mobilise beneficial partnership and collaborate on developing creative solutions. Only through a collaborative effort can any risks associated with CF and burnout in the critical care nurse workforce be identified and mitigated.

All data files will be available from the date of acceptance from the following databases: simtk.org (<https://simtk.org/frs/?group_id=1916>), re3data.org (<https://dx.doi.org/10.5526/ERDR-00000123>), ModelDB database from Yale University (<http://modeldb.yale.edu/263988>), and GitHub (<https://github.com/mcapllonch/SenseBackSim/tree/publication_data/capllonch-juan_sepulveda_2020_01/>). Introduction {#sec001} ============ Artificial sensory feedback is becoming a viable way to substantially improve the life quality of amputees \[[@pcbi.1007826.ref001], [@pcbi.1007826.ref002]\]. The task of providing it through neural interfaces saw a first success with \[[@pcbi.1007826.ref003]\], and later, sensory feedback was provided in a stable form in \[[@pcbi.1007826.ref004]--[@pcbi.1007826.ref006]\], where it helped human subjects to improve their performance at using bidirectional limb prostheses. In very recent years, new encoding strategies \[[@pcbi.1007826.ref002], [@pcbi.1007826.ref007], [@pcbi.1007826.ref008]\]---involving, mostly, electrode placement, resolution, and the modulation of amplitude and frequency in stimulating signals---have facilitated various forms of biomimetic---natural-like---sensory feedback \[[@pcbi.1007826.ref009], [@pcbi.1007826.ref010]\], which help subjects identifying grasped objects quicker \[[@pcbi.1007826.ref010]\], and improve task performance and embodiment \[[@pcbi.1007826.ref009]\]. However, further improvements are still needed towards full, naturalistic sensory restoration for complete rehabilitation of fine motor function and prosthesis embodiment. The quality of artificial sensory feedback greatly depends on the quality of the interface between the artificial sensory device and the patient's peripheral nervous system (PNS). Such neural interface needs to accurately target specific axons in order to elicit the desired sensations. For this, it is necessary to determine the optimal electrical stimulation patterns---in time, frequency, and space---, that maximise selectivity and accuracy during stimulation. Selectivity is the ability of a neural interface to target the desired axons for stimulation, while avoiding recruiting non-targeted axons. Optimising implant selectivity is not trivial and demands the use of in vivo experiments and/or computer simulations. Computer models come, by definition, with limitations in accuracy compared to the results that could be obtained from experiments. On the other hand, they have the advantages of better affordability and usability as they can have quicker and cheaper setups. Simulations using computer models of electrode-PNS interfaces can be used to predict results from electrical stimulation and, ultimately, optimise electrode designs \[[@pcbi.1007826.ref011]\]. This field has successful precedents as in \[[@pcbi.1007826.ref012]--[@pcbi.1007826.ref014]\] or \[[@pcbi.1007826.ref015]\], whereby the ability of the models to predict the selectivity capabilities of the electrodes was experimentally validated \[[@pcbi.1007826.ref016]\]. These works use the innovative method of hybrid modelling, consisting of coupling Finite Element Methods (FEM) to solve the electric potential over a nerve, and neural compartmental modelling (using NEURON \[[@pcbi.1007826.ref017]\]) to solve resulting neural activity. Although these works use detailed geometrical representations of the nerves in their models, they rely purely on axon activation prediction to study selectivity and do not regard the effects that action potential (AP) propagation may have not only on the selectivity of the electrodes, but also on the frequency encoding of the signals that later reach the central nervous system. We believe that a more specific study is needed to assess the extent to which propagation can affect these variables. In order to carry a detailed study of propagation, ephaptic interactions should be taken into account. Ephaptic interactions are normally disregarded for the case of myelinated axons due to the insulating properties of the myelin sheath, which makes the transverse component of the conductivity across the nerve much lower than the longitudinal component \[[@pcbi.1007826.ref018]\]. However, studies such as \[[@pcbi.1007826.ref019]\] and \[[@pcbi.1007826.ref020]\] provide insights about the existence and role of this effect during conduction on myelinated axons. Our hypothesis is that ephaptic interactions between axons in a peripheral nerve are likely to play a role in information processing through alteration of the relative timings between APs from different axons, and possibly, in the selectivity capabilities of the electrodes, in an similar way as in the olfactory nerve in mammals \[[@pcbi.1007826.ref021]\], and by mutual influence between axons during stimulation. Hence, including this type of coupling in the models might provide an improvement onto existing achievements in the predictions of electrodes' fascicle selectivity and information encoding, which would in turn lead to more accurate and more naturalistic artificial sensory feedback in neural interfaces. We have developed three-dimensional EMI-type \[[@pcbi.1007826.ref022]\] models of both realistic and ideal peripheral nerve trunks which use a Resistor Network (RN) in order to simulate stimulation and propagation with ephaptic interactions in a unique simulation, with the ultimate goal of using it towards making predictions of fascicle targeting selectivity, frequency encoding, and overall electrode performance, in order to optimise electrode designs. The significance of this work lies in being the first work, to the best of our knowledge, that studies Ephaptic Coupling (EC) for a nerve model with realistic details and which deviates from restrictive assumptions such as the Mean-Field (MF) model or more regular geometries \[[@pcbi.1007826.ref019]--[@pcbi.1007826.ref021], [@pcbi.1007826.ref023]\], and therefore intends to elucidate the relevance of EC in more realistic conditions. Furthermore, we present a complete and self-consistent EMI model, specific for models of nerves and bundles of cylindrical axons---models of mere bundles of axons or fibers can be distinguished in this work from nerve models by the absence of other elements, such as perineurial membranes separating fascicles---which uses an existing geometrical tessellation technique to model the nearest-neighbour electrical connections between fibers and tissues. In summary, the main novelty of this work is the study of EC: - for nerves and bundles of randomly-located myelinated mammal peripheral axons with varying diameters, following both uniform and natural-like distributions, - that departs from MF assumptions and takes the inter-axonal distances into account through a RN, - that, for this purpose, presents a method to quantify nearest-neighbour electrical connections for any distribution of axons and tissues across the nerve, - in scenarios where the nerve models are stimulated by cuff electrodes. Results {#sec002} ======= Field generated by the electrode {#sec003} -------------------------------- In this subsection, we represent the extracellular potential field, *v*~*E*~, generated by the pulses exerted by one active pad on a cuff electrode on a nerve model. For this and the following subsection, we used a 1 cm long nerve (model named Nerve 1 in Methods) surrounded on part of its length by a stimulating cuff electrode that provided one square stimulating pulse. The cuff model was centered at the middle of the nerve's length. The 0° pad (blue diamond on [Fig 1](#pcbi.1007826.g001){ref-type="fig"}) injected a square pulse with a duration of 200 *μ*s and an intensity of −3 *μ*A. The fiber diameters were randomly chosen following a distribution based on \[[@pcbi.1007826.ref024]\], although the diameters were bounded between 3 and 20 *μ*m. No fibers thinner than 3 *μ*m were taken into account, since low diameter fibers have short internodal lengths and would increase the RN resolution, along with the simulation's computational cost. Considering that the fields obtained here are used in the stimulation studies in the next subsection, it is important to remark that, in order to save computational resources, the RN was connected only in the region under the cuffs. This was considered as a safe assumption since the fields far from the stimulation point were too small to play a relevant role during stimulation. The rest of the nerve's length was left in order to avoid the effects of sealed-end boundary conditions of the axons. {#pcbi.1007826.g001} A cross-sectional view of the absolute value of the field *v*~*E*~ over the nerve can be seen in [Fig 1](#pcbi.1007826.g001){ref-type="fig"}, and three samples of its longitudinal profile (*z*-axis) can be seen in [Fig 2](#pcbi.1007826.g002){ref-type="fig"}. The field, which is negative across the entire domain, has a minimum value of −2413.62 mV at the location of the active pad, but its absolute value is lower than 1000 mV over the rest of the domain. The field can be seen to decrease with the distance from the active pad both in [Fig 1](#pcbi.1007826.g001){ref-type="fig"} and in [Fig 2](#pcbi.1007826.g002){ref-type="fig"}. At the ends of the cuff, the field is effectively zero ([Fig 2](#pcbi.1007826.g002){ref-type="fig"}). {#pcbi.1007826.g002} In simulations where the presence of the axons is merely accounted for by the anisotropy of the endoneurium's resistivity tensor, a smooth dependence of \|*v*~*E*~\| with distance across the *x*-*y* plane from the active pad should be expected. However, in this simulation, axons are explicitly included in the RN. The resulting field ([Fig 1](#pcbi.1007826.g001){ref-type="fig"}) presents deviations from such a smooth dependence, at points where \|*v*~*E*~\| is generally low. This is due to the conductive axoplasm of the axons, which lowers the impedance to ground on their locations. Effects of ephaptic coupling on axon recruitment and selectivity {#sec004} ---------------------------------------------------------------- In order to study the effects of EC on axon recruitment and selectivity during stimulation, we tested the differences in stimulation results from simulations with and without EC. For this, we used the model Nerve 1 under the same conditions as the previous subsection. Two sets of simulations were run for this study: one including EC (labeled as SEC; results in [Fig 1](#pcbi.1007826.g001){ref-type="fig"} for a pulse of −3 *μ*A) and one not including it (SNOEC). SEC simulations were run by modelling the nerve as a RN. SNOEC simulations were prepared in the following way: The axon models are the same as in SEC. However, there is no RN interconnecting the axons, and therefore, no explicit modelling of any extracellular tissue or device. In order to model stimulation, the extracellular fields along all the axons in SEC were captured at the time step following the start of the stimulating pulse, and then used in SNOEC as the extracellular stimulating field on the axons. In order to quantify the effects of stimulation, we measured the axon recruitment in response to the stimulating pulses. The presence of APs on each fiber was detected when the transmembrane potential of the fiber (*v*~*m*~) reached 15 mV. This AP detection method was used throughout this study. We ran pairs of simulations {SEC, SNOEC} for current pulse amplitudes ranging from −0.2 to −4 *μ*A, with steps of −0.2 *μ*A. The method used for the stimulating fields in SNOEC ensures the axons are stimulated with the same field coming from the electrode in SNOEC and SEC. However, results vary substantially between both cases ([Fig 3](#pcbi.1007826.g003){ref-type="fig"}). The recruitment in SEC is, for all fascicles and pulse amplitudes, higher than in SNOEC, and it is also triggered for smaller currents. Recruitment ratios are only equal between SEC and SNOEC in trivial cases: when recruitment is zero and when it is saturated (i.e., the maximum number of axons in a fascicle has been recruited) in both simulations. This is due to the endogenously generated field (or ephaptic field), which adds up to the artificial field from the electrode and generates an increased depth of the total stimulating field over all axons (see [Fig 4](#pcbi.1007826.g004){ref-type="fig"}, where the ephaptic field and membrane voltage are represented for a random axon under a stimulating pulse with amplitude −2 *μ*A). The ephaptic field activates axons by pushing them over their activation thresholds, where the electrode fields alone are not enough. Hence, the ephaptic field effectively reduces the threshold current for axon recruitment. In the simulation with a stimulating pulse amplitude of −2 *μ*A, this ephaptic field is deeper than −50 mV on average (right panel) although it reaches depths in the range between −60 and −80 mV for some axons. There are no axons for which this field is positive throughout the duration of the stimulating pulse. It does, however, become positive after the pulse, likely due to the refractory periods of the axons. {#pcbi.1007826.g003} {#pcbi.1007826.g004} This model contains 658 axons, most of which are firing APs at similar times in SEC for strong enough stimulating pulses. From a MF model perspective, this means that the individual contribution to the ephaptic field from each axon might be in the order of, at least, 10 *μ*V. In cases where an electrode is set to selectively target a group of axons, the collective influence of these on the ephaptic field may be lower, and therefore, the effects on axon recruitment may be lower as well. Nevertheless, we can tell that the magnitude of the effect of EC on the axons response is big enough to be taken into account unless working with much smaller groups. The position of the active pad with respect to Fascicle 1 was assumed to be the optimal for maximising the selectivity for this fascicle. We studied the variation of the selectivity for Fascicle 1 with the presence of EC. We used the inter-fascicular selectivity provided by \[[@pcbi.1007826.ref015]\], and calculated its value for the whole range of stimulating pulse amplitudes. Results ([Fig 5](#pcbi.1007826.g005){ref-type="fig"}) indicate that EC, in the case of Fascicle 1, has the effect of narrowing the range of pulse amplitudes for which the selectivity is optimal by approximately 0.5 *μ*A, and shifting the peak of the selectivity also by approximately 0.5 *μ*A. Also, the maximum selectivity that can be reached is lower than in SNOEC. This can be understood thanks to the increase in axon recruitment in all other fascicles for pulse amplitudes from −0.6 *μ*A and stronger. Recruitment in Fascicle 1 is always higher than in the other fascicles thanks to its proximity to the active pad, and it reaches its maximum recruitment sooner. Therefore, selectivity for Fascicle 1, while using only the current active pad, cannot be negative. The possible effects of EC on the selectivity of other fascicles, however, may be different, since their optimal selectivity configurations vary. {#pcbi.1007826.g005} Effects of ephaptic coupling on propagation {#sec005} ------------------------------------------- We intended to study the effects that EC may have on propagation of APs. For this, we used the same approach: we ran a pair of simulations, SEC and SNOEC, on the same model, using the same stimulation protocol, and their results were compared. Propagation with EC needs to be studied along a longer model than Nerve 1, and for a longer period of time. Increasing the length of Nerve 1 highly increases the computational demands of simulations, so we instead used a thinner mono-fascicular nerve model: Bundle 1 (see [Methods](#sec008){ref-type="sec"}), which is 6 cm long and has a diameter of 100 *μ*m. No perineurial tissue was taken into account. In order to increase the effects of EC, the epineurial walls of the bundle were given the same resistivity as the cuffs, thus providing a virtual quasi-isolation from the surrounding saline bath. The bundle's ends were not covered by this isolating surface, so the tissues were in contact with the paths to ground on those two surfaces. Bundle 1 contains 39 axons whose fiber diameters follow a continuous and uniform distribution, in the range from 9 *μ*m to 10.9 *μ*m, and in steps of 0.05 *μ*m. This range was chosen so that the conduction velocities (CVs) did not vary drastically and thus to facilitate the possibility of signal locking between fibers of similar diameters. An intracellular current injection was given to all axons on their first node of Ranvier, consisting of one square pulse of 10 nA at *t* = 0.01 ms with a duration of 10 *μ*s. Results ([Fig 6](#pcbi.1007826.g006){ref-type="fig"}) show the presence of an effective lock of the APs in SEC during the first 0.5 to 1 ms of the simulation. However, it is apparent that this lock is unstable: After around 1 ms, APs tend to detach from the main group along time and increase their CVs. The first APs in detaching do not belong to the higher diameter fibers, but rather, to mid-to-high diameter fibers. These are then followed by higher diameter fibers. As a first hypothesis to explain this observation, this could be due to the loss of a bond between the higher and lower diameter fibers when the mid-to-high diameter fibers depart. However, the causes of this generalised detachment of trajectories from the main AP lock can be numerous and complex. The weakness---or instability---of the EC between fibers of different diameters could be explained by the differences in the CVs they tend to have in the absence of EC, which would act against locking their APs. The observation that these detachments occur after a certain distance along the *z*-axis suggests the presence of factors that trigger the separation of APs when certain conditions are met. One of this is, potentially, the variation along the *z*-axis of the alignment between nodes of Ranvier of different axons, which would modify the strength of their EC. {#pcbi.1007826.g006} The CVs of the fibers can be directly related with the presence of their APs in or outside the AP lock. APs that separate from the group quickly reach the CVs they have in the absence of EC ([Fig 7](#pcbi.1007826.g007){ref-type="fig"}). At the beginning of the simulation, when all the APs form a locked group, they all have CVs of less than half of the values they have in SNOEC. These CVs in the lock, however, gradually increase along time as APs separate from the lock. {#pcbi.1007826.g007} The same simulation sets were run for Bundle 2, although the stimulating pulse was 20 nA. Bundle 2 follows a natural diameter distribution (starting from 3 *μ*m), and the bundle diameter is larger than in Bundle 1 in order to facilitate the presence of more axons, and hence, a smoother diameter variability within the model. Results ([Fig 8](#pcbi.1007826.g008){ref-type="fig"}) indicate much weaker or nearly nonexistent AP lockings in SEC. This is in contrast with the apparent, although unstable and temporary, locking seen for Bundle 1. This is probably due to the wide range of different diameters in Bundle 2. However, a general slowdown of the CVs is still present in SEC. From these results, it is apparent that the strength of the effects of EC on the propagation of APs is highly dependent on the diameter variability between the fibers in a bundle. {#pcbi.1007826.g008} Dependence of ephaptic interactions with distance {#sec006} ------------------------------------------------- We ran two simulations in which we stimulated one random axon in each with an internal current injection and observed the responses of the other (unstimulated, meaning they were not artificially stimulated) axons transmembrane potentials. We compared these responses to the distances from the artificially unstimulated axons to the artificially stimulated axon. In this study, we used two models in order to study different scenarios, which differ in the presence of fascicles separated by perineurium: - Bundle 3 is a 3 cm long, 250 *μ*m diameter, mono-fascicular nerve filled with 20 *μ*m diameter fibers. This model has a larger diameter than Bundle 1 because we wanted to obtain a characterisation of the strength of EC across a wider cross-sectional distance. As is the case of Bundle 1 and Bundle 2, no perineurium is considered. Also, in this and the model below, the epineurial walls of the models were strongly isolated from the saline bath. - Nerve 2 uses the same epineurial and perineurial profile as Nerve 1---it has the same contours for the nerve and the fascicles cross-section---but it is filled exclusively with 20 *μ*m diameter fibers, as Bundle 3, and is also 3 cm long. Results for Bundle 3 are shown in [Fig 9](#pcbi.1007826.g009){ref-type="fig"} (left), and results for Nerve 2 are shown in [Fig 9](#pcbi.1007826.g009){ref-type="fig"} (right). The responses of the unstimulated axons in Bundle 3 follow a clear decreasing trend with the distance from the stimulated axon. The irregularities can be attributed to the limitations of the RN at modelling three-dimensional space and to inter-axonal ephaptic interactions between unstimulated axons. Nevertheless, the total change of the responses along 150 *μ*m of distance does not vary much above 8 *μ*V. This suggests the acceptability of the application of a MF assumption in cases like this model, since variations on *v*~*m*~ of this order of magnitude would not imply big differences in the results from MF and distance-based EC simulations. It is important to bear in mind, however, that this order of magnitude in the unstimulated axons responses is due to the activity of one stimulated axon only. The combined effect of more axons carrying APs would increase it. {#pcbi.1007826.g009} The responses in Nerve 2 are larger, near 0.2 mV inside the fascicle where the stimulated axon is, and approximately between 0.08 and 0.12 mV for the other fascicles. This is in rough agreement with the order of magnitude estimated in the second subsection of the Results if we have in mind that the nerve's length affects this magnitude (a longer length increases the resistance to the saline bath or ground, which increases this magnitude). Axons belonging to different fascicles are easy to identify in [Fig 9](#pcbi.1007826.g009){ref-type="fig"} (right), since the isolation provided by the perineurium makes the response of all axons inside each fascicle similar between them but notably different to the responses in other fascicles. The order of magnitude of these results could mean that the responses would be in the order of several mV should there be more stimulated axons, as seen in [Fig 4](#pcbi.1007826.g004){ref-type="fig"}. However, the intrafascicular variations are, at least, one order of magnitude lower. This would support a local MF choice for each fascicle. However, this choice would be incompatible with modelling inter-fascicle ephaptic interactions or fields from extracellular electrodes. These observations, especially when considering the activity of many axons taking place in simulations, support the importance of choosing a distance-based model. Discussion {#sec007} ========== The model framework developed in this study permits simulating the stimulation and propagation on a peripheral nerve trunk in a single run. The framework introduces a new method to build nearest-neighbour electrical interactions between fibers which builds up a whole electrical network for the nerve. This network simulates the fields coming from electrodes and from the fibers, thus enabling the integrated simulation of EC. This model has the advantage of being able to simulate the interactions between fibers and electrodes as well as with all other fibers in a nerve, where the nerve may have any reasonable shape, contain any number of fascicles separated by perineurial membranes and randomly located fibers of various diameters. However, running this with a reasonable level of computational efficiency has only been possible, so far, by accepting a series of assumptions and limitations: - Axons are cylindrical and use a 1D cable equation. The effects of the transverse components of polarisation around the membranes are not regarded. Although these effects have been found to play no major role in myelinated axons when studying stimulation \[[@pcbi.1007826.ref025]\], no study has been done on their influence on ephaptic interactions of two very nearby cells. Only \[[@pcbi.1007826.ref026]\] provide simulation results which could provide clues on this, yet it is not their main focus. - Axons are straight. However, tortuosity could affect EC by modifying the nearest-neighbour connections of the axons along the *z*-axis. - Electrical currents through space are only modelled along the *z*-axis and on the *x*-*y* plane. A FEM scheme could simulate these currents more accurately. - No capacitive properties have been regarded for any extracellular tissues. - Unmyelinated axons are not regarded in this model. Although of low relevance for our purposes, a more complete model should take them into account. Further work should assess the effects of EC under the conditions studied here (i.e., stimulation with electrodes and propagation in heterogeneous bundles) for unmyelinated axons, and quantify ephaptic interactions between unmyelinated and myelinated fibers. - The largest nerve model we have used in this work has a diameter of 500 *μ*m, and contains fascicles with a diameter of 156.67 *μ*m. These numbers are smaller than the known physiological ranges for human limb peripheral nerves where stimulation is typically studied \[[@pcbi.1007826.ref027], [@pcbi.1007826.ref028]\]. Also, fiber packing ratios are generally lower than physiological values. Therefore, the models used in this work contain less axons than real nerves. The computation time of the RN is highly sensitive to increasing the number of axons in the model. Hence, using physiologically more plausible numbers of axons would have been unattainable. Further improvements on some of the limitations of this model can be carried out in further work. These range from increasing the variety of axon models in use, to including capacitive properties of tissues, and adding tortuosity. The latter could be achieved by dividing the nerve's length in layers, each layer having its particular arrangement of fiber positions according to their tortuosity and hence, having its particular power diagram. Computational cost is generally a drawback for simulations with this model. Calculations over a RN are expensive and this limits the size and resolution that the model can have in order to get reasonable simulation time investments. Parallelisation of the RN could not be done, to the best of our efforts, without compromising numerical stability. This resolution limitation also compromises the accuracy of the results of simulations with EC, since small changes in the RN resolution or arrangement have large effects on EC. Laguerre tessellations are used for building nearest-neighbour connections between fibers. This method is used for the study of granular structures, like polycrystals and foams \[[@pcbi.1007826.ref029]--[@pcbi.1007826.ref031]\], whose field of application is strikingly different from the applications of this work. Yet, it proves to be a convenient method for modelling these connections, since it provides a general tool which serves any possible packing of cylindrical fibers. Prior to this work, no similar approach has been found for this purpose. Point or line-source approximations \[[@pcbi.1007826.ref032]\] can be used for this. However, even their adaptations to anisotropic media neglect the complexities the nerve may have outside each individual fiber, which can turn into an inaccurate modelling when these complexities are important. Also, using the equations from \[[@pcbi.1007826.ref032]\] in our case of mutual EC between many fibers may lead to numerical instability, as seen in \[[@pcbi.1007826.ref033]\]. Furthermore, no study has been found so far using any distance-based approach for a similar type of nerve model. This has allowed us to simulate stimulation and propagation in a somewhat realistic nerve model. From the numerical simulations presented here, we have found that EC lowers stimulation current thresholds and, overall, drives an increased axon recruitment (compared to simulations that neglect EC) during stimulation with a cuff electrode. The thresholds are lowered by an amount of the order of 100 nA for Fascicles 1, 2, 6 and 7, and of 1 *μ*A for Fascicles 3, 4, and 5. The increase in recruitment has a maximum of 64.9% for the whole nerve, and it is above 60% for all fascicles, except for Fascicle 1 (Fascicle 2: 72.9%; Fascicle 3: 84.8%; Fascicle 4: 79.3%; Fascicle 5: 78.6%; Fascicle 6: 66.3%; and Fascicle 7: 80.2%). Fascicle 1 has a maximum recruitment increase of 45.1%. For all the former fascicles, this maximum seems to be centered around a stimulating pulse of −1 *μ*A, and around −0.6 *μ*A for Fascicle 1, which closely correspond to their respective threshold currents in SNOEC. Therefore, these high peak levels in recruitment difference are mostly resolved from the fact that EC lowers the stimulation thresholds by nearly 1 *μ*A. So, for pulses near the peak, EC drives APs in a large number of axons that lie under their thresholds in simulations without EC. The lower threshold reduction and recruitment difference in Fascicle 1 compared to the others can be explained from the relative value of the ephaptic field with respect to the electrode field: While the ephaptic field has an order of magnitude of 10 mV, and may vary within a range of the same order over the whole nerve (as seen for a pulse of −2 *μ*A in [Fig 3](#pcbi.1007826.g003){ref-type="fig"}), the electrode field has, in general, also an order of magnitude of 10 mV in all fascicles, except Fascicle 1, where it is one order of magnitude stronger (see Figs [1](#pcbi.1007826.g001){ref-type="fig"} and [2](#pcbi.1007826.g002){ref-type="fig"}). Therefore, axons located in distant fascicles can be more sensitive to the ephaptic field. Recruitment difference decreases for stronger (i.e., more negative) pulses than −1 *μ*A for Fascicle 1 and −0.6 *μ*A for other fascicles, even when stimulation has not reached its maximum in SEC, because axons start activating in SNOEC. The inter-fascicular selectivity for Fascicle 1 was studied for a range of pulse amplitudes in order to determine how EC affects selectivity for the fascicle lying nearest to the active pad. It has been found that EC has the effects of 1) narrowing the range of pulse amplitudes resulting in high selectivity by approximately 0.5 *μ*A, 2) shifting the peak of the selectivity toward smaller pulse amplitudes in absolute value by approximately 0.5 *μ*A, and 3) reducing the maximum attainable selectivity from 0.9 to 0.68. We have observed how axons interact between them during stimulation, and although the strength of the individual influence from one axon is generally weak, their collective interactions are determinant to whether axons lying close to their thresholds fire an AP or not. We used a configuration where axons of different diameters are uniformly spread across the nerve's cross-section. This is representative of proximal sections of nerves. However, more distal sections present clustering of fiber types and diameters. This is known to affect the spread of activation thresholds within a fascicle \[[@pcbi.1007826.ref034]\], so further studies would be necessary to assess the validity of these findings in such configurations. The possibility of AP firing due to EC during propagation has not been studied in this work. In the study of the dependence of EC with distance, the observed rise in *v*~*m*~ of axons was due to the activity of only one neighbouring axon. It is inferred, from the orders of magnitude under consideration, and from the observed ephaptic fields in the stimulation study, that the simultaneous activity of many more axons could drive unstimulated axons to fire APs. Although studying this possibility is outside the scope of this present work, it is proposed as further work. By following these considerations, EC should be taken into account in simulations of axon recruitment with electrodes, but if it is to be neglected in favour of lower computational costs, it should at least be held in mind that neglecting it may lead to certain inaccuracies in the results. Ideally, such a study lacking explicitly modelled EC could consider these effects by applying a modifying function to recruitment numbers after a simulation. The results in this work suggest that amplitudes of stimulating pulses necessary for axon recruitment in experimental studies and practical applications should be generally lower than as obtained from models lacking EC. We have observed how, in this work, certain already existing findings \[[@pcbi.1007826.ref019]--[@pcbi.1007826.ref021]\] about the effects of EC on few fibers during propagation---CV reduction and AP locking---also apply for bundles with more numerous and heterogeneous fibers. However, these effects are strongly conditioned by similarity between fibers and compromised by heterogeneity to the point of losing their validity when assumptions of homogeneous fibers are not used. It is apparent that fiber diameter variability in a bundle greatly influences the effects of EC on AP propagation. This implies that the effects of EC on propagation might be weak, and even irrelevant, in proximal sections of nerves, where fiber diameters are homogeneously distributed, but they could be stronger, and forming effective lockings, in more distal regions, where fibers may be clustered by size. The results of this work also have assessed the validity of choosing a MF model: although physically not accurate and unsuitable for studies involving extracellular electrodes, it can be justified for others, especially for small mono-fascicular nerves or locally within fascicles. In summary, a detailed computer model of a peripheral nerve trunk has been developed, which involves the implicit coupling of intra- and extracellular electrical activity in a single simulation. It conveniently uses NEURON with a Python framework that handles all the geometrical methods and wraps the whole model. Specific experimental data for validation would be desirable. However, the model succeeds in behaving within physiologically expected ranges. We hope that this new method provided here brings researchers to use it further in order to study more complex cases of ephaptic interactions, and that the results from this study serve to add more knowledge on the effects of EC in bundles of fibers with different sizes, eventually to determine the extent to which modelling EC for studying sensory feedback is necessary. Methods {#sec008} ======= The fundamental assumptions on which the model is based, the axon models in use and a detailed description of the procedures used to model the nerve's tissues are provided here. Main assumptions and limitations {#sec009} -------------------------------- The model relies on several assumptions to simplify the implementation and computational cost while still keeping an acceptable level of accuracy: 1. Only two types of axon models are used: the double-cable models of McIntyre, Richardson and Grill (MRG) \[[@pcbi.1007826.ref035]\] for motor fibers, and Gaines & al. \[[@pcbi.1007826.ref036]\] for sensory fibers. No unmyelinated or other types of myelinated axons are considered. 2. Axons are straight, with no tortuosity (i.e., with no bends, undulations, or tapering) along their length. 3. All axons are parallel to each other. 4. Following the two above assumptions, the cross-section of the nerve's anatomy is constant along its length. 5. All extracellular tissues are purely ohmic. 6. The volumes of the epineurium and endoneurium are regarded as part of a three-dimensional RN. 7. The endoneurium was modelled as an isotropic tissue, since using its anisotropic tensor from \[[@pcbi.1007826.ref037]\] would imply an over-representation of the axons. 8. The perineurium is regarded as a surface with a nominal thickness influencing the values of the resistances that cross it. 9. The nearest-neighbour electrical connections model defines inter-axonal connections only across the *x*-*y* plane, and inter-compartmental connections along the *z*-axis. This is a limitation with respect to FEM schemes, which can model currents flowing in any direction. 10. The RN is computationally expensive. A very large number of axons in the model can greatly increase the simulation time to days. Therefore, although the typical diameters of human limb peripheral nerves where stimulation is studied are in the order of several mm \[[@pcbi.1007826.ref027], [@pcbi.1007826.ref028]\], we used smaller nerve models and axon bundles (see [Table 1](#pcbi.1007826.t001){ref-type="table"} for more details). Also, fiber packing ratios and axon numbers were kept low. 10.1371/journal.pcbi.1007826.t001 ###### Geometrical and electrical properties of the models. {#pcbi.1007826.t001g} Model Diameter (*μ*m) Number of axons Fiber packing ratio Intracellular to extracellular areas ratio Length (cm) ---------------------- ----------------- ----------------- --------------------- -------------------------------------------- ------------- Nerve 1 500 658 1 Fascicle 1 (Nerve 1) 156.67 82 0.282 0.205 1 Fascicle 2 (Nerve 1) 156.67 118 0.350 0.267 1 Fascicle 3 (Nerve 1) 156.67 99 0.344 0.269 1 Fascicle 4 (Nerve 1) 156.67 87 0.290 0.212 1 Fascicle 5 (Nerve 1) 156.67 98 0.330 0.251 1 Fascicle 6 (Nerve 1) 156.67 83 0.293 0.225 1 Fascicle 7 (Nerve 1) 156.67 91 0.283 0.193 1 Bundle 1 100 39 0.398 0.304 6 Bundle 2 150 110 0.347 0.267 6 Bundle 3 250 69 0.450 0.606 3 Nerve 2 500 192 3 Fascicle 1 (Nerve 2) 156.67 26 0.429 0.555 3 Fascicle 2 (Nerve 2) 156.67 28 0.462 0.634 3 Fascicle 3 (Nerve 2) 156.67 28 0.462 0.634 3 Fascicle 4 (Nerve 2) 156.67 27 0.445 0.593 3 Fascicle 5 (Nerve 2) 156.67 29 0.478 0.677 3 Fascicle 6 (Nerve 2) 156.67 27 0.445 0.593 3 Fascicle 7 (Nerve 2) 156.67 27 0.445 0.593 3 Axon and nerve models {#sec010} --------------------- A number of different models were used in this work in order to run the different studies (see [Table 1](#pcbi.1007826.t001){ref-type="table"} for a detailed list of these models and [Fig 10](#pcbi.1007826.g010){ref-type="fig"} for their fiber diameter distributions). Model named Nerve 1 in this work uses both motor and sensory fiber models, with a proportion of 15% motor and 85% sensory fibers \[[@pcbi.1007826.ref036]\]. All other models use, exclusively, motor fibers. In all cases, we used a temperature of 37°C. Unmyelinated fibers are known to be generally more numerous than myelinated fibers in peripheral nerves \[[@pcbi.1007826.ref038], [@pcbi.1007826.ref039]\]. Also, EC in unmyelinated axons can be relatively strong. However, their use in our model implied a high computational cost due to the higher spatial resolution that they require. Furthermore, they are outside the scope of this study as our focus is on the often neglected EC between myelinated fibers. Therefore, unmyelinated fibers were not included in the models presented here. {#pcbi.1007826.g010} In Nerve 1 and Bundle 2, the fiber diameters were randomly assigned following a distribution according to the results in \[[@pcbi.1007826.ref024]\], although they were bound between 3 *μ*m and 20 *μ*m (smaller diameter fibers were excluded due to their fine spatial discretisation requirements, which led to higher computational costs). Therefore, the nodes of Ranvier of the different axons were not necessarily aligned. The different properties of the fiber morphology that depend on the diameter---internodal length, morphology of the myelin attachment (MYSA) and paranodal (FLUT) regions and number of myelin layers, were fitted to a linear regression each, using the values from \[[@pcbi.1007826.ref035]\]. Variables whose linear regressions yielded negative values were fitted to a quadratic curve, as done in \[[@pcbi.1007826.ref040]\]. The implementation of the axon membrane models was made in the NEURON simulation environment \[[@pcbi.1007826.ref017]\]. For Nerve 1 and Nerve 2, we used a nerve model as a cylindrical body with seven cylindrical fascicles of equal diameter, inspired in the five-fascicle model from \[[@pcbi.1007826.ref012]\]. In all models, the fascicles were filled with axons using a simple circle packing algorithm designed for this purpose. The algorithm consisted of one iterative process for each fascicle where, in each iteration, a random diameter value *D*~*k*~ was chosen from the aforementioned distribution for a circle (a fiber, indexed with *k*). For each circle, a loop for positioning trials was then run. On each trial, a random position for the center of the circle was chosen inside the fascicle (more specifically, inside a circle having a diameter *D*~*F*~ − *D*~*k*~, being *D*~*F*~ the diameter of the fascicle, in order to avoid intersection of the circle with the fascicle's membrane). If the circle at the position had no intersections or contacts with any other circle that had been placed previously in the fascicle, the position was assigned to it and a new random circle was chosen. The algorithm stopped when a circle could not be placed at a suitable position after 10, 000 trials. For this process, a minimum allowed distance between axons was chosen to be 1 *μ*m (which was taken into account at each contact check), so no two axons could be closer to each other than that. [Table 1](#pcbi.1007826.t001){ref-type="table"} summarises the fiber packing results for the different models used in this work. For this, values for each model are shown of the fiber packing ratio, defined as *A*~*F*~/*A*~*T*~, and of the total intracellular to extracellular areas ratio, defined as *A*~*ax*~/*A*~*E*~, where *A*~*F*~ is the sum of the cross-sectional areas of the fibers, including their myelin sheaths, *A*~*ax*~ is the sum of their cross-sectional intracellular areas, *A*~*E*~ is the total cross-sectional extracellular area of the model, and *A*~*T*~ is its total cross-sectional area. This algorithm can fill fascicles of any shape with fibers. The algorithm used here yields fiber packing ratios which are generally lower than the typical values in nerves (see, for instance, \[[@pcbi.1007826.ref041]\] for measured values in human spinal cord). However, these lower ratios prevented us from having a very high number of axons, which would increase the computational cost of the simulations. Three different extracellular tissues were considered in the model ([Table 2](#pcbi.1007826.t002){ref-type="table"}): The epineurium was used for the whole extrafascicular space inside the nerve, the endoneurium was used to account for all the intrafascicular spaces where axons were embedded, or interstitial spaces, and the perineurium was regarded as a surface layer that electrically separated the fascicles from the epineurium. Nevertheless, the epineurium and the endoneurium were given the same electrical properties for the following reasons, respectively: The epineurium was considered to be isotropic as in \[[@pcbi.1007826.ref012], [@pcbi.1007826.ref015]\]. The endoneurium's resistivity taken from the literature \[[@pcbi.1007826.ref037]\] is considered to be anisotropic because it accounts for the longitudinal disposition of the axons. In this RN, however, axons are explicitly represented by implementing their membranes and intracellular resistances as part of the RN. Using the known value from \[[@pcbi.1007826.ref037]\] for the longitudinal component of the endoneurium's resistivity, $\rho_{En}^{L} = 175$ Ω ⋅ cm, is then not suitable for this model, since that would imply an over-representation of the intracellular resistances. Hence, given the lack of knowledge about the value of $\rho_{En}^{L}$, we made the conservative assumption of considering the endoneurium as an isotropic tissue, and used its transverse component of the resistivity, $\rho_{En}^{T}$, as the value for its longitudinal component. 10.1371/journal.pcbi.1007826.t002 ###### Parameters used for the RN. {#pcbi.1007826.t002g} Symbol Value Source Description ----------------- ---------------------- ---------------------------------------------------- ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- *ρ*~*ax*~ 70 Ω ⋅ cm \[[@pcbi.1007826.ref035]\] Axoplasmic resistivity. $\rho_{En}^{L}$ 1211 Ω ⋅ cm \[[@pcbi.1007826.ref037]\] Longitudinal (*z*-axis) component of the resistivity of the endoneurium. See main text to understand the discrepancy with the anisotropic tensor from \[[@pcbi.1007826.ref037]\]. $\rho_{En}^{T}$ 1211 Ω ⋅ cm \[[@pcbi.1007826.ref037]\] Transverse (*x*-*y* plane) component of the resistivity of the endoneurium. $\rho_{Ep}^{L}$ 1211 Ω ⋅ cm \[[@pcbi.1007826.ref012], [@pcbi.1007826.ref015]\] Longitudinal component of the resistivity of the epineurium. $\rho_{Ep}^{T}$ 1211 Ω ⋅ cm \[[@pcbi.1007826.ref012], [@pcbi.1007826.ref015]\] Transverse component of the resistivity of the epineurium. $\rho_{P}^{T}$ 1.136 ⋅ 10^5^ Ω ⋅ cm \[[@pcbi.1007826.ref011]\] Transverse (and only) component of the resistivity of the perineurium (value for 37° see reference). *ρ*~*I*~ 10^9^ Ω ⋅ cm \[[@pcbi.1007826.ref012]\] Resistivity of the insulator. *ρ*~*S*~ 50 Ω ⋅ cm \[[@pcbi.1007826.ref012]\] Resistivity of the saline bath. Δ~*P*~ 4.7 ⋅ 10^−4^ cm \[[@pcbi.1007826.ref042]\] Thickness of the perineurium (3% of the fascicle diameter in Nerve 1; see [Table 1](#pcbi.1007826.t001){ref-type="table"}). Δ~*I*~ 2.4 ⋅ 10^−2^ cm \[[@pcbi.1007826.ref012]\] Thickness of the insulating cuff. Δ~*S*~ 0.85 cm Thickness of the saline bath in the cylindrical container (in the absence of cuffs). Δ~*C*~ 2.2 cm \[[@pcbi.1007826.ref012]\] Cylindrical container's diameter. *D*~*N*~ 0.5 cm Diameter of the nerve. *n*~*H*~ 36 Number of points in the triangulation hull (or number of NAELC on the nerve's membrane). Resistor network model {#sec011} ---------------------- The whole extracellular volume of the nerve is modelled with a RN which uses \[[@pcbi.1007826.ref043]\] as the basic model of the extracellular medium between two cables. Ours is an adaptation from such model that suits any number of myelinated axons and also volumes in the nerve that contain no axons. ### Extracellular space connecting two neighbouring axons {#sec012} The model from \[[@pcbi.1007826.ref043]\] consists of two parallel core-conductor (unmyelinated) axons linked by a grid of resistors. Each axon is coupled to its parallel (longitudinal) extracellular cable through its membrane compartments, and the two longitudinal extracellular cables are linked to each other by transverse resistors *R*~*T*~---perpendicular to the axons, at each compartment's position. Each longitudinal extracellular cable is a series of resistors with value *R*~*L*~ located one at each compartment. Two important adaptations are needed in case the model includes myelinated axons with different internodal lengths and therefore, with misaligned nodes of Ranvier ([Fig 11](#pcbi.1007826.g011){ref-type="fig"}): First, the extracellular cables of the fibers are continuous resistive cables along which transverse resistors can be connected at any location. Second, there are two options for how to connect the transverse resistors: The first one is to locate them at regular intervals along the *z*-axis. The second one consists of connecting them at the locations of the nodes of Ranvier of both axons (this is the case shown in [Fig 11](#pcbi.1007826.g011){ref-type="fig"}). In this case, the set of transverse resistor locations along the *z*-axis between any two fibers *k* and *l* is *Z*~*T*,(*k*,*l*)~, which is the union of the sets of positions of the nodes of the two axons: $$Z_{T,(k,l)} = Z_{k} \cup Z_{l},$$ and therefore, it contains *M*~*k*,*l*~ elements (which means there are *M*~*k*,*l*~ transverse resistors between the two axons; see [Table 3](#pcbi.1007826.t003){ref-type="table"} for a list of all the variables used here), i.e., the sum of the number of nodes of Ranvier of the two axons minus the number of pairs of nodes which share the same location on the *z*-axis (because such case, obviously, means there is only one resistor for two nodes): $$M_{k,l} = N_{k} + N_{l} - \sum\limits_{i = 1}^{N_{k}}\sum\limits_{j = i}^{N_{l}}\delta\left( z_{NR,i} - z_{NR,j} \right),$$ where *z*~*NR*,*i*~ (*z*~*NR*,*j*~) is the position of the *i*-th (*j*-th) node of Ranvier of fiber *k* (*l*). {#pcbi.1007826.g011} The length along the *z*-axis of one transverse resistor *n* is given by: $$c_{k,l}^{n} = \frac{z_{T,(k,l)}^{n + 1} - z_{T,(k,l)}^{n - 1}}{2},$$ being $z_{T,(k,l)}^{n}$ a member of *Z*~*T*,(*k*,*l*)~: $$z_{T,(k,l)}^{n} \in Z_{T,(k,l)}\forall n \mid n \in \left\lbrack 1,M_{k,l} \right\rbrack$$ 10.1371/journal.pcbi.1007826.t003 ###### Variables used for the RN. {#pcbi.1007826.t003g} Symbol Units Description --------------------- -------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------ *a*~*k*,*i*~ None Fraction of cross-sectional area of tissue of type *i* present in polygon *k*. $b_{k,l}^{i}$ None Distance crossed through a tissue of type *i* by the transverse resistor between *k* and *l* as a fraction of the total distance between the membranes of *k* and *l*. *A*~*P*,*k*~ cm^2^ Cross-sectional area of polygon *k*. *A*~*E*,*k*~ cm^2^ Extracellular cross-sectional area inside polygon *k*. *D*~*k*~ cm Diameter of fiber *k* (zero for NAELC). $c_{k,l}^{n}$ cm Length (along the *z*-axis) of the transverse resistor number *n* between cables *k* and *l*. *d*~*C*,(*k*,*l*)~ cm Distance between the centers of fibers *k* and *l*. *s*~*k*,*l*~ cm Length of the segment in common between polygons *k* and *l*. $\rho_{k}^{Lu,L}$ Ω ⋅ cm Longitudinal component of the lumped resistivity for polygon *k*. $\rho_{k,l}^{Lu,T}$ Ω ⋅ cm Transverse component of the lumped resistivity between cables *k* and *l* *r*~*L*,*k*~ Ω/cm Resistance per unit length of the extracellular cable *k*. $R_{T,(k,l)}^{n}$ Ω Value of the transverse resistor *n* between cables *k* and *l* *R*~*G*~ Ω ⋅ cm Resistance to ground from a point on the nerve's membrane per unit length. The resistance per unit length of each longitudinal extracellular cable *r*~*L*,*k*~, equivalent to *R*~*L*~ in \[[@pcbi.1007826.ref043]\], is given by the extracellular cross-sectional area that can be assigned to each fiber. This represents the longitudinal resistance of the volume of extracellular medium surrounding each fiber. This extracellular cross-sectional area is given by the tessellation of the nerve described below (see also Figs [12](#pcbi.1007826.g012){ref-type="fig"} and [13](#pcbi.1007826.g013){ref-type="fig"}). {ref-type="fig"} and [Table 1](#pcbi.1007826.t001){ref-type="table"}).](pcbi.1007826.g012){#pcbi.1007826.g012} {#pcbi.1007826.g013} ### Modelling the extracellular space for any number of axons and extrafascicular regions {#sec013} Our model needs to extend this network to fit any number of randomly distributed axons across the fascicles. Also, the epineurium and regions of the nerve lying outside the fascicles that naturally do not contain axons may be large enough for us to need to model them with a finite-sized mesh with which to capture the electric field resulting from external stimulation with a certain level of detail. The model creates an electrical resistive network among fibers by extending the nearest-neighbour connections model described above. In order to make the modelling of the extrafascicular regions compatible and consistent with this connectivity model, these regions were assigned longitudinal extracellular cables that were not directly attached to any fibers (referred to as non-axonal extracellular longitudinal cables, NAELC, from now on), but which were connected among them using the same method (with regular spacing among transverse resistors in this case). Therefore, the NAELC, all the extracellular cables directly attached to the fibers, and the transverse resistors, form the extracellular part of the RN. The extension of this nearest-neighbour interaction model to the whole RN for an entire nerve requires a method to provide each axon and NAELC with a given extracellular area and to model the values for all the transverse resistors. Such procedure is the following: A cross-sectional 2D slice of the epineurium is filled with points obtained from a Delaunay triangulation \[[@pcbi.1007826.ref044]\] of the nerve's cross-sectional area. Each point falling outside the fascicles corresponds to the location of one NAELC, and any points falling inside the fascicles are removed. These points, together with the circles that define the locations and diameters of the axons, form a packing of non-intersecting circles in a two-dimensional space (the points may be regarded as zero-diameter circles for this purpose). This way, we obtain the set of positions for all fibers and NAELC ([Fig 12](#pcbi.1007826.g012){ref-type="fig"}). A natural way to divide the space in an individual region for each circle of the packing is given by computing its power diagram, described as the Voronoi tessellation in the Laguerre geometry \[[@pcbi.1007826.ref045]\]. This tessellation technique divides the nerve's cross-section into a set of convex polygons ([Fig 12](#pcbi.1007826.g012){ref-type="fig"}), each one containing one circle, thus existing a one-to-one correspondence between polygons and circles, and therefore assigning an extracellular cross-sectional area *A*~*E*,*k*~ to each fiber and NAELC. Polygons containing the points on the nerve's membrane are cropped so that they do not intersect the nerve's outer space. ### Longitudinal resistances of the resistor network {#sec014} The longitudinal resistivity of an extracellular cable is determined using a lumped value of the longitudinal components of the resistivities of the tissues intersecting its polygon. $$\rho_{k}^{Lu,L} = \sum\limits_{i}a_{k,i} \cdot \rho_{i}^{L},$$ where *k* indicates the cable or polygon, *i* indicates the type of tissue and then, *a*~*k*,*i*~ is the cross-sectional area of tissue type *i* present in polygon *k* as a fraction of the total extracellular area enclosed by the polygon (this is, scaled over *A*~*E*,*k*~). In theory, in this study, this sum is made over two types of tissue: endoneurium and epineurium (*i* ∈ {*En*, *Ep*}). However, as mentioned above, we used the same value of $\rho_{i}^{L}$ for both. Nevertheless, this equation serves for any number of tissue types the modeller wishes to include. The resistance per unit length of each extracellular cable is: $$r_{L,k} = \frac{\rho_{k}^{Lu,L}}{A_{E,k}},$$ where *A*~*E*,*k*~ is the aforementioned extracellular cross-sectional area of the polygon. If *A*~*P*,*k*~ is the total area of the polygon and *D*~*k*~ is the diameter of fiber *k*, *A*~*E*,*k*~ is given by: $$A_{E,k} = A_{P,k} - \pi D_{k}^{2}$$ If the polygon does not contain a fiber but a NAELC, $$A_{E,k} = A_{P,k}$$ ### Transverse resistances of the resistor network {#sec015} Sides shared by adjacent polygons in the power diagram represent electrical contacts between the polygons (which is equivalent to surface contacts between polygonal prisms because the polygons are extruded along the *z*-axis) and determine which cables or fibers are coupled by transverse resistors. The weighted Delaunay triangulation dual to the power diagram \[[@pcbi.1007826.ref045]\] (red lines in Figs [12](#pcbi.1007826.g012){ref-type="fig"} and [13](#pcbi.1007826.g013){ref-type="fig"}) indicates these connections. The resistance of such a contact depends directly on the distance *d*~*C*,(*k*,*l*)~ between the centers of the two circles and inversely on the product of its segment's length *s*~*k*,*l*~ (green segment joining the two coloured polygons in [Fig 13](#pcbi.1007826.g013){ref-type="fig"}) times its length along the *z*-axis $c_{k,l}^{n}$. Hence the values of the extracellular transverse resistors between two fibers are: $$R_{T,(k,l)}^{n} = \rho_{k,l}^{Lu,T}\frac{d_{C,(k,l)}}{c_{k,l}^{n} \cdot s_{k,l}},$$ The transverse component of the lumped resistivity $\rho_{k,l}^{Lu,T}$ is computed in the following way: $$\rho_{k,l}^{Lu,T} = \sum\limits_{i}b_{k,l}^{i} \cdot \rho_{i}^{T},$$ where $b_{k,l}^{i}$ is the distance crossed by the resistor within the tissue of type *i*, scaled over *d*~*C*,(*k*,*l*)~. For merely geometrical arrangements, the perineurium is modelled as an infinitely thin layer, so it does not affect the calculations of *r*~*L*~. Yet its nominal thickness was not ignored for the calculations of the resistances of transverse resistors crossing it, since its thickness is known to affect the results of stimulation \[[@pcbi.1007826.ref042]\]. Its thickness was added in the calculation of the corresponding $R_{T,(k,l)}^{n}$ in the following way: $$R_{T,(k,l)}^{n} = \frac{1}{c_{k,l}^{n} \cdot s_{k,l}}\left( \rho_{k,l}^{Lu,T}\left( d_{C,(k,l)} - n_{P}\Delta_{P} \right) + \rho_{P}^{T}n_{P}\Delta_{P} \right),$$ where *n*~*P*~ is the number of perineurial membranes crossed by a resistor (1 between an axon and a NAELC, 2 between two axons in different fascicles, 0 otherwise). NAELC are always discretised in regular intervals, using the shortest internodal length in the nerve for $c_{k,l}^{n}$. Transverse resistors connecting a NAELC and a fiber are located on the nodes of Ranvier of the fiber. Nerve's external environment and electrodes {#sec016} ------------------------------------------- The nerve was centered along the axis of a larger cylindrical container (*z*-axis) filled with a saline bath. The surface of this larger cylinder was connected to ground (zero potential), as done before by \[[@pcbi.1007826.ref012]\]. For modelling purposes, this can be used as a sufficient representation of the animal's body surrounding the nerve, assuming that in a real experiment, the ground would presumably be found on a distant location, right outside the animal's body or in the Central Nervous System. This model framework allows the user to define cuff electrodes for stimulation. We used cuff electrode models based on \[[@pcbi.1007826.ref046]\]. These electrodes are 4.25 mm long and contain four rings separated by 750 *μ*m each. Each ring contains four pads, placed at 0°, 90°, 180° and 270° with respect to the *x*-axis. More details about the geometry and materials of these electrodes can be found in \[[@pcbi.1007826.ref046]\]. In this work, the cuff model was simplified by leaving only one ring in the center, and by adapting the inner diameter to the nerve model diameter. Stimulation from the pads is simulated using current point sources on the nerve's membrane's NAELC in contact with the desired pads. The current path between the points on the nerve's membrane and the container's walls was assumed to be purely radial (hence no longitudinal currents are allowed across the bath or the cuff insulators). For this, all points in the discretised nerve lying on its membrane (which are given by the triangulation hull in the cross-section) were connected to the container's cylindrical wall using radially aligned resistors. The resistance per unit length for each of these resistors was estimated from the geometry of the bath (see Tables [2](#pcbi.1007826.t002){ref-type="table"} and [3](#pcbi.1007826.t003){ref-type="table"} for variables and parameters): $$R_{G} = \frac{\rho_{I}\Delta_{I} + \rho_{S}\Delta_{S}}{\left( {\pi D_{N}/n_{H}} \right)},$$ where: $$\Delta_{I} + \Delta_{S} = \Delta_{C}$$ In the regions of the nerve (along its length) not covered by the cuffs, the membrane was directly in touch with the saline bath and [Eq 12](#pcbi.1007826.e033){ref-type="disp-formula"} then becomes: $$R_{G} = \frac{\rho_{S}\Delta_{C}}{\left( {\pi D_{N}/n_{H}} \right)}$$ All NAELC and extracellular cables of fibers were connected to ground on both ends since they are assumed to be in contact with the container's bases. The ends of the intracellular domains of the fibers, however, are treated as sealed ends and do not have such connections. [^1]: The authors have declared that no competing interests exist.

Introduction {#S0001} ============ Parrot bornaviruses 1--8 (PaBV- 1-8) are the causal agents of a progressive fatal avian neurologic syndrome referred to as Parrot bornavirus syndrome, a complex of clinical problems that can include proventricular dilatation disease or PDD, macaw wasting disease, enteric ganglioneuritis and encephalitis, avian ganglioneuritis, or neurological deficits.[@CIT0001]--[@CIT0005] Parrot bornavirus syndrome primarily affects captive birds of the *Psittacidae* and *Cacatuidae* family, such as cockatoos, cockatiels, lovebirds, conures, parakeets (other than Budgerigars), and especially macaws, but has been diagnosed in over 80 bird species.[@CIT0006]--[@CIT0008] Tissue distribution of the virus following infection is extensive. Parrot Bornavirus and viral RNA are detected in the brain, eye, retinal nerve, spinal cord, heart, adrenal glands, kidneys, and intestines.[@CIT0009],[@CIT0010] The disease is characterized by infection of the central and peripheral nervous system and other organs with PaBV, leading to lymphoplasmacytic infiltration of those tissues, and culminating in nervous system disorders and gastrointestinal malfunctions.[@CIT0008]--[@CIT0014] No effective treatment for PaBV infection or Parrot bornavirus syndrome currently exists. Experimentally, IFN-α inhibits virus infection and reduces viral load in quail cell culture[@CIT0015] and ribavirin inhibits transcription and reduces the viral load in cultured duck embryo fibroblasts.[@CIT0016],[@CIT0017] Symptomatic treatment and management are the only currently recommended therapies for Parrot bornavirus syndrome.[@CIT0007],[@CIT0018] Based on clinical and pathological signs of PaBV infection, disease may result from inflammatory reactions to the virus within the brain, nerves, and other tissues.[@CIT0010]--[@CIT0012] Thus, nonsteroidal anti-inflammatory drugs (NSAIDs) and immunosuppressive drugs could inhibit or reduce the inflammation caused by PaBV infection and lessen the severity of the clinical disease.[@CIT0007],[@CIT0018] The use of NSAIDs is reported to reduce the severity of clinical signs in birds affected with PDD.[@CIT0007] Celecoxib and meloxicam are NSAIDs commonly used for the symptomatic treatment of birds diagnosed with Parrot bornavirus syndrome.[@CIT0018]--[@CIT0021] However, meloxicam did not reduce the clinical signs in cockatiels experimentally infected with PaBV-4 and in fact may have exacerbated the disease progression.[@CIT0022] Due the discrepancies in reported outcomes and the limited controlled studies on the effectiveness of NSAIDs and dearth of research comparing celecoxib and meloxicam in treating PaBV infected birds, the objective of this study was therefore to evaluate the effects of celecoxib and meloxicam treatment on clinical signs, viral shedding, and pathology of cockatiels infected with PaBV-2. We hypothesized that NSAID administration will reduce the severity of disease in infected cockatiels. Materials And Methods {#S0002} ===================== Parrot Bornavirus {#S0002-S2001} ----------------- PaBV-2 was isolated from the brain of experimentally infected cockatiels (*Nymphicus hollandicus*).[@CIT0014] Virus for inoculation was grown as previously described.[@CIT0023] Briefly, duck embryo fibroblast cultures were inoculated with stock virus and maintained in Dulbecco's modified eagle medium (Gibco^®^, Life Technologies Co., Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (Gibco^®^, Life Technologies Co) at 37°C in an atmosphere of 5% CO~2~. After 3 days of incubation, cells were harvested, divided into 1.0 mL aliquots, and stored at −80°C. Virus was confirmed to be PaBV-2 by RNA extraction and RT-PCR analysis followed by sequence analysis of the PCR product, as described below. Birds were inoculated using a combined intranasal and intramuscular administration of infected cells containing 8 × 10^4^ focus forming units of the virus. Nonsteroidal Anti-Inflammatory Drugs {#S0002-S2002} ------------------------------------ A suspension containing 1.0 mg/mL meloxicam was made by crushing three meloxicam 15.0 mg tablets (Lupin, Pharmaceuticals, Inc. Baltimore, MD, USA) using a mortar and pestle, and dissolving the powder in 1 mL deionized water. Ten millilitre of Ora-Plus (Perrigro^®^ Co., Dublin, Ireland) suspending vehicle was added, and then Ora-Sweet (Perrigro^®^ Co.) was added to the suspension to obtain a final volume of 45.0 mL. A suspension containing 10.0 mg/mL celecoxib was made by adding the contents of nine celecoxib 50.0 mg capsules (Pfizer Inc., Mission, KS, USA) to 1.0 mL deionized water. Ten millilitre of Ora-Plus suspending vehicle was added, and then Ora-Sweet added to the suspension to obtain a final volume of 45.0 mL. All solutions were stored at 4ºC and prepared fresh every 30 days. Prior to administration, all solutions were warmed to room temperature. Animals {#S0002-S2003} ------- Twenty-seven cockatiels (*Nymphicus hollandicus*), ranging from 79 to 145 g (mean 101 g), were used. The birds were assessed as healthy by physical examination and medical history. The cockatiels were quarantined for 60 days, during which time each bird was tested three times over 4 weeks for psittacid herpesvirus (genotype 1-4), *Chlamydia spp*. and *Macrorhabdus ornithogaster* (avian gastric yeast). Birds needed to have all tests negative to be included in the study. The cockatiels were housed 14 or 13 birds per cage, with a light-dark cycle of 12 hrs and a room temperature of 23.3 (±5.0)°C, at the Schubot Exotic Bird Health Center aviary, Texas A&M University. Birds were fed a 1/6 cup per bird of premium daily FruitBlend with natural fruit flavors (ZuPreem^®^, Shawnee, KS, USA), and had access to tap water ad libitum. An animal use protocol detailing the experimental protocol was reviewed and approved by the Texas A&M University Office of Research Compliance, complying with guidelines included in the National Research Council of the National Academies' publication *Guide for the Care and Use of Laboratory Animals*, 8th edition. Experimental Protocol {#S0002-S2004} --------------------- Birds were matched with respect to historical shedding of PaBV, weight, and sex, and randomly assigned into three groups of nine birds each: Group 1 birds (placebo) were inoculated with PaBV-2 and beginning 23 days post-inoculation were treated orally, once daily with only the delivery solution (water, Ora-plus, and Ora-Sweet). Group 2 birds (meloxicam treated) were inoculated with PaBV-2 and beginning 23 days post-inoculation were treated orally, once daily with 1.0 mg/kg meloxicam. Group 3 birds (celecoxib treated) were inoculated with PaBV-2 and beginning 23 days post-inoculation were treated orally, once daily with 10.0 mg/kg celecoxib. All treatments were administered prior to morning feeding. The dosage of drug administered was recalculated after each weekly weighing. The drug treatment administered to each group was unknown to the drug administrator/evaluator, assay technician, and pathologist until completion of the study. Birds were observed daily for clinical signs and deviations from normal behavior: these included feather fluffing, bowed head, overly quiet, almond-shaped eyes, over-eating, lethargy, and reluctance to fly. Prior to inoculation, cloacal swabs were collected twice; weights and body condition scores (BCS) were assessed once. After viral inoculation, birds were weighed and BCS was assessed weekly; cloacal swabs were collected every third week and stored at −80ºC until assayed by RT-PCR. Body condition scores were determined by assessing the region of the keel and pectoral muscle. Body condition scores ranged from 1 to 5, thin to obese, respectively.[@CIT0024] A bird was withdrawn from the study and euthanized if it met the following pre-established criteria: weight loss \>20% of the initial weight; BCS of 1; or as recommended by the attending veterinarian. On day 173 (±2) following experimental inoculation \[day 150 (±2) of NSAID treatment or placebo treatment\], surviving birds were anesthetized with 5% isoflurane in 100% oxygen; weight, BCS, cloacal swab, and urine were collected. Urine was collected as previously described by Heatley et al.[@CIT0025] Immediately after sample collection while the bird was still in deep anesthesia, the bird was humanely killed by chamber exposure to 100% CO~2~. A complete necropsy was performed immediately and gross lesions were recorded. Paired samples of heart, liver, feather follicle, spleen, crop, proventriculus, ventriculus, intestine, gonad, pancreas, adrenal gland, kidney, lung, spinal cord, brain, eye, aqueous humor, optic nerve, brachial plexus, and sciatic nerve were collected and stored at −80ºC for later analyses by RT-PCR. The remainder of each organ was placed in 10% neutral-buffered formalin for histologic examination and immunohistochemistry (IHC) testing. RT-PCR {#S0002-S2005} ------ Tissue, urine, and cloacal swabs were tested for the presence of viral RNA by RT-PCR as previously described.[@CIT0023] All samples were tested in duplicate for both the matrix protein and phosphoprotein. Samples were considered negative for cycle threshold (C~T~) ≥37.0. If a sample was positive for only one of the two proteins, the sample was retested. Results were analyzed using the Sequence Detection System, Version 2.4.1 (SDS 2.4) software (Life Technologies, Thermo Fisher, Carlsbad, CA, USA). Histopathology & Immunohistochemistry {#S0002-S2006} ------------------------------------- Tissue samples were fixed in 10% neutral-buffered formalin, processed overnight, embedded in paraffin, sectioned at 4 µm, stained with haematoxylin and eosin (H & E), and examined by light microscopy according to standard procedure. Immunohistochemistry, to demonstrate the presence of PaBV nucleoprotein, was performed on tissues obtained at necropsy according to previously described methods.[@CIT0026] The reviewing pathologist assigned a semiquantitative viral score to the tissue sample using the following ordinal scale: none detected (-), small quantity detected (+), moderate quantity detected (++), large quantities detected (+++). Statistical Analysis {#S0002-S2007} -------------------- The Gehan--Breslow method was used to analyze differences in survival between the treatment groups. Two-way repeated measures Analysis of Variance (ANOVA) was used to compare weight changes between the treatment groups and day over the experimental time course. The Kruskal--Wallis One Way ANOVA on ranks was used to ascertain differences in histological findings and IHC results. A *P* ≤ 0.05 was considered statistically significant. Sigma Plot version 10.0.1 was used for performing all statistical analyses (Systat Software, Inc., San Jose, CA, USA). Results {#S0003} ======= Clinical Observations {#S0003-S2001} --------------------- Survival did not differ significantly between the 3 study groups ([Figure 1](#F0001){ref-type="fig"}). In the placebo group (group 1), 2 of 9 birds were euthanized prior to the end of the study on 54 and 61 days post-inoculation. In the meloxicam-treated group (group 2), 2 of 9 birds were euthanized or found dead on 33 and 79 days post-inoculation. In the celecoxib-treated group (group 3), 4 of 9 birds were euthanized or found dead on 37, 43, 45, and 74 days post-inoculation. Of the 8 birds that died prior to study completion, 2 were found dead, one each in the meloxicam and the celecoxib-treated groups, while the remaining 6 showed neurological or gastrointestinal signs characteristic of PaBV infection ([Table 1](#T0001){ref-type="table"}).Table 1Cockatiels Euthanized Or Found Dead Prior To Study's EndTreatmentBird IDDied Or Euthanized (Day^a^)BCSWeights (gms)ObservationsPlacebo154395.0Loss of balance and flying into cage wall261274.8Regurgitating with a distended cropMeloxicam1033176.0Found dead11792117.6Regurgitation, enlarged crops, feather fluffing, head drooping and tilted, off balanced, lethargic, spends most of the time on the cage floorCelecoxib1937297.0Found dead2043274.5Eyes closed, unstable, head tilt, lethargic2145383.0Eyes closed, unstable, head tilt, lethargic2274164.0Enlarged crop, eyes closed, unstable, head tilt, lethargic, feces dark in color[^1] Figure 1Survival analysis of cockatiels (*Nymphicus hollandicus*) inoculated with PaBV-2 and treated with either placebo, meloxicam (1.0 mg/kg), or celecoxib (10.0 mg/kg).**Notes:** The Gehan--Breslow statistic for survival curve was used to generate the survival analysis. Birds were experimentally inoculated with PaBV-2 on day 0. On day 23 post-inoculation (dashed arrow), birds started once daily, oral administration with the following: placebo control group (solid black line), meloxicam (dotted grey line) and celecoxib (dashed grey line). Body weights did not differ significantly between the study groups over the study period. Within each treatment group, significant daily differences in weight were noted. All groups had a reduction in weight on day 7 after treatment, with or without NSAID, was administered. Between 7 and 47 days, the body weights of the NSAID-treated groups were significantly lower than the weights prior to inoculation or the commencement of the NSAID treatment; however, from 54 days after treatment until study's end, there was no significant difference between their weight prior to inoculation or treatment. Body condition scores between or within the groups did not differ significantly during the study period. Most birds that died or were euthanized early tended to have lost weight and BCS over the course of the study. A single bird from the meloxicam group was an exception. At the time of euthanasia, this bird had a greatly increased weight compared to other birds, but had a low BCS. Necropsy findings of a profoundly dilated crop and proventriculus that were full of feed were most likely the cause of the increased weight ([Figure 2B](#F0002){ref-type="fig"}).Figure 2Gross necropsy findings in cockatiels *(Nymphicus hollandicus)* inoculated with PaBV-2 and treated with either placebo, meloxicam (1.0 mg/kg), or celecoxib (10.0 mg/kg). (**A**) Bird 15, meloxicam-treated group: euthanized on day 173 post-inoculation. Moderate dilation of proventriculus (yellow arrow). (**B**) Bird 21, celecoxib-treated group: euthanized on day 45 post-inoculation. Mild dilation of proventriculus (yellow arrow) with undigested seed present in the intestines (grey arrows). (**C**) Bird 22, celecoxib-treated group: euthanized on day 74 post-inoculation. Severe dilation of crop (red arrow), mild dilation of proventriculus (yellow arrow), and blacken intestinal content (grey arrows). Detection Of PaBV RNA {#S0003-S2002} --------------------- The results of viral RNA detection in cloacal swab samples are summarized in [Table 2](#T0002){ref-type="table"}. Prior to experimental inoculation, cloacal swab testing for viral RNA was negative for all birds, with the exception of one cockatiel that had a positive cloacal swab on day −17 of inoculation, but was negative on days −31, 13, and 20 post-inoculation. PaBV shedding was first detected 42 days post-inoculation in groups 2 and 3. At study end, all birds that had not died or been euthanized early for humane concerns had positive cloacal swabs, with the exception of one bird in group 2, which was negative throughout the study period. The cumulative number of birds with positive cloacal swabs by the end of the study was 8/9, 6/9, and 7/9 in groups 1, 2, and 3, respectively.Table 2Detection Of Viral RNA In Cloaca Samples From Cockatiels *(Nymphicus hollandicus)* Inoculated With PaBV-2 And Treated With Placebo, Meloxicam (1.0 mg/kg), Or Celecoxib (10.0 mg/kg)TreatmentBird IDDay Post-Inoculation−31/-17132033/374243 - 616374/7984103125146173 (±2)Placebo1^a^−/−−−\--2^a^−/−−−-**+**3−/−−−\--**+++++**4−/−−−-**++++++**5−/−−−-**++++++**6−/−−−-**++++++**7−/−−−-**++++++**8−/−−−-**++++**−**+**9−/−−−-−**+++++**Meloxicam10^a^−/−−−−11^a^−/−−−-−−12−/−−−-**++++++**13−/−−−-−**+++++**14−/−−−-**++++++**15−/−−−-−\--**+**-**+**16−/−−−-**++++++**17−/**+**−−**+++++++**18-/-−−-−\-\-\-\--Celecoxib19^a^−/−−−−20^a^−/−−−-−21^a^−/−−−**+**−22^a^−/−−−**+**−**+**23−/−−−-**++++**-**+**24−/−−−**+++++++**25−/−−−-−**+++++**26−/−−−**+++++++**27−/−−−-**++++++**[^2] At the study's end, viral RNA was detected in the midbrain, hindbrain, cerebellum, forebrain, kidney, and urine of all surviving birds, except for one. There was no significant difference in the amount of detectable viral RNA between the treatment groups. Necropsy And Histopathology {#S0003-S2003} --------------------------- The predominant gross abnormalities seen at necropsy were dilation of the crop and proventriculus and an enlarged, dilated heart with thin walls ([Figure 2A](#F0002){ref-type="fig"}--[C](#F0002){ref-type="fig"}). However, these abnormalities were not consistently present nor of consistent severity. Birds that were euthanized or died early in the study had more pronounced crop and proventricular dilatation than birds that survived until day 173 (±2) post-inoculation. Black intestinal contents were present in 2 birds in group 2 and 2 birds in group 3; these 4 birds had been euthanized prior to the study's endpoint ([Figure 2B](#F0002){ref-type="fig"} and [C](#F0002){ref-type="fig"}). Other abnormalities such as liver mottling, enlarged spleen, mild intestinal distension, and pale pancreas were occasionally noted, with no predilection for any group. Histopathological changes occurred in many tissues, but no significant differences in the severity of the lesions occurred based on group ([Table 3](#T0003){ref-type="table"}). The lesions observed included: lymphoplasmacytic myenteric ganglioneuritis in the crop, proventriculus, ventriculus and intestines; multifocal dilation of tubules/interstitial inflammation, fibrosis and/or mineralization, and scattered lymphoid nodule formation in the kidney; lymphoplasmacytic infiltration within the epicardial, myocardial, and/or Purkinje cells of the heart; and, lymphoplasmacytic perivascular cuffing in the central nervous system ([Figure 3](#F0003){ref-type="fig"}). Though infrequent, histopathological changes were also seen in the liver, pancreas, lung, spleen, optic nerves, and adrenal glands.Table 3Distribution Of Histological Lesions In Cockatiels *(Nymphicus hollandicus)* Inoculated With PaBV-2 And Treated With Placebo, Meloxicam (1.0 mg/kg), Or Celecoxib (10.0 mg/kg)TreatmentTissueBrainHeartIntestinesProventriculusPancreasKidneyOcular nerveSpinal cardLiverCropVentriculusSpleenLungAdrenalPlacebo1^a^**--+----------------+--**2^a^**------+++++----+----**3**----+--++++++++--**4**+--+--+--++--++----**5**----+--+--++----+----**6**+++----+++--++----+**7**++++--+++--+++**8**----+--+--++--+----**9**+--+++++++++----**Meloxicam10^a^**------+----+------+----**11^a^**++++++++--++--**12**--------++++--++--**13**----+++--+++--+----+**14**------+----------++--++**15**+++------++----++++**13**--+--+----++----------+**17**+++--++++----++++**18**+----+----++----+----**Celebrex19^a^**+++++++++--++**20^a^**+--++++++----+--**21^a^**+++--++++----+--**22^a^**+++++++++++**24**+--+--+--++--++----+**25**----++++++--+++--+**26**+--+--++++--++----**27**----+--------------+----**28**------+----------++--**[^3] Figure 3Histologic findings in cockatiels *(Nymphicus hollandicus)* inoculated with PaBV-2 and treated with either placebo, meloxicam (1.0 mg/kg), or celecoxib (10.0 mg/kg). (**A**) Lymphoplasmacytic infiltration of serosal ganglia of the crop. Bird 3, placebo, euthanized 173 (±2) days post-inoculation. (**B**) Moderate lymphoplasmacytic infiltration of subserosal ganglia in the proventriculus. Bird 9, placebo, euthanized 173 (±2) days post-inoculation. (**C**) Moderate lymphoplasmacytic infiltration of subserosal ganglia in the ventriculus. Bird 17, meloxicam treated, euthanized 173 (±2) post-inoculation. (**D**) Lymphocytic infiltration in the interstitium of kidney with formation of a lymphoid nodule. Bird 3, placebo, euthanized 173 (±2) days post-inoculation. (**E**) Lymphoplasmacytic infiltration in the epicardial ganglia of the heart. Bird 25, celecoxib treated, euthanized 173 (±2) days post-inoculation. Immunohistochemistry {#S0003-S2004} -------------------- Distribution or amount of viral nucleoprotein did not differ statistically between the three groups ([Table 4](#T0004){ref-type="table"}). Virus (PABV-2) was predominantly detected in the brain, heart, gastrointestinal tract and kidneys but also detected in liver, pancreas, lung, spleen, adrenal optic nerve, uropygial gland, cloaca, gonads, and skin/feather follicles. Skeletal muscle was the only tissue consistently negative for the virus. No virus was detected in any tissues of four birds: one in group 1, two in group 2, and one in group 3.Table 4Tissue Distribution And Relative Amount Of PaBV Nucleoprotein (N-protein) As Detected By ImmunohistochemistryTreatmentBird IDTissueBrainHeartLiverIntestineCropProventriculusVentriculusPancreasKidneyLungplacebo1^a^----2^a^+++--3+++++--+++++++++++++++++4++++++++++++++++++5+++++++++++6++++++++++++++--+7++++++++++++++++++++8++--++++++++++++++++9+++++++++++++++++++--+Meloxicam10^a^----11^a^+++--12++--+++++++--13++--++++++++++++--14+++++++++++++++++++++15+++----++--16++++++++++----17++++++++++++++++++18------------Celecoxib19^a^+--20^a^----21^a^++--22^a^+++++23+++--+++++++++++++24++++++++++++++++++++25+++++++++++++++++++++26+++++++++++++27+++++++++++++++[^4] Discussion {#S0004} ========== Oral administration of meloxicam, 1.0 mg/kg, or celecoxib, 10.0 mg/kg, once daily for 150 days failed to show differences in the clinical presentation, viral shedding, gross lesions, viral distribution, nor histopathology in cockatiels experimentally inoculated with PaBV-2 when compared to untreated birds. These results agree with previously published experimental study on meloxicam usage,[@CIT0022] but conflict with reports on the treatment of clinically affected birds.[@CIT0019]--[@CIT0021] Bird species differences, viral genotype differences, and evaluation criteria may account for the lack of agreement between our studies and previous reports on clinical cases. In this study, 4 cockatiels treated with NSAIDs had black intestinal material that may have been autolyzed blood, and all 4 were euthanized after 10 to 56 days of NSAID treatment. In a prior study that treated PaBV inoculated cockatiels with meloxicam, 1 bird had blood-filled, blackened intestine, while another bird had black material that may have been autolyzed blood in its intestines.[@CIT0022] In mammals, gastrointestinal abnormalities, such as ulcers and bleeding, and renal necrosis due to ischemia are major features of NSAID toxicity; however, these have not been reported as major side effects of NSAID use in birds.[@CIT0027]--[@CIT0035] The combined effect of PaBV induced pathological changes and NSAID treatment may exacerbate gastrointestinal irritation and toxicity of the NSAID and may increase the risk for gastrointestinal bleeding. The administration of NSAIDs to birds infected with PaBV did not affect their weight nor BCS. Birds that displayed severe clinical signs and either were removed early or died, had decreased weight and BCS. This is consistent with the gastrointestinal dysfunction that leads to starvation in the birds.[@CIT0007],[@CIT0014],[@CIT0022],[@CIT0036] It is interesting to note that while most birds infected with PaBV decreased in weight, one bird in the meloxicam group had gained weight at the time of its removal from the study. Upon necropsy, the crop and proventriculus were distended with feed. Presumably, the weight gain was due to the proventricular contents. This reinforces that bird body weight alone should not be used to assess disease progression or severity. Survival and early removal of the birds from the study were not statistically different between the groups; however, the number of birds with clinical conditions warranting early removal was twice as high in the celecoxib-treated group as compared to the meloxicam-treated or placebo group ([Figure 1](#F0001){ref-type="fig"}). In addition to the NSAIDs having no apparent effect on overall mortality, the time at which cockatiels started to show clinical signs and had to be removed early (33--79 days post-PaBV inoculation) was not affected by NSAID treatment and was consistent with the time period seen in other studies of PaBV-2 experimentally inoculated birds.[@CIT0003],[@CIT0009],[@CIT0014],[@CIT0022],[@CIT0036],[@CIT0037] Gross pathology showed no significant difference between the three treatment groups and all groups had clinical presentations indicative of Parrot bornavirus syndrome, with a dilated proventriculus being most represented, as described in the literature.[@CIT0003],[@CIT0009],[@CIT0014],[@CIT0022],[@CIT0036],[@CIT0037] NSAID treatment in cockatiels inoculated with PaBV-2 was expected to decrease lesion distribution and tissue inflammatory response; however, in this study treatment lacked any significant impact. Shedding of PaBV-2 was not affected by the administration of NSAIDs. In fact, treated birds tended to shed earlier than the placebo group, though there was no significant difference. Shedding was first detected on day 42 post-inoculation and became more consistent by day 63 post-inoculation, a time period that corresponded with previous work in our lab. At the end of the study, only one bird had not shed virus. Previous research has shown that there is not a 100% correlation between histopathology and virus detection between and among tissues and fluids.[@CIT0025],[@CIT0038] One bird in the meloxicam-treated group was negative for the presence of PaBV by RT-PCR and IHC throughout the study. However, at necropsy, its proventriculus was dilated and contained undigested seed. Histopathology showed perivascular cuffing with increased glial cells in the brain and lymph nodules in the mucosa, ganglia, and serosa of the ventriculus. Tissues may be negative for viral antigens by IHC but the animal can still have a subclinical or asymptomatic infection.[@CIT0038] *Cryptosporidium* was identified in the proventriculus of this bird which may have been a factor in causing some of these lesions.[@CIT0039] Conclusion {#S0005} ========== The administration of meloxicam or celecoxib failed to alter the progression or severity of clinical signs, gross or histopathological changes, viral shedding, or distribution of viral RNA in cockatiels infected with PaBV-2. Caution should be stressed when prescribing NSAIDs in birds with gastrointestinal dysfunction, such as may occur with PaBV infection, due to the potential for gastrointestinal irritation, bleeding, or other unwanted side effects of NSAIDs. The authors thank Debra Turner and Dr. Jordan Gentry for technical assistance. Supported in part by the Schubot Exotic Bird Health Center. The open access publishing fees for this article have been covered by the Texas A&M University Open Access to Knowledge Fund (OAKFund), supported by the University Libraries and the Office of the Vice President for Research. Disclosure {#S0006} ========== All authors declare no conflicts of interest relevant to the research performed. [^1]: **Notes:** ^a^Is the day post-inoculation. [^2]: **Notes:** RT-PCR testing identified PaBV matrix and phosphoprotein RNA. -Negative (cycle threshold ≥37.0); +Positive detection for viral RNA; ^a^Euthanized prior to study's endpoint of 173 (±2) days post-inoculation. [^3]: **Notes:** -No histopathologic lesions noted; +Histopathologic lesion present; ^a^Euthanized prior to the end of treatment. [^4]: **Notes:** -No PaBV-2 N-protein antigen detected; +Mild amount of PaBV-2 N-protein antigen detected; ++Moderate amount of PaBV-2 N-protein antigen detected; +++Large amount of PaBV-2 N-protein antigen detected; ^a^Euthanized prior to the end of treatment.

Introduction {#Sec1} ============ The *PDCD4* (*Programmed cell death 4*) gene encodes a highly conserved nuclear-cytoplasmic shuttling protein that acts as a tumor suppressor (for recent reviews see refs. ^[@CR1],[@CR2]^). PDCD4 contains two highly structured MA-3 domains located in the central and C-terminal parts of the protein, which mediate protein-protein-interactions with the translation initiation factor eIF4A. A putative unstructured domain at its N-terminal has been shown to mediate protein-protein- and protein-RNA-interactions^[@CR3]--[@CR10]^. PDCD4 was initially shown to suppress tumor development in an *in-vitro* mouse keratinocyte model of tumor promotion^[@CR11]^, but has since been implicated as a tumor suppressor in a broad spectrum of human tumors^[@CR12]--[@CR19]^. Down-regulation of PDCD4 expression in tumor cells occurs by different mechanisms. *PDCD4* mRNA is targeted by several microRNAs, most prominently oncogenic microRNA miR-21, whose over-expression in cancer cells down-regulates *PDCD4* expression^[@CR20],[@CR21]^. On the protein level, p70(S6K) kinase-mediated phosphorylation of PDCD4 triggers its ubiquitination by the E3 ubiquitin ligase complex SCF(βTRCP) and its subsequent degradation^[@CR22]^. A large body of work has suggested that down-regulation of *PDCD4* expression contributes to tumor development by stimulating the mobility and the metastatic potential of tumor cells^[@CR18]--[@CR20],[@CR23]--[@CR25]^. Furthermore, silencing of *PDCD4* has been shown to affect the cellular DNA-damage response, suggesting that decreased PDCD4 expression might compromise genomic stability and contribute to tumor development^[@CR26],[@CR27]^. PDCD4 has emerged as a critical regulator of protein translation due to its ability to interact with and inhibit the function of the eukaryotic translation-initiation factor eIF4A, a RNA helicase that promotes the unwinding of mRNA secondary structures present in the 5′-untranslated regions (UTRs) of certain mRNAs^[@CR3],[@CR4],[@CR19],[@CR28]^. PDCD4 is therefore thought to suppress the cap-dependent translation of mRNAs with 5′-structured UTRs. This was supported by studies showing that PDCD4 suppresses the translation of RNAs containing engineered 5′-hairpin structures^[@CR3],[@CR4]^ as well as by the identification of specific mRNAs regulated by this mechanism^[@CR19],[@CR28]^. However, alternative mechanisms of translational suppression involving direct RNA-binding of PDCD4 to the coding regions of specific mRNAs have also been described^[@CR29],[@CR30]^. Our current understanding of the function of human PDCD4 derives mostly from work carried out with transformed tumor cells. Here, we have used a telomerase-immortalized human epithelial cell line to study the effect of PDCD4 silencing on the cell cycle, gene expression and mRNA translation. Our work reveals a novel role of PDCD4 in the regulation of the cell cycle and provides a more complete picture of its cellular functions. Results {#Sec2} ======= PDCD4 is required for the G1/S-transition in RPE cells {#Sec3} ------------------------------------------------------ Our current understanding of PDCD4′s role in human cells is largely based on studies using transformed tumor cell lines. Such studies have provided insight into the function of PDCD4 as a tumor suppressor but may not reveal an unbiased picture of its cellular roles due to the aberrant nature of these cells. To study the function of human PDCD4 in normal cells we have used the telomerase-immortalized hTERT-RPE-1 cell line (referred to as RPE hereafter) as a model of untransformed epithelial cells. Expression of PDCD4 was effectively silenced by two different siRNAs (Fig. [1a](#Fig1){ref-type="fig"}). The cells did not show obvious changes of their spindle-shaped fibroblast-like morphology when viewed under the microscope. To explore whether PDCD4 knockdown disrupts the cell cycle we examined the cell cycle distribution of asynchronous cultures of RPE cells treated with PDCD4-specific or control siRNAs by flow cytometry. The cell cycle profiles of the control and PDCD4 knock-down cells were different. Specifically, the abundance of S- and G2-phase cells was strongly decreased in cultures treated with the two different PDCD4-specific siRNAs compared to the control cells (Fig. [1b](#Fig1){ref-type="fig"} and Supplementary Table [S1](#MOESM1){ref-type="media"}). Both siRNAs yielded similar results suggesting that the partial G1 arrest is induced by PDCD4 knockdown and not by off-target effects.Figure 1PDCD4 knockdown affects the cell cycle and growth properties of RPE cells. (**a**) Silencing of PDCD4 expression in RPE cells with PDCD4-specific siRNA-1 and -2. (**b)** Cell cycle distribution of RPE cells treated with control or PDCD4-specific siRNA-1 and -2. G1 and G2/M peaks are marked. (**c**) Equal numbers of RPE cells treated with control siRNA or PDCD4 siRNA-1 or -2 were plated onto replicate tissue culture plates. The growth of the cells was followed over several days by fixing one of the replicate plates at each indicated day of culture with formaldehyde. After 5 days of culture all plates were stained simultaneously with crystal violet. (**d**) RPE cells treated with siRNAs as in A. The cells were then incubated in medium supplemented with 10 μCi/ml 3H-thymidine for 1 hour. Subsequently, the radioactivity incorporated into DNA was determined by TCA-precipitation and liquid scintillation counting. The bars indicate the percentage of DNA synthesis (with standard deviation) of the PDCD4 siRNA treated cells relative to control cells. Asterisks indicate statistical significance (\*\*p \< 0.01; \*\*\*p \< 0.001; students-t test). (**e)** RPE cells were treated for 24 h with control siRNA or PDCD4-specific siRNA-1 and -2. The cells were then arrested in the late G1 phase by incubation for 24 hours in the presence of 0.5 mM mimosine. Cells were then processed immediately for flow cytometry analysis or were washed with fresh medium lacking mimosine and cultivated for additional 10 or 20 hours before being analyzed by flow cytometry. G1 and G2/M peaks are marked. Based on this observation we hypothesized that PDCD4 knockdown decreases the proliferation rate of the cells. To test whether this is the case, we monitored the growth of the cells over a period of 5 days following knockdown with PDCD4-specific or control siRNA. We used a qualitative assay of cell proliferation by plating equal numbers of cells on replicate culture plates and visualized their proliferation by crystal violet staining (Fig. [1c](#Fig1){ref-type="fig"}). We found that the intensity of staining of cells transfected with PDCD4-specific siRNAs was decreased compared to the control cells, suggesting decreased proliferation upon Pdcd4 knockdown. As in the previous experiment, both Pdcd4-specific siRNAs had similar effects. To substantiate the finding that PDCD4 knockdown reduces the proliferation of RPE cells we quantified their DNA-synthesis activity as an independent measure of proliferation. We performed ^3^H-thymidine incorporation assays by incubating equal numbers of control- and PDCD4-knockdown cells for 1--2 hours in the presence of radiolabeled thymidine and determined the amount of radioactivity incorporated into TCA-precipitable high molecular weight DNA. RPE cells treated with the PDCD4-specific siRNAs displayed significantly reduced DNA synthesis activity compared to cells treated with control siRNA (Fig. [1d](#Fig1){ref-type="fig"}). Thus, in comparison to the control cells the number of cells in S-phase was significantly reduced in cultures treated with PDCD4-specific siRNA. This is consistent with our cell cycle measurements where almost no S-phase cells were visible after PDCD4 knockdown (Fig. [1b](#Fig1){ref-type="fig"}). Overall, these experiments suggested a slowed-down entry of cells into S-phase when PDCD4 levels are low. To demonstrate the effect of PDCD4 knockdown on the G1/S-transition more directly we silenced PDCD4 expression and synchronized the cell population by an additional treatment for 24 hours with 0.5 mM mimosine to block DNA-replication^[@CR31]^. This leads to a reversible arrest of most of the cells at the G1/S boundary. The cells were then released into S-phase by washing them with medium without mimosine, followed by an analysis of the cell cycle profile immediately after the release of the cell cycle block and after 10 and 20 hours (Fig. [1e](#Fig1){ref-type="fig"} and Supplementary Table [S1](#MOESM1){ref-type="media"}). Most of the G1-arrested, control siRNA-treated cells had entered into S-phase within 10 hours after removal of mimosine. At 20 hours there was a distinct G2-peak and an increased G1 peak, suggesting that a fraction of the cells had progressed through mitosis to reach the subsequent G1-phase. In contrast, only a small fraction of the cells treated with either of the PDCD4-specific siRNAs had entered into S-phase even after 20 hours, indicating that PDCD4 knockdown had strongly blocked the G1/S-transition. Defective G1/S-checkpoint control overrides the requirement for PDCD4 to undergo G1/S-transition {#Sec4} ------------------------------------------------------------------------------------------------ The finding that PDCD4 is required for the G1/S-transition seems counterintuitive considering that PDCD4 expression is often decreased in tumor cells^[@CR1],[@CR2]^. We therefore hypothesized that defective G1/S-checkpoint control, which is a hallmark of many tumor cells, might circumvent the requirement for PDCD4 to pass the G1/S-boundary. To test whether defective G1/S-checkpoint control can override the requirement for PDCD4 for cells to enter into S-phase we investigated the effect of PDCD4 knockdown in HEK293T cells. These cells express the adenoviral E1A protein and the SV40 large T antigen, both of which sequester the retinoblastoma protein and allow transcription factor E2F to be active independently of cyclin/Cdk-induced phosphorylation, thereby inactivating the G1/S-checkpoint^[@CR32],[@CR33]^. Although PDCD4 expression was silenced effectively in these cells (Fig. [2a](#Fig2){ref-type="fig"}) the cell cycle distribution of an asynchronous culture of HEK293T cells is almost indistinguishable, as judged by the slightly reduced height of the G2-cell peak and the almost similar height of the plateau of S-phase cells between the G1 and G2 peaks when comparing the control and the Pdcd4-knockdown cell populations (Fig. [2b](#Fig2){ref-type="fig"} and Supplementary Table [S1](#MOESM1){ref-type="media"}). Moreover, we observed no significant change in the incorporation of ^3^H-thymidine into DNA (Fig. [2c](#Fig2){ref-type="fig"}). Furthermore, we used mimosine to block HEK293T cells in the cell cycle. Because mimosine arrests cells at the G1/S-boundary as well as cells that have already entered S-phase, mimosine-treatment of HEK293T cells resulted in a peak of G1-cells with a pronounced shoulder towards a higher DNA-content. Importantly, the cell cycle profiles recorded at 9 and 20 hours after removal of mimosine showed that PDCD4-silenced HEK293T cells had entered into S-phase upon removal of the drug similar to the control cells (Fig. [2d](#Fig2){ref-type="fig"} and Supplementary Table [S1](#MOESM1){ref-type="media"}). This indicates that PDCD4 knockdown does not affect the G1/S-transition in HEK293T cells.Figure 2The G1/S-transition is independent of PDCD4 expression in HEK293T cells. (**a**) Silencing of Pdcd4 expression by treatment of HEK293T cells with PDCD4-siRNA-2. (**b**) Cell cycle distribution of HEK293T cells treated with control or Pdcd4-specific siRNA-2. G1 and G2/M peaks are marked. (**c)** HEK293T cells treated for 72 hours with control or PDCD4-specific siRNA as in A were incubated for 1 h in medium supplemented with 10 μCi/mL ^3^H-thymidine. The radioactivity incorporated into DNA was determined by TCA-precipitation and liquid scintillation counting. The bars indicate the percent DNA synthesis (with standard deviation) of the PDCD4 siRNA treated cells relative to control cells. (**d)** HEK293T cells treated for 24 hours with control or PDCD4-specific siRNA were analysed for the G1/S-transition as in Fig. [1e](#Fig1){ref-type="fig"}. Positions of the G1 and G2 peaks are marked. Knockdown of PDCD4 activates the G1/S-checkpoint in RPE cells by increasing the expression of p21^WAF1/CIP1^ {#Sec5} ------------------------------------------------------------------------------------------------------------ Taken together, our data support the concept that PDCD4 knockdown activates the G1/S cell cycle checkpoint in RPE cells, thereby delaying cell cycle progression at the G1/S boundary. We have previously reported that knockdown of PDCD4 increases the activity and expression of p53 and thereby stimulates the expression of the p53 target gene *CDKN1A*^[@CR26],[@CR28]^. *CDKN1A* encodes the Cdk-inhibitor p21^WAF1/CIP1^ that plays a key role at the G1/S-checkpoint. DNA damage induces p21^WAF1/CIP1^ expression via p53, which then inhibits Cdk activity and causes a G1/S cell cycle arrest^[@CR34],[@CR35]^. Therefore, we hypothesized that the requirement for PDCD4 to enter the S-phase was due to its ability to balance or counteract the basal activity of p53 in unstressed cells. This would suggest that decreasing the activity of p53 by an inhibitor would relieve the requirement for PDCD4 expression for S-phase entry. To test this possibility, we employed pifithrin-α (PFT-α), an inhibitor that suppresses the p53-dependent activation of p53 target genes^[@CR36]^. We knocked down PDCD4 both in the presence or absence of PFT-α and analyzed the expression of p21^WAF1/CIP1^ by western blot. Interestingly, we found a strong increase of p21^WAF1/CIP1^ expression following knockdown of PDCD4 in the absence of PFT-α (Fig. [3a](#Fig3){ref-type="fig"}), consistent with our earlier studies^[@CR26]^ and demonstrating that silencing of PDCD4 increases p21^WAF1/CIP1^ expression also in RPE cells. As expected, in the presence of 30 μM PFT-α the increase of p21^WAF1/CIP1^ expression was strongly suppressed. To measure S-phase entry of the cells we performed ^3^H-thymidine-labeling experiments. This showed that knockdown of PDCD4 in the absence of PFT-α strongly reduced DNA-synthesis activity (Fig. [3b](#Fig3){ref-type="fig"}), whereas the inhibition of DNA-synthesis activity by PDCD4 knockdown was less strong in the presence of PFT-α. It is possible that the DNA synthesis activity of PDCD4 knockdown cells did not reach the level of control cells in the presence of PFT-α because there was still a residual increase of p21^WAF1/CIP1^ in the presence of PFT-α. This could be due to a limiting concentration of the inhibitor, or could reflect a minor contribution of a p53-independent mechanism of stimulation of p21^WAF1/CIP1^ expression by PDCD4 silencing, as proposed recently^[@CR37]^. Overall, our data show that the G1/S-cell cycle block induced by PDCD4 knock-down is caused, at least to a significant part, by the p53-dependent increase of p21^WAF1/CIP1^ expression. PDCD4, therefore, plays a crucial role in counteracting basal p53 activity in unstressed cells.Figure 3PDCD4 is linked to the G1/S checkpoint via the p53-p21^WAF1/CIP1^ axis. (**a)** Knockdown of PDCD4 increases the expression of p21^WAF1/CIP1^. RPE cells were transfected with control siRNA or PDCD4-specific siRNA-2 in the absence or presence of 30 μM PFT-α. Total cell extracts were then analyzed by western blotting for expression of PDCD4, p21^WAF1/CIP1^ and β-actin. (**b)** RPE cells were labeled with ^3^H-thymidine for 1 hour, followed by TCA precipitation and liquid scintillation counting to determine their DNA synthesis activity. The bars indicate the percentage of DNA synthesis (with standard deviation) of the PDCD4 siRNA treated cells relative to control cells. Asterisks indicate statistical significance (\*\*\*p \< 0.001; students-t test). (**c,d)** DNA damage induced down-regulation of PDCD4 expression. RPE cells were exposed to UV light for the indicated times, using a germicidal UV-C lamp in a tissue culture hood or were cultivated in the presence of the indicated concentrations of mitoxantrone. Cells were incubated for 16 hours and total cell extracts were analyzed by western blotting for expression of PDCD4, the DNA double strand break marker γ-H2AX, and β-actin. Previously, we had observed that DNA damage down-regulates PDCD4 expression in HepG2 cells, suggesting a role of PDCD4 in the DNA-damage response^[@CR28]^. This prompted us to examine whether the expression of PDCD4 is also decreased in response to DNA damage in RPE cells. We employed UV-irradiation and the topoisomerase inhibitor mitoxantrone to induce DNA-damage, which we monitored by the DNA double strand break marker γ-H2AX^[@CR38]^. The increase of g-H2AX staining between untreated cells (first lanes in panels C and D) and cells UV-irradiated or incubated with mitoxantrone confirmed that both treatments caused DNA damage, which was accompanied by virtually complete loss of PDCD4 expression (Fig. [3c,d](#Fig3){ref-type="fig"}). This suggested that PDCD4 does not act as an antagonist of p53 in the presence of genotoxic stress. Silencing of PDCD4 in RPE cells affects the abundance and translation of multiple mRNAs {#Sec6} --------------------------------------------------------------------------------------- To obtain an integrated view of the functions of PDCD4, we employed RNA-Seq and ribosome profiling. This allowed us to explore the effect of PDCD4 knockdown in RPE cells on transcriptome-wide mRNA abundance and translation (Supplementary Fig. [1a](#MOESM1){ref-type="media"}). First, we used PDCD4 siRNA-2 and control siRNA in RPE cells (Supplementary Fig. [1b](#MOESM1){ref-type="media"}) and subjected them to RNA-Seq. We found that 496 genes were significantly upregulated and 750 genes were down-regulated by PDCD4 silencing. The heat map (Fig. [4a](#Fig4){ref-type="fig"}) shows all genes that are up- or down-regulated two-fold or more. We validated these changes by quantitative real-time PCR to confirm the expression of representative up- and down-regulated mRNAs (Fig. [4b](#Fig4){ref-type="fig"}).Figure 4Pdcd4 knockdown induces transcriptome-wide changes of mRNA expression. (**a**) Heat map of all mRNAs whose expression levels were significantly altered (padj value \< 0.05) after silencing of PDCD4 by a log2-fold change \> 0.5 or \<−0.5. The individual columns represent the results of three independent samples from cells transfected with control siRNA (control) and two independent samples of cells transfected with PDCD4 siRNA-2 (si-2). Selected genes are marked on the right side. (**b**) Real-time PCR analysis of selected up- and down-regulated RNAs. The columns indicate mRNA abundance in control siRNA (black bars) and PDCD4 siRNA-2 (grey bars) treated RPE cells. Individual expression levels determined from three independent biological replicates are marked by white dots. Asterisks indicate statistical significance (\*p \< 0.05; \*\*p \< 0.01; \*\*\*p \< 0.001; students-t test). To investigate whether PDCD4-dependent changes of mRNA expression affect specific biological processes we performed gene set enrichment analysis (GSEA^[@CR39]^). mRNAs suppressed by PDCD4 silencing were strongly enriched in genes that are implicated in DNA replication, E2F targets and cell cycle regulation (Fig. [5a](#Fig5){ref-type="fig"} and Supplementary Table [S2](#MOESM2){ref-type="media"}), substantiating our previous findings. Genes bound by the DREAM complex, a transcriptional regulatory complex playing a key role in cell cycle regulation^[@CR40]--[@CR42]^, and genes with a peak of expression at the G1/S-checkpoint were strongly downregulated upon PDCD4 silencing. Further gene ontology (GO) term analysis of genes repressed by PDCD4 knockdown confirmed that these genes were involved in various DNA-related processes and aspects of cell cycle regulation. Genes that were up-regulated by PDCD4 knockdown were enriched in processes related to immune responses, aspects of extracellular matrix organization, cytokine signalling and motility (Fig. [5b](#Fig5){ref-type="fig"}). Overall, these findings suggest that decreased expression of PDCD4, as seen in many tumor cells, similarly affects a plethora of cellular processes. Furthermore, the data underline the notion that PDCD4 plays a crucial role in cell cycle regulation, particularly at the G1/S-phase transition and the subsequent S-phase.Figure 5Gene set enrichment analysis of genes affected by PDCD4 knockdown. (**a)** Examples of GSEA charts revealing the role of PDCD4 in cell cycle regulation. (**b)** GO-term (biological process) enrichment analysis of genes up- and downregulated by PDCD4 silencing. Significantly differentially expressed gene sets were identified by GSEA and reduced to most significant terms by REVIGO. Positive and negative enrichment scores, respectively, indicate up- and down-regulation in PDCD4 knockdown cells. Numbers indicate counts of genes in each gene set. The asterisks indicate FDR q-values (\*\<0.05; \*\*\<0.01; \*\*\*\<0.001; \*\*\*\*\<0.0001; \*\*\*\*\* \< 0.00001). To assess the global effects of PDCD4 on mRNA translation we performed ribosome profiling^[@CR43],[@CR44]^ using RPE cells transfected with PDCD4-specific siRNA-2 or control siRNA. In this approach, polysomes of control and PDCD4 knockdown RPE cells are nuclease digested, ribosome-protected mRNA fragments (RPF) isolated and used for deep sequencing to generate "snapshots" of global translation. By combining these data with RNA-Seq data it is possible to determine the effect of PDCD4 silencing on the translation efficiency of individual mRNAs (Fig. [6a](#Fig6){ref-type="fig"}). By comparing the RNA-Seq and ribosome profiling data from control and PDCD4 knockdown cells we identified 496 transcripts that were significantly induced and 750 genes that were downregulated, while 1688 genes remained unchanged by PDCD4 silencing ("RNA-Seq" in Fig. [6b](#Fig6){ref-type="fig"}). The RPF analysis showed that 592 and 728 genes had increased or decreased RPF levels, while 4388 genes did not exhibit significant translational changes upon PDCD4 silencing ("RFP" in Fig. [6b](#Fig6){ref-type="fig"}). For the majority of transcripts the changes in translation levels correlate with altered mRNA abundance following PDCD4 silencing. mRNAs that are translationally regulated by PDCD4 knockdown ("translationally changed" in Fig. [6b](#Fig6){ref-type="fig"}) were defined as transcripts that exhibit altered RPF levels, while mRNA levels remained unaffected in response toPDCD4 silencing. This resulted in the identification of 34 mRNAs (Supplementary Table [S3](#MOESM3){ref-type="media"}). Since PDCD4 has been implicated in the translational suppression of mRNAs with structured 5′-UTRs^[@CR3],[@CR4]^, we used mfold^[@CR45]^ to examine the potential of the 5′-UTRs of the selected mRNAs to form secondary structures. We plotted the ΔG-values predicted for the folding of the 5'-UTRs separately for those mRNAs that showed increased or decreased translation after PDCD4 knockdown (Fig. [6c](#Fig6){ref-type="fig"} and Supplementary Table [S3](#MOESM3){ref-type="media"}). This indicated that mRNAs whose translation was increased by PDCD4 silencing had more negative predicted ΔG-values, reflecting a higher secondary structure potential of their 5′-UTRs, than those mRNAs whose translation was decreased by the PDCD4 knockdown. We further identified 85 mRNAs that remained stable at the level of translation but were altered at the mRNA levels upon PDCD4 silencing and, hence, were also differentially translated between control and PDCD4 knockdown cells (Supplementary Table [S3](#MOESM3){ref-type="media"}). We determined the predicted ΔG-values for folding of the 5′-UTRs of the selected mRNAs whose translation was increased or decreased by PDCD4 silencing (Fig. [6d](#Fig6){ref-type="fig"} and Supplementary Table [S3](#MOESM3){ref-type="media"}). Similar to our previous analysis this showed that mRNAs whose translation was more effective after PDCD4 knockdown have lower ΔG-values for folding of their 5′-UTRs than mRNAs whose translation was decreased. Combining these different sets of mRNAs, we identified 62 translationally up-regulated and 57 down-regulated mRNAs upon PDCD4 silencing ("translationally changed" in Fig. [6b](#Fig6){ref-type="fig"}), of which mRNAs with increased translation after knockdown of PDCD4 possess more highly structured 5′-UTRs than mRNAs whose translation is decreased when PDCD4 is silenced. (Fig. [6e](#Fig6){ref-type="fig"}). Although we cannot exclude indirect effects of PDCD4 silencing on the translation of specific mRNAs, our analyses are consistent with the concept that PDCD4 suppresses the translation of mRNAs that contain structured 5′-UTRs. Besides the identification of mRNAs that are potential targets of translational suppression by PDCD4, our work has also revealed mRNAs that show decreased translation upon PDCD4 knockdown. The identification of groups of mRNAs whose translation is either positively or negatively regulated by PDCD4 sets the stage for future work to understand the role of PDCD4 in translation regulation in more detail.Figure 6PDCD4 silencing affects the translational landscape of RPE cells. (**a)** Scatter plot showing the log~2~-fold changes on the mRNA (y-axis) and ribosome footprint (x-axis) levels in PDCD4 knockout RPE cells relative to control knockout cells. Genes altered only in one parameter by PDCD4 silencing are represented as colored dots (translationally changed genes in B). (**b)** Changes in RNA-Seq, RPF, and translationally changed genes between control and PDCD4 knockout cells. For RNA-seq and RPF data, "up" and "down" includes genes with a log~2~-fold change \> 0 or \<0, respectively, and an adjusted p-value \< 0.05. For translationally changed genes, "up" and "down" includes genes with an adjusted p-value \< 0.05 for mRNA or RPF and a corresponding adjusted p-value for unchanged hypothesis testing \< 0.1. **(c--e)** Box-plots showing the distribution of ΔG values for RNA secondary structures in the 5′-UTRs of mRNAs translationally suppressed or activated by PDCD4 silencing. Discussion {#Sec7} ========== PDCD4 is a multifunctional protein initially described as a transformation suppressor in a murine keratinocyte transformation model^[@CR11]^. Subsequent work has strongly suggested that PDCD4 acts as a tumor suppressor in a broad spectrum of human tumor types^[@CR1],[@CR2]^ and has shown that decreased expression of human PDCD4 contributes to tumor development in various ways, for example by enhancing the motility and invasiveness of the tumor cells^[@CR18]--[@CR20],[@CR23]--[@CR25]^. Most of the studies addressing the function of human PDCD4 have employed various tumor cells, raising the question whether these studies fully reflect the function of human PDCD4 in normal cells. Therefore, we have used a telomerase-immortalized human epithelial cell line to highlight novel aspects of PDCD4′s function. Our work shows for the first time that PDCD4 is required for the G1/S-phase transition. We observed that siRNA-mediated down-regulation of PDCD4 expression strongly impaired the entry of the cells into S-phase, decreased DNA synthesis activity and reduced cell proliferation rate. Our results suggest that the role of PDCD4 as a G1/S cell cycle regulator is linked to the activity of p53. More specifically, our work supports the notion that PDCD4 is required to counteract the activity of p53, preventing the activation of the G1/S-checkpoint in unstressed cells and permitting them to enter into S-phase. In this scenario, knockdown of PDCD4 leads to increased p53-dependent expression of p21^WAF1/CIP1^ and concomitant activation of the G1/S-checkpoint. Using HeLa cells, we have previously observed increased p21^WAF1/CIP1^ expression after knockdown of PDCD4^[@CR26]^. However, unlike the work reported here, knockdown of PDCD4 in HeLa cells only showed aberrant cell behaviour in the presence of DNA damage but did not result in overt cell cycle defects. This might be due to the defective nature of the G1/S-checkpoint in these cells caused by the sequestration of the RB protein by the human papilloma virus E7 protein expressed in HeLa cells^[@CR46]^. HEK293T cells also have a defective G1/S checkpoint (resulting from the expression of the adenovirus E1A and E1B proteins). We found that the requirement for PDCD4 expression for S-phase transition is indeed absent in these cells. Overall, our work identifies a novel role of PDCD4 as a cell cycle regulator that balances p53 activity in unstressed cells, presumably to prevent G1/S-checkpoint activation. Interestingly, we also showed before^[@CR28]^ and confirmed here that induction of DNA damage leads to down-regulation of PDCD4 expression, which suggests that this function of PDCD4 is abolished under conditions of genotoxic stress. The identification of a pro-proliferative role for PDCD4 in the cell cycle is somewhat unexpected in the light of its function as a tumor suppressor. At first glance, low expression of PDCD4 in tumor cells would be expected to impede the cell cycle, however, many tumor cells have a defective G1/S checkpoint as a result of p53 or other mutations, neutralizing the inhibitory effects of low PDCD4 expression on the G1/S-transition. Transcription profiling has revealed a large number of genes that are up- or down-regulated upon PDCD4 knockdown, providing an unbiased view of the cellular processes that are affected by PDCD4. Consistent with previous studies showing increased motility and invasiveness^[@CR18]--[@CR20],[@CR23]--[@CR25]^, GO-term analysis for the biological function of the genes up-regulated by PDCD4 knockdown identifies functions related to extracellular matrix organization and cell adhesion amongst others. GO-term analysis of genes down-regulated by PDCD4 knockdown identifies a plethora of cell cycle- and DNA-related functions that are inhibited when PDCD4 expression is low, such as DNA-replication, DNA-recombination, DNA-repair, telomere organization, chromosome segregation and chromatin remodelling, amongst others. This suggests that decreased PDCD4 expression contributes to tumor development and progression by compromising genomic integrity. Finally, our ribosome profiling analysis shows that the translation of the majority of transcripts was not affected by silencing of PDCD4 because changes in the abundance of ribosome footprints correlated with changes in the expression levels of these mRNAs. By focussing on transcripts that were affected in only one parameter (i.e. mRNA expression level or the frequency of RPF reads) in response to PDCD4 knockdown, we have identified several mRNAs whose translation was moderately increased following PDCD4 knockdown, suggesting that they might be translational targets of PDCD4. These RNAs exhibit an increased potential to form stable secondary structures in their 5′-UTRs compared to mRNAs showing decreased translation after PDCD4 silencing, consistent with the notion that Pdcd4 preferentially inhibits translation of RNAs with structured 5′-UTRs^[@CR3],[@CR4]^. Similarly, PDCD4 knockdown stimulates the translation of RNAs that lack secondary structure in their 5′-UTR or that have short 5′-UTRs. Whether the translation of these RNAs is suppressed by binding of PDCD4 to their coding regions, as already reported for certain mRNAs^[@CR29],[@CR30],[@CR47]^, or whether PDCD4 affects their translation indirectly remains to be addressed by future studies. In addition to the identification of potential target mRNAs for translational repression by PDCD4 we have also discovered mRNAs whose translation is positively affected by PDCD4. Whether PDCD4 mediates these effects directly or indirectly and whether this reflects a novel aspect of the function of PDCD4 in translation, remains to be investigated in future work. Overall, our study is the first analysis of genome-wide changes of mRNA abundance and translation induced by PDCD4 silencing in an immortalized human epithelial cell line. This sets the stage for more detailed studies on the role of PDCD4 in the future. Materials and Methods {#Sec8} ===================== Cells and siRNA transfections {#Sec9} ----------------------------- hTERT-RPE-1 is a line of telomerase-immortalized human retina pigment epithelial cells^[@CR48]^. The cells were grown in DMEM/Ham's F12 medium supplemented with 10% fetal calf serum. PDCD4 expression was silenced with siRNA duplexes targeting the sequences CACCAAUCAUACAGGAAUA (PDCD4 siRNA-1) or GCUUCUUUCUGACCUUUGU (PDCD4 siRNA-2). SiRNA targeting Renilla luciferase (AAACAUGCAGAAAAUGCUG) was used as negative control. siRNAs (100 nM) were reversely transfected using Lipofectamine^®^ RNAiMax (ThermoScientific), according to manufacturer's protocols. Cells were harvested 48 to 72 h after transfection. Cell cycle analysis {#Sec10} ------------------- Cells were trypsinized, fixed with 70% ice-cold ethanol in PBS for 1 h or longer at −20 °C, washed with PBS (+0.5% BSA) and stained with propidium iodide (50 μg/mL PI and 25 μg/mL RNase A in PBS) for 1 h at room temperature. In some experiments, cells were synchronized by incubation for 24 h in growth medium containing 0.5 mM mimosine. To release the cells into the cell cycle they were washed twice with grown medium lacking mimosine. Flow cytometry analysis was performed using a Beckman-Coulter Cytomics FC500 flow cytometer. 10 000 to 15 000 cells were counted per condition in every experiment. ### Antibodies {#Sec11} Western blotting of PDCD4 was performed using a rabbit anti PDCD4 antiserum raised against the N-terminus of human Pdcd4^[@CR26]^. Antibodies against p21^WAF1/CIP1^ (05--345, Millipore), γ-H2AX (GTX61796, Genetex) and β-actin (AC15, Sigma-Aldrich) were obtained from commercial sources. ### Quantitative real-time PCR {#Sec12} Total cellular RNA was isolated with TRIzol^TM^ Reagent (Invitrogen), as recommended by the manufacturer. Total RNA (2 μg) was reverse transcribed with the First Strand cDNA Synthesis Kit (K1612, ThermoScientific) using OligoT primers in 20 μL according to the manufacturer's instructions. Real-time RT-PCR reactions were carried out in 96-well plates using Power SYBR Green PCR Master Mix (Applied Biosystems). Reactions were performed using a StepOnePlus RT-PCR instrument (Applied Biosystems) and the following parameters: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Each experiment included a no-template control. PCR reaction specificity was confirmed by melting curve analysis of the products. Primer sequences are given in Supplementary Table [S4](#MOESM1){ref-type="media"}. Relative gene expression was calculated by the ΔΔC~T~ method:^[@CR49]^ First, ΔC~T~ values were calculated by subtracting the C~T~-values obtained for individual mRNAs from those obtained for β-actin mRNA. Then, ΔΔC~T~ values were calculated by subtracting the ΔC~T~ values of Pdcd4 siRNA-treated cells from those of control siRNA-treated cells. All experiments were conducted with at least three biological replicates. ^3^H-thymidine labeling {#Sec13} ----------------------- Cells were incubated with growth medium supplemented with 10 μCi/ml 3H-thymidine for 1 h. The cells were then washed with PBS, lysed in PBS containing 1% SDS and heated to 95 °C to reduce the viscosity. Aliquots were then spotted on Whatman filter paper and washed 2 times for 15 min with 10% trichloracetic acid (TCA) and once with ethanol. The filter paper was dried and the radioactivity was determined in a scintillation counter. To correct for differences in the cell number between Pdcd4-specific and control knockdown samples aliquots of the lysed cells were spotted on nitrocellulose membrane and hybridized to a ^32^P-labeled probe of total human DNA. Alternatively, aliquots of the lysed cells were analyzed by SDS-PAGE and western blotting for expression of β-actin to determine the relative number of cells. RNA-seq {#Sec14} ------- For RNA-seq of poly(A)-selected RNA, RPE cells were incubated for 24 h after transfection with siRNA (Pdcd4 siRNA-2 or control siRNA), and cells were directly lysed in TRIzol^TM^ reagent (Invitrogen). Total RNA was extracted with 1-bromo-3-chloropropane, precipitated with EtOH, resuspended in milliQ water and treated with TURBO DNase (Ambion) for 30 min at 37 °C, 1400 rpm. RNA was extracted again with acidic phenol to remove DNase. The quality of the RNA was examined with an Agilent Bioanalyzer. Sequencing libraries of poly(A)-enriched RNA were finally generated with the TruSeq Stranded mRNA LT Kit (Illumina). Ribosome profiling {#Sec15} ------------------ Ribosome profiling was carried out as previously described^[@CR44]^. RPE cells were incubated for 24 h after transfection with siRNA (PDCD4 siRNA-2 or control siRNA). 2 h before harvesting the culture medium was replaced with fresh medium. To stabilize elongating ribosomes, cells were treated with 100 µg/mL cycloheximide (CHX) for 5 min at 37 °C, following a washing step with ice cold PBS (containing 100 µg/mL CHX). Cells were then lysed in lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM MgCl~2~, 100 mM NaCl, 1% Triton, 1 mM DTT, 100 µg/mL CHX) per sample. Each sample consisted of cells from 8 tissue culture dishes (10 cm diameter), and cell debris was pelleted at 4 °C, 10 000 x g for 3 min. 10 OD~260~ units of cell extract were then supplemented with 900 U RNase I (Ambion) and 0.5% deoxycholate and treated for 20 min at 22 °C and 800 rpm in a thermomixer. The reaction was stopped by addition of 240 U SUPERase In (Ambion)( + 0.5% deoxycholate) and extracts were fractionated by centrifugation at 4 °C, 35 000 rpm for 3 h in a SW-41 Ti swinging-bucket rotor (Beckman Coulter) on 10--50% sucrose density gradients (20 mM Tris-HCl (pH 7.5), 10 mM MgCl~2~, 100 mM NH~4~Cl, 2 mM DTT, 100 µg/mL CHX). Gradients were fractionated at 0.75 mL/min with continuous monitoring of the OD~254~ using a Biocomp Instruments Gradient Station (Teledyne Isco). Monosome fractions were collected and, following addition of 1% SDS, flash-frozen and stored at −80 °C. RNA was isolated from gradient fractions by the hot acid phenol method (1-bromo-3-chloropropane used instead of chloroform), and ribosome footprints were purified from monosome RNA by size selection of 28--30 nt fragments (excluding a major band around 31 nt) on 15% polyacrylamide, 8 M urea, 1xTBE gels. Sequencing libraries from ribosome-protected footprints were generated by 3′-end dephosphorylation, followed by 3′-adapter ligation, reverse transcription, and circularization as described in^[@CR44]^. Sequencing data analysis {#Sec16} ------------------------ The analysis of the ribosome profiling and RNA-seq datasets was essentially performed as described in^[@CR44]^. Briefly, libraries for ribosome profiling and RNA-Seq were sequenced on an Illumina NextSeq sequencer. Ribosome-profiling reads were processed by clipping adapter sequences and trimming of the 4 randomized nucleotides in the linker with the FASTX-Toolkit version 0.0.13 (<http://hannonlab.cshl.edu/fastx_toolkit>). After processing, residual rRNA sequences were remove from ribosome-profiling datasets using bowtie version 1.0.0.^[@CR50]^. Ribosome-profiling and raw RNA-Seq reads were mapped to hg38 transcripts (UCSC canonical transcripts extended 18 bp into the UTRs). Count tables of mapped reads were generated using custom scripts and differential expression determined with DESeq. 2^[@CR51]^. Differential expression was scored using an adjusted p-value of 0.05 for the hypothesis of a changed gene (res) and unchanged expression was additionally scored using an adjusted p-value of 0.1 for the hypothesis of an unchanged gene (resLA). Transcripts translationally changed were defined with an adjusted p-value for mRNA or ribosome profiling data \>0.05 (res) with the corresponding adjusted p-value in the other category (resLA) \< 0.1. Gene set enrichment analysis (GSEA) {#Sec17} ----------------------------------- GSEA was carried out using the GSEA preranked tool (<http://software.broadinstitute.org/gsea/index.jsp>) with 1,000 gene set permutations. The genes in the expression dataset were ranked by their log~2~ fold change. Redundant terms were removed with REVIGO^[@CR52]^. Calculation of ΔG values for 5′-UTR secondary structure formation {#Sec18} ----------------------------------------------------------------- The 5′UTR sequences of the relevant mRNAs were retrieved from the NCBI nucleotide sequence data base and truncated immediately after the start codon. If several mRNA sequences were available sequence variant 1 was chosen. The sequences were then submitted to the RNAfold web server (<http://rna.tbi.univie.ac.at>) to predict the mimimum free energy of the optimal secondary structure (Supplementary Table [S5](#MOESM1){ref-type="media"}). Data access {#Sec19} ----------- The RNA-seq and ribosome profiling data from hTERT-RPE-1 cells (PDCD4 siRNA2 and control siRNA) have been submitted to the NCBI Gene Expression Omnibus (GEO; [www.ncbi.nlm.nih.gov/geo/](http://www.ncbi.nlm.nih.gov/geo/)) under accession number GSE138533 (<https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE138533>). Supplementary information ========================= {#Sec20} Supplementary data. Supplementary tabel S2. Supplementary tabel S3. **Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary information ========================= is available for this paper at 10.1038/s41598-020-59678-w. This work was supported by the Deutsche Forschungsgemeinschaft to K.-H.K. and S.A.L. and by the Open Access Publication Fund of the University of Münster. A.H., B.S.N., S.A.L. and K.-H.K. designed and performed experiments. A.H., B.S.N. S.A.L. and K.-H.K. wrote the paper. All authors approved the final version of the manuscript. The authors declare no competing interests.

Introduction {#s1} ============ The tree genus *Gymnocladus* (Leguminosae) comprises five species [@pone.0087287-Roskov1] distributed in the Eastern North America and Eastern Asia [@pone.0087287-Lee1], and considered to have originated in the Eastern Asia during the Eocene and migrated across the Bering land bridge to North America [@pone.0087287-Sanjappa1]. *G. diocus* (L.) K. Koch is restricted to North America, while *G. angustifolius* (Gagnep.) J.E. Vidal is confined to Vietnam. The remaining three species, *G. chinensis* Baill., *G. assamicus* Kanjilal ex P.C. Kanjilal and *G. burmanicus* Parkinson are distributed in the region bordering India, China and Myanmar (Burma). The geographical ranges of *G. assamicus* and *G. chinensis* within India are restricted to the Northeastern states [@pone.0087287-Schnabel1]. *G. assamicus* is a critically endangered tree species with declining populations [@pone.0087287-Choudhury1]. Extensive field surveys and environmental niche modelling (ENM) studies revealed existence of only a few remnant populations of the species confined to moist areas on hilly slopes and stream banks in the West Kameng and Tawang districts of Arunachal Pradesh [@pone.0087287-Menon1]. The mating system of the genus *Gymnocladus* has been broadly described as polygamous, unisexual, bisexual or dioecious [@pone.0087287-Dezhao1]--[@pone.0087287-Watson1]. Although *G. chinensis* is considered as a polygamous species [@pone.0087287-Dezhao1], no detailed studies on floral biology or the breeding system of *Gymnocladus* species exist. We studied floral biology of *G. assamicus* and discovered an androdioecious mating system where male and hermaphrodite flowers are produced on separate trees. Androdioecy is a rare mating system [@pone.0087287-Charlesworth1]--[@pone.0087287-Pannell2], and since Darwin\'s [@pone.0087287-Yampolsky1] original report on the androdioecy, no reliable evidence for the occurrence of androdioecy was reported until 1922 [@pone.0087287-Darwin1]. To date, about 50 plants and 36 animal species have been described as androdioecious [@pone.0087287-Weeks1]. In contrast, dioecy (occurrence of male and female plants) and gynodioecy (occurrence of female and hermaphrodite plants) are known to occur in approximately 6% [@pone.0087287-Heilbuth1] and 10% [@pone.0087287-Delannay1] of angiosperms respectively. Since the first confirmed report of androdioecy in *Datisca glomerata* (Datiscaceae) [@pone.0087287-Liston1], several reports of androdioecy have been published [@pone.0087287-Pannell2], [@pone.0087287-Fritsch1]--[@pone.0087287-Weeks2]. During the last two decades, several plant species have been described as androdioecious on the basis of morphology. However, detailed studies have revealed that many such species are functionally dioecious or cryptic dioecy with sterile pollen in morphologically hermaphroditic flowers [@pone.0087287-Mayer1]. Only few plant species including *Datisca glomerata* [@pone.0087287-Liston1], *Mercurialis annua* [@pone.0087287-Pannell3], *Schizopepon bryoniaefolius* [@pone.0087287-Akimoto1] and *Morinda umbellata* subsp. *boninensis* [@pone.0087287-Nishide1] have been confirmed to be functionally androdioecious, where both male and hermaphrodite flowers produce fertile pollen. In the present study, we demonstrate the functional androdioecy in *Gymnocladus assamicus*, a less known and endangered tree species in the Eastern Himalayan region of Northeast India. We studied the reproductive biology of two plant morphs of *G. assamicus* through (1) quantifying the proportions of male and hermaphrodite plants in populations, (2) analysing their reproductive and vegetative phenology, (3) examining floral features, (4) assessing male and hermaphrodite functions of each sexual phenotype, and (5) determining the breeding system through controlled pollination experiments. Materials and Methods {#s2} ===================== We declare that no specific permissions were required for field studies as study locations were not privately owned or not in protected areas. This study was sponsored by Department of Science and Technology, Government of India (Sanction no. SR/SO/PS-16/2002 to MLK) and no further permission was required. Study species {#s2a} ------------- Since *G. assamicus* is a critically endangered tree species with a declining population size, only a limited number of flowering individuals were available for the present study. The individuals studied were located in high altitudinal (1500--2200 m asl) forests in the village of Dirang (27°15′ to 27°10′N; 92°12′ to 19°14′E) in the West Kameng district of Arunachal Pradesh, India. The climate of the area is subtropical to wet temperate with an annual rainfall of 1752 mm of which 75 percent is normally received during the rainy season between June and October. Average monthly temperature ranges from 0°C during the winter (November--February) to 34°C during the summer (May--June). Out of the total of 28 mature trees included in the present study ([Table 1](#pone-0087287-t001){ref-type="table"}), nine trees were recorded to bear hermaphrodite flowers and produced fruits regularly. The trees were either solitary or occurred as small groups within fragmented forests in an area of about 10 km^2^ ([Figure 1](#pone-0087287-g001){ref-type="fig"}). The number of trees in each site ranged from one to six and majority of sites had no seedlings or saplings suggesting a poor regeneration potential of the species. {#pone-0087287-g001} 10.1371/journal.pone.0087287.t001 ###### Location of *G. assamicus* in and around Dirang. {#pone-0087287-t001-1} Site Locality No. of individuals Male/Female ratio ------- ------------------------------- -------------------- ------------------- ----- MS Moishing 1 1 1∶1 DB Dirang Basti \- 1 0∶1 CM Changfu Moon 4 \- 4∶0 YV Yewang Village 1 1 1∶1 DM Dambla Basti 2 5 2∶5 JN Jyotinagar 1 \- 1∶0 RV Runkung Village \- 1 0∶1 LV-I Lishpa Village I (Rama Camp) 5? -- LV-II Lishpa Village II (Rama Camp) 5? -- (? = unknown). Phenology and floral biology {#s2b} ---------------------------- Eighteen trees were regularly monitored for their phenology during the period between 2004--2007 and included in the detailed study of floral biology, while nine individuals from two sites namely Moishing (MS) and Dambla Basti (DM) were used for mating system studies through controlled pollination experiments. Ten individuals in Lishpa Village (LV) I and II did not flower during the study period. Phenological events including bud break, leaf flushing, leaf shedding, flowering, fruiting and fruit set were recorded during the study period. Daily observations were made during the peak flowering period to record various stages of floral development. Subsequent events such as fruit set and fruit maturation were recorded on weekly basis. The number of flowers per inflorescence was counted from 20 randomly selected inflorescences per tree and the morphological details of floral parts were observed using a hand lens. Flower longevity was determined by observing 20 marked flowers per tree from the time of flower opening until wilting. Growth and development of various floral parts were recorded twice daily, at around 0600 hrs and 1800 hrs. The volume of nectar produced (µl) in both floral morphs at various developmental stages was measured using graduated micropipettes. The nectar secretion patterns were studied in 20 randomly selected flowers from 20 bagged inflorescences of each floral morph. Measurements were carried out at 0600 hrs and 1800 hrs daily during the entire lifespan of the flower. The floral morphometric measurements were made using a digital calliper (Mitutoyo Japan). In the present study, the floral ontogeny was divided into six stages as given in [Table 2](#pone-0087287-t002){ref-type="table"}. The pre-anthesis bud stage with purple perianth and closed tepals ready to open was designated as stage I (time: 0 h). The anthesis initiation and opening of tepals (time: 24 h) was considered as stage 2, and almost open flowers with ca. 15--16 mm wide opening of tepals (time: 48 h) were considered as stage 3. At the stage 4, flowers were fully open, and emerged anther lobes were at the level of the stigma. At this stage pollen grains were highly fertile (58--60 percent), and in hermaphrodite flowers, the stigma was highly receptive (time: 72 h). The stage at which flowers begin to wilt, tepals start to curl and reproductive parts enter the drying phase (time: 96 h) was considered as stage 5. The onset of floral senescence, when pollen fertility ceases and the perianth tubes falls was considered as stage 6. 10.1371/journal.pone.0087287.t002 ###### Stages of floral development of *G. assamicus.* {#pone-0087287-t002-2} Stages Dimension of flower Stigma receptivity Pollen fertility (%) Nectar volume (µl) (M) -------- --------------------- -------------------- ---------------------- ------------------------ -------- -------- -------- S1 8.15 2.45 X X X X X S2 9.44 2.74 X X X X X S3 15.75 3.12 Modest 8--10 10--12 2.5--3 2--2.5 S4 16.52 3.74 Maximum 58--60 58--62 16--18 14--15 S5 16.08 3.86 Weak 6--8 8--10 10--12 9--10 S6 15.98 3.33 X X X X X Flower visitors {#s2c} --------------- The visitors to flowers of trees at MS and DM sites were recorded throughout the day during the peak flowering period. The insect visitors were trapped with sweep nets and classified into general groups such as bees, beetles, moths, and butterflies and further identified using reference manuals and assistance from specialists at the Zoological Survey of India (ZSI) in Itanagar, Arunachal Pradesh. Honeybees were identified to the level of species using taxonomic keys, and bird species were identified using photographs, field observations and consulting ornithological experts of the region (Dr. Anwaruddin Choudhury, Rhino Foundation, Guwahati, India). Pollen viability and stigma receptivity test {#s2d} -------------------------------------------- The pollen viability was assessed as a percentage of pollen grains germinated using the sitting drop culture method [@pone.0087287-Shivanna1]. Pollen grains were collected from 10 randomly harvested flowers from different individuals of both morphs at different developmental stages, germinated in Brewbaker and Kwack\'s medium [@pone.0087287-Brewbaker1] and counted under a compound microscope. Five replicates each from hermaphrodite and male trees were studied. Pollen was considered viable when clear pollen tube growth was visible under the microscope. Stigma receptivity was tested using H~2~O~2~ following the method of Dafni [@pone.0087287-Dafni1]. Emission of bubbles from the stigma surface at high rate was considered strong receptivity, while slow emission of bubbles was considered as weak stigma receptivity. Pollen morphology {#s2e} ----------------- The pollen morphology was studied using a scanning electron microscope (SEM). Dried pollen samples were mounted on metal stubs, gold coated and observed under a scanning electron microscope (LEO 1430VP, Karlzeiz, Germany). Mating system analyses {#s2f} ---------------------- Mating system experiments were conducted on nine trees located at MS and DM sites. The following controlled pollination experiments were carried out and percent fruit set was recorded. a. Open pollination -- fruit set under natural condition in 302 non-manipulated flowers. b. Spontaneous self pollination -- fruit set in 396 unopened flowers bagged for excluding flower visitors. c. Controlled pollination: freshly opened flowers were emasculated using fine forceps and bagged for artificial pollination. The flowers at other developmental stages in each chosen inflorescence were removed before bagging. The controlled pollinations were carried out *in situ* during the stigma receptivity period (Stage 4; [Table 2](#pone-0087287-t002){ref-type="table"}). To determine the autogamous (pollen from the same flower) fruit set, hand pollination was carried out in 143 flowers while that of geitonogamous pollination (pollen from another flower of the same tree) was carried out in 160 flowers. The xenogamous pollination (pollen from a different tree) was carried out between male versus hermaphrodites in 134 flowers and hermaphrodites versus hermaphrodites in 147 flowers. d. Agamospermic and parthenocarpic fruit set was determined using 93 bagged and emasculated hermaphrodite flowers without pollination. Equilibrium male frequency in the population {#s2g} -------------------------------------------- In order to assess the congruence between observed frequency of males and the theoretically expected frequency of males in a population under various levels of selfing (s = 0.1, 0.2, and 0.5) and inbreeding depression [@pone.0087287-Charlesworth2], we plotted the range of predicted male frequency values against inbreeding depression. We used K values slightly lower (K = 2.5) and higher (K = 3.5) than the observed K value (K = 2.9) based on the overall differences in flower production between male and hermaphrodite trees. Results {#s3} ======= Phenology {#s3a} --------- Individual *G. assamicus* trees remained leafless for over two months during the winter (January--February). The mature pods persisted on trees until the next flowering season. New leaves appeared in early March followed by flowering in April, which lasted for 15--20 days. Phenological patterns of vegetative phases were similar in both plant morphs. Flowers of male trees bloomed between the last week of March and second week of April, whereas blooming of trees with hermaphrodite flowers peaked in April ([Figure 2](#pone-0087287-g002){ref-type="fig"}). {#pone-0087287-g002} Floral morphology {#s3b} ----------------- Both male and hermaphrodite flowers were borne on terminal racemose inflorescences with fine pubescence, and flowers were tubular in shape, purple in color, odorless and lasted for about 96 hours. Male inflorescences were 13--16 cm long and 5--6 cm wide with nearly whorled lateral branches, which emerged from about 15--20 nodes on the inflorescence axis ([Figure 3A](#pone-0087287-g003){ref-type="fig"}; [Table 3](#pone-0087287-t003){ref-type="table"}). Hermaphrodite inflorescences were shorter and ranged from 4--6 cm in length with fewer nodes, and fewer numbers of flowers than male inflorescences ([Figure 3B](#pone-0087287-g003){ref-type="fig"}; [Table 3](#pone-0087287-t003){ref-type="table"}). The developmental stages of both male and hermaphrodite flowers were similar and produced similar amounts of nectar ([Table 2](#pone-0087287-t002){ref-type="table"}). The pollen grains of both morphs were viable and fertile. {#pone-0087287-g003} 10.1371/journal.pone.0087287.t003 ###### Floral characteristics of *G. assamicus.* {#pone-0087287-t003-3} Features Inflorescence Length (cm) Inflorescence Breadth (cm) No. of flower per inflorescence Average flower production Biomass at fully opened stage (gm) Length of corolla tube (mm) Width of corolla tube (mm) Length of pistil (mm) Length of anther lobe (mm) --------------- --------------------------- ---------------------------- --------------------------------- --------------------------- ------------------------------------ ----------------------------- ---------------------------- ----------------------- ---------------------------- --------------- Male 12 (±0.81) 2.92 (±0.14) 72.50 (±4.01) 238227.92 0.049 (±0.002) 5.50 (±0.027) 3.43 (±0.038) \-\-\-\-\-\-\-- 8.99 (±0.065) 8.80 (±0.015) Hermaphrodite 3.38 (±0.11) 2.26 (±0.06) 23.40 (±1.41) 80521.98 0.088 (±0.004) 9.20 (±0.017) 3.74 (±0.014) 10.84 (±0.054) 9.67 (±0.050) 9.07 (±0.025) ±S.E. n = 20. Male flowers {#s3c} ------------ Among 28 mature *G. assamicus* trees included in the present study ([Table 1](#pone-0087287-t001){ref-type="table"}), the overall percentage of male trees was 32.14. The male flowers were pedicellate and cylindrical, and male individuals produced as much as 80500 flowers per tree, almost three times than hermaphrodite trees (24000 flowers per hermaphrodite tree; [Table 3](#pone-0087287-t003){ref-type="table"}). The perianth tube of five united tepals was narrow at the base and gradually widened at the tip. Each flower had ten stamens with five longer and five shorter alternatively arranged filaments. Vestigial carpels are visible at the base of the perianth tube ([Figure 3C](#pone-0087287-g003){ref-type="fig"}). Male flowers open between 0900--1100 hrs and anthers dehisce after 2000--2400 hrs. Pollen grains collected from freshly opened flowers showed 8--10 percent germination and reached to 58--60 percent germination at the stage 4, and then decreased with the age of the flower reaching to no viability at stage 6 ([Table 2](#pone-0087287-t002){ref-type="table"}). Hermaphrodite flowers {#s3d} --------------------- The hermaphrodite flowers were long-pedicellated and larger than male flowers at anthesis ([Figure 3D](#pone-0087287-g003){ref-type="fig"}; [Table 3](#pone-0087287-t003){ref-type="table"}). The anthers were didynamous at full maturity with the longer anther lobes reaching above the receptive surface of the stigma, while the shorter ones remained at the level of the stigma ([Figure 4, A--F](#pone-0087287-g004){ref-type="fig"}). The pollen viability percentages were similar to those of male flowers at corresponding developmental stages ([Table 2](#pone-0087287-t002){ref-type="table"}). Flowers opened between 1400--1600 hrs and maximum pollen germination (58--62 percent) was observed in one-day old flowers, and then gradually decreased over time ([Figure 5](#pone-0087287-g005){ref-type="fig"}). The pistil in the hermaphrodite flowers was well demarcated into stigma, style and ovary. The flower length at the anthesis was about 10 mm, and the style was straight, green in color, moderately thick and compressed with an oblique stigma. The stigma surface was slightly slanted, papillate with wet sticky exudates ([Figure 6](#pone-0087287-g006){ref-type="fig"}). The unilocular superior ovary contained 7.7±0.23 (n = 20) ovules. Detailed floral morphometric data are given in [Table 3](#pone-0087287-t003){ref-type="table"}. {#pone-0087287-g004} {#pone-0087287-g005} {#pone-0087287-g006} Nectar production {#s3e} ----------------- Flowers started production of nectar when they attained the stage 2 ([Table 2](#pone-0087287-t002){ref-type="table"}). The maximum nectar production was at stage 4 ([Table 2](#pone-0087287-t002){ref-type="table"}) and then gradually decreased and dried up at stage 6. Although the nectar secretion pattern was uniform among different flowers, nectar volume varied significantly (p\<0.05) between the two flower morphs ([Table 2](#pone-0087287-t002){ref-type="table"}). Pollen morphology {#s3f} ----------------- Scanning electron microscopic (SEM) observation of the pollen grains of both morphs revealed that pollen grains were of polycolpate (multiple colpi with elongated apertures) and psilate (lacking ornamentation). SEM also revealed that pollen grains of male and hermaphrodite flowers were morphologically similar in size and shape ([Figure 7: A, B](#pone-0087287-g007){ref-type="fig"}). {#pone-0087287-g007} Mating system {#s3g} ------------- The manual self pollination of hermaphrodites within flowers (autogamous) or between flowers of the same tree (geitonogamous) increased the fruit set considerably as compared to open pollination fruit set ([Table 4](#pone-0087287-t004){ref-type="table"}). The xenogamous pollination of hermaphrodite flowers with pollen from males yielded slightly lower fruit set (not statistically significant) than pollinated with pollen from hermaphrodite flowers. Four out of the nine study sites had only hermaphrodite trees, which produced fruits and seeds consistently under natural conditions suggesting that hermaphrodite flowers set fruits in the absence of male trees in the neighborhood. 10.1371/journal.pone.0087287.t004 ###### Results of controlled pollination experiments. {#pone-0087287-t004-4} Treatment Number of flowers pollinated Number of flowers produced fruit Average Fruit set percentage (± SE) [\*](#nt103){ref-type="table-fn"}Probability level of fruit set ------------------------------ ------------------------------ ---------------------------------- ------------------------------------- ----------------------------------------------------------------- Open pollination 302 146 48.31 (±2.75) F = 10.325, P\<0.001 Spontaneous self pollination 396 157 36.64 (±2.48) Hand pollination 143 107 74.91 (±2.92) Geitonogamy 160 105 65.59 (±2.82) Xenogamy with male 134 78 57.50 (±3.85) Xenogamy with hermaphrodite 147 99 68.40 (±3.53) \*Differences in percent fruit set among treatments were highly significant. Pollinators {#s3h} ----------- Altogether nine species of anthophilous insects, which included social bees, beetles and butterflies were collected from flowering *G. assamicus* trees. The social bees were composed of two species namely *Apis cerana* and *Apis dorsata*. *Apis dorsata* was more common than *Apis cerana.* Three species of leaf cutter bees (*Megachile* spp.), one species of beetle and two species of butterflies were also recorded. Among vertebrates, only one nectarivorous bird, the 'Green-tailed Sunbird or Nepal Yellow-backed Sunbird' (*Aethopyga nipalensis*) frequently visited flowers during the peak flowering period ([Table 5](#pone-0087287-t005){ref-type="table"}). 10.1371/journal.pone.0087287.t005 ###### Flower visitors of *G. assamicus.* {#pone-0087287-t005-5} Sl. No. Common name Order Family Scientific name --------- ---------------------- --------------- -------------- ------------------------ 1 Honey bee Hymenoptera Apidae *Apis cerana* Fabr. 2 Giant honey bee Hymenoptera Apidae *Apis dorsata* Fabr. 3 Bumblebee Hymenoptera Apidae *Bombus* sp. 4 Leaf cutter bees Hymenoptera Megachilidae *Megachile* sp. 5 Leaf cutter bees Hymenoptera Megachilidae *Megachile* sp. 6 Leaf cutter bees Hymenoptera Megachilidae *Megachile* sp. 7 Flower chaffer Coleoptera Scarabaeidae Unidentified 8 Moth/Butter fly Lepidoptera Unidentified Unidentified 9 Moth/Butter fly Lepidoptera Unidentified Unidentified 10 Green-tailed Sunbird Falconiformes Accipitridae *Aethopyga nipalensis* Equilibrium male frequency in the population {#s3i} -------------------------------------------- The observed male frequency was slightly higher than the expected equilibrium frequency of males under theoretical expectations with high level of inbreeding depression ([Figure 8](#pone-0087287-g008){ref-type="fig"}). {#pone-0087287-g008} Discussion {#s4} ========== Although vegetative phenology of male and hermaphrodite trees of *G. assamicus* was similar, male trees start flowering earlier than the hermaphrodites, which provides more pollination opportunities to males [@pone.0087287-Stephenson1] as observed in many dioecious plants [@pone.0087287-Lloyd1], [@pone.0087287-Bawa1]. In addition, male trees produce almost three time more flowers than hermaphrodite trees increasing the pollination success of male trees and enhances the male fitness [@pone.0087287-Dai1] and outcrossing rates [@pone.0087287-Dorken1]. Moreover, the observed nearly two-fold increase (97%) in overall nectar production in male trees may play a significant role in attracting pollinators [@pone.0087287-Paccini1] to secure pollination success [@pone.0087287-Dixon1] and increase male fitness [@pone.0087287-Feinsinger1]. This increased fitness of male trees is crucial for the evolution and maintenance of androdioecy [@pone.0087287-Pannell2], [@pone.0087287-Lloyd2] and dioecy [@pone.0087287-Charlesworth1], [@pone.0087287-Bawa2]. The predicted equilibrium male frequencies under various levels of inbreeding and selfing rates based on Charlesworth & Charlesworth [@pone.0087287-Charlesworth2] indicated that high level of inbreeding depression is required to maintain the observed frequency of males in the population ([Figure 8](#pone-0087287-g008){ref-type="fig"}). The data on flower production over a long duration, empirical selfing rates and the level of inbreeding depression of *G. assamicus* are needed to assess the congruence of observed data with theoretical prediction to infer equilibrium frequency of males in *G. assamicus* populations. Most likely, the realized K value may be higher than the estimated K value based on the number of flowers produced in male and hermaphrodite trees. The mating system analyses through controlled pollination experiments revealed that hermaphrodite individuals of *G. assamicus* are self-compatible and autogamous in nature. Moreover, four out of nine sites had only hermaphrodite trees, which produced fruits and seeds under natural conditions. This supports the view that hermaphrodite flowers are complete and can set fruit in the absence of male trees. The xenogamous crosses with pollen from male trees resulted in fruit set confirming the fertility of pollen from male trees. Our results from controlled pollination experiments ([Table 4](#pone-0087287-t004){ref-type="table"}) provided convincing evidence that *G. assamicus* individuals are cosexual with both male and female functions as well as co-occurrence of male and hermaphrodite trees in natural populations. This observation is in agreement with the results of well-studied animal androdioecious taxa where mixed populations of males and self-compatible hermaphrodites co-occur [@pone.0087287-Philbrick1]--[@pone.0087287-Weeks3]. Our observation of multiple pollinator species in *G. assamicus* trees also suggests that pollinators are relatively non-specialized as in dioecious plants [@pone.0087287-Matsuyama1] that are pollinated by unspecialized pollinators such as small bees, flies, and other dipteran species [@pone.0087287-Opler1], [@pone.0087287-Muenchow1]. Evolution and maintenance of androdioecy in *G. assamicus* {#s4a} ---------------------------------------------------------- Maintenance of an androdioecious breeding system requires complete outcrossing or low selfing rate, and high inbreeding depression [@pone.0087287-Charlesworth1], [@pone.0087287-Bawa3]. Theoretical modelling has shown that males can invade a cosexual population only if the fitness of males is greater than twice than the cosexual individuals, and a fertility advantage is necessary for the evolution of androdioecy mating system [@pone.0087287-Fritsch1], [@pone.0087287-Pannell3]. Thus, the cosexual individuals in *G. assamicus* may have evolved by breakdown of a dioecious ancestral system where females gained some male function. Significantly lower flower production in hermaphrodites than that of males suggests that male function in hermaphrodites are weak. Similar observations in other functionally androdioecious species such as *Datisca glomerata* [@pone.0087287-Philbrick1] and *Morinda umbellata* subsp. *boninensis* [@pone.0087287-Nishide1] support these predictions [@pone.0087287-Turner1]--[@pone.0087287-Weeks3], [@pone.0087287-Chasnov1]. The high numbers of flowers on male than hermaphrodite trees, and early flowering of male trees imply that, when males are present, the selfing rate of hermophrodites is probably low. Carlson [@pone.0087287-Carlson1] demonstrated this phenomenon in a protandrous herb *Chrysothemis friedrichsthaiana*. Did *G. assamicus* hermaphrodites evolve from a dioecious ancestor? {#s4b} ------------------------------------------------------------------- Androdioecy is considered as an intermediate step towards the evolution of dioecy [@pone.0087287-Charlesworth1], [@pone.0087287-Lloyd2], [@pone.0087287-Bawa2], [@pone.0087287-Wolf1], [@pone.0087287-Astrop1] or may have evolved from dioecy as result of selection for male function in females for reproductive assurance during colonization [@pone.0087287-Pannell3], [@pone.0087287-Akimoto1], [@pone.0087287-Rieseberg1], [@pone.0087287-Wolf2]. In *G. assamicus*, hand pollination increases the fruit set, suggesting pollination limitation for fruit set. Phylogenetic studies in conjunction with mating system analyses of related species are needed to discern if androdioecy in *G. assamicus* evolved from hermaphrodites or dioecious progenitors. We thank B. Basumatary, and J. Tsering for the assistance with pollination experiments, NRC Yak (ICAR) for technical support, Central Instrumentation Facility, Indian Institute of Technology, Guwahati for the Scanning Electron Microscopy support and anonymous reviewers for invaluable comments that improved the manuscript. [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: BIC MLK. Performed the experiments: BIC. Analyzed the data: BIC. Contributed reagents/materials/analysis tools: MLK. Wrote the paper: BIC MLK SD. [^3]: Current address: Department of Botany, Dr. Harisingh Gour Central University, Sagar, Madhya Pradesh, India

***Background.*** Children with underlying chronic conditions are sub-optimally vaccinated against influenza, even if influenza vaccines (IV) are publicly funded. Since 2012, the province of Quebec has recommended live-attenuated IV (LAIV) as the preferred vaccine for children with non-immunocompromising conditions. For a second consecutive year, an influenza vaccination clinic was set up in our pediatric tertiary care hospital. We aimed to compare this clinic\'s results to last year\'s and evaluate patients\' preference for LAIV vs trivalent inactivated IV (TIV). ***Methods.*** Between October 15 and December 24 of each year (2012 and 2013), a vaccination clinic was opened weekdays during working hours and staffed with vaccine nurses. TIV and LAIV were available. Parents were asked to fill a pre-piloted questionnaire. Descriptive statistics were used. ***Results.*** During both years, we reached 630 patients with chronic illnesses accounting for 9% of the total outpatient visits for that time of year. In 2013, 264 (44%) were between 2 and 9 years, 257 (43%) were aged 9 to 18 years and 75 (13%) of vaccinated patients were immunosuppressed. Of the 623 participants for whom the information was available, 378 (61%) had received their IV in the previous year and in 49% (172/348), had received it in our vaccination clinic. For 122 of 603 parents who answered (20%), the presence of this vaccination clinic was instrumental in their child receiving their IV. In 2012, 437 of 588 patients aged 2 years and over (74%) were vaccinated with LAIV, compared to 348 of 512 (68%) this year (p = 0.02). These represented 79% of LAIV eligible children (n = 442). Of 360 patients previously vaccinated, 103 (29%) had received LAIV the year before and 88% chose to be vaccinated again with LAIV this year. LAIV was preferred by caregivers (n = 341) because it was perceived as less painful (49%), no needle was involved (41%) and in 42% because of their physician\'s recommendation. Additionally, 487 household members were vaccinated in 2013. ***Conclusion.*** Yearly influenza vaccination coverage in children with chronic illnesses can be improved with a vaccination clinic on site at a tertiary centre. Household members are also reached through this strategy. When LAIV is not contra-indicated, it remains caregivers\' and patients\' preferred vaccine. ***Disclosures.*** **All authors:** No reported disclosures. [^1]: **Session:** 99. Adult and Pediatric Vaccines [^2]: Friday, October 10, 2014: 12:30 PM

The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. Introduction {#s1} ============ Pterygium is a common ocular surface disease with the characteristics of triangle shape pathologic tissue of fibrovascular neoformation, which originates from conjunctiva, eventually invades cornea and will block the vision in severe cases. Pterygium often happens in the specific geographic regions with strong ultraviolet light, such as, South-East Asia, South-East China, Australia, and so on. Extensive research has been done on the pathogenesis of pterygium. Oxidative stress is considered a major pathogenesis of pterygium, there are other causes, for example, ultraviolet radiation-induced DNA injury, limbal stem cells deficiency (LSCD) [@pone.0108859-Cimpean1]--[@pone.0108859-Chui1] while the mechanism of pterygium is not fully understood. Meanwhile, there is no effective medication to treat pterygium or prevent the development of pterygium, the current main treatment is to remove the pterygium by surgery and the relapse rate after surgery is high [@pone.0108859-Ozgurhan1], [@pone.0108859-Nakasato1]. Multiple recent investigations suggest that the epithelial cells of pterygium are highly proliferative, with tumor cell like cell property [@pone.0108859-Chowers1]--[@pone.0108859-Liang1]. This high cell proliferation leads to the rapid development and high rate of relapse of pterygium in the clinic. It needs better elucidation on the mechanism of pterygium and exploration of new inhibitory agents to hamper the development of pterygium. SERPINA3K is a member of the family of serine proteinase inhibitors. SERPINA3K is expressed in the liver, kidney, and ocular tissues. SERPINA3K was first identified as a specific inhibitor of tissue kallikrein, also known as kallikrein-binding protein, since it specifically binds with tissue kallikrein to form a covalent complex and inhibits its proteolytic activities [@pone.0108859-Chai1]. We recently reported that SERPINA3K has antiinflammatory, antiangiogenic and antioxidant activity in the corneal epithelium [@pone.0108859-Liu1], [@pone.0108859-Zhou1]. SERPINA3K is also believed to be an inhibitor of Wnt signaling pathway [@pone.0108859-Zhang1]. In this present study, we, for the first time, investigated the inhibitory effects of SERPINA3K on the epithelial cells of pterygium and the underlying mechanism by focusing on reactive oxygen species (ROS) system and Wnt signaling pathway. Methods {#s2} ======= Patients {#s2a} -------- Seventy-six primary pterygium patients were recruited, irrespective of sex (18 cases of men and 58 cases of women) and age (25--76 years old, mean of age: 50±3.4). The conjunctiva samples were collected from 10 strabismus patients, irrespective of sex and age (2--18 years old). All cases were carefully diagnosed clinically with routine examinations and slit-lamp observation. The patients were not found any severe ocular complications, for example, corneal ulcer, and so on, when recruited. The patients underwent surgery at Xiamen Eye Center. All investigations were conducted in accordance with the tenets of the Declaration of Helsinki and were approved by the Ethics Committee of Xiamen Eye Center (an affiliated hospital of Xiamen University). A written informed consent was acquired from all participating patients. The head part of the pterygium tissue, that is, the part invading cornea, was excised for the cell culture experiment. Materials {#s2b} --------- The CCK-8 assay kits were purchased from Dojindo (Tokyo, Japan). The antibodies of anti-NOX4, anti-NQO1, anti-NRF2, anti-β-catenin, anti-nonphospho-β-catenin, and anti-LRP-6 were purchased from Abcam (Cambridge, MA). AlexaFluor488-conjugated IgG was purchased from Invitrogen (Carlsbad, CA). Purification of SERPINA3K {#s2c} ------------------------- The SA3K/pET28 construct was introduced into Escherichia coli strain BL21. The purification procedure of SERPINA3K has been previously reported [@pone.0108859-Liu1]. The purity of recombinant SERPINA3K was examined by SDS PAGE. Endotoxin concentration was monitored by using a limulus amebocyte kit. Activity was checked by MTT assay with HUVEC cells. Cell Culture {#s2d} ------------ The culture experiments were performed within two hours after surgical removal of pterygium or collection of conjunctiva samples. Fresh head part of pterygial specimens was dissected and conjunctiva tissue was collected. The dissected specimens were then cut into small pieces (1--2 mm in diameter), washed in keratinocyte serum-free defined medium (KSF-M) (GIBCO, Carlsbad, CA) and placed in a culture dish. The culture dish was placed in a CO~2~-regulated incubator with 5% CO~2~. The KSF-M medium was replaced every 2 days for about 15 days until the appearance of an outgrowth of pterygial or conjunctival epithelial cells. The procedures of pterygium epithelial cell culture and identification were previously described and followed [@pone.0108859-Xu1]. Experimental Procedures {#s2e} ----------------------- First passage of pterygial epithelial cells (PECs) or conjunctival epithelial cells collected from explant culture were cultured and used in the formal experiments until the cells were cultured to 75% confluency for cell viability measurement and 90% confluency for the cell migration test, respectively. SERPINA3K at concentrations given was added in the media for 12 or 24 hours in the treatment groups before experimental measurements and assays. Cell Viability {#s2f} -------------- The cultured pterygial epithelial cells (PECs) and conjunctival epithelial cells were used. Cell viability or cell proliferation of PECs and conjunctival epithelial cells was measured by the CCK-8 assay. CCK-8 assay was conducted with the protocol of the manufacturer. Briefly, after incubation in conditional media for 24 hours, the media were replaced by CCK-8 constituted in culture media, followed by incubation for 4 hours at 37°C in the dark. The CCK-8 containing medium was detected directly after incubation. The absorbance was measured spectrophotometrically at 570 nm with a Bio Tek ELX800 microplate reader (Bio-Tek Instruments, Winooski, VT). Cell Migration {#s2g} -------------- A scratch wound test was conducted to detect the cell migration of PECs. Briefly when PECs cells were cultured to 90% confluency, a scratch was applied in the center of the culture dish. The cell images were recorded at 0 and 12 hours. The length of the unmigrated or uninvading area was measured and analyzed to represent the cell migration of PECs. Western Blot {#s2h} ------------ Total cellular proteins of the harvested PECs cells were extracted. The standard Western blot protocol was applied. The specific primary antibodies of anti-NOX4, anti-NQO1, anti-NRF2, anti-β-catenin, anti-nonphospho-β-catenin, anti-LRP-6, and a horseradish peroxidase--conjugated secondary antibody were used. Finally, the specific bands were visualized by enhanced chemiluminescence reagents and recorded on film. Quantitative Real-time Polymerase Chain Reaction (PCR) Assay {#s2i} ------------------------------------------------------------ Total RNA was extracted from the cultured PECs cells by using TRIzol reagent (Invitrogen, Carlsbad, CA.). Reverse transcription was performed with Oligo 18T primers and reverse transcription reagents according to the manufacturer\'s protocol (TaKaRa, Shiga, Japan). Quantitative real-time polymerase chain reaction (PCR) was performed with mRNA special primers. The following primers were used for the PCR assay: for NOX4, 5′-TATCCAGTCCTTCCGTTGGTT-3′ (forward) and 5′-CTGAGGTACAGCTGGATGTTGA-3′ (reverse); for SOD2, 5′-GAGAAGTACCAGGAGGCGTTG-3′ (forward) and 5′-GAGCCTTGGACACCAACAGAT-3′ (reverse) and for NRF-2, 5′-AAACCAGTGGATCTGCCAAC-3′(forward) and 5′-GACCGGGAATATCAGGAACA-3′(reverse). PCR reactions were performed on a BIO-RAD CFX-96 Real Time system with SYBR Premix Ex Taq (TaKaRa, Shiga, Japan) at 95°C for 10 minutes, followed by 45 cycles of 95°C for 10 seconds, 57°C for 30 seconds, and 75°C for 10 seconds, after which melt curve analysis was performed at once from 65°C to 95°C. All reactions were performed in triplicate and the average cycle threshold (Ct) values greater than 38 were treated as negative. The level of GAPDH mRNA was used as an internal control. Statistical Analysis {#s2j} -------------------- One-way analysis of variance test (ANONA) was conducted to analyze the data of CCK-8 assay, scratch wound test, Western blot, and quantitative real-time PCR assay followed by a post hoc analysis Tukey test to compare the differences between the groups or a Student\'s t-test. A value of p\<0.05 was considered statistically significant. Results {#s3} ======= SERPINA3K Suppressed Cell Viability and Migration of Pterygium Epithelial Cells {#s3a} ------------------------------------------------------------------------------- Since the epithelial cells of pterygium are highly proliferative [@pone.0108859-Chowers1]--[@pone.0108859-Liang1], we first evaluated the inhibitory effects of SERPINA3K (SA3K) on the cell viability or cell proliferation of cultured pterygial epithelial cells (PECs) and compared PECs with cultured human conjunctival epithelial cells. It demonstrated that SA3K statistically significantly suppressed the cell proliferation of PECs at concentration of 80, 160 and 320 nM after treatment of 24 hours ([Figure 1B](#pone-0108859-g001){ref-type="fig"}). Meanwhile, SA3K at same concentrations did not influence the cell proliferation of cultured human conjunctival epithelial cells significantly ([Figure 1A](#pone-0108859-g001){ref-type="fig"}), indicating that SERPINA3K may selectively and specifically suppress the cell proliferation of pterygial epithelial cells. We next investigated the effects of SA3K on the cell migration of cultured PECs using scratch wound test. SA3K at concentration of 160 nM and 320 nM significantly inhibited the cell migration of PECs after treatment of 12 hours. ([Figure 1C, 1D](#pone-0108859-g001){ref-type="fig"}) These data indicated that SERPINA3K has inhibitory effects on pterygium. {#pone-0108859-g001} SERPINA3K Regulated the ROS Generation Enzyme and Antioxidants {#s3b} -------------------------------------------------------------- Oxidative stress is the major pathogenesis of pterygium [@pone.0108859-Cimpean1]--[@pone.0108859-Chui1], we recently reported that SERPINA3K plays an antioxidant role in the corneal epithelium [@pone.0108859-Zhou1], we then identified if SERPINA3K inhibited PECs through targeting the reactive oxygen species (ROS) system, for example, regulations of the key enzyme of the ROS generation system: NADPH oxidase 4 (NOX4) [@pone.0108859-Bedard1]--[@pone.0108859-Block1] and the antioxidants of the ROS system: such as, NAD(P)H dehydrogenase (quinone 1) (NQO1), NF-E2--related factor-2 (NRF2) and superoxide dismutases (SOD2) [@pone.0108859-Siegel1]--[@pone.0108859-Kaspar1]. We first examined the effect of SERPINA3K on the ROS generation enzyme NOX4 by Western blot with specific anti-NOX4 antibody. It showed that SA3K significantly downregulated the protein level of NOX4 after treatment of 24 hours ([Figure 2A, 2B](#pone-0108859-g002){ref-type="fig"}). In addition, we also detected the gene expression of NOX4 by quantitative real-time PCR assay. SA3K significantly inhibited the gene expression of NOX4 in the PECs after treatment of 24 hours. ([Figure 2C](#pone-0108859-g002){ref-type="fig"}). {#pone-0108859-g002} On the other hand, we evaluated the effects of SA3K on the antioxidants of ROS system: NQO1, NRF2 and SOD2. It was demonstrated by Western blot that SA3K significantly increased the protein level of NQO1 after treatment of 24 hours ([Figure 3A, 3B](#pone-0108859-g003){ref-type="fig"}). We also detected the changes of gene expression and protein level of antioxidant NRF2 in the PECs after treatment of SA3K for 24 hours. The Western blot results revealed that SA3K significantly increased the protein level of NRF2 in the PECs ([Figure, 3C, 3D](#pone-0108859-g003){ref-type="fig"}). Furthermore, it was also shown by quantitative real-time PCR assay that the gene expression of NRF2 was significantly increased in the PECs ([Figure 3E](#pone-0108859-g003){ref-type="fig"}). Moreover, SA3K also statistically significantly elevated the gene expression of another antioxidant SOD2 in the PECs after treatment of 24 hours ([Figure 3F](#pone-0108859-g003){ref-type="fig"}). {#pone-0108859-g003} These data suggested that SERPINA3K protects against oxidative stress of PECs via blocking the ROS generation enzyme and increasing the antioxidants. SERPINA3K Downregulated Wnt Signaling Pathway {#s3c} --------------------------------------------- We previously demonstrated that SERPINA3K is a Wnt signaling pathway inhibitor via binding and blocking the Wnt pathway upstream effector: low-density lipoprotein receptor-related protein 6 (LRP6) [@pone.0108859-Zhang1], we then determined if the inhibitory effects of SERPINA3K on the PECs are associated with the downregulation of Wnt signaling pathway in the present study. To evaluate the downregulation of Wnt pathway in the PECs, we conducted Western blot of the key effectors of Wnt pathway: β-catenin and LRP6. The Western blot data showed that the protein levels of β-catenin and LRP6 were decreased in the PECs after treatment of SA3K for 24 hours ([Figure 4A, 4B, 4C](#pone-0108859-g004){ref-type="fig"}). To further confirm the association of SA3K with the Wnt pathway in the PECs, we also measured another key Wnt pathway effector: nonphospho-β-catenin by Western blot. SA3K at concentration of 80, 160 and 320 nM statistically significantly decreased the protein level of nonphospho-β-catenin after treatment of 24 hours ([Figure 4D, 4E](#pone-0108859-g004){ref-type="fig"}). These results suggested that SA3K downregulates the Wnt signaling pathway. {#pone-0108859-g004} Discussion {#s4} ========== Pterygium is a pathologic ocular surface tissue with highly proliferative cells [@pone.0108859-Chowers1]--[@pone.0108859-Liang1] and it still lacks of effective medication except the surgical removal. In this study, we, for the first time, demonstrated that SERPINA3K, a serine proteinase inhibitor, suppresses the cell proliferation and migration of pterygial epithelial cells. Moreover, we also revealed that the underlying mechanism of SERPINA3K is through regulations of ROS system and the blockade of Wnt signaling pathway. Our experimental data may contribute to the exploration of new therapeutic agents to antagonize the formation and development of pterygium. SERPINA3K has been demonstrated to have antiinflammation and antioxidatant activities in the eyes including corneas and retina [@pone.0108859-Liu1], [@pone.0108859-Zhou1], [@pone.0108859-Zhang2], [@pone.0108859-Zhang3]. It has not been reported about the effect of SERPINA3K on the pterygium, which is associated with oxidative stress and inflammation in pathogenesis. The experimental evidence from this study supported our previous findings of antiinflammatory and antioxidant effects of SERPINA3K in another ocular pathologic tissue and status. It suggested that SERAPINA3K, as an endogenous protein with multiple functions including antiinflammatory, antiangiogenic and antioxidant effects, may have an advantage to treat pterygium and prevent the development of pterygium compared with the current application of antiinflammatory medication after surgical removal of pterygium. A pterygium originates from conjunctiva and it invades cornea in many cases, it is necessary to determine if SERPINA3K selectively and specifically targets the pterygial epithelial cells, by comparison of the effects of SERPINA3K on conjunctival epithelial cells and corneal epithelial cells. It was revealed by the present investigation that SERPINA3K did not suppress the cell proliferation of cultured human conjunctival epithelial cells. In addition, we previously demonstrated that SERPINA3K at various concentrations did not inhibit the cell proliferation of human corneal epithelial cells (HCEs) [@pone.0108859-Liu1]. These data suggest that SERPINA3K can specifically suppress the cell proliferation of pterygial epithelial cells. On the other hand, as a pterygium is composed of epithelial layer and the basal layer, which contains lots of fibroblasts or stroma cells, we just demonstrated that SERPINA3K has inhibitory effects on the cell proliferation and migration of epithelial cells of pterygium in this study, it needs further investigation to elucidate the effect of SERPINA3K on pterygial fibroblasts or stroma cells. Due to the limitation of application of SERPINA3K on human pterygium patients in clinic and the lack of good in vivo animal models of pterygium at present time, we only performed the investigation of SERPINA3K in the cultured pterygial cells, a further investigation on the efficacy of local application of SERPINA3K on animal model of pterygium in vivo and human pterygium should be conducted when available. Furthermore, we mainly focused on the effect of SERPINA3K in primary pterygium in this study, it is of worth to elucidate the effect of SERPINA3K on recurrent pterygium in the future. Oxidative stress is believed to play a vital role in the pathogenesis and development of pterygium [@pone.0108859-Cimpean1]--[@pone.0108859-Chui1]. Oxidative stress is also a major pathogenesis of other eye diseases, for example, other ocular surface diseases, age-related eye diseases, and so on. [@pone.0108859-Giacco1]--[@pone.0108859-Buddi1] Oxidative stress is a pathologic state of excessive ROS production or abnormal balance of the ROS system or ROS pathway [@pone.0108859-Wasserman1], [@pone.0108859-Kietzmann1]. In this study, we demonstrated that SERPINA3K suppresses the generation enzyme of ROS: NOX4, which is a key enzyme of ROS generation [@pone.0108859-Bedard1]--[@pone.0108859-Block1], meanwhile, SERPINA3K also elevated the levels of various antioxidant factors, such as NQO1, NRF2 and SOD2 [@pone.0108859-Siegel1]--[@pone.0108859-Kaspar1]. This present study is consistent with our previous report about the antioxidant activity of SERPINA3K in the corneal epithelium [@pone.0108859-Zhou1]. Since we mainly focused on the antioxidant role of SERPINA3K in pterygium, it is necessary to make a further comparison of the antioxidant activities of SERPINA3K between the pterygium, conjunctiva and corneas with same range and stages of age to better understand and evaluate the antioxidant role of SERPINA3K in the formation and development of pterygium. It is reported that SERPINA3K is an inhibitor of Wnt signaling pathway, the target and binding site of SERPINA3K is the upstream effector of Wnt pathway: LRP6 [@pone.0108859-Zhang1]. Thus we evaluated the alteration of Wnt pathway after treatment of SERPINA3K in the cultured pterygial epithelial cells, we demonstrated that SERPINA3K down-regulated the Wnt pathway, including the effectors: LRP6, β-catenin and nonphospho-β-catenin. This result also supports the hypothesis that SERPINA3K is an inhibitor of Wnt signaling pathway in different ocular tissue and cells, while the more detailed molecular mechanistic needs further elucidated, for example, how SERPINA3K affects the activities of downstream factors of Wnt pathway in the pterygial epithelial cells, such as transcription factor 4 (TCF4), and so on. It also requires a better understanding of the role of Wnt pathway in the formation and development of pterygium and to compare the effects of SERPINA3K on Wnt pathway between pterygium, conjunctiva as well as cornea, while there are multiply reports indicating that Wnt signaling pathway is highly expressed or activated in the pterygium [@pone.0108859-Zhou2], [@pone.0108859-Kato1]. Taken together, we provided novel experimental evidence that SERPINA3K may inhibit the formation and development of pterygium and SERPINA3K has antioxidant activity in pterygium, indicating that SERPINA3K is of the potential to be used as a therapeutic agent in the treatment of pterygium or to prevent the relapse of pterygium after surgical removal in clinic. [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: CZ FP YZ. Performed the experiments: CZ LG JZ LC TZ RZ FP. Analyzed the data: CZ FP ZT XX JxM ZL YZ. Contributed reagents/materials/analysis tools: ND MY. Wrote the paper: CP YZ.

Biological context {#Sec1} ================== The *Bacillus subtilisxpt*-*pbuX*-mRNA guanine riboswitch, which is representative of all known purine riboswitches, regulates transcription by binding guanine with high specificity (Mandal et al. [@CR5]). mRNA transcription can be completed when no guanine is present, however mRNA transcription is prematurely aborted when guanine is present and binds to the aptamer part of the riboswitch, which is partially pre-arranged to facilitate ligand binding (Ottink et al. [@CR6]). Guanine binding promotes the formation of a stable terminator hairpin, which interacts with the RNA polymerase, signalling the protein to abort the transcription (Mandal et al. [@CR5]). In contrast to the highly conserved binding pocket of the riboswitch's aptamer part, variations in length and base pairing have been observed in the terminator hairpin, resulting in variations in stability. These differences in stability, and consequently differences in structure play an important role in the kinetic trapping mechanism employed by the guanine riboswitches, where during transcription a decision between termination and antitermination is made. Methods and experiments {#Sec2} ======================= The RNA sequence of the terminator hairpin (Fig. [1](#Fig1){ref-type="fig"}) was prepared as previously described (Girard et al. [@CR3]). The unlabelled NMR sample (0.44 mM) was prepared in 10 mM Na-phosphate buffer (pH 6.7) containing 0.1 mM EDTA. All NMR spectra were recorded at 15°C on an 800 MHz Varian Inova spectrometer equipped with a cryo-probe. NOESY spectra in water (93% H~2~O, 7% D~2~O) were recorded at 15°C with 100, 200, and 300 ms mixing times. The water signal was suppressed with a jump-return pulse (Plateau and Gueron [@CR8]) in combination with a Watergate water suppression scheme (Piotto et al. [@CR7]). In 100% D~2~O, NOESY spectra were recorded with 100, 300 and 500 ms mixing times. In addition, DQF-COSY spectra and proton decoupled natural abundance ^1^H-^13^C-HMQC spectra were recorded to further aid in the assignment process (Wijmenga and Van Buuren, [@CR9]; Cromsigt et al., [@CR1]). Imino protons and cytosine amino protons were assigned from the spectra measured in H~2~O, whereas the aromatic protons and carbons and the sugar C/H1′ and C/H2′ resonances were assigned using the spectra measured in D~2~O. All acquired data were processed with NMRPipe (Delaglio et al. [@CR2]). Peak picking and assignment of the spectra was performed with the Sparky software (Kneller and Kuntz [@CR4]).Fig. 1Natural abundance ^1^H-^13^C-HMQC spectra of the 35-nucleotide guanine riboswitch terminator hairpin. **a** C8H8 region of purine residues; the C8H8 signal of loop residue A138 is shifted just out of displayed spectral window (142.6 ppm, 8.264 ppm); the relatively high ppm value of H8 indicates de-stacking of this loop residue, which is also evident from the relatively high H2 ppm value seen in *panel B*. Also the sequence and secondary structure of the terminator hairpin is shown; numbering is according to Mandal et al. 2003 **b** C2H2 region of adenine residues **c** C5H5 region of cytosine residues **d** C5H5 region of uracil residues Assignments and data deposition {#Sec3} =============================== The assignments have been deposited into the BMRB database under number 16479. The nearly complete base assignment (^1^H: 100%; ^13^C: 97%) and partial sugar assignment (^1^H: 21%; ^13^C: 18%) was achieved using high-quality 2-dimensional homonuclear and heteronuclear NMR spectra (Fig. [1](#Fig1){ref-type="fig"}a--d) recorded from one unlabelled RNA sample in H~2~O and one in D~2~O each of moderate concentration (0.44 mM). From an imino-imino sequential walk in the NOESY spectra recorded in H~2~O, all imino protons could be assigned except the one from G121 (the closing GC base pair, which is partially opened due to end fraying and whose imino exchanges with H~2~O), and from the loop residues U136 and U140, which are only weakly base paired or not base paired at all. The iminos from loop residues U135 and U141 could be detected in the NOESY with 100 ms NOE mixing time, showing that they do form a base pair. From the H~2~O-NOESYs, the cytosine-amino protons were also assigned. Aromatic (C/H2, C/H5, C/H6, C/H8) and sugar (C/H1′, C/H2′) chemical shifts were determined from the spectra recorded in D~2~O. Due to heavy overlap in the spectra of this relatively large RNA, the C/H2′ resonances could only partially be assigned and the other sugar shifts (C/H3′, C/H4′, C/H5′, C/H5″) could not be determined. A full H1′--H6/8 sequential walk, continuing throughout the entire stem and loop could be obtained from the NOESY spectra recorded in D~2~O. Using these spectra, combined with a DQF-COSY spectrum and natural abundance ^1^H-^13^C-HMQC spectra (Fig. [1](#Fig1){ref-type="fig"}), nearly all aromatic and sugar-H1′ chemical shifts of stem-residues could be assigned and all resonances of the loop residues. The only exception is the H1′ of C134, which could not be assigned unambiguously due to heavy spectral overlap. The H2's of C2′-endo puckered sugars could readily be determined from the observable H1′--H2′ coherences in the COSY spectrum (Girard et al. [@CR3]). From the NOESY spectra, this assignment could be extended with 6 more H2′ resonances. Using the ^1^H-^13^C-HMQC spectra, nearly all C2, C5, C6, C8 and C1′ resonances could be assigned. Due to spectral overlap, the carbon assignments could not fully be completed. The H8 and H2 of A138 show relatively high chemical shift values, 8.26 ppm and 8.10 ppm, respectively; the experience little ring current, indicating, as expected, a lack of stacking interactions for this loop residue. This research was supported by NWO, The Netherlands (SW). **Open Access** This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Introduction ============ Aminoglycosides (AG) play an important and adjunctive role in the treatment of life-threatening infections owing to their synergetic and broad-spectrum activity against both gram-positive and gram-negative bacteria. This group of antibiotics bind to the ribosomes of the bacteria thereby leading to inhibition of protein synthesis and consequent bacterial cell death. Their extensive use has resulted in development and dissemination of resistance to this class of antimicrobials. The mechanisms of aminoglycoside resistance are diverse. The most common mechanism is the inactivation of the antibiotic by a family of enzymes named aminoglycoside modifying enzymes (AME). [@JR_1] The AMEs catalyze the modification of the AGs at--OH or--NH ~2~ groups of the 2-deoxystreptamine nucleus or sugar moieties via acetyltransferases, nucleotidyltransferases, and phosphotransferases [@JR_2] which modify the drug, resulting in poor binding to the ribosome thereby allowing the bacteria to survive in the presence of the drug. [@JR_3] Besides the AMEs, other resistance mechanisms include change in bacterial membrane permeability, expression of efflux pumps, and methylation through 16S ribosomal RNA methyltransferases. These 16S rRNA methyltransferases are often encountered in multidrug-resistance gram negatives especially among *New Delhi Metallo betalactamase* producers. [@JR_4] There are more than 85 AMEs reported in both gram-positive and gram-negative bacteria. A few among them, particularly ant(2′)-I, aac(6′)-I, aac(3′)-I to IV, and aac(3′)-VI, undergo continuous mutation [@JR_5] leading to the generation of new AME variants which utilize a variety of AGs as substrates, cleave them, and make them ineffective. These AMEs virtually spread to all bacterial types through conjugative plasmids, natural transformation, or transduction. [@BR_6] Dissemination of AMEs via plasmids has been reported in developed countries. [@JR_7] Since there is a paucity of information from India regarding the prevalence and type of AMEs in gram-negative bacteria, the present study was undertaken to determine their prevalence among clinical isolates of gram-negative bacteria in a tertiary care center. There are some AMEs which were originally detected in gram-positive (GP AMEs) bacteria and characteristically occur in them. They are aph(3′)-IIIa that is the most prevalent and a bifunctional AME aac(6′)-Ie-aph(2′)-Ia. They confer resistance to a broad spectrum of AGs. There have been reports of their presence in gram-negative clinical isolates from Slovakia and Germany. [@JR_8] Hence, this study was aimed to detect their presence in gram-negative bacteria. Materials and Methods ===================== Ethical Approval ---------------- The study was approved by the Institutional Ethical Committee (IEC-NI/15/APR/6/18). Bacterial Isolates ------------------ A total of 386 amikacin-resistant gram-negative bacteria which were clinically significant and nonduplicate were collected over a period of 3 years from June 2015 to September 2018. All the isolates were speciated based on conventional or VITEK-2 system (Vitek-2 GN-card; BioMerieux).The bacterial isolates included were obtained from different clinical sources such as blood (14), urine (176), exudate (162), and respiratory secretion s(34) Antibiotic Susceptibility Testing --------------------------------- Disk diffusion test was performed in accordance with the Clinical Laboratory Standard Institute (CLSI, 2016). [@BR_9] The AGs tested were amikacin (30 µg), gentamicin (10 µg), tobramycin (10 µg), and netilmicin (10 µg) (HiMedia Laboratories). Polymerase Chain Reaction ------------------------- Nine sets of uniplex and two sets of multiplex polymerase chain reactions (PCRs) were performed for AMEs using previously described primers and conditions. [@JR_10] [@JR_11] [@JR_12] [@JR_13] [@JR_14] The primers used for different sets of genes, their annealing temperatures, and the amplicon sizes are listed in [Table 1](#TB_1){ref-type="table"} . ###### Primers used for performing PCR for amplifying AME genes --------------------------------------------------------------------------------------------------------------------------- Simplex set Gene Primer Annealing temperature\ Amplicon size\ (°C) (bp) -------------- ------------------------------ ----------------------------------- ------------------------ ---------------- 1 aac(6′)-lb F-TTG CGA TGC TCT ATG AGT GGC TA\ 60 482 R-CTC GAA TGC CTG GCG TGT TT 2 aph(3′)-VI F-ATGGAATTGCCCAATATTATT\ 55 780 R-TCAATTCAATTCATCAAGTTT 3 aac(3′)-I F-TTC ATC GCG CTT GCT GCY TTY\ 56 239 R-GC CAC TGC GGG ATC GTC RCC RTA 4 aac(3′)-II F-GCG CAC CCC GAT GCM TCS ATG G\ 58 370 R-GGC AAC GGC CTC GGC GTA RTG SA 5 aac(3′)-III F-GAC AAT GGC GTG CTA SCS GAR T\ 58 241 R-C CAG ATG CTC GGC ATG RTG SAG 6 aac(3′)-IV F-GAC GAC GAG CCG TTC GAY CC\ 58 280 R-C CT CAA CTC GGC AAG ATG SAG 7 aac(3′)-VI F-GCC CT CCC GAC GCA TCS ATG G\ 55 780 R-CGC CAC CGC TTC GGC ATA RTG SA 8 ant(2′)-I F-TGG GCG ATC GAT GCA CGG CTR G\ 58 428 R-AA AGC GGC ACG CAA GAC CTC MAC 9 ant(4′)-IIb F TAT CTC GGC GGT CGA GT\ 60 364 R CAC GCG GGG AAA CGC GAG AA Multiple × 1 aac(6′)-Ie-aph(2′)-Ia F-CAGGAATTTATCGAAAATGGTAGAAAAG\ 55 369 R-CACAATCGACTAAAGAGTACCAATC aph(3′)-IIIa F-GGCTAAAATGAGAATATCACCGG\ 523 R-CTTTAAAAAATCATACAGCTCGCG Multiple × 2 aph(2′)-Ib F-CTTGGACGCTGAGATATATGAGCAC\ 58 867 R-GTTTGTAGCAATTCAGAAACACCCTT aph(2′)-Ic F-CCACAATGATAATGACTCAGTTCCC\ 444 R-CCACAGCTTCCGATAGCAAGAG aph(2′)-Id F-GTGGTTTTTACAGGAATGCCATC\ 641 R-CCCTCTTCATACCAATCCATATAACC --------------------------------------------------------------------------------------------------------------------------- Seven sets of uniplex PCRs were performed for 16SrRNA methyltransferase using primers established in our earlier study. [@JR_15] Each reaction volume contained 2 µL of the deoxyribonucleic acid (DNA) template added to the master mix which includes 10 pmol of the forward and reverse primers (Sigma-Aldrich), 10 mm deoxyribonucleotide triphosphate (Takara), 5U Taq polymerase (Takara), and 10× buffer with MgCl ~2~ (Takara). Amplification of the reactions was performed under the following conditions: initial denaturation at 95°C for 4 minutes, followed by 32 cycles of denaturation at 94°C for 30 seconds, annealing based on the primer employed for 30 seconds with an extension at 72°C for 50 seconds, and a final extension for one cycle at 72°C for 5 minutes. The PCR product was then run on a 1.5% agarose gel for detection of the amplified fragment. Template DNA Preparation ------------------------ A single bacterial colony was inoculated into Luria-Bertani broth (HiMedia Laboratories) and incubated overnight at 37°C, and it was then centrifuged at 10,000 rpm for 10 minutes. The pellet was re-suspended in 250 µL of Millipore water, boiled at 100°C for 10 minutes, and cooled and centrifuged at 10,000 rpm for 10 minutes. The supernatant served as the template DNA. [@JR_16] DNA Sequencing -------------- PCR-positive amplicons were purified and sequenced. The sequenced strains served as positive controls. Sequencing was done by BigDye 3.1 cycle sequencing kit using Sanger AB13730 XL DNA analyzing instrument (AgriGenome). The nucleotide sequences were aligned using the Bioedit sequence program (product version 7.0.5.3) and were compared with the basic alignment search tool available at the National Center for Biotechnology Information website ( [www.ncbi.nIm.nih.gov](www.ncbi.nIm.nih.gov) ). Conjugation Assay ----------------- Bacterial conjugation was performed at 37°C for the clinical isolates which harbored either one of the GP AMEs \[aph(3′)-IIIa and aac(6′)-Ie-aph(2′)-Ia\]. Azide-resistant *Escherichia coli* J53 served as recipient. The transconjugants were selected on MacConkey agar plate containing 100 μg of sodium azide (HiMedia Laboratories) along with 4 μg of amikacin. [@JR_17] The transferability of the AMEs through plasmid in transconjugants was confirmed by PCR. Result ====== The study isolates includes *E. coli* ( *n* = 79), *Klebsiella pneumonia* ( *n* = 149), *Klebsiella oxytoca* ( *n* = 4), *Citrobacter freundii* ( *n* = 2), *Enterobacter cloacae* ( *n* = 11), *Proteus mirabilis* ( *n* = 5), *Proteus vulgaris* ( *n* = 2), *Providencia rettgeri* ( *n* = 16), *Morganella morganii* ( *n* = 12), *Pseudomonas aeruginosa* ( *n* = 63), *Pseudomonas fluorescens* ( *n* = 4), *Pseudomonas putida* ( *n* = 1), and *Acinetobacter baumannii* ( *n* = 38). All the study isolates exhibited resistance to all the tested AGs as determined by disk diffusion method. PCR identification revealed the prevalence of 16SrRNA methyltransferases and AMEs, of which all the clinical isolates carried one or more than one 16SrRNA methyltransferases (data were not disclosed in this study). Of the study isolates, 46.63% harbored single AME and 38.86% harbored more than one AME. The distributions of these enzymes among the different gram-negative species were tabulated ( [Table 2](#TB_2){ref-type="table"} ). ###### Distribution of AME genes and their combination among different gram negatives ------------------------------------------------------------------------------------------------------------------------------------------------ Aminoglycoside modifying enzyme genes Total number of gram negatives ( *n* = 386) ---------------------------------------------------------- --------------------------------------------- ---- ---- ---- ---- ---- ---- ---- ---- aac(6′)-lb ( *n* = 135) 22 59 2 5 4 4 6 21 12 aac(3′)-I ( *n* = 24) 7 4 -- 1 3 3 1 4 1 aph(3′)-VI ( *n* = 13) -- 5 -- -- -- -- 1 7 -- aac(3′)-VI ( *n* = 5) -- -- -- 3 -- -- -- -- 2 aac(3′)-II ( *n* = 3) -- 3 -- -- -- -- -- -- -- aac(6′)-lb+ aac(3′)-I( *n* = 68) 30 24 -- 1 -- 1 -- 10 2 aac(6′)-lb+ aph(3′)-VI ( *n* = 47) 6 24 -- 1 -- -- 2 7 7 aac(6′)-lb+ aac(3′)-II ( *n* = 8) 3 5 -- -- -- -- -- -- -- aph(3′)-VI + aac(3′)-I ( *n* = 3) -- -- -- -- -- -- -- -- 3 aac(6′)-lb+ aph(3′)-VI + aac(3′)-I\ 2 7 -- -- -- -- -- 7 4 ( *n* = 20) aac(6′)-lb+ aac(3′)-I+ aac(3′)-II\ 1 -- -- -- -- -- -- -- -- ( *n* = 1) aac(6′)-lb+ ac(3′)-I+ aac(3′)-II + aph(3′)-VI ( *n* = 3) 3 -- -- -- -- -- -- -- -- ------------------------------------------------------------------------------------------------------------------------------------------------ Interestingly GP AMEs namely aph(3′)-IIIa and aac(6′)-Ie- aph(2′)-Ia were identified in this study isolates. They were detected in 3.88 and 8.03% of the study isolates, respectively ( [Table 3](#TB_3){ref-type="table"} ). Co-occurrence of both of these enzymes was encountered in 7.77%. ###### Study isolates associated with AMEs prevalent in gram-positive bacteria ------------------------------------------------------------------------------------------------------------------------- Gram-positive\ Total number of gram negatives ( *n* = 386) AMEs ------------------------------------ --------------------------------------------- ---- ---- ---- ---- ---- ---- --- ---- aph(3′)-IIIa ( *n* = 15) -- 7 -- -- -- -- -- 8 -- aac(6′)-Ie- aph(2′)-Ia ( *n* = 31) 4 5 -- -- 2 5 2 8 5 aph(3′)-IIIa +\ 6 13 -- 3 -- -- -- 8 -- aac(6′)-Ie- aph(2′)-Ia ( *n* = 30) ------------------------------------------------------------------------------------------------------------------------- However, AMEs such as ant(2′)-I, ant(4′)-IIb, aac(3′)-III, aac(3′)-IV, aph(2′)-Ib, aph(2′)-Ic, and aph(2′)-Id were not encountered in any of the study isolates. Conjugation assay was successful in all the clinical isolates tested which harbored the GP AMEs. Discussion ========== AGs play a vital role as monotherapy and in combination for the treatment of majority of bacterial infection. The resistance to AGs in bacteria is predominantly due to the AMEs. [@JR_1] All the 386 clinical isolates were resistant to all the tested AGs. They did not exhibit any substrate-specific hydrolyzing profile which is commonly encountered in AME. This is attributable to the presence of 16S rRNA methyltransferases which confer resistance to all AGs. [@JR_4] The prevalence of aac(6′)-lb singly and in combinations with other AME is higher ( [Table 2](#TB_2){ref-type="table"} ) when compared with previous reports from Iran, China, and Spain which had 31. 6, 19.6, 4.2% of aac(6′)-lb, respectively. [@JR_18] [@JR_19] [@JR_20] The AME aac(3′)-I was the second most prevalent gene singly and in also combination(6.21 and 17.61%). This enzyme has been reported in large number of gram-negative clinical isolates previously. [@JR_2] The enzyme aph(3′)-VI, first identified in *A. baumannii* in 1988, [@JR_21] was the third most prevalent gene. AMEs such as ant(2′)-I, ant(4′)-IIb, aac(3′)-III, and aac(3′)-IV were not encountered in our study isolates but their presence was widely reported in countries like Iran, [@JR_22] France [@JR_23] and China. [@JR_24] The GP AMEs aph(2′)-Ib, aph(2′)-Ic, and aph(2′)-Id were not encountered in our study; however, their presence was significantly reported in *Enterococci* and *Staphylococcus* . [@JR_14] [@JR_25] This significant difference in their presence of AMEs may be due to usage of antibiotics and other geographical factors involved. [@JR_19] The bifunctional enzyme aac(6′)-Ie-aph(2′)-Ia that confers high-level resistance to gentamicin, amikacin, tobramycin, netilmicin and is considered more prevalent in *Enterococci* [@JR_26] has been identified in the present study; there are two previous reports citing its presence and transferability in gram-negative bacteria. [@JR_8] The prevalence of these AMEs in this study is 19.68% and their transfer indicates their location on conjugative plasmids. However, the prevalence rate is significantly less compared with their rate of occurrence in *Enterococci* (38.20%). [@JR_27] Conclusion ========== Our findings throw light on the distribution of the different AMEs and their combination among the clinical isolates of gram-negative bacteria. To the best of our knowledge, this is the first report to study the presence of GP AMEs in gram-negative bacteria from India. Considering the transferability potential of these resistance genes between gram-positive and gram-negative bacteria frequent surveillance studies are required to study the changing pattern and evolution of resistance among bacteria. **Conflict of Interest** None.

All relevant data are within the paper and its Supporting Information files. Introduction {#sec001} ============ *Staphylococcus aureus* infections of the bloodstream or deep wound are a serious complication of major surgeries, including cardiothoracic and orthopedic surgery, resulting in significant morbidity and mortality \[[@pone.0147767.ref001], [@pone.0147767.ref002]\]. *S*. *aureus* is also the most commonly isolated microorganism from patients in intensive care units, which have mortality rates that reach 60% \[[@pone.0147767.ref003]\]. Due to methicillin-resistant *S*. *aureus* infections, up to one third of patients diagnosed with *S*. *aureus* bacteremia succumb even when treated with appropriate antibiotic therapy \[[@pone.0147767.ref004]\]. Therefore, a vaccine that provides rapid protection against *S*. *aureus* during the post-operative or intensive care period would address an important unmet medical need. We recently developed a four-component *S*. *aureus* vaccine (4C-Staph) consisting of Hla~H35L~, EsxAB, FhuD2, and Csa1A recombinant proteins \[[@pone.0147767.ref005]\]. Hla~H35L~ is a genetically detoxified mutant of α-hemolysin (Hla) \[[@pone.0147767.ref006]\], a highly conserved exotoxin that plays a prominent role in early stages of invasive infections disrupting epithelial and endothelial barriers, contributing to pathogen-associated mortality \[[@pone.0147767.ref006], [@pone.0147767.ref007]\]. Immunization with Hla~H35L~ partially protected mice against staphylococcal pneumonia, peritonitis, and skin infections inducing functional antibodies neutralizing the lytic activity of native Hla \[[@pone.0147767.ref008]--[@pone.0147767.ref010]\]. Remarkably, however, Hla neutralization was not sufficient to eradicate *S*. *aureus* infection, suggesting that additional antigens are required for an efficacious vaccine \[[@pone.0147767.ref009]\]. EsxAB is a fusion of the two ESAT-6-like secreted virulence factors EsxA and EsxB associated to abscess formation, which may facilitate persistence and spread of the pathogen in the infected host \[[@pone.0147767.ref011]--[@pone.0147767.ref013]\]. FhuD2 is a lipoprotein involved in iron uptake and in early stages of invasive *S*. *aureus* infection \[[@pone.0147767.ref014]--[@pone.0147767.ref016]\], while Csa1A is a putative lipoprotein whose role in pathogenesis is under investigation \[[@pone.0147767.ref017]\]. We have recently shown that two doses of 4C-Staph vaccine formulated with alum protected against a panel of epidemiologically relevant *S*. *aureus* strains in kidney abscess, peritonitis, skin, and pneumonia mouse models of *S*. *aureus* infection \[[@pone.0147767.ref005]\]. Adjuvants enhance and accelerate adaptive immune responses toward a co-administered antigen, while also directing the quality of the immune response \[[@pone.0147767.ref018], [@pone.0147767.ref019]\]. Three major types of cell-mediated effector immunity meant to optimally respond to distinct threats have been identified: type 1 protects against intracellular pathogens and comprises IFN-γ-producing cells (e.g. Th1 cells), type 2 protects against helminths and comprises IL-4/IL-13-producing cells (e.g. Th2), while type 3 protects against extracellular bacteria and fungi and comprises IL-17-producing cells (e.g. Th17) \[[@pone.0147767.ref020]\]. Aluminum salts-based adjuvants, which are included in many licensed vaccines, preferentially induce type 2 responses while agonists of Toll-like receptors (TLRs), a family of receptors that recognize pathogen-associated molecular patterns \[[@pone.0147767.ref021], [@pone.0147767.ref022]\], induce mainly type 1 responses \[[@pone.0147767.ref018]\]. The potential of small molecule immune potentiators (SMIPs) targeting TLR7, an endosomal TLR that recognizes single-stranded RNAs, as vaccine adjuvants has been shown in pre-clinical settings, especially for Imiquimod. However, systemic activation induced by these SMIPs has posed safety issues \[[@pone.0147767.ref023]\]. The rational design of SMIP.7-10, a novel TLR7 agonist that can be stably adsorbed to alum (T7-alum), minimized systemic exposure and inflammation while retained *in vivo* potency \[[@pone.0147767.ref024]\]. Therefore, in this study, we investigated whether single immunization with 4C-Staph/T7-alum conferred prompt protection against *S*. *aureus* infection. We found that this was indeed the case and we elucidated the immune mechanisms involved. Materials and Methods {#sec002} ===================== Vaccine formulations and animal immunizations {#sec003} --------------------------------------------- Vaccine antigens, Hla~H35L~, EsxAB, FhuD2, and Csa1A, were amplified by PCR from *S*. *aureus* NCTC8325 strain and cloned as tagless constructs. Antigens were purified and adsorbed to alum by incubation with aluminum hydroxide (alum) with or without SMIP-7.10 (T7), at pH 6.5--7.0 and osmolality 0.308±0.060 Osm/Kg, with slow stirring for a few hours at room temperature (RT) \[[@pone.0147767.ref005]\]. One dose of vaccine (100 μl) consisted in 10 μg of each antigen adsorbed to 2 mg/ml alum alone (4C-Staph/alum) or together with 50 μg SMIP-7.10 (4C-Staph/T7-alum). Formulations were adjusted to physiological ranges of pH and isotonicity and had an endotoxin content ˂1.5 European U/ml. Five-week old female BALB/c (Charles River Laboratories) or J~H~ (Taconic) mice were used. For active immunization, mice were immunized i.m. (50 μl/hind leg quadriceps). Control mice received equal amounts of alum, or T7-alum. For passive immunization, pools of sera were prepared from BALB/c mice immunized once with 4C-Staph/T7-alum (immune serum) or T7-alum (control serum) by 32 days. Five-week-old naïve BALB/c mice were injected with 150 μl of immune or control serum into the tail vein the day before challenge with *S*. *aureus*. Bacterial inoculum and challenge {#sec004} -------------------------------- *S*. *aureus* Newman strain (kindly provided by Professor Schneewind, University of Chicago) was grown in tryptic soy broth (Difco Laboratories) at 37°C shaking at 250 rpm to an optical density at 600 nm (OD~600~) of 2. After centrifugation at 3,320 x g for 10 min., bacteria were washed with 50 ml of phosphate-buffered saline (PBS, pH 7.4; Invitrogen) and then diluted in PBS to obtain the required infectious dose/mouse. Inocula were verified experimentally by plating on tryptic soy agar and enumeration of the colony forming units (CFU) the day after. Kidney abscess model: mice were challenged i.v. with 100 μl of a sublethal dose of bacteria (\~ 2×10^7^ CFU). After 4 days, mice were euthanized; both kidneys were removed, homogenized in pool in 2 ml PBS, 2-fold serially diluted, and plated in duplicate for CFU counts. Peritonitis model: mice were challenged i.p. with 100 μl of a LD~80~ of bacteria (\~5 x 10^8^ CFU). Mice were monitored daily for 15 or 30 days, as indicated. Mice that survived were euthanized and CFU in kidneys were enumerated as described above. Quantification of 4C-Staph-specific and Hla-neutralizing antibodies {#sec005} ------------------------------------------------------------------- Vaccine-specific antibody titers were measured by Luminex (Luminex^®^ 200 TM) at a fixed serum dilution. Hla~H35L~, FhuD2, EsxAB and Csa1A purified proteins were covalently conjugated to the free carboxyl groups of xMAP multi-analyte microspheres (Luminex Corporation). Antigen-specific antibodies were revealed using PE-labeled secondary antibodies. For total IgG, median fluorescence intensity (MFI) values were converted into relative Luminex units (RLU)/ml using a mouse serum as standard. Hla neutralizing antibodies were quantified with a rabbit red blood cells (RBCs)-based functional assay. Serial 2-fold dilutions of pooled mouse sera (n = 8/pool) were incubated with 12.5 nM Hla (Sigma) in triplicates. After 30 min at 37°C in agitation (350 rpm), RBCs (Emozoo Snc.) were added and incubation prolonged for 30 min without shaking. Then plates were centrifuged for 5 min at 1,000 x g and the OD~540~ of supernatants was determined with a SpectraMax^®^ 340PC384 Absorbance Microplate Reader (Molecular Devices). Percent hemolysis was calculated using as denominator the OD~540~ obtained following lysis of rabbit RBCs without mouse serum. Hla neutralizing titers were expressed as the reciprocal of the serum dilution inhibiting 50% of Hla hemolysis (ED~50~) given by native Hla, calculated using a \"log (agonist) vs. normalized response\" nonlinear regression curve (GraphPad Software, Inc.). Intracellular cytokine staining {#sec006} ------------------------------- Splenocytes were isolated 12 days after vaccination, plated at 1-2x10^6^ cells/well in 96-well plates, and stimulated with vaccine proteins (Hla~H35L~, EsxAB, FhuD2 and Csa1A, 10 μg/ml each) together with anti-CD28 and anti-CD49d mAb (2 μg/ml each, BD Biosciences) at 37°C for 16--18 h. Brefeldin A (5 μg/ml) was added for the last 4 h. The cells were then stained with Live/DeadYellow (Invitrogen), fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences), washed in Perm/Wash buffer (BD Biosciences), incubated with anti-CD16/CD32 Fc block (BD Biosciences) for 20 min at RT, and stained with the following fluorochrome-conjugated mAbs anti: CD3-PerCP Cy5.5, CD4-V500, IFN-γ-PE, IL-2-APC, TNF-AF700, CD44-V450 (BD Pharmingen), CD8-PE Texas Red (Invitrogen), IL-17A-PE Cy7, IL-4-AF488 and IL-13-AF488 (eBioscience) in Perm/Wash buffer (BD Biosciences) for 20 min at RT, washed twice in Perm/Wash buffer, suspended in PBS. Samples were acquired on a LSRII special order flow cytometer (BD Biosciences) and T-cell responses were analyzed using FlowJo software (TreeStar) applying the gating strategy described in [S1 Fig](#pone.0147767.s001){ref-type="supplementary-material"}. Depletion of effector CD4^+^ T cells {#sec007} ------------------------------------ To deplete effector CD4^+^ T cells, mice were vaccinated on day 0, injected twice i.p. with 100 μg of a rat anti-CD4 mAb (clone GK1.5; Areta International) or isotype-matched control antibody (isot. ctr., rat IgG2b; R&D System) on day 6 and 8, and challenged with *S*. *aureus* on day 10. CD4^+^ cell depletion efficiency was evaluated by flow cytometry on heparinized blood taken the day before infection (day 9). For this purpose, RBCs were lysed with RBCs lysis buffer (Biolegend). White blood cells were stained with Live/Dead Yellow (Invitrogen), incubated with anti-CD16/CD32 Fc block (BD Biosciences), and stained with: anti-CD3-PerCP Cy5.5, (BD Pharmingen), anti-CD8-PE TexasRed (Invitrogen), and anti-CD4-Pacific Blue (Invitrogen) in PBS 0.1% BSA for 20 min at room temperature. Cells were fixed with Cytofix (BD Biosciences), suspended in PBS and analyzed by LSR II flow cytometer (BD Biosciences). No CD4^+^ T cells were detected in the peripheral blood indicating that depletion occurred efficiently ([S2 Fig](#pone.0147767.s002){ref-type="supplementary-material"}). *In vivo* cytokine neutralization {#sec008} --------------------------------- To neutralize IL-17A and/or IFN-γ *in vivo*, mice immunized with 4C-Staph/T7-alum or T7-alum were injected i.p. with 100 μg of a neutralizing mouse anti-IL-17A mAb (clone 17F3; BioXCell) and/or a neutralizing rat anti-IFN-γ mAb (clone XMG 1.2; BioXCell) every other day starting from 3 h prior to *S*. *aureus* challenge (day 12 after vaccination) until sacrifice. Control mice received the same amount of isot. ctr. antibodies (mouse IgG1 and/or rat IgG1; BioXCell). Statistical analysis {#sec009} -------------------- Log-rank test was used to evaluate differences in survival of infected mice. One-way ANOVA and Sidak's multiple comparisons test were used to evaluate differences in the bacterial load in kidneys of infected mice and differences in CD4^+^ T-cell responses to vaccination when more than two groups were compared. Comparisons between frequencies of cytokine-producing CD4^+^ T cells in response to 4C-Staph/alum or 4C-Staph/T7-alum vaccination were performed using a Student\'s *t* test and a partial permutation test. For total IgG titers, Kruskall-Wallis and Dunn\'s multiple comparisons tests were used to evaluate differences in vaccine-specific antibody titers. Statistical analyses were performed using GraphPad Prism 6 software (GraphPad Software, Inc.) and SPICE version 5.1 (<http://exon.niaid.nih.gov>). Ethics statement {#sec010} ---------------- All animal studies were carried out in compliance with current Italian legislation on the care and use of animals in experimentation (Legislative Decree 26/2014, authorizations 185/2011-B and 249/2011-B approved by the Italian Ministry of Health) and with the Novartis Animal Welfare Policy and Standards (authorizations AWB 201105 and AWB 201106). After infection, mice were monitored daily using a dedicated clinical score system based on the following parameters: piloerection, hunched posture, decreased mobility, rolling/twisting movements and weight loss. Mice were euthanized by cervical dislocation when they exhibited either one of the following humane endpoints: a loss of 20% bodyweight and/or moderate to severe signs of rolling/twisting movements that were pre-established in agreement with Novartis Animal Welfare Policies. All animals were provided environmental enrichment and food and water were offered *ad libitum*. Results {#sec011} ======= One dose of 4C-Staph/T7-alum vaccine confers rapid and efficacious protection against *S*. *aureus* {#sec012} --------------------------------------------------------------------------------------------------- We tested the efficacy of one dose of adjuvanted 4C-Staph vaccines in the mouse kidney abscess and peritonitis models. For this purpose, BALB/c mice were vaccinated i.m. with 4C-Staph/T7-alum or 4C-Staph/alum vaccines. Control groups of mice received T7-alum or alum. Twelve days after immunization, mice were challenged with *S*. *aureus* Newman strain. In the kidney abscess model, mice were challenged i.v. with a sublethal dose of bacteria (2x10^7^ CFU). After 4 days mice were sacrificed and CFU in kidneys were enumerated. As shown in [Fig 1A](#pone.0147767.g001){ref-type="fig"}, 4C-Staph/T7-alum-vaccinated mice showed a 1.55 log~10~ reduction in CFU/kidneys compared to T7-alum-treated control mice (4.66 ± 0.28 and 6.21 ± 0.23 log~10~, mean ± SEM CFU/kidneys, respectively, *p* = 0.0003), and a 1.14 log~10~ reduction in CFU/kidneys compared to 4C-Staph/alum-immunized animals (5.80 ± 0.28 log~10~ CFU/kidneys, *p* = 0.0080). Remarkably, 4 out of 32 mice vaccinated with 4C-Staph/T7-alum had no detectable bacteria in kidneys compared to 0 out of 31 mice in the 4C-Staph/alum group. {#pone.0147767.g001} In the peritonitis model, which can be used to study the spread of bacteria into the bloodstream \[[@pone.0147767.ref009]\], mice were challenged i.p. (5x10^8^ CFU) with a lethal dose of *S*. *aureus* and their survival was monitored for 30 days. One month after infection, 75% of mice immunized with 4C-Staph/T7-alum survived *S*. *aureus* challenge compared to 41% of mice immunized with 4C-Staph/alum (*p* = 0.0058, [Fig 1B](#pone.0147767.g001){ref-type="fig"}). Furthermore, mice vaccinated with 4C-Staph/T7-alum that survived had significantly less bacteria in kidneys (1.7 log~10~ reduction, *p* = 0.0091) and 91% of them had no detectable bacteria in kidneys as compared to survivors in the 4C-Staph/alum group (54%, [Fig 1C](#pone.0147767.g001){ref-type="fig"}). Taken together, these data showed that single dose of 4C-Staph/T7-alum vaccine considerably reduced bacterial load in kidneys upon *S*. *aureus* i.v. challenge and not only allowed the vast majority of mice to survive after i.p. challenge, but also promoted efficient control of bacteria in kidneys of survivors. 4C-Staph/T7-alum vaccine rapidly induces functional and protective antibodies {#sec013} ----------------------------------------------------------------------------- The induction of antibodies specific for each component of the vaccine by 4C-Staph/T7-alum and 4C-Staph/alum was evaluated 12 and 32 days after vaccination. IgG specific for Hla~H35L~, EsxAB, FhuD2, and Csa1A were detected in sera from all mice vaccinated with 4C-Staph/T7-alum by day 12 and further increased by day 32, with the exception of one mouse that had no detectable anti-Csa1A IgG ([Fig 2A](#pone.0147767.g002){ref-type="fig"}). In contrast, only Hla~H35L~-specific IgG were detected in all sera from mice immunized with 4C-Staph/alum 12 days after vaccination, while EsxAB- and FhuD2-specific IgG were detected only in some sera, and Csa1A-specific IgG were not found. By day 32, Hla~H35L~- and EsxAB-specific IgG were found in sera from all 4C-Staph/alum vaccinated mice, while FhuD2- and Csa1A-specific IgG were found only in some sera. Higher levels of IgG1 and IgG2a specific for each vaccine component were found in sera of mice vaccinated with 4C-Staph/T7-alum as compared to sera of mice vaccinated with 4C-Staph/alum ([Fig 2C](#pone.0147767.g002){ref-type="fig"}), while no differences in vaccine-specific IgM levels were observed ([Fig 2D](#pone.0147767.g002){ref-type="fig"}). In addition, Hla neutralizing titers were measurable in sera from mice vaccinated with 4C-Staph/T7-alum by day 12 (ED~50~ = 41) and increased by 5-fold at day 32 (ED~50~ = 215), while Hla-neutralizing activity was detected in sera from mice vaccinated with 4C-Staph/alum at d32 (ED~50~ = 39) but not at day 12 (ED~50~ \< 6, [Fig 2B](#pone.0147767.g002){ref-type="fig"}). Altogether, these data show that single dose of 4C-Staph/T7-alum vaccine readily induced not only seroconversion of vaccinated mice against each 4C-Staph component but also Hla-neutralizing antibodies. {#pone.0147767.g002} To determine if antibodies induced by 4C-Staph/T7-alum vaccination were protective against a lethal challenge with *S*. *aureus*, sera collected from mice 32 days after immunization with 4C-Staph/T7-alum (or T7-alum, as control) were passively transferred into naïve mice one day before i.p. infection. All mice that received immune serum were protected from death during the first 3 days after infection ([Fig 3A](#pone.0147767.g003){ref-type="fig"}). Thereafter, the survival gradually declined to become 28% at day 15, when all survivors had bacteria in kidneys ([Fig 3B](#pone.0147767.g003){ref-type="fig"}). In contrast, 100% of mice that received control sera died by day 6 after infection. Finally, we showed that 4C-Staph/T7-alum vaccination required B cells to protect mice against *S*. *aureus* infection by using J~H~ mice, which lack mature B cells and antibodies \[[@pone.0147767.ref025]\]. Within two days after i.p. challenge, 96% of J~H~ mice vaccinated with 4C-Staph/T7-alum succumbed to the infection while 88% of BALB/c mice were still alive 15 days after infection (*p* \< 0.0001, [Fig 3C](#pone.0147767.g003){ref-type="fig"}). {#pone.0147767.g003} Overall these data suggest that antibodies induced by 4C-Staph/T7-alum vaccine are necessary and sufficient to protect against early death caused by *S*. *aureus* i.p. infection. 4C-Staph/T7-alum induces vaccine-specific Th1 and Th17 cells {#sec014} ------------------------------------------------------------ Both human clinical observations and animal models support the concept that CD4^+^ T cells and Th17 cells in particular play a role in protection against *S*. *aureus* infection \[[@pone.0147767.ref026], [@pone.0147767.ref027]\]. Therefore, we evaluated the magnitude and the quality of vaccine-specific CD4^+^ T-cell responses induced by 4C-Staph/T7-alum and 4C-Staph/alum. For this purpose, splenocytes from mice immunized by 12 days were stimulated with 4C-Staph proteins *in vitro*. Vaccine-specific CD4^+^CD44^high^ T cells were identified and characterized at the single-cell level by polychromatic intracellular flow cytometry based on their ability to produce IL-2, TNF, IL-4/IL-13, IFN-γ and/or IL-17A. As shown in [Fig 4A](#pone.0147767.g004){ref-type="fig"}, the frequencies of CD4^+^CD44^high^ T cells producing any of these cytokines alone or in combination (total CYT^+^ cells) were higher in 4C-Staph/T7-alum than in 4C-Staph/alum vaccinated mice. Analyses of the quality of vaccine-specific CD4^+^CD44^high^CYT^+^ T cells revealed comparable frequencies of IL-2^+^ and IL-2^+^TNF^+^ cells amongst the two vaccines, while 4C-Staph/T7-alum induced higher percentages of TNF^+^ cells. In addition, 4C-Staph/T7-alum induced higher frequencies of Th1 cells (IFN-γ^+^ and TNF^+^IFN-γ^+^) and Th17 cells (IL-17A^+^), but lower percentages of Th2 cells (IL-4^+^/IL-13^+^, IL-2^+^IL-4^+^/IL-13^+^ and IL-2^+^TNF^+^IL-4^+^/IL-13^+^) than 4C-Staph/alum ([Fig 4B](#pone.0147767.g004){ref-type="fig"}). Interestingly, 4C-Staph/T7-alum also induced higher percentages of polyfunctional IL-2^+^TNF^+^IFN-γ^+^ CD4 T cells. {ref-type="supplementary-material"} for gating strategy). The response of unstimulated cells was subtracted from that of stimulated cells. Data are the merge of four independent experiments. (**A**) Percentages of CD4^+^CD44^high^ T cells producing any combination of IL-2, TNF, IL-4/IL-13, IFN-γ or IL-17A in response to vaccine protein stimulation (CD4^+^CD44^high^ total CYT^+^ cells) were calculated applying Boolean gates. Bars represent mean ± SEM. \**p* \< 0.05, \*\**p* \< 0.01, \*\*\**p* \< 0.001 by one-way ANOVA and Sidak post-test. (**B**) Percentages of CD4^+^CD44^high^ T cells producing IL-2, TNF, IL-4/IL-13, IFN-γ and/or IL-17A in each of the possible combinations (CD4^+^CD44^high^ CYT^+^) in response to vaccine proteins stimulation calculated applying Boolean gates. No cells expressing more than 3 cytokines at once were detected. Bars represent mean ± SEM. \**p* \< 0.05 by unpaired Student *t* test, two-tailed, and a partial permutation test.](pone.0147767.g004){#pone.0147767.g004} These results demonstrated that one dose of 4C-Staph/T7-alum induced higher frequencies of vaccine-specific cytokine-producing CD4^+^ T cells and polarized the CD4^+^ T cells more towards a Th1/Th17 phenotype as compared to 4C-Staph/alum. CD4^+^ T cells induced by 4C-Staph/T7-alum contribute to the protection against *S*. *aureus* {#sec015} --------------------------------------------------------------------------------------------- To assess if vaccine-specific CD4^+^ T cells play a role in protection, mice were treated with an anti-CD4 mAb 6 and 8 days after immunization with 4C-Staph/alum or 4C-Staph/T7-alum to allow T cell-dependent antibody production, but to eliminate CD4^+^ T cells with effector function before *S*. *aureus* challenge ([S2 Fig](#pone.0147767.s002){ref-type="supplementary-material"}). In the kidney abscess model, the depletion of CD4^+^ T cells abolished the protection conferred by 4C-Staph/T7-alum, as evidenced by the numbers of CFU found in kidneys that was not different in the T7-alum vaccine group (*p* = 0.145, [Fig 5A](#pone.0147767.g005){ref-type="fig"}). In contrast, CD4^+^ T cell depletion did not impact the efficacy of 4C-Staph/alum (statistically significant decrease in CFU in kidneys vs. mice vaccinated with alum, *p* = 0.002). In the peritonitis model, depletion of CD4^+^ T cells significantly decreased the survival of mice vaccinated with 4C-Staph/T7-alum (58% survival vs. 79% of mice treated with isotype control antibody, *p* = 0.034), but not of mice vaccinated with 4C-Staph/alum (13% survival vs. 31% of mice treated with isotype control antibody, *p* = 0.207, [Fig 5B](#pone.0147767.g005){ref-type="fig"}). In addition, depletion of CD4^+^ T cells in mice vaccinated with 4C-Staph/T7-alum caused an increase of 1.74 log~10~ in the CFU/kidneys of survivors as compared to isotype control antibody-treated mice (*p* = 0.0372, [Fig 5C](#pone.0147767.g005){ref-type="fig"}). {#pone.0147767.g005} Overall, these data show that effector CD4^+^ T cells contribute to the protection conferred by one dose of 4C-Staph vaccine adjuvanted with T7-alum both in the kidney abscess and peritonitis models, although other mechanisms of protection were involved, in agreement with the role of humoral immunity that we have shown in [Fig 3](#pone.0147767.g003){ref-type="fig"}. Neutralization of IL-17A increases the bacterial load in kidneys of mice vaccinated with 4C-Staph/T7-alum upon i.p. *S*. *aureus* infection {#sec016} ------------------------------------------------------------------------------------------------------------------------------------------- Depletion of effector CD4^+^ T cells had no effect on protection conferred by vaccination with 4C-Staph/alum in either the kidney abscess or the peritonitis models ([Fig 5A and 5B](#pone.0147767.g005){ref-type="fig"}) indicating that the quality of effector CD4^+^ T cells induced by 4C-Staph/T7-alum vaccination is important. Since 4C-Staph/T7-alum induced a vaccine-specific CD4^+^ T cell response more polarized towards Th1 and Th17 cells as compared to 4C-Staph/alum ([Fig 4B](#pone.0147767.g004){ref-type="fig"}), we neutralized IL-17A and IFN-γ, separately or together, immediately before and repeatedly after i.p. challenge to block the effects of these cytokines produced in response to *S*. *aureus*. Treatment with neutralizing mAb specific for IL-17A and/or IFN-γ had no effect on survival compared to treatment with isotype-matched control antibody ([Fig 6A--6C](#pone.0147767.g006){ref-type="fig"}, left panels). However, neutralization of IL-17A alone or in combination with IFN-γ caused a 1.85 log~10~ (*p* = 0.019) and a 1.66 log~10~ (*p* = 0.0002) CFU increase, respectively, in kidneys of mice that survived the infection compared to survivors injected with isotype-matched control antibody ([Fig 6A and 6C](#pone.0147767.g006){ref-type="fig"}, right panels). Neutralization of IFN-γ alone did not cause a significant increase in CFU/kidneys (*p* = 0.253, [Fig 6B](#pone.0147767.g006){ref-type="fig"}, right panel). These data indicated that IL-17A produced in mice vaccinated with 4C-Staph/T7-alum in response to *S*. *aureus* i.p. infection is important to control dissemination of staphylococci to organs distant from the infection site. {#pone.0147767.g006} Discussion {#sec017} ========== A vaccine that elicits a fast protective response against *S*. *aureus* infection would be highly desirable for patients with planned surgery or in intensive care units \[[@pone.0147767.ref001]--[@pone.0147767.ref003]\]. Appropriately adjuvanted 4C-Staph vaccine might achieve this goal. Therefore, we formulated 4C-Staph with a novel small molecule targeting TLR7 adsorbed to alum \[[@pone.0147767.ref024]\] to give 4C-Staph/T7-alum, and tested this vaccine formulation as single dose in *S*. *aureus* kidney abscess and peritonitis mouse models. We showed that 4C-Staph/T7-alum vaccine reduced by more than 100-fold the bacterial burden in kidneys of mice infected i.v. and protected roughly 80% of vaccinated mice from the lethal outcome associated with an i.p. challenge, outperforming 4C-Staph/alum formulation. Remarkably, 91% of 4C-Staph/T7-alum vaccinated mice that survived the i.p. infection had no detectable staphylococci in kidneys as compared to 54% of 4C-Staph/alum vaccinated mice. These observations on one hand showed that 4C-Staph/T7-alum promoted a more efficient control of bacteria than 4C-Staph/alum, and on the other hand highlighted that considering just survival rates as an index of *S*. *aureus* vaccine efficacy can lead to overestimated results. Indeed, the dichotomy between survival and bacterial burden has been recently highlighted by an elegant study by Maurer *et al*. \[[@pone.0147767.ref028]\]. The immunologic correlates of protection induced by 4C-Staph/T7-alum vaccine consisted in faster: (*i*) seroconversion of nearly 100% of vaccinated mice; (*ii*) production of antibodies specific for each of the vaccine antigens; (*iii*) induction of Hla-neutralizing antibodies; (*iv*) induction of vaccine-specific CD4^+^ T cells, a higher percentage of which produced IFN-γ, IL-17A, and TNF than those induced by 4C-Staph/alum. Protection conferred by 4C-Staph/T7-alum was mediated by antibodies, as demonstrated by the lack of efficacy of the vaccine in B cell-deficient mice and the passive protection against *S*. *aureus* challenge achieved by transferring immune sera. Furthermore, depletion of effector CD4^+^ T cells in mice vaccinated with 4C-Staph/T7-alum, but not 4C-Staph/alum, resulted in an increase in renal bacterial burden upon i.v. or i.p. challenge as well as in reduction of survival rate upon i.p. challenge, indicating that effector CD4^+^ T cells' polarization towards Th1 and Th17 induced by 4C-Staph/T7-alum (but not 4C-Staph/alum) vaccination is likely to be crucial. Indeed, neutralization of IL-17A, alone or together with IFN-γ (but not IFN-γ alone) resulted in a 100-fold increase in bacterial load in kidneys of 4C-Staph/T7-alum-vaccinated mice. While not statistically significant, there was a trend for an increase in CFU in IFN-γ-neutralizing antibody-treated mice and a more marked increase in CFU in mice treated with the combination of IL-17A- and IFN-γ-neutralizing antibodies compared to IL-17A-treated mice, suggesting that IFN-γ might also play a role in protection. Neutralization of either cytokine, alone or in combination, had no effect on survival. This result together with the partial effect on survival of effector CD4^+^ T cell depletion as opposed to the dramatic effect of the lack of B cells/antibodies in J~H~ mice indicated that antibodies are required at the moment of intraperitoneal infection to control bacterial growth and toxicity, giving time to effector CD4^+^ T cells to expand and exert their protective role. The importance of the humoral response in protection against *S*. *aureus* infection is shown by preclinical as well as clinical data (reviewed in \[[@pone.0147767.ref026]\]). Passive transfer of antibodies raised against different staphylococcal antigens conferred partial protection against *S*. *aureus* in mouse models \[[@pone.0147767.ref008], [@pone.0147767.ref014], [@pone.0147767.ref029]\]. In humans, circulating antibodies to several *S*. *aureus* antigens are commonly found, particularly in colonized subjects, which present milder disease outcomes to systemic infections as compared to non-colonized patients (reviewed in \[[@pone.0147767.ref026]\]). In this study, passive vaccination protected all animals from death in the first days after i.p. challenge, while survival rates decreased afterwards, suggesting that a continuous supply of functional antibodies is needed, and/or mechanisms other than antibodies contribute to 4C-Staph/T7-alum-induced protection. The concept that antibodies alone are insufficient to protect against *S*. *aureus* infection is supported by the lack of efficacy of several passive and active immunization strategies in phase 3 clinical trials, despite high antibody titers were achieved \[[@pone.0147767.ref026], [@pone.0147767.ref030]\]. The role of cell-mediated immunity and in particular of IL-17-producing cells, belonging either to the innate immunity (e.g. γδ T cells) or to the adaptive immunity (i.e. Th17), in protection against *S*. *aureu*s is well documented both in patients with genetic defects that affect the IL-23/IL-17 immune axis and mice deficient in *il-17a*, *il-17f* or *il-17ra* \[[@pone.0147767.ref026], [@pone.0147767.ref031]--[@pone.0147767.ref033]\]. IL-17A and IL-17F act on many non-immune cells, including epithelial cells, inducing: (*i*) production of chemokines that in turn mobilize and recruit neutrophils and macrophages to the site of infection; and (*ii*) production of anti-microbial peptides \[[@pone.0147767.ref034]\]. Both mechanisms promote *S*. *aureus* clearance (reviewed in \[[@pone.0147767.ref027]\]). In addition, human Th17 can kill bacteria, including *S*. *aureus*, through the anti-microbial action of IL-26 that they produce \[[@pone.0147767.ref035]\]. In mice, IL-17 produced by γδ T cells was protective against *S*. *aureus* infection \[[@pone.0147767.ref036]--[@pone.0147767.ref039]\], while IL-17 produced by Th17 in response to vaccination with Als3p, ClfA, and IsdB contributed to protection against *S*. *aureus* \[[@pone.0147767.ref040]--[@pone.0147767.ref042]\], supporting our own observations with 4C-Staph/T7-alum. In the peritonitis model, IL-17A neutralization and CD4^+^ effector T cell-depletion equally increased the bacterial load in kidneys of mice vaccinated with 4C-Staph/T7-alum, suggesting that Th17 are the main source of IL-17A in these settings. The contribution of IL-17-producing cells other than Th17 deserves further investigation. The fact that effector CD4^+^ T cell depletion, but not IL-17A (or IFN-γ) neutralization, affected also mice survival indicated that other cytokines produced by CD4^+^ effector T cells might be important. IL-17F, which was produced by vaccine-specific CD4^+^ T cells (data not shown) and was not neutralized by the anti-IL-17A antibody, could be such a cytokine. IL-17F contributes to control *S*. *aureus* infection since double *il-17a*^-/-^*il-17f*^-/-^ deficient mice, but not single *il-17a*^-/-^ or *il-17f*^-/-^ deficient mice, were more susceptible to opportunistic *S*. *aureus* infections \[[@pone.0147767.ref043]\]. TNF could also be important since it can not only synergize with IL-17A and IL-17F, which on their own are poor activators of signaling \[[@pone.0147767.ref044], [@pone.0147767.ref045]\], but also prime neutrophils, rendering them faster and more efficient against pathogens \[[@pone.0147767.ref046], [@pone.0147767.ref047]\]. In summary, we showed that one dose of 4C-Staph/T7-alum vaccine elicits a fast and efficacious protection against *S*. *aureus* systemic as well as peripheral infection through the induction of vaccine-specific functional antibodies, CD4^+^ effector T cells, and IL-17A. Cooperation between humoral and cell-mediated immunity is likely to be required to achieve efficacious protection against the broad spectrum of pathologies induced by *S*. *aureus* infections. Supporting Information {#sec018} ====================== ###### Gating-tree for phenotypic and functional characterization of vaccine-specific CD4^+^ T cells by polychromatic intracellular flow cytometry. Splenocytes from single mice immunized by 12 days were stimulated or not with vaccine antigens (10 μg/ml each) *in vitro*. Splenocytes were then stained and analyzed by intracellular cytokine staining. Live cells were identified based on Live/Dead staining. Lymphocytes were gated based on their forward side scatter (FSC) vs. side scatter (SSC) profile. Singlets were gated based on their SSC properties. CD4^+^CD44^high^ T cells were identified based on CD3, CD4, and CD44 expression. Inside the CD4^+^CD44^high^ T-cell population, cells producing IL-2, TNF, IL-4/IL-13, IFN-γ or IL-17A were identified setting gates on non-stimulated cells (not shown). The dot plots refer to splenocytes of a representative mouse immunized with 4C-Staph/T7-alum stimulated *in vitro* with vaccine proteins. (TIF) ###### Click here for additional data file. ###### Depletion efficacy of CD4^+^ T cells in peripheral blood of mice treated with anti-CD4 or isot. ctr. demonstrated by flow cytometry analyses. Blood was collected from individual mice 9 days after vaccination and CD4^+^ T cells were identified in live white blood cells based on the expression of CD3, CD4 and CD8 markers. Representative dot plots are shown. (TIF) ###### Click here for additional data file. We wish to thank the staff at Novartis Animal Research Center, particularly Marco Tortoli and Stefania Torricelli for skilled animal handling, and Benn Reeves for the dedicated *S*. *aureus* clinical score system; Sara Marchi, Claudia Facciotti, and Elena Cartocci for the supply of 4C-Staph proteins; Luisa Galli-Stampino for contributing to intracellular cytokine staining set up; Alessandra Anemona for statistical analysis support. [^1]: **Competing Interests:**The authors have read the journal\'s policy and have the following competing interests: Salaries for all authors were provided by Novartis Vaccines and Diagnostics S.r.l. during completion of the experimental studies contained herein. This does not alter the authors' adherence to all PLoS One policies on sharing data and materials. [^2]: Conceived and designed the experiments: FM EM MRF GB GG RR EDG FB ES SB. Performed the experiments: FM EM GL AT MB LA DL GT SR-P ES. Analyzed the data: FM EM AT GT MRF BG MP SR-P GB SN ES SB. Contributed reagents/materials/analysis tools: ST CS. Wrote the paper: FM FB ES SB. [^3]: Current address: GlaxoSmithKline Vaccines S.r.l., Siena, Italy [^4]: Current address: Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts, United States of America [^5]: Current address: Centre for Integrative Biology, University of Trento, Trento, Italy

Introduction ============ Sepsis-associated encephalopathy is defined as a diffuse cerebral dysfunction induced by the systemic response to the infection without clinical or laboratory evidence of direct infectious involvement of the central nervous system \[[@B1]\]. Previous clinical observations have shown that the brain is often the first organ to be affected by sepsis, preceeding the clinical symptoms of other organ manifestations. According to the studies of Wilson and colleagues and Young and colleagues, electroencephalogram (EEG) may be abnormal in 87% of patients with bacteriemia. They diagnosed 70% with disturbance of consciousness of differing severity ranging from somnolence to coma \[[@B1]-[@B3]\]. Ebersoldt and colleagues, reviewing sepsis-associated delirium, reported on a prevalence ranging from 9 to 71% \[[@B4]\]. The exact pathomechanism involved is not yet fully understood. It is believed that microcirculatory alterations, disturbance of cerebral autoregulation, damage of the blood-brain barrier, branched chain/aromatic amino acid inbalance and the direct effect of the inflammatory process (e.g. free radicals, oxydative stress, cytokines, excitotoxicity apoptosis) on glial cells may play a decisive role. Sepsis-related encephalopathy is most likely to be a multifactorially determined syndrome \[[@B5]\]. When assessing cerebral microvascular contributing factors, in previous human investigations Matta and Stow \[[@B6]\] found cerebral autoregulation and carbon dioxide reactivity to be normal in patients with sepsis, whereas Terborg and colleagues reported on severely disturbed vasomotor reactivity (VMR) \[[@B7]\]. In the past two decades, different stimuli have been used to test cerebral autoregulation and metabolic regulation, such as altering arterial partial pressure of carbon dioxide (pCO~2~) either by inhalation of carbon dioxide or by changing respiratory rate (carbon dioxide reactivity), breath holding test (carbon dioxide reactivity), decreasing systemic blood pressure and therewith cerebral perfusion pressure (cerebral autoregulation) and intravenous injection of acetazolamide. Acetazolamide, the reversible inhibitor of the enzyme carbonic anhydrase, has been used to test cerebral VMR in various diseases and conditions \[[@B8]\]. Disturbed cerebrovascular reactivity (CVR) as a sign of cerebral microvascular alterations has been demonstrated in patients with diabetes mellitus \[[@B9],[@B10]\], arterial hypertension \[[@B11]\], systemic lupus erythematosus \[[@B12]\], in subjects hemodynamically significant stenoses and occlusions of the carotid arteries \[[@B13]\]. With respect to the debated involvement of the above cerebral microvascular alterations, in the present study we intended to test whether acetazolamide-induced cerebral VMR is altered in patients with sepsis-associated encephalopathy. To the best of our knowledge this is the first study that uses the transcranial Doppler-acetazolamide test to assess cerebral VMR in sepsis-related encephalopathy. Materials and methods ===================== The study was approved by the local Medical Ethics Committee of the Debrecen University Health and Medical Science Centre. Patients fulfilling the criteria of clinical sepsis according to the guidelines of the American College of Chest Physicians/Society of Critical Care Medicine (ACCP/SCCM) Consensus Conference Committee \[[@B14]\] were enrolled in the study. Those with hemodynamic instability, in need of hemodynamic support or with signs of hypoperfusion of the different organs were excluded. Patients were not under mechanical ventilation prior to or during the study. Patients were selected and screened during daily rounds on the postoperative surgical wards or from the multidisciplinary surgical ICU. Sepsis-related encephalopathy was defined as a combination of the following: patients had to meet the criteria of clinical sepsis and had to show disturbance of consciousness or alertness of any severity. Any other metabolic causes of conscious disturbance were excluded (hypoxemia, hyper-or hypoglycemia, increased serum urea, creatinine or ammonia levels). A certified neurologist (BF) performed a detailed neurological assessment of all the patients in order to exclude direct infectious involvement of the central nervous system (such as meningitis or encephalitis). Sedative drugs were not administered before the neurological assessment. Consciousness/alertness disturbance was graded by two scales: the Richmond Agitation-Sedation Scale (RASS) and the Ramsay scores. The different categories of these scoring systems are described elsewhere in detail \[[@B15]\]. As septic patients suffered from altered consciousness, their nearest relatives were asked to give informed consent. When sepsis and encephalopathy were diagnosed, patients were transferred to the ICU and a continous monitoring of arterial blood pressure, echocardiography, pulse oxymetry was initiated. This made it possible to perform arterial blood gas analysis every five minutes after acetazolamide administration. Transcranial Doppler measurements were performed in the supine position using a Rimed Digilite Transcranial Doppler sonograph (Rimed Ltd, Raanana, Israel). A 2 MHz probe was used for insonation, and sample volume, gain and power were kept constant during the investigation. Temporal window was used for insonation, probes were fixed by LMY-2 probe holder (Rimed Ltd, Raanana, Israel). The device enabled the assessment of the best available signal of the middle cerebral artery between the depths of 45 to 55 mm. Systolic, diastolic and mean blood flow velocities were registered, and pulsatility indices were calculated by the device. After a blood flow velocity measurement was performed at rest, 15 mg/kg acetazolamide (Diamox, Lederle Pharmaceuticals, Carolina, Puerto Rico, USA) was injected intravenously. As proposed in previous studies \[[@B8]\], blood flow velocities were continously registered until 20 minutes after injection of the vasodilatory stimulus. CVR was defined as the percentage increase of the middle cerebral artery mean blood flow velocity after administration of acetazolamide. CVR was calculated as follows: where MCAV~ACZ~is the middle cerebral artery mean blood flow velocity measured at 5, 10, 15 and 20 minutes after acetazolamide, and MCAV~rest~is the middle cerebral artery mean blood flow velocity measured at rest. Cerebrovascular reserve capacity (CRC; the maximal percentage increase of the blood flow velocity after acetazolamide administration), was calculated as follows: where MCAV~ACZmax~is the highest mean blood flow velocity in the middle cerebral artery within 20 minutes after administration of acetazolamide. Transcranial Doppler measurements were performed in 20 age- and sex-matched persons, who were free of sepsis, diabetes mellitus, hypertension, significant stenoses of the cerebral arteries or any known diseases which, according to our present knowledge, could have influenced CVR testing. These subjects served as controls for the study. In these subjects arterial sampling for blood gas analysis was only performed at resting state, because inserting a radial artery catheter or serial arterial sampling during the whole study was considered unethical. Statistical analysis -------------------- Means and standard deviations were reported for all values. Before performing statistical comparisons of the parameters, a normality test was used. Parameters with normal distribution were compared with the appropriate unpaired t-tests. Repeated measure analysis of variance was used to detect differences in MCAV and CVR values after acetazolemide administration. When significant differences were detected, pairwise comparisons were performed between the groups using the Mann-Whitney U test. Differences were accepted as statistically significant if *P*value was less than 0.05. Results ======= Fourteen patients with sepsis-associated encephalopathy and 20 control persons were enrolled. Blood pressure values assessed by arterial blood pressure did not change during the acetazolamide testing. During the study, slight hyperventilation was observed, but any deterioration of the patients\' status did not occur during or after acetazolamide. The results of the most important clinical and laboratory data of septic patients and controls are summarized in Table [1](#T1){ref-type="table"}. From these data it can be seen that blood pressures and blood gas analysis parameters were comparable in the two groups at rest. In septic patients, pH slightly decreased, while pCO~2~and partial pressure of oxygen slightly increased during the acetazolamide test. The distribution of the Ramsay scales were in the septic groups as follows: Ramsay 1 = 6 cases, Ramsay 3 = 4 cases, Ramsay 4 = 4 cases. There were five cases with RASS +1 and a further eight cases with RASS -1. Thus, in all cases either a sepsis-related delirious state or somnolence was present. ###### Results of the most important clinical or laboratory parameters before in septic and in control patients Sepsis Control *P*value --------------------------- -------------- ------------- ---------- **Systolic BP (mmHg)** 117.9 ± 10.3 113.5 ± 8.7 0.20 **Diastolic BP (mmHg)** 69.7 ± 5.9 75.0 ± 5.4 0.01 **Mean BP (mmHg)** 84.7 ± 7.6 87.8 ± 5.3 0.21 **Arterial pH** 0 minutes 7.39 ± 0.04 7.40 ± 0.03 0.48 5 minutes 7.38 ± 0.04 NA \- 10 minutes 7.37 ± 0.03 NA \- 15 minutes 7.37 ± 0.04 NA \- 20 minutes 7.37 ± 0.04 NA \- **Arterial pCO~2~(mmHg)** 0 minutes 36.8 ± 3.4 38.9 ± 1.96 0.11 5 minutes 38.2 ± 3.5 NA \- 10 minutes 41.0 ± 4.4 NA \- 15 minutes 40.8 ± 3.9 NA \- 20 minutes 41.3 ± 4.9 NA \- **Arterial pO~2~(mmHg)** 0 minutes 87.0 ± 9.7 83.7 ± 3.46 0.07 5 minutes 91.5 ± 11.2 NA \- 10 minutes 91.5 ± 9.3 NA \- 15 minutes 91.2 ± 8.9 NA \- 20 minutes 90.0 ± 9.0 NA \- **WBC count (G/l)** 15.1 ± 6.4 5.93 ± 1.84 \< 0.001 **PCT** 8.89 ± 8.7 NA \- Means and standard deviations are shown. BP: blood pressure; NA: not available; PCO~2~: partial pressure of carbon dioxide; PCT: procalcitonin; PO~2~: partial pressure of oxygen; WBC: white blood cell count. The results of the transcranial Doppler measurements are summarized in Table [2](#T2){ref-type="table"}. Resting systolic blood flow velocities did not differ, but the mean and the diastolic blood flow velocities were lower in the group with sepsis-associated encephalopathy. It has to be noted that pulsatility indices were higher at the resting state in patients with sepsis-related encephalopathy and this difference remained unchanged after administration of acetazolamide. Absolute blood flow velocities after the vasodilator drug were higher among control subjects than in septic patients. In a further analysis we checked the time-course of the vasomotor reaction to acetazolamide. As shown in Figure [1](#F1){ref-type="fig"}, patients with sepsis-associated encephalopathy reacted slower to the vasodilatory stimulus than control persons. When assessing the maximal vasodilatory ability of the cerebral arterioles to acetazolamide during 20 minutes of vasomotor testing, we found that patients with sepsis-associated encephalopathy reacted to the drug to a lesser extent than control subjects. The results are depicted in Figure [2](#F2){ref-type="fig"}. {#F1} {#F2} ###### Systolic, diastolic and mean blood flow velocities (cm/s) and pulsatility indices before and after administration of acetazolamide in control persons and in patients with sepsis-associated encephalopathy ----------------------------------------------------------------------------- Time after acetazolamide (minutes) Sepsis\ Control\ *P*value (n = 14) (n = 20) ------------------------------------ -------------- -------------- ---------- **0** 85.4 ± 20.7 85.9 ± 13.7 0.94 **5** 99.6 ± 31.6 114.1 ± 20.5 0.15 **10** 96.5 ± 24.2 118.5 ± 19.5 \< 0.05 **15** 101.9 ± 27.1 124.4 ± 17.5 \< 0.05 **20** 102.0 ± 27.7 121.9 ± 17.4 \< 0.05 **Diastolic blood flow velocity** **0** 32.5 ± 12.3 45.6 ± 8.8 \< 0.01 **5** 35.9 ± 12.5 61.9 ± 12.6 \< 0.001 **10** 40.1 ± 13.3 64.2 ± 13.9 \< 0.001 **15** 43.2 ± 17.4 64.4 ± 11.7 \< 0.001 **20** 40.0 ± 12.6 80.4 ± 14.3 \< 0.001 **Mean blood flow velocity** **0** 47.9 ± 14.5 58.2 ± 12.0 \< 0.05 **5** 55.4 ± 18.2 77.8 ± 17.1 \< 0.01 **10** 56.4 ± 16.0 79.3 ± 16.6 \< 0.001 **15** 59.4 ± 19.4 64.4 ± 11.7 \< 0.01 **20** 58.7 ± 17.5 80.4 ± 14.3 \< 0.001 **Pulsatility index** **0** 1.15 ± 0.35 0.85 ± 0.20 \< 0.01 **5** 1.21 ± 0.26 0.80 ± 0.16 \< 0.001 **10** 1.01 ± 0.32 0.70 ± 0.16 \< 0.01 **15** 0.98 ± 0.34 0.76 ± 0.15 \< 0.05 **20** 1.06 ± 0.24 0.74 ± 0.14 \< 0.01 ----------------------------------------------------------------------------- Means and standard deviations are shown. Discussion ========== In the present study we found that cerebral VMR is impaired in patients with sepsis-associated encephalopathy. It is also clear from our results that not only maximal vasodilative capacity (CRC) but also the time-course of the vasodilative effect (CVR) is affected after administration of acetazolamide in septic patients. Thus, the reaction of the cerebral arterioles to the vasodilatory stimulus is not only lower in magnitude, but also occurs slower in patients with sepsis-associated encephalopathy. When analyzing absolute blood flow velocities in the middle cerebral artery, it is clear that they are lower in patients with sepsis-associated encephalopathy compared with non-septic control persons after acetazolemide stimulation. A decrease in the blood flow velocity measured within the middle cerebral artery may theoretically be explained in two ways: either the large and medium-size vessel (the middle cerebral artery) is dilated or there is a vasoconstriction at the level of resistance arterioles of its corresponding territory. Although this question cannot be answered based only on the absolute blood flow velocity values, taking the pulsatility indices into account, the higher pulsatility index among patients with sepsis-associated encephalopathy is more likely to indicate vasoconstriction of the cerebral arterioles. It has been shown previously that an increase in resistance distal to the site of insonation results in an increased blood flow pulsatility \[[@B16]\]. Thus, based on our results, decreased cerebral blood flow velocities along with higher pulsatility indices in patients with sepsis-associated encephalopathy can be ascribed to the vasoconstriction of the resistance arterioles. These results are in accordance with previous studies stating that cerebral blood flow is reduced and cerebrovascular resistance is increased in sepsis-associated encephalopathy \[[@B1],[@B17]\]. It seems that general vasodilation does not affect the brain circulation in sepsis; instead a vasoconstriction of the resistance arterioles occurs. This is the explanation for the findings of Matta and Stow, who found that sepsis-induced vasoparalysis does not involve the cerebral vasculature \[[@B6]\]. There are numerous factors in sepsis that may contribute to the vasoconstriction of the brain resistance arterioles. First, in animal experiments it has been demonstrated that the blood-brain barrier, which normally maintains a homeostatic environment for brain cells, becomes leaky within the first hours of endotoxemia. Disruption of the blood-brain barrier allows high levels of endogenous catecholamines to directly influence cerebrovascular resistance \[[@B18]\]. Second, it is believed that cytokines and ILs produced during the course of the sepsis cascade may alter the activity of the endothelial nitric oxide synthase. The inhibition of endothelial nitric oxide synthase leads to the impairment of the microcirculation of the brain by causing vasoconstriction \[[@B1]\]. Finally, alterations of the coagulation system resulting in microthromboses and microinfarctions as seen in sepsis may also contribute to the microvascular dysfunction \[[@B19]\]. The goal of cerebral autoregulation and metabolic vasoreactivity testing is to see whether the brain circulation is able to adopt to sudden and critical changes of blood pressure (autoregulation) or metabolic demands (metabolic regulation). From the previous clinical investigations and animal experiments it is clear that cerebral arterioles of 40 to 200 μm in diameter are common actors of both autoregulatory and metabolic response of the brain circulation. Different stimuli have been used to test cerebral autoregulation and metabolic regulation, such as altering pCO~2~(carbon dioxide reactivity), breath holding test (carbon dioxide reactivity), decreasing systemic blood pressure and therewith cerebral perfusion pressure (cerebral autoregulation) and intravenous injection of acetazolamide. Basically, there are two main factors to take into account during VMR tests: the maximal vasodilative capacity (CRC) and the time-course of the reaction (CVR) \[[@B8]\]. In the present study we used intravenous acetazolamide to assess the cerebral vasomotor response. For the sake of clarity we intend to explain the concept of transcranial Doppler acetazolamide tests. Acetazolamide is a reversible inhibitor of the carbonic anhydrase, which is located at the surface of the erythrocytes. The enzyme catalyses the following reaction: CO~2~+ H~2~O → H~2~CO~3~→ H^+^+ HCO~3~). It also induces a slight temporary hypercapnia lasting for approximately 20 minutes, which results in vasodilation of the cerebral arterioles, most probably through inducing nitric oxide synthesis \[[@B8]\]. As described above, cerebral arterioles are key actors in cerebral autoregulation and metabolic regulation. Dilation of these vessels results in a decrease of cerebrovascular resistance. As shown in Figure [3](#F3){ref-type="fig"}, transcranial Doppler measurements can be performed at the level of the middle cerebral artery and cerebral arterioles cannot be directly assessed. When an arteriolar vasodilation occurs, the cerebrovascular resistance of the corresponding arterial territory decreases, resulting in an increase of the cerebral blood flow velocity measured in the middle cerebral artery. Thus, cerebral arteriolar function cannot be directly measured. Only changes of the cerebrovascular resistance induced by acetazolamide can be indirectly assessed by measuring cerebral blood flow velocities in the middle-sized arteries of the corresponding territory. It has to be noted that there are some limitations of our study. Transcranial Doppler does not measure cerebral blood flow. It measures cerebral blood flow velocity, the changes of which are not equal, but only proportional to changes of cerebral blood flow. A further limitation is the lack of arterial pCO~2~monitoring in the control group. {#F3} In our study, a less intensive CVR was detected in patients with sepsis-associated encephalopathy, that is cerebral arterioles reacted to the vasodilator stimulus slower and to a lesser extent. Besides a slower vasodilation after acetazolamide administration, the maximal dilation of the cerebral arterioles (CRC) was also lower in septic patients. These results are in accordance with those of Terborg and colleagues, who also demonstrated dysfunction in patients with severe sepsis and septic shock \[[@B7]\]. Similarly, animal studies have showed decreased carbon dioxide-induced VMR in streptococcal sepsis \[[@B20]\]. In recent animal models it has been shown that microcirculatory dysfunction in the brain precedes changes in evoked potentials \[[@B21]\]. Taking the absolute blood flow velocities and pulsatility indices in the present study into account, it is conceivable that vasoconstriction of the cerebral arterioles may be responsible for the impaired VMR. As shown in Table [2](#T2){ref-type="table"}, pulsatility indices were higher throughout the entire course of the acetazolamide test among septic patients compared with control persons, suggesting vasoconstriction of the resistance vessels. Although there was a slight difference between diastolic pressures of septic and control persons, it has to be noted that mean arterial pressures in the two groups were similar and therefore the significance of this BP difference during transcranial doppler sonography (TCD) -acetazolamide testing most probably did not influence the results. Conclusions =========== The clinical signficance of the present study may be summarized as follows. First, the results of the transcranial Doppler acetazolamide test may help to better understand the pathophysiology of septic encephalopathies. Second, as we mentioned above, cerebral autoregulation and metabolic regulation occur at the same level of the cerebral circulation (resistance arterioles). In our series of septic patients without hemodynamic compromise or need of hemodynamic support, the ability of the brain resistance arterioles to dilate was decreased. If it is considered that sepsis-associated shock situations and sudden decreases of cerebral perfusion pressure evoke a strong autoregulatory response, an already reduced vasodilatory capacity should limit both the static and dynamic autoregulatory response of the cerebral arterioles. One of the most important functions of cerebral autoregulation is to ensure constant cerebral blood flow (and therewith oxygen delivery) during changes in systemic blood pressure. Further studies are needed to clarify the importance of hemodynamic monitoring and proper hemodynamic support in early phases of sepsis (and sepsis-related encephalopathy is an early warning sign), in order to prevent critical blood pressure changes in the cerebral vascular bed and thus the progression of brain damage. Key messages ============ • Cerebral arteriolar function is altered in sepsis-associated encephalopathy • Cerebral arterioles of patients with SAE react lesser extent to vasodilatory stimuli • Cerebral hemodynamic changes may be involved in the early pathogenetic phases of SAE Abbreviations ============= CRC: cerebrovascular reserve capacity; CVR: cerebrovascular reactivity; ECG: echocardiogram; EEG: electroencephalogram; MCAV: middle cerebral artery mean blood flow velocity; PCO~2~: partial pressure of carbon dioxide; RASS: Richmond Agitation-Sedation Scale; VMR: vasomotor reactivity. Authors\' contributions ======================= SS and TV performed the transcranial Doppler tests. ÁC and MC participated in the design of the study. JH and IT drafted the manuscript. BF performed neurological examinations. BF and MC participated in planning the design of the study, performing the statistical analysis, and completing the manuscript. All authors read and approved the final manuscript. Competing interests =================== The authors declare that they have no competing interests.

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Introduction ============ Glioblastoma multiforme (GBM) is the most common and most aggressive primary brain tumor, accounting for more than 50% of all gliomas. Despite aggressive surgery, chemotherapy and radiotherapy, the mean overall survival achieved for GBM patients is less than 18 months [@B1]. Consequently, substantial efforts have been made in recent years towards the development of new, improved therapies directed against specific features of the pathology on an individual patient basis. GBM exhibits a highly developed neovasculature and is one of the most vascularized solid tumors. The neovasculature in GBM is heterogeneous, abundant, hyperpermeable, and disorganized with deformed vessels of irregular diameter [@B2]. The high neovascularization may contribute to the aggressive behavior of GBM, and the inhibition of tumor angiogenesis is a promising therapeutic strategy. A vast network of pro-angiogenic signaling molecules and their cognate receptors have been identified and characterized [@B3],[@B4]. Among them, members of the vascular endothelial growth factor (VEGF)/VEGFR family are considered the prime regulators of both physiological and pathological angiogenesis [@B2]. VEGFR2, which is primarily expressed in vascular endothelium, is a key mediator of VEGF-induced pro-angiogenic activities [@B5], playing a pivotal role in the regulation of endothelial cell biology [@B6]. VEGFR2 is upregulated in tumor vasculature compared to normal vasculature [@B7], thus emerging as an attractive antiangiogenic target. Many promising new antiangiogenic therapies targeting the VEGF-VEGFR axis with different mechanisms of action have been evaluated in recent years. Examples include VEGFA-specific agents (e.g., bevacizumab, ranibizumab, VEGF-trap), neutralizing VEGFR2-specific antibodies/antibody fragments (e.g., ramucirumab and CDP791), and small molecule tyrosine kinase inhibitors that block VEGFR signaling (e.g., sunitinib, axitinib, sorafenib) (see an overview of clinical studies in [@B8]). However, not all patients respond to antiangiogenic therapy [@B9]-[@B11]. It is increasingly evident that the successful implementation of these therapies requires improved diagnostic tools that can identify the patient population that is most likely to respond and match it with the optimal treatment [@B12]. A noninvasive imaging technique for monitoring the status of tumor angiogenesis that could provide a prompt readout of posttreatment response is therefore highly desirable [@B13]. For this purpose, imaging agents for single photon emission computed tomography (SPECT) and positron emission tomography (PET) targeting VEGFRs and their ligands could enable a detailed characterization of the molecular status of the endothelium in the tumor. Efforts to image the VEGF-VEGFR axis have so far been focused either on tracing the ligand distribution with anti-VEGFA monoclonal antibodies or on imaging of VEGFR2 expression using natural/mutated VEGF isoforms. Nagengast et al. were the first to demonstrate the potential of ^111^In- and ^89^Zr-labeled bevacizumab for noninvasive in vivo imaging of VEGFA [@B14]. Bevacizumab was subsequently labeled with iodine-125, yttrium-86 and copper-64 [@B15]-[@B17]. However, imaging of VEGFA is challenging due to its complex and dynamic nature. VEGFA is often sequestered in the extracellular matrix through its interaction with heparan sulfate and is released by proteolytic activity in the tumor microenvironment [@B18], [@B19]. Moreover, an important limitation of using monoclonal antibodies as imaging probes is that they circulate in the blood for several days requiring long times after injection to achieve reasonable contrast. In addition, nonspecific uptake of antibodies in tumors due to the enhanced permeability and retention (EPR) effect might lead to false-positive findings and limit therapy monitoring [@B20]. VEGFA isoforms have also garnered increasing attention as imaging agents during the last two decades. VEGF165 and VEGF121 have been labeled with various radionuclides, such as iodine-123 and -125, copper-64, gallium-68, technetium-99m [@B21]-[@B25], and an engineered single-chain version of VEGFA selective to VEGFR2 (scVR2) has been labeled with zirconium-89 [@B26], showing encouraging imaging results. However, these findings have been tempered by the inherent angiogenic activity of such agents and the costs associated with their large-scale synthesis. Affibody molecules are a class of scaffold proteins with short blood circulation time, high stability and high target affinity [@B27],[@B28]. Recently, Fleetwood et al. reported the selection of several anti-VEGFR2 affibody molecules with affinities to VEGFR2 below 10 nM [@B29]. Affibody molecules were selected for binding to human VEGFR2 (hVEGFR2) but also demonstrated cross-reactivity to murine VEGFR2 (mVEGFR2) in the low nanomolar range. Although all selected affibody molecules blocked VEGFA-binding to VEGFR2 *in vitro*, they recognized nonoverlapping epitopes on the receptor and could bind simultaneously [@B29]. Furthermore, they inhibited VEGFA-induced phosphorylation and cell proliferation [@B30]. Binders with low picomolar affinity are desirable for targets with low expression/density. In the case of HER2 targeting, affibody molecules with an affinity of 160 pM had 6.5-fold higher tumor uptake and 2-fold slower wash-out compared to counterparts with 3.8 nM affinity in tumors with low receptor density (4×10^3^ receptors/cell) [@B31]. We hypothesized that i) the fusion of two anti-VEGFR2 affibody molecules targeting nonoverlapping epitopes (so-called biparatopic dimer) should increase affinity due to the avidity effect by increasing the binding area, and ii) the increase in protein size should not play a negative role since receptor expression is primarily limited to endothelial cells and tissue penetration is not essential. Here, we used the strategy of fusing two anti-VEGFR2 affibody molecules targeting nonoverlapping epitopes with affinities in the nanomolar range to create a biparatopic affibody dimer denoted Z~VEGFR2~-Bp~2~, which showed increased target affinity. The unique C-terminal cysteine residue enables site-specific conjugation of the NODAGA chelator for labeling with radiometals. In this feasibility study, indium-111 (t~½~ = 2.8 days) was used as the label since it permits the long experiments that are required for accurate characterization of the new binder, e.g., determination of affinity, cellular processing and following the biodistribution profile of the new conjugate at later time points. We show that NODAGA-Z~VEGFR2~-Bp~2~ can be exploited for noninvasive visualization of tumor angiogenesis in preclinical GBM. Methods ======= Detailed descriptions of materials, equipment and methods used in this study are given in [Supplementary Material](#SM0){ref-type="supplementary-material"}. Production and characterization of anti-VEGFR2 affibody ------------------------------------------------------- The new antagonistic biparatopic affibody conjugate was designed and produced in *E. coli* BL21 Star (DE3) cells. Purity and size of the purified protein were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS). A maleimide derivative of 1,4,7-triazacyclononane,1-glutaric acid-4,7 acetic acid (NODAGA) was site-specifically conjugated to the unique C-terminal cysteine of the protein. The conjugated protein was purified by semipreparative reversed-phase high-performance liquid chromatography (RP-HPLC). Correct protein mass was confirmed by LC/ESI-MS. The purity of the conjugated protein was determined by analytical RP-HPLC. The secondary structure and thermal stability of the final NODAGA-Z~VEGFR2~-Bp~2~ conjugate (HEHEHE-Z~VEGFR2~-Bp~2~-Cys-Maleimide-NODAGA) were analyzed by circular dichroism (CD) spectroscopy. Binding of the new conjugate was detected by flow-cytometric analysis and by surface plasmon resonance-based biosensor assay. Radiolabeling and characterization of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ -------------------------------------------------------------------------- NODAGA-Z~VEGFR2~-Bp~2~ was incubated with indium-111 in ammonium acetate buffer, pH 5.5, at 85 °C for 30 min. The radiochemical yield of the conjugate in the crude mixture was determined by instant thin layer chromatography (ITLC). The stability of the compound was tested in 1000-fold molar excess of ethylenediaminetetraacetic acid (EDTA) and in PBS at a pH of 7.4. Purification of the radiolabeled conjugate for in vivo studies was performed using size exclusion NAP5-columns. The radiochemical purity was analyzed by radio-ITLC and verified by SDS-PAGE. In vitro characterization of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ ----------------------------------------------------------------- The temperature-sensitive SV40T-transformed pancreatic islet endothelial cell line Mus EC MS1 mouse endothelial (MS1) used for in vitro characterization was a kind gift from Dr. Jack L. Arbiser, Children\'s Hospital, Harvard Medical School, Boston, MA [@B32]. In vitro binding specificity and cellular processing were studied according to published methods [@B33]. Association to and dissociation of ^111^In-labeled NODAGA-Z~VEGFR2~-Bp~2~ from VEGFR2 were investigated in MS1 cells by quantitative real-time binding measurements using LigandTracer Yellow Instruments. The resulting data were analyzed in TraceDrawer™ to calculate the association rate (k~a~) and dissociation rate (k~d~) constants, as well as the dissociation constant K~D~. To estimate if \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ can detect changes in VEGFR2 expression in response to endothelial cell-targeted therapy, in vitro binding of the tracer to MS1 cells treated with heat shock protein 90 (HSP90) inhibitor 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin, Alvespimycin) was measured. Untreated cells were used as a control. The results of the binding experiments were correlated with the cell survival fraction, determined as described [@B34]. Small animal studies -------------------- All animal experiments were planned and performed in accordance with national legislation on laboratory animals\' protection and were approved by the Ethics Committee for Animal Research in Uppsala. In vivo stability of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ was studied in NMRI mice 15 min after intravenous injection of 4 µg (10 MBq) of protein in the tail vein and by comparison of the biodistribution of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ and \[^111^In\]In-acetate 2 h after intravenous injection. Targeting specificity, biodistribution of activity over time and imaging properties of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ were studied in Balb/c *nu/nu* mice bearing subcutaneous MS1 tumors. Balb/c nu/nu mice with subcutaneous PC-3 (prostate carcinoma) xenografts were used to verify in vivo targeting of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ to human VEGFR2. C57BL/6 mice with intracranial GL261 glioblastoma tumors were used to study the imaging properties of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~. Groups of 3-4 mice were used per data point if not stated otherwise. Mice bearing MS1 tumors were injected intravenously in the tail with 1, 4 and 20 µg of ^111^In-labeled compound (30 kBq), and biodistribution was studied 2 h post injection (pi). Additionally, biodistribution was studied 6 and 24 h pi with 4 µg of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ (30 kBq). The PC-3 xenografted mice were injected with 4 µg of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ (30 kBq), and biodistribution was studied at 2 h pi. For whole body SPECT/CT imaging, mice bearing MS1 tumors were injected intravenously with 4 µg of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ (10 MBq). The animals were sacrificed at 2 h pi, and SPECT/CT scans were carried out with a nanoScan SPECT/CT. Mice with intracranial murine glioblastoma GL261 tumors were monitored under anesthesia on a 3T nanoScan PET/MRI instrument every third day starting from day 12 after tumor inoculation. The mice with MRI-confirmed orthotopic GL261 glioma tumors were injected intravenously with \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ (1-40 µg, 6 MBq/µg). The animals were euthanized at 2 h pi, and SPECT/CT scans were carried out using a nanoScan SPECT/CT as described above. To confirm targeting specificity, an additional group of mice was injected with 4 µg of ^111^In-labeled Z~taq~-Z~taq~. SPECT raw data were reconstructed using the Tera-Tomo™ 3D algorithm. The CT raw files were reconstructed using Filter Back Projection. SPECT data were reconstructed for attenuation and scatter corrections with their respective CTs. SPECT-CT data were fused and analyzed in PMOD 3.510. SPECT images were presented as MIP (maximum intensity projection) on the RGB color scale, and CT were presented as MIP in grayscale. For the tumor and cerebellum, spheres of 4 mm radius were drawn on fused SPECT-CT images. Relative uptake of tracer (tumor-to-cerebellum) was evaluated in unmasked whole brain images. Regions of interests (ROIs) were drawn on fused SPECT-CT images along the trans-axial plane to create a mask of the whole brain volume. The whole brain mask was used to exclude pixels outside the selected area to visualize the distribution of activity in the brain among groups. Masking was performed only for visualization of the tumors as MIP. Data were analyzed with unpaired, two-tailed t-tests to determine if differences were significant (p\<0.05). Whole extracted brains were snap-frozen after imaging, and 20 µm sections were cut through the brain. The activity distribution in brain sections was studied using digital macroautoradiography with a Cyclone Storage Phosphor System and was analyzed using OptiQuant image analysis software. The signal intensities in the ROIs in tumor implants and in intact brain were compared. Further sections were processed for H&E staining. Additional brain sections were immunofluorescently stained for VEGFR2 and CD31. Results ======= Production and characterization of Z~VEGFR2~-Bp~2~ -------------------------------------------------- The Z~VEGFR2~-Bp~2~ protein (see **Figure [1](#F1){ref-type="fig"}A** for a schematic outline) was produced in *E. coli* and purified using IMAC. After site-specific conjugation of a maleimide derivative of the NODAGA chelator, the resulting protein was purified by semipreparative RP-HPLC. Analysis using RP-HPLC demonstrated a purity of over 96% (**Figure [1](#F1){ref-type="fig"}B**). Mass determination on LC/ESI-MS showed identical experimental mass compared with the theoretical value, 14,951 kDa (**Figure [1](#F1){ref-type="fig"}C**). CD spectroscopy demonstrated an alpha-helical content comparable to typical affibody molecules and high refolding capability after heating up to 90 °C (**Figure [1](#F1){ref-type="fig"}D**). The melting temperature of the new biparatopic affibody conjugate was determined to be 45 °C (**Figure [1](#F1){ref-type="fig"}E**). The interaction between NODAGA-Z~VEGFR2~-Bp~2~ and mVEGFR2 was analyzed using a surface plasmon resonance (SPR)-based biosensor assay to verify that conjugation of the NODAGA chelator had no negative influence on the binding to the receptor. The previously studied Z~VEGFR2~-Bp~2~-ABD [@B30] was included in the assay for comparison. The results from the assay showed similar binding to mVEGFR2 for NODAGA-Z~VEGFR2~-Bp~2~ compared with Z~VEGFR2~-Bp~2~-ABD, which suggested that the NODAGA conjugation did not appreciably affect association/dissociation of the interaction (**Figure [S1](#SM0){ref-type="supplementary-material"}**). A flow cytometry-based assay was performed to investigate the ability of Z~VEGFR2~-Bp~2~ to bind to the murine cell lines, MS1 (SV-40 transformed endothelial), PC-3 (prostate cancer) and GL261 (glioblastoma). Z~VEGFR2~-Bp~2~ was produced and purified in fusion with an albumin-binding domain (ABD) to allow detection [@B30]. Ramucirumab (anti-human VEGFR2 monoclonal antibody) and DC101 (anti-mouse VEGFR2 monoclonal antibody) were included for comparison. Cell binding of Z~VEGFR2~-Bp~2~ was detected using fluorescently labeled human serum albumin (HSA) for Z~VEGFR2~-Bp~2~ and Alexa Fluor® 647 anti-rat or anti-human IgG antibody for Ramucirumab and DC101. A clear shift in the fluorescent signal was observed for the MS1 cell line for both Z~VEGFR2~-Bp~2~ and DC101. For the GL261 and PC-3 cell lines, no significant shift in fluorescence was observed for Z~VEGFR2~-Bp~2~, Ramucirumab or DC101 compared to the negative controls (**Figure [2](#F2){ref-type="fig"}A**), strongly indicating that these tumor cell lines do not express VEGFR2. Radiolabeling of NODAGA-Z~VEGFR2~-Bp~2~ with indium-111, identity and stability of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ ----------------------------------------------------------------------------------------------------------------------- NODAGA-Z~VEGFR2~-Bp~2~ was successfully labeled with indium-111 with a radiochemical yield of 97±1% as determined by ITLC (n=34). After size exclusion purification, the radiochemical purity of the product was 100%. SDS-PAGE analysis of the labeled compound showed a single peak well-separated from the low molecular weight control peak represented by the free ^111^In acetate. Apparent specific activities up to 8.6 MBq/µg (apparent molar activity = 124 GBq/µmol) were obtained. The compound was stable in PBS and in excess EDTA up to 4 h (98±1% of activity associated with protein for PBS and 97±1% for EDTA challenge) (**Table [S1](#SM0){ref-type="supplementary-material"}**). In blood samples taken 15 min after injection of the tracer in mice, no traces of free indium-111 or ^111^In-labeled protein fragments were detected (data not shown). Comparison of biodistribution patterns of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ and free indium-111 2 h after injection (**Figure [S2](#SM0){ref-type="supplementary-material"}**) showed that the blood clearance of radiolabeled conjugate was much more rapid than for free indium-111, i.e., the activity concentration in blood was 24-fold lower for \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~. In contrast, activity uptake in liver was 15-fold lower after injection of free indium-111 than after injection of the radiolabeled conjugate. In vitro characterization of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ ----------------------------------------------------------------- The*in vitro* binding specificity assay demonstrated specific binding of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ to VEGFR2-expressing MS1 cells. Presaturation of receptors by adding a large molar excess of non-labeled protein, the natural ligand VEGF, or the anti-mVEGFR2 antibody DC101 resulted in a significant reduction of cell-associated activity (**Figure [2](#F2){ref-type="fig"}B**). \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ was rapidly internalized by MS1 cells (**Figure [2](#F2){ref-type="fig"}C**). After 8 h of incubation at 37 °C, 55% of cell-associated activity was internalized. Both the cell-associated activity and the internalized fraction plateaued after the 8 h time-point. The kinetics of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ binding to VEGFR2 were studied in MS1 cells in real time. The interaction followed a 1:1 Langmuir adsorption model (**Figure [2](#F2){ref-type="fig"}D**). The calculated dissociation constant at equilibrium was in the low picomolar range (K~D~ = 3±2 ×10^-11^ M) with a k~a~ of 8±1 ×10^4^ M^-1^s^-1^ and a k~d~ of 3±1 ×10^-6^ s^-1^. Treatment of endothelial MS1 cells with 17-DMAG was utilized as a model for initial assessment of the potential use of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ for therapy monitoring. The results showed that continuous incubation of cells with 25 nM 17-DMAG for 14 days resulted in complete cell eradication. Two hours of incubation of MS1 cells with 25 nM 17-DMAG resulted in a significant (p \< 5×10^-6^) decrease in \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ binding (**Figure [2](#F2){ref-type="fig"}E**). Incubation of MS1 cells with 25 nM 17-DMAG over 48 h resulted in a significant (p \< 0.0001) decrease in survival compared to untreated control (**Figure [2](#F2){ref-type="fig"}F**). Characterization of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ in small animal models ------------------------------------------------------------------------------- Targeting properties of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ were studied in BALB/c *nu/nu* female mice bearing MS1 tumors. Initially, the biodistribution and tumor targeting were studied 2 h pi for three different protein doses: 1, 4 and 20 µg/animal (**Figure [3](#F3){ref-type="fig"}A** and **Table [S2](#SM0){ref-type="supplementary-material"}**). The clearance of activity from the blood was efficient for all tested protein doses. There were no significant differences in the biodistribution patterns of activity after the injection of 1 and 4 µg. High activity uptake was found in kidneys, lungs, liver, spleen, and bones. In these organs (except for bones), the activity uptake was dose-dependent. In liver, lungs and spleen, the activity uptake decreased significantly (*p*\<0.05) with increased protein dose. Together with the decrease in activity uptake in receptor-expressing organs, the activity uptake increased in kidneys due to more protein being excreted via the renal pathway. The two injected protein doses of 1 and 4 µg provided similar values of tumor uptake. The significantly lower tumor uptake for the 20 µg dose compared to the lower doses indicated partial saturation of VEGFR2 binding and demonstrated in vivo specificity of the radiotracer. Tumor-to-nontumor ratios were higher for the injected protein dose of 4 µg than for 1 µg (**Figure [3](#F3){ref-type="fig"}B** and **Table [S2](#SM0){ref-type="supplementary-material"}**). However, for all tested doses, tumor activity uptake did not exceed the uptake in liver, spleen, and kidneys and was similar to the uptake in bones and lungs. The biodistribution of activity after the injection of 4 µg of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ was further studied 6 and 24 h pi (**Figure [3](#F3){ref-type="fig"}C** and **Table [S3](#SM0){ref-type="supplementary-material"}**). With time, the activity uptake decreased significantly in blood, lung, liver, spleen, and tumors. Simultaneously, the activity uptake increased significantly in the kidney (major excretory organ for affibody molecules). In other studied organs and tissues, the activity uptake was stable over time. Tumor-to-nontumor ratios significantly decreased over time with the exception of tumor-to-blood ratio (**Figure [3](#F3){ref-type="fig"}D** and **Table [S3](#SM0){ref-type="supplementary-material"}**). Very low activity uptake was found in brain tissue for all tested protein doses and times after injection. MicroSPECT/CT imaging of mice bearing MS1 tumors was performed at 2 h pi (**Figure [4](#F4){ref-type="fig"}A**). Tumors were visualized with prominent contrast. High activity uptake was observed in liver and kidneys as expected from ex vivo data. The activity uptake in bones was heterogeneous with a higher uptake in the joints. Uptake of activity was also observed in flat bones (sternum, ribs, and hip) and could be assumed in lymph nodes (axillary, inguinal, popliteal, and lymph nodes of head and neck area) and the pineal gland in brain. To confirm these findings, samples of skin with axillary lymph nodes, breast chest with ribs, femur, kidney, brain and tumor were dissected directly after microSPECT/CT imaging. On autoradiography images (**Figure [4](#F4){ref-type="fig"}B**), elevated activity uptake was detected in lymph nodes, ribs and joints. The activity uptake in kidneys was concentrated in the cortex. The activity uptake in the pineal gland was confirmed. Additionally, uptake was detected in lateral ventricles. The activity uptake in tumors was heterogeneous, in agreement with the SPECT/CT images. VEGFR2 targeting was also assessed in mice bearing PC-3 xenografts. Distribution of activity in healthy organs and tissues was in good agreement with the data obtained for the mice bearing MS1 tumors (**Table [S4](#SM0){ref-type="supplementary-material"}**). However, the activity uptake in tumors of epithelial origin was three-fold lower than for tumors of endothelial origin. This difference translated into lower tumor-to-nontumor ratios for the PC-3 model: tumor-to-blood and tumor-to-muscle, 3; tumor-to-brain, over 20 (**Table [S4](#SM0){ref-type="supplementary-material"}**). The growth of intracranial GL261 tumors was monitored using microMRI starting from day 14, when the tumor diameter was approximately 2 mm, to day 21, when the diameter had increased two-fold (**Figure [5](#F5){ref-type="fig"}A** and **Figure [S3](#SM0){ref-type="supplementary-material"}**). Activity uptake in intracranial tumors was analyzed as tumor-to-normal brain ratios on microSPECT/CT brain images and on ex vivo autoradiography. Activity uptake in tumors 2 mm in diameter was confirmed by macroautoradiography, but visualization using SPECT was not convincing (**Figure [S3](#SM0){ref-type="supplementary-material"}**). Based on results from macroautoradiography, the optimal injected protein dose of 4 µg was confirmed (**Figure [S4](#SM0){ref-type="supplementary-material"}**). It should be noted that according to macroautoradiography data, the activity uptake was more homogeneous in smaller tumors than in tumors with diameters suitable for microSPECT/CT imaging. Intracranial tumors of 4 mm diameter were visualized on SPECT/CT images (**Figure [5](#F5){ref-type="fig"}B-C**). In the whole brain masked images, the tumor was easy to delineate when 4 µg of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ injected (**Figure [5](#F5){ref-type="fig"}D** and**Figure [S5](#SM0){ref-type="supplementary-material"}**). The tumor-to-cerebellum ratios after injection of 4 µg \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ were significantly higher than the ratios observed for the 40 µg injected dose and for the non-targeting conjugate (**Figure [5](#F5){ref-type="fig"}E**). Microautoradiography of cryosectioned CNS tissue was in good agreement with SPECT/CT images. A high activity uptake was detected in the tumor area (subsequently confirmed by H&E staining) as observed on MRI in the mice injected with 4 µg of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~, and a lower uptake was observed in the mice injected with a blocking amount of targeting or the same dose of non-targeting dimer (**Figure [6](#F6){ref-type="fig"}A**). H&E staining visualized tumor areas (**Figure [6](#F6){ref-type="fig"}B**). Tumor-to-brain ratios based on autoradiography results were significantly higher for the mice injected with 4 µg \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ compared to the tumor-to-brain ratios obtained for the 40 µg injected dose and for the non-targeting conjugate (average for 26-29 slides) (**Figure [6](#F6){ref-type="fig"}C**). Immunofluorescent staining for the endothelial markers platelet-endothelial cell adhesion molecule-1 (PECAM-1; CD31) and VEGFR2 identified abundant vasculature in the glioblastomas (**Figure [7](#F7){ref-type="fig"}**), validating the availability of the target. Discussion ========== Bevacizumab, a VEGFA-targeting antibody blocking ligand binding to VEGFR2, was approved by FDA for recurrent GBM in 2009. It prolongs median progression-free survival after the primary diagnosis and improves progression-free survival and overall survival in after recurrence [@B9]. However, the assessment of therapeutic effects in GBM and the radiologic evaluation criteria remain challenging and even controversial, in particular distinguishing true progression from treatment-related changes (pseudoprogression or pseudoresponse) [@B35]. Thus, there is an urgent need for advanced techniques for noninvasive visualization of tumor angiogenesis in GBM to guide therapeutic design and for therapy monitoring. It has been shown in preclinical studies that bevacizumab decreases neovasculature formation, reduces microvessel density, and normalizes vasculature [@B36]. These effects have also been observed in GBM patients with decreased VEGFR2 expression during treatment [@B11],[@B37]. Preclinical and clinical evidence thus point to VEGFR2 expression as a possible predictive or pharmacodynamic biomarker for antiangiogenic therapy in GBM. For example, it was found in a Phase II trial that GBM patients with high VEGF/VEGFR2 ratios (high VEGF and low VEGFR2) had worse outcomes when treated with bevacizumab [@B10]. Thus, radionuclide molecular imaging of VEGFR2 expression in GBM might provide important diagnostic information and make antiangiogenic therapy of GBM more personalized. It is conceivable that the initial elevated expression would predict response to therapy, and its downregulation during treatment would be a marker of response. Here, we present the binding affinity and specificity, internalization rate, optimal injected protein dose and optimal imaging window for a new VEGFR2-specific reagent. The properties of this new tool indicate that it has important potential for clinical monitoring of GBM. The labeling of NODAGA-Z~VEGFR2~-Bp~2~ with indium-111 was successful, with high apparent molar activities of up to 124 GBq/µmol that may be required for imaging of targets with low density when limitations on the injected mass of the imaging probe could be an issue [@B38]. \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ showed excellent stability and retained binding specificity to VEGFR2 in vitro and in vivo. The new binder competed with the binding of the natural ligand VEGFA to VEGFR2, a feature that could be exploited for monitoring of receptor occupancy during antiangiogenic treatment. The potential of VEGFR2 imaging using \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ to predict response to neovasculature-targeted therapy was assessed in vitro using the HSP90 inhibitor 17-DMAG. HSP90 regulates many aspects of tumor angiogenesis, and its inhibition downregulates VEGFR2 expression [@B39]. Binding of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ to MS1 cells decreased after treatment with 17-DMAG (**Figure [2](#F2){ref-type="fig"}E-F**). \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ demonstrated a somewhat higher internalization rate compared to affibody conjugates targeting other tyrosine kinase receptors (e.g., HER2 [@B33]) that warrants the use of a residualizing radiometal label. Ligand-receptor binding kinetics measured in real-time showed dissociation constant values in the low picomolar range (30 pM). The affinity of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ for binding to VEGFR2 was two orders of magnitude better than affinities of either parental affibody monomers. This finding corroborates our hypothesis that fusion of two affibody binders with moderate affinity towards different epitopes situated in close proximity should improve the binding affinity of the biparatopic molecule. The low dissociation rate constant of 3×10^-6^ s^-1^ together with the internalization should be beneficial for targets exposed to blood flow where the ligand can be easily cleared if dissociated from the receptor. In conclusion, Z~VEGFR2~-Bp~2~ has properties that indicate that it is particularly well suited for detection of neovasculature in CNS tumors. Finding the optimal injected protein dose and the optimal imaging window was the focus of the initial in vivo characterization. The overall biodistribution pattern of the new agent was in good agreement with other VEGFR2-targeting agents ([@B40]-[@B43], as shown particularly in Figure 8 in [@B44]). The in vivo data showed a pronounced influence of injected protein dose on the imaging contrast (**Figure [3](#F3){ref-type="fig"}A-B**). The blood clearance of activity was fast, and the tumor-to-blood ratios 2 h pi were approximately 10 for MS1 (endothelial) and 3 for PC-3 (epithelial) tumors, reflecting different target density in a tumor composed of endothelial cells compared to an epithelial tumor with VEGFR2 expression confined to the host-derived tumor vasculature. Remarkably, the tumor-to-blood ratios provided by \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ in both tumor models were superior to ^64^Cu-labeled ramucirumab (\~1 at 48 h pi), ^61/64^Cu-labeled VEGF121 (\~1 at 8 h pi for ^61^Cu and \~2 at 16 h pi for ^64^Cu), ^68^Ga -labeled VEGF121 (\<1 at 4 h pi), and scVR2 (\<2 at 2 h pi) [@B26],[@B40]-[@B43]. A high tumor-to-blood ratio is important for imaging. It determines the imaging contrast in vivo as blood-associated activity contributes to activity in all organs. Increasing the injected protein dose from 1 to 4 µg increased the tumor-to-nontumor ratios, in agreement with earlier studies where the increase of injected affibody mass resulted in increased imaging contrast for ubiquitously expressed molecular targets, e.g., HER1 and HER3 [@B45],[@B46], likely due to partial saturation of receptors in normal tissues and reduced sequestering of radiolabeled conjugate. High and partially blockable activity uptake was seen in liver, lungs and spleen (**Figure [3](#F3){ref-type="fig"}A**). This finding could possibly be explained by the low expression of VEGFR2 in epithelial cells. However, a more likely explanation would rest on the assumption that the small size of the affibody probe would allow it to cross the vessel wall, e.g., at endothelial junctions in the peripheral vasculature and to bind to VEGFR2 expressed on the basolateral aspect of the endothelial vessel lining. In contrast, in the CNS, the tight vessel barrier (blood-brain-barrier) would prevent extravasation of the affibody, explaining the better tumor-to-organ ratio observed for glioblastoma compared to brain tissue [@B47]. Thus, the high uptake in liver, lung and spleen could, to some extent, represent specific binding of the affibody to the vasculature in these organs. It should be noted that the same organs demonstrated high uptake of ^61/64^Cu- and ^68^Ga-labeled VEGFA isoforms, VEGFR121, in earlier studies [@B41]-[@B43]. High activity uptake was also found in kidneys and bones. Bone uptake is most likely due to VEGFR2 expression in the vasculature and on certain leukocyte populations in the red bone marrow [@B48], while the high kidney uptake could partially reflect the high reabsorption of the affibody conjugate after renal excretion [@B49]. However, specific binding to VEGFR2 expressed in nonendothelial cell types cannot be ruled out [@B50]. The high uptake in the liver is particularly challenging for imaging, as the liver is a large and well-perfused organ with a well-fenestrated vasculature. The high capacity for liver entrapment could reduce the bioavailability of the tracer. Therefore, finding the optimal injection dose would be even more challenging. The overall higher tumor-to-organ ratios for the 4 µg dose in the MS1 model indicate that this dose might be preferred for investigation of biodistribution over time. The relatively rapid washout of activity from the tumor could be explained by dissociation of the affibody-receptor complex despite the strong affinity of Z~VEGFR2~-Bp~2~ to VEGFR2 (low picomolar range). This finding supports our hypothesis that high binding affinity is required for targeting of receptors that are exposed to blood flow. Consequently, the overall tumor-to-organ ratios decreased over time (**Figure [3](#F3){ref-type="fig"}D**), suggesting that the optimal time point for image acquisition should be shortly after administration. SPECT/CT images acquired 2 h pi with 4 µg \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ were in good agreement with the data obtained in ex vivo measurements (**Figure [4](#F4){ref-type="fig"}A**). The overall biodistribution pattern indicates that the diagnostic potential of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~, as well as other VEGFR2 binders, will be limited in most cancers due to high uptake in major metastatic sites, such as liver, lungs, lymph nodes and bones. The tumor-to-normal brain ratios seen for \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ in vivo (\~80 at 2 h pi) were much higher than reported for ^64^Cu-labeled ramucirumab (\~10 at 48 h pi) and ^61^Cu-labeled VEGF121 (\~5 at 8 h pi) \[40.41\], suggesting that the conjugate could be used for VEGFR2 imaging in GBM. For imaging of GBM in a murine model, GL261 was chosen as this glioma model is known to replicate phenotypic characteristics of the human disease, such as invasive growth [@B51]. Preliminary autoradiography results on brain slides of mice injected with \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ demonstrated low activity uptake in the brain limited to the pineal gland and lateral ventricles (**Figure [4](#F4){ref-type="fig"}B**). Using SPECT/CT and post-imaging autoradiography in mice bearing GL261 tumors, the optimal injected protein dose (4 µg/mouse) and required tumor size (4 mm in diameter) were verified (**Figure [S3](#SM0){ref-type="supplementary-material"}** and **Figure [S4](#SM0){ref-type="supplementary-material"}**). SPECT/CT images showed a high accumulation of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ in the area of the brain containing the tumor implants detected in MR images (**Figure [5](#F5){ref-type="fig"}**). Imaging of VEGFR2 in the orthotopic GBM murine model was VEGFR2-specific considering that both the increase in injected protein dose and the use of non-targeting size-matched affibody conjugate resulted in significantly decreased tumor-to-brain ratios (**Figure [5](#F5){ref-type="fig"}E**). In SPECT images, tumor-to-cerebellum ratios were two-fold higher for animals injected with 4 µg of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ compared to animals injected with lower apparent specific activity or a size-matched, non-targeting affibody. The difference in activity uptake in tumors and normal brain tissue was even more pronounced when autoradiograms of brain slices were analyzed: tumor-to-normal brain ratios were three-fold higher for the cohort injected with 4 µg compared to the 40 µg group and six-fold higher compared to the non-targeted group (**Figure [6](#F6){ref-type="fig"}C**). This discrepancy between SPECT imaging and autoradiography can be explained by several factors: the relatively small size of the tumors that both limited delineation accuracy and contributed to a partial volume effect, heterogeneous activity uptake within tumors, and the limited quantification accuracy of SPECT imaging. The results of this study demonstrate that our strategy for the development of a high-affinity biparatopic anti-VEGFR2 affibody conjugate by dimerization of two distinct epitope binders was successful. Often, the dimerization of affibody molecules results in decreased tumor uptake. This phenomenon is a consequence of the reduction in tumor penetration that is not compensated by the increased binding affinity and has been observed for anti-HER2 and anti-HER1 affibody molecules [@B52],[@B53]. However, conceivably, targeting of VEGFR2 does not require deep tissue penetration as the target is located on endothelial cells. In this study, we demonstrated that the anti-VEGFR2 biparatopic affibody conjugate is potentially suitable for molecular targeting in GBM for patient stratification to antiangiogenic therapies and monitoring of therapy response. In clinical practice, imaging of VEGFR2 expression will not be used for tumor detection, but rather for target detection, concentrating on the areas with already confirmed tumors, e.g., by MRI. Our clinical experience with detection of HER2 expression in breast cancer patients using ^68^Ga-labeled affibody molecule ABY-025 demonstrated that even tumors with low activity uptake due to low receptor expression can be successfully visualized [@B54] and that absorbed doses to liver and kidneys were less than the maximum allowed to a single organ in a healthy adult after administration of a clinically relevant amount of gallium-68 [@B55]. Moreover, the tumor-to-blood and tumor-to-brain ratios for the new imaging tracer were superior to those recorded for the natural ligand to VEGFR2 and anti-VEGFR2 monoclonal antibodies. An additional advantage of affibody-based tracers is the less expensive production by prokaryotic cells [@B56]. Selection of an optimal imaging strategy is essential for the development of a novel imaging probe. We had to take into account that imaging 24 h or later might provide higher contrast, as has been observed for anti-HER2 affibody molecules \[^111^In\]In-ABY-025 [@B57]. Therefore, the use of some long-lived radionuclide would be desirable for the initial evaluation. Furthermore, advantages of clinical PET over SPECT (better resolution and better attenuation correction providing more accurate quantification) are not valid for small animal scanners. The positron range in tissues results in noticeable degradation of the resolution of small animal PET compared to SPECT. Our scanner, the nanoScan SPECT, has around 2-fold better spatial resolution compared to the nanoScan PET [@B58],[@B59]. This difference is translated into a smaller partial volume effect in quantitative imaging of small lesions, such as the orthotopic glioblastoma model. The uncertainty in attenuation correction is much lower in preclinical SPECT when the size of the attenuating tissue is much smaller. Dosimetry considerations are not important for small animal imaging, which permits injection of higher activity and compensation for reduced registration efficiency. Therefore, we selected the long-lived single-photon emitter ^111^In for this initial study. Our findings indicate that the optimal imaging time window for this agent is shortly after administration. This finding is compatible with the half-life of a short-lived generator-produced positron-emitting gallium-68, thus allowing the use of PET in clinics, which would provide higher imaging sensitivity and more accurate activity quantification. We foresaw such a possibility when designing the tracer. Therefore, we selected the NODAGA chelator, which is suitable for ^68^Ga as well. NODAGA, a cyclic tri-aza chelator provides stable labeling of peptides and scaffold proteins with ^68^Ga for PET and ^111^In for SPECT [@B60]-[@B62]. Some reoptimization of labeling conditions (temperature, pH and time) and preclinical validation should permit the use of \[^68^Ga\]Ga-NODAGA-Z~VEGFR2~-Bp~2~ in clinical PET. Our studies demonstrated that activity concentration in blood was less than 0.5% ID/g, even by 2 h pi for all tested protein doses and appreciably decreased with time, which was in good agreement with data for other dimeric affibody molecules [@B52],[@B53], and 15-fold lower than after injection of free indium-111. This finding indicated high in vivo stability of the ^111^In-NODAGA complex. Possible transchelation of indium-111 to transferrin (the most abundant blood protein with chelating properties) should cause elevated activity concentration in blood with very slow elimination, which was not observed. Cross-reactivity between murine and human VEGFR2 is critical for clinical translation and has been a focus in the development of the binders from the initial selection of the first-generation candidates [@B29]. That selection effort yielded two variants that bound human VEGFR2 and were also cross-reactive for murine VEGFR2. After affinity maturation and construction of the biparatopic high-affinity binder (Z~VEGFR2~-Bp2), we again confirmed cross-reactivity between human VEGR2 and murine VEGFR2 [@B29],[@B30]. The Z~VEGFR2~-Bp2 hence binds both orthologs with similar affinity and the affinity measurements presented here are in agreement with the previously published results, indicating a strong position for future clinical translation. In conclusion, the high-affinity anti-VEGFR2 affibody conjugate \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ specifically targeted VEGFR2 in vivo and visualized VEGFR2 expression in glioblastoma in a murine orthotopic model. Tumor-to-blood ratios for \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ were higher compared to other VEGFR2 imaging probes. \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ appears to be a promising probe for in vivo, noninvasive visualization of tumor angiogenesis in GBM. Further studies should concentrate on the development of imaging probes suitable for PET to evaluate the imaging potential of the new agent, such as therapy monitoring. Supplementary Material {#SM0} ====================== ###### Supplementary methods, figures and tables. ###### Click here for additional data file. Dr. Jack L. Arbiser, Children\'s Hospital, Harvard Medical School, Boston, MA, is acknowledged for the kind gift of the temperature-sensitive SV40T-transformed pancreatic islet endothelial cell line MS1. The authors would also like to thank Marie Hedlund, Department of Immunology, Genetics and Pathology, Uppsala University, for expert technical assistance. The molecular imaging work in this publication was supported by the Wallenberg infrastructure for PET-MRI (WIPPET), a Swedish nationally available imaging platform at Uppsala University, Sweden, financed by Knut and Alice Wallenberg Foundation and Science for Life Laboratory (SciLifeLab pilot infrastructure grant). This work was further supported by the Swedish Cancer Society (grants CAN 2017/649 (JL), CAN2013/586 and CAN 2016/463 (SS), CAN2014/474, CAN 2017/425 (AO), CAN2015/350 (VT), and CAN2016/585 (LCW)), the Swedish Research Council (grants 621-2012-5236 (SS), 2015-02509 (AO), and 2015-02353 (VT)), the Swedish Agency for Innovation VINNOVA (grants 2016-04060 (AO) and 2017-02015 (SS, JL)) and the Wallenberg Center for Protein Technology (SS, JL). CNS : central nervous system GBM : glioblastoma multiforme HER : human epidermal growth factor receptor %ID/g : percent injected dose per gram NODAGA : maleimide derivative of 1,4,7-triazacyclononane,1-glutaric acid-4,7 acetic acid PET : positron emission tomography SPECT : single photon emission computed tomography VEGF : vascular endothelial growth factor VEGFR2 : vascular endothelial growth factor receptor-2. {#F1} ![**(A)** Flow-cytometric analysis of binding of Z~VEGFR2~-Bp~2~ to different cell lines, MS1, PC-3 and GL261. Binding of Z~VEGFR2~-Bp~2~-ABD to cells was detected by Alexa Fluor (AF) 647-labeled HSA. AF-647 labeled HSA was included as negative control. Ramucirumab (anti-hVEGFR2) and DC101 (anti-mVEGFR2) were included as controls for binding to human (PC-3) or mouse (MS1 and GL261) cell lines. Blue histograms indicate affibody/antibody samples, and red histograms are negative controls. The experiment was performed in duplicate. **(B)** In vitro binding specificity of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ tested on MS1 cells in the presence or absence of non-labeled Z~VEGFR2~-Bp~2~, VEGFA, or anti-mVEGFR2 antibody DC101. The cell-associated activity is presented as a percentage of the total added activity (average value from three cell dishes ± SD). **(C)** Binding and internalization of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ by MS1 cells. Data are presented as average values from three cell dishes ± SD. Error bars might not be visible because they are smaller than point symbols. **(D)** Real-time binding data of the \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ interaction. Arrows indicate the concentration change. **(E)** Binding of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ to MS1 cells after 2 h treatment with 25 nM 17-DMAG. An equal amount of cells per culture dish was seeded, and equal \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ activity was added to each cell culture dish. The data are presented as the average value for three cell dishes ± SD. Uptake by treated cells was significantly (p \< 5×10^-6^) lower than by untreated controls. **(F)** Survival of MS1 cells after treatment with 25 nM 17-DMAG over 48 h. The data are presented as the average value for three cell culture flasks ± SD.](thnov08p4462g002){#F2} ![**(A, C)** Comparative biodistribution of different injected protein doses (1, 4 and 20 µg/animal) of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ in BALB/C nu/nu in mice bearing MS1 tumors 2 h pi and biodistribution over time 2, 6 and 24 h pi (4 µg/animal). **(B, D)** Tumor-to-normal-tissue ratios 2 h pi (1, 4, 20 µg), 6 h pi (4 µg), and 24 h pi (4 µg).](thnov08p4462g003){#F3} ![**(A)** Coronal MIP SPECT/CT images showing tracer distribution in BALB/C nu/nu mice bearing MS1 tumors. The animal used for imaging was injected with 4 µg of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ and euthanized 2 h pi. LN - lymph node, L - liver, K - kidneys, FB - femur and bone, PG - pineal gland, R - ribs, T - tumor. **(B)** Digital images (right) showing activity distribution detected with macroautoradiography in tissues dissected directly after SPECT/CT imaging.](thnov08p4462g004){#F4} ![**(A)** In vivo MRI (prior to the injection) and SPECT/CT **(B)** coronal and **(C)** transaxial imaging of animals with intracranial GL261 tumors at 2 h pi of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~. Animals were injected with 4 µg (left column), 40 µg (middle column) of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~, or 4 µg of \[^111^In\]In-NODAGA-Z~tag~-Z~taq~ (right column). **(D)** Coronal MIP SPECT images showing tracer distribution in the brains of mice bearing intracranial GL261 tumors. For masking, brain VOI was defined using CT images of fused SPECT/CT. The range of the RGB color scale was 0.1-0.6 for Z~VEGFR2~-Bp~2~ and 0.3-0.7 for Z~tag~-Z~taq~ for comparability. **(E)** Tumor-to-brain ratios based on imaging analysis.](thnov08p4462g005){#F5} ![**(A)** Macroautoradiography and **(B)** H&E staining of brain slices of mice bearing intracranial GL261 tumors. Animals were injected with 4 µg (left column) or 40 µg (middle column) of \[^111^In\]In-NODAGA-Z~VEGFR2~-Bp~2~ or 4 µg of \[^111^In\]In-NODAGA-Z~tag~-Z~taq~ (right column) and were euthanized 2 h pi. **(C)** Tumor-to-brain ratios based on macroautoradiography analyses (n = 26-29).](thnov08p4462g006){#F6} {#F7} [^1]: \* These authors contributed equally [^2]: Competing Interests: VT, JL, AO, and SS are the members of the scientific advisory board of Affibody AB. SS, VT and AO are minority share owners of Affibody AB. Affibody AB holds intellectual property rights and trademarks for Affibody molecules. BM, RG, FPR, EL, RKS, FF, SSR, LCW declare no potential conflict of interest.

Introduction {#Sec1} ============ Global seaweed production, largely derived from aquaculture (96.5 percent by volume of the wild-collected and cultivated aquatic plants combined), has changed considerably since 2005^[@CR1]^. This production increased from 13 million in 2005 to reach 30 million tons (live weight) in 2016. Moreover, 99% of the world's production comes from Asian countries, notably China (47.9%), Indonesia (38.7%), Philippines (4.7%), Republic of Korea (4.5%), Democratic People's Republic of Korea (1.6%), Japan (1.3%) and Malaysia (0.7%). Furthermore, trade in aquatic plants increased from USD 60 million in 1976 to more than USD 1 billion in 2016, with Indonesia, Chile and the Republic of Korea the major exporters, and China, Japan and the United States of America the leading importers. Accordingly, red seaweed production accounts for 53% of the world production^[@CR1]^. The most exploited red algae, expressed in a million tons year^−1^, are *Euchema* seaweeds nei and *Eucheuma* spp. (10.5), *Gracilaria* spp. (4.1) and *Porphyra* spp. (1.3). In contrast, the red alga *Gracilaria* spp. was barely farmed at all in 1990. However, the increase in *Gracilaria* spp. production, by aquaculture, is mainly due to the growing demand for agar. Consequently, *Gracilaria* spp. aquaculture has been initiated by many countries, in different regions of the world, such as Thailand, Chile, Vietnam, Portugal, Australia, Brazil and India^[@CR2]^. The red alga *Gracilaria gracilis*, which grow in Asian coasts, was introduced into the Mediterranean Sea^[@CR3]^, and established in the lagoons^[@CR4]--[@CR7]^. Consequently, the alga was found all year round, but is the most component of the BL flora between April and June^[@CR4]^. In Tunisia, the quantity of seaweeds harvested, was restricted along the Bizerte Lagoon and the Tunis Lake, which is inadequate to supply the raw material requirement of the industries. Hence, *G. gracilis* aquaculture was indicated as the main solution^[@CR4]^. However, the BL was chosen to initiate many cultivation attempts of *G. gracilis*. As a result, a low biomass supply was obtained due to the interaction with environmental factors (Grazing, epiphitism, hydrodynamism, etc...) and limited surface suitable for the benthic culture methods. As a consequence, these culture methods were applied only in a depth less than 2 m, which represent a 10% of the BL surface^[@CR8]^. Furthermore, agar obtained (gel strength less than 400 g cm^−2^) was of low quality compared to that extracted from *Gelidium latifolium* (gel strength higher than 800 g cm^−2^), which considered as the highest and used in many industrial applications^[@CR9]^. Hence, no large-scale culture of *G. gracilis* is being developed outside the Asian region. That's why suspended culture method should be an alternative to enhance biomass production in lagoon and sea^[@CR8],[@CR9]^. In addition, when we move toward the lagoon depth and sea, we will look for the possibilities to improve the alga quality. Around the world *Gracilaria* biomass was used usually for agar extraction. In contrast, it contains a wide variety of valuable compounds, such as proteins and pigments^[@CR10]^. The cascading biorefinery, as an alternative to single product extraction approach, aim to extract all components present in the algae biomass^[@CR11],[@CR12]^. According to^[@CR13],[@CR14]^, it is financially attractive to firstly extract R-PE, and then agar will be extracted from the by-product obtained. Furthermore, the agar by-product can be treated as waste to produce biofuels. Consequently, it is necessary to study the ecophysiology responses of *G. gracilis* to the various factors, which affect their growth and chemical composition, such as light intensity, nutrients, salinity and temperature variations. In Tunisia, no attempt has been done to understand *G. gracilis* behavior in deeper lagoon and sea. Therefore, the purpose of this study was to provide new insights on *G. gracilis* growth capacities and biochemical composition (agar, proteins and R-PE) in the BL and the BB. In addition, it may also provide knowledge on algal biomass uses. Materials and methods {#Sec2} ===================== Preparation of experimental material {#Sec3} ------------------------------------ *Gracilaria gracilis* was collected between 0.5 and one meter depth from the Bizerte Lagoon (Fig. [1](#Fig1){ref-type="fig"}), North Tunisia (37°13′N; 9°55′E) at the end of Februray 2016. The collected thalli were transported to the laboratory in a cool environment to reduce stress. Collected samples were subjected to a series of washing steps using filtered sea water to eliminate diatoms contamination, epiphytes and other competing organisms. Then, the cleaned thalli, were placed in a tank with physicochemical parameters (dissolved oxygen, temperature and salinity) similar to those of the lagoon. Homogenous thalli with bright-red color, similar lengths and branches were selected.Figure 1(**a**) Situation on Tunisia map within the Mediterranean region. (**b**) *Gracilaria gracilis* culture area. (**c**) Culture site location in the Bizerte Lagoon and Bay. Cultivation of gracilaria gracilis {#Sec4} ---------------------------------- The *G. gracilis* culture experiments were conducted from March to May 2016 (90 days), in the Bizerte Lagoon (BL; 37°13N, 9°51′W) and Bay (BB; 37°15N, 9°59 W) in North Tunisia (Fig. [1](#Fig1){ref-type="fig"}). The experimental sites were located within sites specifically devoted to aquaculture with average water depths of 8 and 20 m for the BL and the BB, respectively. The suspended culture in the two sites was carried out using lantern nets (Fig. [2](#Fig2){ref-type="fig"}). The lantern net is formed by solid steel rings and crossbars coated with anti-corrosive. The crossbars were used to fix the *G. gracilis* thalli. Each lantern net is 3.75 m in length and contained fifteen experimental baskets (40 cm of diameter and 0.25 m in height) enclosed by monofilament netting of 6 mm mesh size. Each compartment has an opening through which thalli can be inserted or removed. Lantern nets loaded with thalli were hung down into the water column from long lines with buoys, which were placed in 1.3 m intervals. Lantern nets followed the tide without changing their position relative to the water surface during tidal cycles; get a vertical suspension due to a heavy-duty chain length. A synthetic rope of 36 mm in thickness and threefold the water depth in length was attached to the chain and rose to the marker buoys. Prepared tufts (500 g) were placed in the basket, being attached to the crossbars by a braid wire. Every lantern net, as well as baskets, was marked in such a way that each tuft represents an experimental unit. Three lantern nets were used in each site. As a result, at each depth, there were three experimental samples for statistical analyses. Lantern nets were then immersed in the filtered sea water, whose salinity was gradually increased into that of the BB to prevent thallus loss until they were transferred to the culture site.Figure 2Lantern nets hang down into the water column in Bizerte Bay. Weights of tufts were recorded at the beginning and at the end of the experiments to quantify algae growth. DGR was calculated by the formula in^[@CR15]^:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\rm{DGR}}( \% \,{{\rm{day}}}^{-1})={\rm{In}}({\rm{Wf}}/{\rm{W}}0)/{\rm{t}}\times 100$$\end{document}$$where Wf is the final fresh weight after the t days of culture, and W0 is the initial fresh weight. At the end of the culture period, a percentage tufts loss (PTL) was calculated using the following formula:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\rm{PTL}}( \% )=({\rm{Nf}}/{\rm{N}}0)\times 100$$\end{document}$$where Nf and N0 were the final and initial number of tufts. Environmental measures {#Sec5} ---------------------- Three water samples were collected every month (March, April and May) from the BL and the BB, in acid-washed 1 L plastic bottles from a depth of 0.5 to 1 m using a Van Dorn water sampler three liters. For each sample taken, three replicates were analysed. Water samples fixed with 0.5 mL of H~2~SO~4~ 4N. In the laboratory, triplicate samples (3 L) were filtered using Whatman® Grade GF/C Glass Microfiber filters to determine NO~3~^−^, NH~4~^+^ and PO~4~^−^. Water samples were analysed for Ν and Ρ according to^[@CR16],[@CR17]^ methods. During the culture period Secchi depth, temperature, salinity and dissolved oxygen were measured. Three measures of Secchi depth per month were done according to the recommendations of^[@CR18]^, by the same person at the same time of the day. Water temperature, oxygen and salinity were measured *in situ* using Hack multi-parameter (HQ40D), at the same time in the day, according to the recommendations of^[@CR19]^. Agar extraction and quality determination {#Sec6} ----------------------------------------- At the end of the culture period, dry-cleaned seaweed samples were first washed with tap water to remove salts. Then, they were placed in 400 mL of a 5% H~2~SO~4~ solution for 1 h at room temperature and further rinsed thoroughly using tap water. Agar extraction was performed in acid-washed 500 mL glass bottles at 100 °C for 90 min using a 2.5 (% w/v) of dried alga and distilled water. The heated solution was then filtrated using a bûchner filter and a vacuum pump. The filtrate obtained was then transferred to a flat steeled recipient until it was cooled at room temperature for 20 min and then frozen overnight at −18 °C. Next day, the filtrate was thawed at room temperature until a thin agar film was formed. The agar yield was calculated as follows:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\rm{Agar}}\,{\rm{yield}}( \% )=({\rm{Wa}}/{\rm{Ws}})\times 100$$\end{document}$$where Wa is the dry agar weight and Ws is the dry seaweed weight (g). Sulfaphate content in polysaccharides was determined turbidimetrically using Barium chloride after acid hydrolysis as described by^[@CR20]^. First, a barium chloride-gelatin solution was prepared by dissolving 600 mg of gelatin in 200 mL heated osmosis water (60--70 °C) and placed for 1 to 2 h at room temperature after cooling to 4 °C during 16 h. Then, 2 g of Barium chloride was dissolved in the resulting gelatin solution and left to stand for 2--3 h. Next, a hydrochloric acid solution (HCL 0.5N) was prepared. Thereafter, a sulfate standard curve was generated from a series of K~2~SO~4~ solutions (3 mg mL^−1^) containing between 0.02 and 0.20 mg mL^−1^. Lastly, agar (10 mg) was weighed and dissolved in 0.5 mL of hydrochloric acid for two hours at 100 °C. The hydrolyzate was compensated to 10 mL and centrifuged at 5000 G for 10 minutes. Then, 1 mL was placed in a test tube with the presence of 1 mL of HCl (0.5N) and 0.5 mL of barium chloride-gelatin solution, then compensated by 9 mL of osmosis water and mixed by vortex. After 15 min at room temperature, the absorbance was determined at λ = 360 nm with UV-visible spectrophotometer (Jenway 6405 type) and the absorbance value was obtained. Hydrochloric acid solution was used as a blank. The 3,6-AG content was determined by the colorimetric method of^[@CR21]^, using the resorcinol--acetal reagent and fructose as standard. The acetal solution was prepared by diluting 1 mL of acetal in 100 mL of deionized water. The resorcinol solution was prepared by diluting 150 mg of resorcinol in 100 mL of deionized water. Then, 9 mL of resorcinol solution was added to 1 mL of acetal solution diluted to 1/25. Next, 2 mL of agar solution (0.02 mg mL^−1^) was transferred into a test tube containing 10 mL of cold resorcinol-acetal reagent, mixed thoroughly and allowed to cool in ice bath for 3 min. Then, the tube was placed into water bath (80 °C) for 10 min. After cooling (15 mn) at room temperature, the absorbance was determined at λ = 555 nm with UV-visible spectrophotometer (Jenway 6405 type). A standard curve was prepared using D-fructose solution concentration, ranging from 0.018 to 0.09 mM. The 3,6-AG content was calculated and expressed as the percentage (dry weight basis). Experiments were performed in triplicate. Determination of crude protein and R-PE {#Sec7} --------------------------------------- Crude protein was determined according to the^[@CR22]^ method. Fresh alga (1 g) was placed in 20 mL of deionized water. A seaweed extract sample (1 mL) was putted in a hemolytic tube to which was added 2 mL of Coomassie Blue reagent and then homogenized. Absorbance was determined at λ = 595 nm after 5 min, using an UV-visible spectrophotometer (Jenway 6405 type). Curve calibration was performed using a Bovine Serum Albumin (BSA) solution between 0 and 2.0 mg mL^−1^. Protein content Q (mg) in seaweed samples was calculated as follows:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\rm{Q}}={\rm{V}}\ast {\rm{C}}$$\end{document}$$where C is the protein concentration (mg g^−1^ fresh weight), obtained using the calibration curve; V = initial sampling volume (mL). Results are presented as percentage of dry weight (15%). The R-PE content was determined as described by^[@CR13]^ and the absorbance was measured at 565 nm, which is the maximum of R-PE absorbance. Beer-Lambert law established the absorbance at 565 nm as follows:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\rm{A}}={\rm{\varepsilon }}\ast {\rm{L}}\ast {{\rm{C}}}_{1}=({\rm{\varepsilon }}\ast {\rm{L}}\ast {{\rm{C}}}_{2})/({\rm{MV}})$$\end{document}$$where C~2~ was calculated by the following equation:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\rm{C}}}_{2}={\rm{A}}\ast \frac{260000}{2000000}=0.13\ast {\rm{A}}$$\end{document}$$ A: absorbance at 565 nm, ε: R-PE extinction coefficient (2.10^6^ M^−1^ cm^−1^), L: optic length (=1 cm), C~1~: molar concentration of R-PE (M), C~2~: Concentration of R-PE (mg mL^−1^), MW: molecular weight of R-PE (260000 Da). Statistical analyses {#Sec8} -------------------- All results were expressed as mean ± Standard deviation. The Daily growth rate (% day^−1^), Agar yields (%), sulphate (%), 3,6-AG (%), protein (%) and R-PE (mg g^−1^ fw) obtained in different depths and sites were examined using the statistical package Statistica, version 5.1^[@CR23]^, as shown in Table [1](#Tab1){ref-type="table"}. After verification of the homogeneity of the variances and the normality of the data, the results, were subjected to two-way ANOVA analysis to assess the impacts of sites and depths according to the GLM procedure. When ANOVA proved to be significant, the Duncan's test was used to compare averages; the significance level of 5% was retained.Table 1Physico-chemical parameters at the Bizerte Lagoon and Bay water. Values are means ± SD; n = 9 for physical parameters and n = 18 for chemical parameters. Means in same row followed by same superscript letter are not significantly different according to the Duncn's test (P \> 0.05).BLBB**Chemical parametres**Nitrate (μ mol L^−1^)21.87^a^ ± 2.171.64^b^ ± 0.59Ammonium (μ mol L^−1^)13.32^a^ ± 2.8011.73^a^ ± 1.91Orthophosphate (μ mol L^−1^)4.53^a^ ± 0.493.54^a^ ± 0.71**Physical parametres**Secchi depth (m)2.37^a^ ± 0.254.50^b^ ± 0.50Salinity (psu)35.86^a^ ± 0.0436.90^b^ ± 0.36Temperature (°C)17.45^a^ ± 3.5719.43^a^ ± 1.25Dissolved oxygen (mgL^−1^)7.98^a^ ± 0.028.28^a^ ± 0.71pH8.18^a^ ± 0.078.36^a^ ± 0.16BL: the Bizerte lagoon; BB: the Bizerte bay; Means in same row followed by same superscript letter are not significantly different (P \> 0.05). Ethical statement {#Sec9} ----------------- This article doesn't contain any studies with animals performed by any of the authors. Results {#Sec10} ======= The physicochemical characteristics of water in the BL and the BB, during the culture period, are shown in Table [1](#Tab1){ref-type="table"}. Consequently, the water transparency (m), salinity (psu) and nitrate concentration (µmol L^−1^) varied significantly between the BL and the BB. In contrast, the water temperature (°C), dissolved oxygen (mgL^−1^), pH, ammonium (µmol L^−1^) and orthophosphate concentration (µmol L^−1^) were similar. The BL water was less transparent compared to the BB (2.37 ± 0.25 m vs 4.50 ± 0.50 m). The average salinity were different between the BL and the BB (35.86 ± 0.04 psu vs 36.90 ± 0.36 psu). The nitrate showed the higher concentrations in the BL compared to the BB (21.87 ± 2.17 μmol L^−1^ vs 1.64 ± 0.59 μmol L^−1^). Results of DGR (%), agar yields (%),3,6-AG (%), sulphate (%), proteins (%) and R-PE (mg g^−1^ fresh wt) were shown in Tables [2](#Tab2){ref-type="table"}, [3](#Tab3){ref-type="table"}, [4](#Tab4){ref-type="table"} and Supplementary Fig. [S1](#MOESM1){ref-type="media"}. Hence, *G. gracilis* grows differently (P \< 0.05) between the site and the culture depth (Table [2](#Tab2){ref-type="table"}). Accordingly, the DGR recorded in the BL was higher than that of the BB (1.42 ± 0.65% day^−1^ vs 1.19 ± 0.34% day^−1^). In the BL, we recorded a decrease of DGR from the surface to 3.75 m. Consequently, a highest DGR were recorded between the surface and 1 m. As a result, DGR recorded at 1 m is the highest (2.33 ± 0.03% day^−1^). Whereas, a lowest DGR ( \<1%) was recorded over 3 m. In addition, the DGR at 3.5 m does not exceed 0.5% day^−1^ and showed null values at 3.75 m, by far the lowest, due to thallus degeneration. In contrast, DGR recorded in the BB were homogenous in the studying depths (p \> 0.05) and varied from 0.86 ± 0.32% day^−1^ to 1.57 ± 0.42% day^−1^ (Table [3](#Tab3){ref-type="table"}). In the BL, the thalli loss was insignificant from the water surface to 1.5 m. Consequently, the PTL ranges between 1% and 2%. In contrast, in the BB, up to 1.5 m, we recorded the higher PTL, especially near the water surface (10%).Table 2Daily Growth Rate (DGR %), Agar yield (%), Sulfate (%), 3,6-Anhydrogalactose (3,6-AG %), Proteins **(%)** and R-phycoerythrin **(**R-PE mg g^−1^fw) obtained from *Gracilaria gracilis* samples cultured at different depths in two sites (Bizerte Lagoon and Bizerte Bay, Northeast Tunisia) analyzed by ANOVA at 95% confidence level.Factors*SSdfMSFp***DGR (%)**Intercept154.071154.071557.88\<0.05Site1.2211.2212.36\<0.05Depth7.85140.565.67\<0.05Site\*Depth10.24140.737.39\<0.05Error5.93600.10**Agar (%)**Intercept40636.38140636.386314.463\<0.05Site382.751382.7559.475\<0.05Depth110.93147.921.231\>0.05Site\*Depth58.34144.170.648\>0.05Error386.13606.44**Sulfate (%)**Intercept4932.62214932.6223507.418\<0.05Site176.0841176.084125.207\<0.05Depth54.125143.8662.749\<0.05Site\*Depth74.098145.2933.763\<0.05Error84.380601.406**3,6-AG(%)**Intercept97159.40197159.4036228.44\<0.05Site6518.9116518.912430.75\<0.05Depth853.101460.9422.72\<0.05Site\*Depth1196.311485.4531.86\<0.05Error160.91602.68**Proteins(%)**Intercept41150.41141150.41979.3834\<0.05Site36.62136.620.8715\>0.05Depth976.251469.731.6596\>0.05Site\*Depth661.061447.221.1238\>0.05Error2521.006042.02**R-PE(mgg**^**−1**^**fw)**Intercept31.92131.92535.76\<0.05Site6.3016.30105.83\<0.05Depth1.15140.081.38\>0.05Site\*Depth0.23140.020.27\>0.05Error3.57600.06Table 3Daily growth rate and proximate biochemical composition of *Gracilaria gracilis* cultivated in Bizerte lagoon (BL) and bay (BB) at different depth (from 0.25 m to 3.75 m). Values are means ± SD; n = 9. Means with the same superscripts are not significantly different (*p* = 0.05).Depth (m)DGR (%)Agar (%)Sulfate (%)3,6-AG(%)Proteins (%)R-PE (mgg^−1^fw)BL0.252.33^a^ ± 0.0318.40^a^ ± 1.447.97^e^ ± 0.5432.41^a^ ± 1.3922.06^a^ ± 10.720.25^a^ ± 0.05BL0.501.96^ab^ ± 0.4618.07^a^ ± 2.258.70^bcde^ ± 0.8332.79^a^ ± 1.8117.10^a^ ± 6.960.24^a^ ± 0.04BL0.752.06^ab^ ± 0.3220.70^a^ ± 0.268.43^cde^ ± 0.5432.98^a^ ± 2.3020.13^a^ ± 7.760.25^a^ ± 0.03BL1.001.96^ab^ ± 0.1919.43^a^ ± 2.638.13^e^ ± 0.4040.68^a^ ± 3.3016.36^a^ ± 6.580.31^a^ ± 0.09BL1.251.75^abc^ ± 0.2718.90^a^ ± 1.658.28^de^ ± 0.2929.98^a^ ± 0.5718.50^a^ ± 7.420.28^a^ ± 0.03BL1.501.58^bc^ ± 0.1321.23^a^ ± 3.968.39^cde^ ± 0.1731.47^a^ ± 1.3416.56^a^ ± 6.300.31^a^ ± 0.06**BL**1.751.34^bc^ ± 0.3917.43^a^ ± 2.679.42^abcd^ ± 0.2432.64^a^ ± 1.1521.86^a^ ± 9.560.36^a^ ± 0.16BL2.001.49^bc^ ± 0.2719.27^a^ ± 1.369.58^abc^ ± 0.1731.93^a^ ± 1.8621.77^a^ ± 8.460.37^a^ ± 0.19BL2.251.42^bc^ ± 0.1121.57^a^ ± 3.719.42^abcd^ ± 0.2930.42^a^ ± 1.1022.64^a^ ± 10.590.37^a^ ± 0.17BL2.501.53^bc^ ± 0.1418.50^a^ ± 2.117.52^e^ ± 0.5235.97^a^ ± 5.9523.97^a^ ± 8.640.41^a^ ± 0.19BL2.751.43^bc^ ± 0.1619.17^a^ ± 2.109.69^ab^ ± 0.4636.43^a^ ± 0.6329.63^a^ ± 8.230.43^a^ ± 0.21BL3.001.05^c^ ± 0.1518.00^a^ ± 2.558.62^bcde^ ± 0.4030.73^a^ ± 0.4627.18^a^ ± 6.520.45^a^ ± 0.19BL3.251.05^cd^ ± 0.0718.80^a^ ± 4.169.96^a^ ± 0.5035.04^a^ ± 1.8426.05^a^ ± 2.790.30^a^ ± 0.03BL3.500.57^d^ ± 0.2519.07^a^ ± 0.957.97^e^ ± 0.3727.26^a^ ± 0.5322.30^a^ ± 3.090.32^a^ ± 0.05BL3.75−0.16^e^ ± 0.6119.27^a^ ± 1.829.96^a^ ± 0.7032.12^a^ ± 1.5224.20^a^ ± 4.050.32^a^ ± 0.05BB0.250.86^a^ ± 0.3221.13^a^ ± 1.447.74^ab^ ± 2.6045.82^abc^ ± 1.2715.38^a^ ± 5.690.63^a^ ± 0.13BB0.501.03^a^ ± 0.4824.03^a^ ± 2.255.38^ab^ ± 0.7546.12^ac^ ± 2.7815.47^a^ ± 3.850.64^a^ ± 0.11BB0.750.99^a^ ± 0.4322.47^a^ ± 0.267.40^ab^ ± 2.3840.78^bcd^ ± 0.8127.74^a^ ± 0.470.65^a^ ± 0.09BB1.000.91^a^ ± 0.7820.87^a^ ± 2.635.91^ab^ ± 0.4641.31^abcd^ ± 0.4116.30^a^ ± 4.700.73^a^ ± 0.22BB1.251.57^a^ ± 0.4222.73^a^ ± 1.655.42^ab^ ± 1.3739.94^bd^ ± 1.5115.45^a^ ± 2.760.77^a^ ± 0.09BB1.501.32^a^ ± 0.3923.40^a^ ± 3.968.28^a^ ± 1.8540.05^d^ ± 1.9325.39^a^ ± 6.420.81^a^ ± 0.15**BB**1.751.27^a^ ± 0.1521.27^a^ ± 2.677.90^ab^ ± 1.7946.96^a^ ± 4.5218.14^a^ ± 3.040.82^a^ ± 0.41BB2.001.50^a^ ± 0.2925.23^a^ ± 1.364.58^ab^ ± 0.7139.83^bcd^ ± 0.3019.23^a^ ± 6.000.82^a^ ± 0.49BB2.251.38^a^ ± 0.0725.50^a^ ± 3.715.84^ab^ ± 2.8937.96^d^ ± 0.3528.88^a^ ± 10.190.82^a^ ± 0.43BB2.501.23^a^ ± 0.0122.47^a^ ± 2.115.30^ab^ ± 0.8642.19^abcd^ ± 2.9417.06^a^ ± 1.090.93^a^ ± 0.50BB2.751.18^a^ ± 0.1122.93^a^ ± 2.108.39^b^ ± 0.2945.74^ac^ ± 1.5220.87^a^ ± 5.090.96^a^ ± 0.56BB3.001.17^a^ ± 0.2123.60^a^ ± 2.554.84^ab^ ± 1.3236.74^d^ ± 2.2923.44^a^ ± 4.410.97^a^ ± 0.49BB3.251.18^a^ ± 0.2222.90^a^ ± 4.163.40^b^ ± 1.0438.61^d^ ± 5.0218.61^a^ ± 2.911.07^a^ ± 0.07BB3.501.09^a^ ± 0.2324.90^a^ ± 0.955.19^ab^ ± 1.9439.41^d^ ± 0.3720.86^a^ ± 7.371.12^a^ ± 0.13BB3.751.19^a^ ± 0.2426.23^a^ ± 1.824.50^ab^ ± 1.0039.06^d^ ± 1.4428.38^a^ ± 5.291.16^a^ ± 0.12BL: the Bizerte Lagoon; BB:the Bizerte Bay; DGR: Daily Growth Rate;3,6-AG(%):3,6-anhydrogalactose; R-PE: R-phycoerythrin; SD: Standard deviation.Table 4Daily growth rate and proximate biochemical composition of *Gracilaria gracilis* cultivated in Bizerte Lagoon and Bizerte Bay. Values are means ± SD of results obtained at different depth (from 0.25 m to 3.75 m); n = 45. Means in same row followed by same superscript letter are not significantly different according to the Duncn's test (P \> 0.05).BLBBDGR(%)1.42^a^ ± 0.651.19^b^ ± 0.34Agar(%)19.19^b^ ± 2.3223.31^a^ ± 2.64Sulfate(%)8.80^a^ ± 0.866.00^b^ ± 2.003,6-AG(%)23.30^b^ ± 5.4041.36^a^ ± 3.68Proteins(%)20.74^a^ ± 7.2222.02^a^ ± 6.34R-PE(mgg^−1^fw)0.33^b^ ± 0.120.86^a^ ± 0.31BL: the Bizerte Lagoon; BB: the Bizerte Bay; DGR: Daily Growth Rate;3,6-AG(%):3,6-anhydrogalactose; R-PE: R-phycoerythrin; SD: Standard deviation. The agar yields (Tables [3](#Tab3){ref-type="table"} and [4](#Tab4){ref-type="table"}), which depend (p \< 0.05) on the site of production, were 19.19 ± 2.32% and 23.31 ± 2.64% for the BL and the BB respectively. Inversely, the effect of the culture depth was not significant (p \> 0,05) and homogeneous distribution in the whole studying water column was recorded. The 3,6-AG content and sulphate degree of agar varied significantly between the sites and the depths (p \< 0.05). In the BB, the 3,6-AG content of agar was twice as high as that obtained in the BL (41.37 ± 3.68% vs 23.30 ± 5.40%) (Table [4](#Tab4){ref-type="table"}). In addition, in the BL, the highest 3,6-AG content was recorded at 3.25 m, the lowest at 3.50 m and an irregular distribution between the other depths (Table [3](#Tab3){ref-type="table"}). However, in the BB the highest amount of 3,6-AG (\> 45%) was found near the water surface (\<0.5 m), followed by a stationary phase between 0.5 m and 3 m; values varied between 40% and 45%. The lowest values (\<40%) were recorded over 3 m (Table [3](#Tab3){ref-type="table"}). The algae cultivated in the BL has the higher sulphate degree than those cultivated in the BB (6 ± 2.00% vs 8.80 ± 0.86%) (Table [3](#Tab3){ref-type="table"}). Furthermore, in the BL the highest sulphate degree (10%) was attained over 3 m, and the lowest (7.50%) at 2.50 m. Nevertheless, in the BB, the lowest sulphate degree (3.40%) recorded at 3.25 m (Table [3](#Tab3){ref-type="table"}). The site and the depth does not affect the protein content of the alga (p \> 0.05). Accordingly, the contents recorded were 22.02 ± 6.34% and 20.74 ± 7.22% for the BL and the BB respectively (Table [4](#Tab4){ref-type="table"}). The R-PE contents in the BL (0.33 ± 0.12 mg.g^−1^ fwt) was lower (p \< 0.05) than that obtained in the BB (0.86 ± 0.31 mg. g^−1^ fwt) and values were homogeneously distributed across the depths in both sites (Table [3](#Tab3){ref-type="table"}). Discussion {#Sec11} ========== The physicochemical parametres of culture site {#Sec12} ---------------------------------------------- The lower Secchi depth (2 m) obtained in the BL is due to the turbidity. However, the muddy bottom and the wind increase the turbidity of the water. Likewise, the lagoon receives several urban and industrial discharges from the around cities, other than the sediments from the rivers^[@CR24]^. Unlike the BL, Secchi depth obtained in the BB occurs in the water depths greater than 20 m as indicated by^[@CR25]^. The salinity value in the BL is similar to that recorded by^[@CR26],[@CR27]^. The water temperature values reported in our study were similar to that obtained by^[@CR26]--[@CR28]^, which were (15--23 °C), (15--25 °C) and (19--20 °C) respectively. In the BL, there is no vertical gradient of salinity or temperature as indicated for other lagoons^[@CR29],[@CR30]^, probably due to the shallow depth. The salinity and temperature values recorded in the BB between March and May are consistent with those reported previously (19 °C and 37 psu)\[84\]. The nutrient concentrations in the BL are comparable to those of a Mediterranean Sea Lagoon, Lake Burullus in Egypt^[@CR31]^. Accordingly, the authors reported nutrient concentrations in spring as follows: nitrate (7.41--28.90 µM), ammonium (7.34--34.30 µM) and phosphates (8.59--24.30 µM). In addition, a result of a physicochemical assessment of the Nador Lagoon water quality (Northeast Morocco), indicates a varied nitrate (1.08--29.28 µM) and phosphates (1--7 µM) concentrations^[@CR32]^. Furthermore, a nitrate (0.35--52.4 µM) and phosphate (0.41--2.24 µM) concentrations were determined in Mar Chiquita, a coastal lagoon in Argentina (South Atlantic Ocean)^[@CR33]^. According to^[@CR34]^, there is no vertical stratification of different nutrients (ammonium, nitrate and orthophosphate) in the BL. The lagoon receives in winter and spring an important flow of nutrients (liquid and solid) from their watershed, containing a very high quantity of nitrate compared to the BB^[@CR35]^. In conclusion, during the culture period, the BB has a higher transparency but a lower nitrogen (nitrate + ammonium) concentration compared to the BL. Growth {#Sec13} ------ For *G. gracilis* growth, the DGR recorded in the BL and the BB are in the range of those recorded in outdoor culture, in Tunisia or in others regions, which generally varied between 1% and 4% day^−1^ ^[@CR8],[@CR9],[@CR36],[@CR37]^. In contrast, indoor culture the DGR attained 10% day^−1^ or higher^[@CR38]^. The difference between the results could be due to the nitrogen concentration of the medium (nitrate + ammonium), which falls within the range of outdoor concentrations found in other studies. However, in the BL and the BB, the nitrogen concentration is too low (\<50 µM) to sustain the high seaweed DGR required for biomass production as indoor culture (\>1000 µM) due to the highly nitrophilic character of *G. gracilis*. Hence, the higher growth rate in the BL can be attributed to the nitrogen enrichment due to surface run off into the lagoon, which allows the alga to meet their nitrogen requirements compared to the BB. Moreover, the water transparency, act differently on *G. gracilis* growth^[@CR39]^. Consequently, lower light and higher nitrogen in the BL enhance DGR in contrast to lower nitrogen and higher light in the BB, which reduce algae growth. Hence, the difference between the findings could be due to the significant effect of light and nitrogen interaction on *G. gracilis* growth as indicated by^[@CR39]^. In addition, the lower DGR (\<3%) obtained in the BL and the BB could be due to the temperature and salinity values, which were out of the optimum growth range. Wide temperature (0--35 °C) and salinity (10--40 psu) tolerance of *Gracilaria* spp. has been reported but the optimum growth has been recorded in a restricted ranges (20 °C--28 °C; 25 psu −30 psu)^[@CR40]--[@CR42]^. In contrast, the water temperature and salinity values in the BL and the BB, during the experimental period, are outside the optimum range of alga growth (\<20 °C, \>30 psu) or near to their lower growth limit. Finally, based on the DGR obtained, the plants reached a harvestable size after 90 days in the BL and 110 days in the BB, but it is only of 30 days in Chilika Lake in India^[@CR43]^. The difference between results may be attributed to the physicochemical characteristics of the water in both sites. Our data show a markedly difference in DGR of *G. gracilis*, across the depths in both sites. However, light intensity is a relevant factor, which affects algae growth. In the BL, the highest DGR values were obtained in the shallowest depth (\<1 m) and the lowest in deeper one (\>3 m). Our findings are consistent with those of^[@CR44]--[@CR47]^. Consequently, they indicate that the lower DGR of *Gracilaria* spp. obtained is explained by the reduced light intensity due to the high turbidity essentially in eutrophic lagoon. In contrast, the DGR of *G. gracilis* in the BB was not affected in a depth of 4 m, suggesting the availability of enough light quantities in the studying depths. According to^[@CR48],[@CR49]^, *Gracilaria* can grow in the depths between 8 m and 10 m, but over 4 m their growth is largely affected; especially in a turbid environment that do not let light through. In our study the PTL is low than that recorded in the same lagoon and culture period, using bottom planting methods^[@CR50]^. The studies on *G. gracilis* farming carried out in the BL, by these methods, revealed that the PTL constituted a detrimental factor^[@CR50]^. However, algae losses are related to difficulties in inserting tufts on ropes^[@CR50]^, epiphytism^[@CR51]^ and associated fauna, which contain zoological groups that use the genus *Gracilaria* for habitat and food^[@CR52]^. In our study no epiphytes or epifauna associated to *G. gracilis* in both sites, which could explain the lower PTL. The problems of grazing and entanglement by other epiphytes were controlled upto higher level by the lantern net, which covered all sides of the thallus. The maximum loss was recorded at the shallow depths (\<1.5 m) due to fragmentation of the thalli into small parts (\<1 cm). However, this size facilitates their escape through the mesh of the net in addition to the decomposition of those that remain trapped. The *Gracilaria* culture like any other mariculture activity has an impact on the marine environment. According to^[@CR53],[@CR54]^, there was a decrease in the growth rate of *Zostera japonica* but an increase in the abundance and diversity of invertebrates in the community under the cultivated ropes of *Gracilaria* spp. In the BL, there is no development of seaweeds over 5 m, which avoids any interaction between *Gracilaria* and other species. Lantern nets developed for our study have more advantages than the benthic or the suspended culture methods using thalli inserted on the ropes^[@CR50]^. However, *G. gracilis* productivity is higher and the PTL is lower. This advantage is essentially linked to the attenuation of wave effects on *G. gracilis* thalli, which depend on the site and the depth. However, near the sea bottom, the wave velocity decrease (\<0.2 m s^−1^), whereas near the surface increase (0.38 ms^−1^), which can explain the higher PTL recorded in this part of the water column. In addition, in our study an improvement in attachment to nets covering the hoops (0.5 m^2^) is a mechanism that enables alga to survive the wave action and currents occur in this zone, which result in higher biomass production. In the BL, the marine wave velocity is low (\<0.2 m s^−1^), which could explain their little shearing actions on the thallus^[@CR24],[@CR55]^. In conclusion, the lower PLT obtained, the better we need for *G. gracilis* culture success in these regions. Based on DGR, after one month when we start from a stocking density of 7.5 kg m^−2^, we will reach a final density of 11.5 kg m^-2^, generating a multiplication factor of 1.5. However, this value is relatively higher than that obtained by the benthic culture system used in the BL^[@CR50]^ but was similar to those obtained by the suspended culture system in the sea, for other *Gracilaria* species. Accordingly, the multiplication factor values obtained were 1.2 for *Gracilaria caudata*^[@CR45]^; 2 for *Gracilaria chilensis*^[@CR56]^; 2.03 for *Gracilaria chilensis*^[@CR57]^; 2.16 for *Gracilaria chilensis*^[@CR58]^ and 6.36 for *Gracilaria sp*.^[@CR47]^. Agar yields and composition {#Sec14} --------------------------- Agar yields of *G. gracilis* obtained in the BL and the BB were comparable to those previously found for *G. gracilis* and others species, which varied from 20% to 30%^[@CR59]--[@CR61]^. In contrast, a lower agar yield of *G. gracilis* was obtained in a shallow part of the BL^[@CR9]^. However, not all studies agree with this finding due to the difference between species and culture sites. The multiple environmental factors, such as nutrient status, light, salinity and water temperature, could affect agar yield^[@CR2],[@CR39]^. The higher agar yields obtained in the BB, could be due to the high salinity level, light quantity and a lower nitrate concentrations than that recorded in the BL^[@CR39]^. The culture depth of *G.gracilis* in the BL and the BB didn't affect the agar yields in contrary to previous findings in the BL by^[@CR9]^, which indicated a higher agar yields at a depth of 2 m compared to those obtained at 0.5 m. Unlike the previous work, this study was performed in the eastern part of the lagoon, characterized by a higher turbidity (Secchi depth \<0.5 m). Consequently, the amount of light is insufficient (\<70 µmol m^2^ s^−1^) to interact with other factors and allowing the alga to produce higher agar yields^[@CR39],[@CR62]^. Accordingly, in the BL the lower light effect on *G.gracilis* at a depth over 2 m, which superior to a critical value indicated above, may be alleviated by the interaction of light and other factors. However, when agar yield was affected, interactions between abiotic factors (light, salinity and nitrogen) alleviate the negative impact and maintain the yield similar to that obtain at the shallow depths^[@CR39]^. In contrast, light amount in the BB, which is independent of the depths, can explain the homogeneity of the agar yields along water column. Agar composition (sulphate degree and 3,6-AG content) obtained in both sites (the BL and the BB) was consistent with those of^[@CR63]^, which indicate that higher agar yields was accompanied by higher 3,6-AG content and lower sulphate degree. The ideal structure of agarocolloid is a non-substituted galactan backbone composed of repeating units of (1,3)-linked-D-galactose and (1,4)-linked 3,6-anhydro-α-L-galactose. However, native agarocolloids are generally a mixture of neutral, sulphated, methylated and pyruvated agarose, which influences their rheological properties. According to^[@CR64],[@CR65]^, the gel properties (gel strength, gelling temperature and melting temperature), which are the most important criteria to evaluated agar, are highly dependent on the amount of sulphate groups as well as the 3,6-AG content. Accordingly, the higher the 3,6-AG content and the lower the sulphate degree, the better agar gel strength. However, the quality of agar from the BB (41.36 ± 3.68% of 3,6-AG and 6.00 ± 2.00% of sulphate) approach that of agarose (48% of 3,6-AG and 2% of sulphate). In contrast, the agar obtained in the BL is of lower quality due to the lower percentage of 3,6-AG and higher sulphate degree (23.30 ± 5.40% of 3,6-AG and 8.80 ± 0.86% of sulphate). Similar results were obtained in the BL^[@CR9]^. Proteins and R-PE contents {#Sec15} -------------------------- The protein contents of *G. gracilis* (\>25%), are lower than that obtained by^[@CR10],[@CR66]^ but are higher than those obtained by^[@CR67],[@CR68]^, which indicates a value varying between 11% and 20%. The difference between results could be due to the environment factors and extraction methods. Hence, in both sites (the BL and the BB) ammonium is a limiting factor, which affects positively chlorophyll and proteins contents but negatively the carbohydrate^[@CR69]^. The ammonium concentration in the lagoon and the bay does not meet the *G. gracilis* requirement to produce proteins contents higher than 20%. In addition, the higher proteins contents were obtained at a salinity lower than 30 psu. The similar proteins contents obtained in both sites despite the difference in nitrate concentration and light quantity, were due to the interaction between them^[@CR39]^. In addition, the differences between the results could be attributed to the extraction methods. However, enzyme-assisted extraction of *G. gracilis* produces a high proteins contents compared to the native method^[@CR13]^. The R-PE is the most abundant phycoerythrins in Rhodophyta^[@CR70]^, which does not exceed 10 mgg^−1^ in *Gracilaria* species^[@CR71]^. However, their concentrations were inversely proportional to the growth rate and vary considerably with environmental factors; essentially light^[@CR39],[@CR72]^. The lower pigments contents of *G. gracilis* in the BL compared to the BB, can be attributed to their higher growth. In addition, the difference between the results could be due to the amount of quantity of light available. However, the inverse relationship between R-PE contents and light intensity is well established^[@CR72]--[@CR74]^. Accordingly, a decrease in light intensity enhances the alga pigments accumulation^[@CR75]^. Red algae grow under intense light, accumulate few phycobilisomes and lower phycobiliproteins content compared to those grow under a low light intensity^[@CR76],[@CR77]^. However, the Secchi depth in the BB was higher than the depth of *G.gracilis* culture, which indicates that the amount of light is homogeneous. In contrast the homogeneity of R-PE in the BL despite the Secchi depth (\<2.5 m) can be attributed to the effects of others factors. For this purpose, light effect on *G.gracilis* R-PE contents depend mainly on nitrogen concentration, salinity and their interaction^[@CR39],[@CR78],[@CR79]^. Probably without the interaction mechanisms, there will be differences in the amount of R-PE between the depths. Aquaculture and biotechnology relevance of results {#Sec16} -------------------------------------------------- We stated that the lagoon was characterized by a higher nitrogen concentration and a lower amount of light. In contrast the opposite was happened in the bay. The red alga *G. gracilis* grow well under higher nitrogen concentrations and light quantity, as in many coasts of Asian countries. Accordingly, the DGR will be limited in our conditions compared to those in India coasts^[@CR43]^. However, in those countries, with the same period of culture, we obtain two production cycles (between 30 and 45 days each one) and only one in Tunisia (between 90 and 110 days). In addition, in the Asian regions there are two growth periods of this alga; it is feasible to obtain four production cycles per year^[@CR43]^. Contrarily, in Tunisia and the Mediterranean region in general, there is only one period of growth (spring) and only one development cycle per year^[@CR4],[@CR8],[@CR50]^. Accordingly, what we presented as a result allows us to prepare a pilot scale up, which is necessary before starting *Gracilaria* aquaculture in the Mediterranean region to boost their productivity by suspended methods. The pilot scale up can spend two years (one cycle/year) and a socioeconomic study can be started. If we consider this to be our ultimate goal, the results obtained in this study allowed us to gather maximum information's on the subject and facilitates the starting alga culture in an industrial way in the near future. In the world, the market price of *Gracilaria* is related to the agar yields and quality. The *Gracilaria gracilis*, is used in several industrials sectors mainly food due to their agar yields. As the agar yields of alga harvested from the BB was higher with better quality, compared to the BL, the total economic value might remain the same. If the physicochemical parameters of the BL and the BB generate a low DGR, which limit the alga quantities, we propose to improve the total economic value of the biomass by the extraction of others interested molecules such as proteins and R-PE. In our study protein content can be compared to other high-protein foods such as soya (30%), beef (25%) or salmon (20%)^[@CR80]^. In addition, a valuable phytochemical may be co-extracted with proteins such us polyphenols, pigments and enzymes having higher additional values, which may be of interest^[@CR13],[@CR14],[@CR81]^. A sequential extraction of agar and other compounds was developed with fresh *G. verrucosa*^[@CR14]^, but it is possible to use dried algae instead of fresh. Increasing interest is being given in the last decades to *Gracilaria* drying. Consequently, a drying technologies were developed allowing the recovery of proteins, R-PE and agar in sufficient quantity^[@CR82]--[@CR84]^. Tunisian aquaculture sector have highly improved during the last two decades; indeed, the national production passed from 2000 tons in 2000 to 20 000 tons in 2017 and the number of aquaculture societies was ten times higher^[@CR85]^. The marine fish farming production represents 90% of the total production. However, we have interested to minimize negative environmental impacts of this activity. The integrated aquaculture systems (IMTA) based on integrated culturing of finfish, seaweed and mussels, have to contribute to the sustainability of aquacultures, but their development requires further research to optimize the technique, which depends on the selected seaweed species and the system of fish farming. In IMTA, the red algae *Gracilaria* utilize photosynthesis to convert inorganic nutrients into organic molecules and reduce the negative environmental impact of aquaculture activity. Consequently, based on the results of growth and biochemical composition obtained in the BB, *Gracilaria gracilis* could be used in IMTA systems due to the homogenous growth and chemical composition obtained. Furthermore, it can absorb a higher quantity of nitrogen, under changing abiotic factors, which can be produced by the system^[@CR39]^. Conclusions {#Sec17} =========== Our results highlight that *G. gracilis*, cultivated in the BL and the BB using suspended method, possess active growth and interesting concentrations of chemical components (agar, proteins, and R-PE). However, we obtain a better productivity, a higher tufts recovery and larger exploited surface than the culture on the substrate. Furthermore, no direct interaction between the alga and the flora. The light was the key factor; the water depth acts differently. However, in the BL the DGR was affected over the depth of 1.5 m but in the BB is not affected up to 4 m. The choice of the depth and site culture may depend on the growth and the final use of biomass (agar, proteins and R-PE). With the higher agar yields in the BL and the BB, suspended culture of *G. gracilis* in the BB is much more attractive due to the higher protein content, R-PE amount and the better agar quality. Supplementary information ========================= {#Sec18} Dataset 1. **Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary information ========================= is available for this paper at 10.1038/s41598-020-66003-y. This study was funded by Ministry of Higher Education and Scientific Research, Tunisia. Special thanks to Superior Institute of Fishing and Aquaculture of Bizerte responsible, for sharing facilities and logistic support. The authors would like to thank the head of B^3^Aqua laboratory, INSTM -- Salammbo, for providing the reagent. We are grateful to Hammami wahiba for preparing all algae culture activities. F. Mensi wrote the main manuscript text. F. Mensi, S. Nasraoui and S. Bouguerra were responsible for the experimental design. F. Mensi, S. Nasraoui, S. Bouguerra and M. Chalghaf were responsible for the field experimentation. S. Nasraoui, S. Bouguerra and A. Ben Ghedifa carried out the biochemical analyses. All authors reviewed the manuscript. The datasets generated during the current study are available on request to the corresponding author. The authors declare no competing interests.

Introduction {#Sec1} ============ Some of the low level radioactive waste (LLW) from reprocessing of spent nuclear fuel is planned to be disposed of in a purpose built repository at a depth of more than 300 m in the geosphere (geological disposal). In such LLW, nitrates can be present in the form of soluble salts and change to various redox states, such as N(V) as nitrate (NO~3~^--^), N(III) as nitrite (NO~2~^--^), N(0) as nitrogen gas (N~2~), and/or N(−III) as ammonia (NH~3~). It is therefore necessary to understand the fate of NO~3~^--^ and any changes it can provide to the redox environment of the geological disposal system (Japan Atomic Energy Agency and The Federation of Electric Power Companies of Japan [@CR14]). In earlier works of some of the current authors, experimental investigations of the chemical reaction between NO~3~^--^ species and coexisting metallic materials (mainly carbon steel) were used to underpin numerical modeling of the NO~3~^--^ reduction reaction in combination with the corrosion of carbon steel (Honda et al. [@CR11];Honda et al. [@CR12];Honda et al. [@CR13]). The role played by microorganisms in the NO~3~^--^ reduction reaction has been recognized as being an important factor in conditioning the underground chemical environments relevant to geological disposal (Chapelle [@CR2];Pedersen [@CR17];Hallbeck & Pedersen [@CR7];Eydal et al. [@CR5];Nielsen et al. [@CR16];Pedersen [@CR18];Pedersen [@CR19]). Previous studies of the underground environment have identified the existence of microorganisms below depths of 300 m, including some NO~3~^--^ reducing microorganisms (Nielsen et al. [@CR16]). In order to establish a proper safety case for the geological disposal system, it is therefore important to construct an evaluation model of NO~3~^--^ behavior including the denitrification process mediated by microorganisms. The purpose of this study is to adopt a bio-denitrification process to the numerical modeling developed for the environment with metals (Honda et al. [@CR11];Honda et al. [@CR12];Honda et al. [@CR13]). In the repository areas, high pH (\>12) due to cementitious barriers as well as extremely low water activity due to extremely high nitrate concentrations (about 7 mol/dm^3^) might prevent bioactivities. Therefore, the bio-denitrification model of this study is mainly focused on the near field of the repository. For the near field environment, pH was assumed to be around 8 based on the hypothetical groundwater called \"Fresh Reducing High pH\" in the report (Japan Atomic Energy Agency and The Federation of Electric Power Companies of Japan [@CR14]). Mathematical models have been developed for NO~3~^--^ reduction by microorganisms (Henze et al. [@CR9];Duthy [@CR4];Clement [@CR3];Andre et al. [@CR1];Mcguire et al. [@CR15]). Among such models, the activated sludge model (ASM) is reasonably well-known, focusing on the industrial and practical use of managing the denitrification process of municipal sewage (Henze et al. [@CR9]). In the ASM, like other proposed models, under anaerobic conditions, the NO~3~^--^ reduction reaction is understood as the coupling of an oxidation reaction of organic materials (electron donors) and a reduction reaction of NO~3~^--^ (electron acceptor) instead of gaseous oxygen. One of the experimental studies on the NO~3~^--^ reduction, in an *in-situ* subsurface environment, was carried out at a site denoted the \"MICROBE laboratory\" (Pedersen [@CR17];Hallbeck & Pedersen [@CR7];Eydal et al. [@CR5]; Nielsen et al.[@CR16];Pedersen [@CR18];Pedersen [@CR19]) at the Äspö Hard Rock Laboratory (HRL) in Sweden. The HRL is an underground facility for demonstrating, testing and researching high level radioactive waste disposal concepts. The HRL is situated under the island of Äspö on the Baltic coast of Sweden, approximately 400 km south of Stockholm. The HRL extends to a depth of 460 m in crystalline igneous rock, where the MICROBE laboratory was established at a depth of 450 m (Pedersen [@CR17]). In the current study, two kinds of organic materials were selected to investigate NO~3~^--^ reduction under geological disposal conditions. A super plasticizer used in the construction of cementitious components was selected as being representative of the geological disposal system and humic acid was selected as being representative of pre-existing organic materials in the bedrock. Firstly, a matrix of *in-vitro* experiments was conducted using these organic materials and typical microorganisms to identify key controlling parameters in the denitrification process. Data obtained from the *in-vitro* experiments were used to develop a NO~3~^--^ reduction reaction model. The NO~3~^--^ reduction reaction model was verified by simulating the data obtained from *in-situ* experiments at the Äspö MICROBE laboratory. Methods {#Sec2} ======= Microorganism {#Sec3} ------------- Activated sludge sampled from municipal waste water and *Azoarcus tolulyticus* (*A. tolulyticus*), an underground bacteria identified as having NO~3~^--^ reduction activity (Zhou et al. [@CR23]), were used as representative microorganisms. Activated sludge was selected in addition to *A. tolulyticus* because the activated sludge is a mixture of various kinds of microorganisms, there should be at least one bacterium that can acclimatize to any given environment, in this case, conditions relevant to geological disposal. Activated sludge was used after the removal of contaminants by washing five times with physiological saline solution. *A. tolulyticus*, isolated from a subsurface aquifer, was provided from the American Type Culture Collection (ATCC) through Summit Pharmaceuticals International Corporation (SPI). The strain (ATCC-51758) was cultivated in a liquid medium under aerobic conditions following ATCC\'s instructions. After repeating the above procedure five times, solutions for the experiments were prepared. Turbidity (absorbance at 610 nm) was adjusted to the same value as that of 1000 mg solution of the activated sludge. The concentration of the so obtained solution of *A. tolulyticus* was defined as 1000 mg/dm^3^. For cultivation, typical media used for the evaluation of denitrification activity was used, replacing ammonium ion by sodium ion in order to avoid uncertainty in material balance analysis of nitrogen. Variation of microorganism concentration was evaluated by Lumitester of Kikkoman Corporation, by which microorganism concentration is evaluated as intensity of luminescence released when the ATP (adenosine triphosphate) is transformed to AMP (adenosine monophosphate) in the presence of luciferaze. In this study, the intensity of luminescence of the initial solution was set to 1.0 and the relative intensity of the sample was evaluated as the variation of microbial population (relative growth rate). Samples and reagents {#Sec4} -------------------- Two kinds of humic acid were provided by the Japanese Humic Substances Society (JHSS) (Watanabe et al. [@CR22]). Humic acids from Dando and Inogashira provided by JHSS were mixed at a mass ratio 1:1. The other humic acid was a reagent grade sodium salt from Aldrich and used as received. A super plasticizer used for the *in-vitro* experiments was Mity 3000S from Kao Corporation, consisting of polyether compounds containing carboxylic groups as main components. The concentration of total organic carbon (TOC) of the solution was adjusted to a designated value by dilution. In order to respond to a recent view that the molecular weight distribution of organic compounds originated from super plasticizers in the solution of cement pores tends to decrease in comparison with that of the original plasticizers (Suguro et al. [@CR21];Fujita et al. [@CR6]), the super plasticizer solution for the *in-situ* experiments was prepared by the following procedure. A super plasticizer, Reobuild SF8LS from BASF, was mixed with a Portland cement to adsorb to the cement materials. The cement slurry was made up by mixing the Portland cement and distilled water (mass ratio of cement/water was 2), followed by the removal of soluble alkaline materials (principally Na and K) by replacing the supernatant with fresh distilled water after overnight storage of the slurry. After replacing the supernatant three times, the soluble alkaline component was removed and a cement hydrate containing 50 wt % water was obtained. This cement hydrate was mixed with solution containing 2 wt % of the super plasticizer and 0.2 wt % of ethylene glycol. The mass ratio of the cement hydrate to the solution containing organic materials was 100 to 20. After this mixture was stored for 7 days under ambient conditions, the supernatant was separated by centrifugation to provide the sample solution containing the extracts of the super plasticizer. The molecular weight of organic materials in the solution was 200 to 300, which was similar to that of organics in the solution of cement pores reported previously (Suguro et al. [@CR21];Fujita et al. [@CR6]). The solution was then bubbled with carbon dioxide to fix the pH to 8 with the precipitate of calcium carbonate removed by filtration using 0.45 μm membrane filter. The final concentration of the solution was set to a designated concentration by removing water through evaporation. A NO~3~^--^ solution was prepared by dissolution of NaNO~3~ into pure water and pH was adjusted to 8 (except Run1 and Run2, pH7) by NaOH. NaNO~3~ and NaOH used were JIS special grade chemicals from Wako Chemicals Corporation. *In-vitro* experiments {#Sec5} ---------------------- In vitro experiments were carried out using ampule experiments and vial experiments described below. To ensure long-term anaerobic conditions and to analyze nitrogen contents, while avoiding air contamination, the so-called ampule experiment (Honda et al. [@CR10]) was conducted (details shown in Table [1](#Tab1){ref-type="table"}). A test solution was made up by mixing of solution containing microorganism and solution containing the nitrate and organics in a glove box (Argon \[Ar\] atmosphere, oxygen concentration \< 0.1 ppm) followed by Ar gas sparging to remove dissolved oxygen from the solution. The solution was then placed in the designated glass tube (volume ≈ 0.08 dm^3^) and the spout fusion sealed to prevent any atmospheric contamination. The sealed glass tubes were then taken out of the glove box and stored in a thermostat oven at 35°C for a designated time of 1--50 days. After storage, the ampule seal was broken in a vacuum chamber connected to the injection port of a gas chromatograph (GC), so that the composition of the production gas could be analyzed without air contamination. For tests to investigate time dependence, ampules were prepared for every experiment run. Shortly after the opening the ampules, NO~3~^--^ and NO~2~^---^ of the solution were analyzed by ion chromatography, NH~3~ by UV spectrometer and TOC by furnace-infrared method. The solid fraction was separated by filtration using a 0.2 μm membrane filter and its C, H and N content was analyzed by a CHN coder (Yanako MT700 CHN).Table 1**Experimental condition of*in-vitro*tests**SeriesRunMethodInitial Condition^\*1^MicroorganismElectron DonorDurationpHConc. of NO~3~^--^Activated Sludge***Azoarcus Tolulyticus***GlucoseSuper PlasticizerMixture of HA^\*2^Sodium Salt of HA^\*3^(mmol/dm^3^)(mg/dm^3^)(mg/dm^3^)(mg/dm^3^)(mg/dm^3^)(mg/dm^3^)(mg/dm^3^)(d)A1Ampule7201000\--0-502Ampule7201000\--3000-6--50B3Ampule8101000\-\--100-104Ampule8501000\-\--100-105Vial8101000\-\--1-0--306Vial8101000\-\--10-0--307Vial8101000\-\--100-0--30C8Ampule850-1000\-\--0119Ampule850-1000\-\--101110Ampule850-1000\-\--1001111Ampule850-1000\-\--100011D12Vial850-1000100\-\--0.2--713Vial850-1000\-\--1000.2--7E14Vial85010\-\-\--01--5015Vial85010\-\-\--101--50^\*1^ In every experiments, temperature was controlled at 35 degree-Celcius and sealed gas was Argon. ^\*2^ Humic acids (HA) from Japan Humic Substance Association, ^\*3^ Reagent grade humic acids from Aldrich. In addition to the ampule tests, vial tests were also conducted to investigate the effects of using different microorganisms and organic materials (details shown in Table [1](#Tab1){ref-type="table"}). In the vial tests, gases in the liquid and gaseous phase in the vial tubes were replaced by Ar and spouts were sealed with silicon rubber to keep the interior of the tubes anaerobic. The volume of the vial tubes was 0.05 dm^3^ and solution volume was 0.03 dm^3^. After storage for a designated period of 1--50 days at room temperature, the gaseous phase was transferred to a gas bag through a syringe and N~2~ analyzed by GC. Analysis of the liquid phase used the same methods as the ampule tests described above. As shown in Table [1](#Tab1){ref-type="table"}, ampule and vial tests were carried out in the combination of variation of organic materials and microorganisms in series A to E. *In-situ* experiments {#Sec6} --------------------- A series of *in-situ* experiments were conducted at the MICROBE Laboratory situated at the Äspö HRL (Pedersen [@CR17]). The flow system setup in the MICROBE Laboratory consists of four flow cells; details can be found in Pederson (Pedersen [@CR17]). Flow cells were dried after sterilize with 70% ethanol. Each cell was subsequently filled with crushed (2--4 mm) and heat sterilized (433K for 5 hrs) rocks from the drill core of the test site, to give a total crushed rock mass = 440 g. Groundwater was then fed into the flow cell. The ground water was introduced from drill core into the inlet of the flow cells, drawn from outlet and circulated to drill core. This circulation was carried out for 30 days at a flow rate of approximately 20 ml/min, a pressure of 2.5 MPa and a temperature of 17°C until bio-films had grown on the crushed rock surface. After the one-through circulation was shutoff, the groundwater was circulated in the flow cells. The solution containing NO~3~^--^ and organic materials was injected. The organic materials were extracts from the mixture of super plasticizer and cementitious materials; described in sample and reagents. The solution was sampled periodically and NO~3~^---^ concentration and number of microorganism was analyzed. Sampling periods were 0, 0.9, 3.9, 8.9, 17 and 31 day. Solution sampled through 0.2 μm sterilized filters was used for analyses of chemical species as a microorganism free solution. Total number of cells of microorganisms (TNC) was determined using the acridine orange direct count method (Pedersen & Ekendahl [@CR20]). The quantitative polymerase reactions (Q-PCR) were run in duplicate. Primers for the gene NarG (Henry et al. [@CR8]) were used. The PCR mixture contained 1.0 μL of the primer (10 pmol/μL), 16 ng of DNA, 12.5 μL Stratagene Brilliant SYBR II Q-PCR Mastermix 2X (AH Diagnostics AB, Skarholmen, Sweden), and sterile water to a final reaction volume of 25 μL. Amplification was carried out on a Stratagene Mx35005P Q-PCR thermal cycler (AH Diagnostics AB). The primers were temperature optimized and the products with the standard samples were checked on agarose gels to verify the size of the fragments. The dissociation curves (melting curves) were also checked to evaluate the specificity of the primers. *Pseudomonas* fluorescence was used as standard. Results {#Sec7} ======= *In-vitro* experiments {#Sec8} ---------------------- Analytical data obtained from the *in-vitro* experiments are shown in Table [2](#Tab2){ref-type="table"} (ampule tests) and Table [3](#Tab3){ref-type="table"} (vial tests). The results from each series are described separately below.Table 2**Analytical results of*in-vitro*tests (glass ampule tests)**SeriesRunTime (d)Liquid phaseGas phaseSolid phaseRelative growth rate of microorganism^\*1^pHTOCT-NNO~3~^--^NO~2~^--^NH~3~N~2~CO~2~NH~3~CHNmmol/dm^3^mmol/dm^3^mmol/dm^3^A1507.73.2226.22.0195.4NDND18443.80.67266.9130138.60.73.43.4NDND24443.20.632287.21402.0NDND3.49.0NDND20382.40.32507.31402.4NDND3.48.4NDND24443.00.44B3108.72.09.43.00.22.83.6NDND17385.20.324108.52.050441.62.62.8NDND18362.00.27C818.32.247480.40.1\-\--2.65.20.60.538118.33.047442.20.70.4NDND0.91.40.20.04918.31.747460.40.1\-\--1.62.80.20.859118.43.247442.20.70.4NDND0.60.50.20.0551018.45.049480.20.1\-\--1.32.00.20.7410118.07.647424.01.10.5NDND2.22.40.60.0581118.3305050ND0.2\-\--1.01.70.10.6311118.53449460.41.10.5NDND3.25.00.30.013\*1 Population of microorganisms relative to the initial solution.Table 3**Analytical results of vial tests**SeriesRunTime (d)00.2137101420223050ItemB5pH8.07.87.87.98.08.0NO~3~^--^ (mmol/dm^3^)0.5000.350.250.250.200.15Relative growth rate ^\*1^1.02.50.50.90.30.36pH8.07.77.87.97.98.1NO~3~^--^ (mmol/dm^3^)0.500.350.300.300.200.15Relative growth rate1.01.80.50.80.30.27pH8.07.77.87.87.98.0NO~3~^--^ (mmol/dm^3^)0.550.350.300.250.200.10Relative growth rate1.01.10.30.40.20.2D12pH8.07.08.08.38.8NO~3~^--^ (mmol/dm^3^)5043322Relative growth rate1.03.103.300.100.3013pH8.07.98.48.78.9NO~3~^--^ (mmol/dm^3^)5043454338Growth rate1.00.590.871.900.16E14pH8.09.28.98.78.68.68.38.78.6NO~3~^--^ (mmol/dm^3^)1010.410.19.5410.510.410.410.110.4Relative growth rate1.001.200.610.430.280.280.170.090.07N~2~ (mmol/dm^3^)0.1415pH8.09.19.08.88.88.78.78.88.8NO~3~^--^ (mmol/dm^3^)1010.310.610.310.610.510.410.310.7Relative growth rate1.001.260.710.460.340.270.180.110.08N~2~ (mmol/dm^3^)0.24\*1 Population of microorganisms relative to the initial solution. In series A, Run 1 was run as a blank and Run 2 contained super plasticizer in order to elucidate the potential of super plasticizer as an electron donor on the behavior of NO~3~^--^ reduction using activated sludge as the microorganism. The temporal changes in the composition of nitrogen compounds in Run 1 and Run 2 are shown in Figure [1](#Fig1){ref-type="fig"}. Although NO~3~^--^ was consumed in the blank test (Run 1), more NO~3~^--^ was reduced to produce more N~2~ by the presence of super plasticizers (Run 2). The consumption of NO~3~^--^ in the blank test will be discussed in more detail later. The material balance of nitrogen classified according to species (NO~3~^--^, NO~2~^--^, NH~3~, N~2~ or solid phase \[Nsolid\]) is shown in Figure [2](#Fig2){ref-type="fig"}. Nsolid was calculated from nitrogen contents of the solid fraction. In the Figure [2](#Fig2){ref-type="fig"}, 2N~2~ was used for direct comparison in atomic nitrogen base. In Run 2, NO~3~^--^ initially accounted for all nitrogen not in the solid phase. The production of NH~3~ and the appearance of NO~2~^--^ was limited to the first 6 days of reaction. Consequently, the NO~3~^--^ decrease could be broadly correlated with the production of N~2~.Figure 1**Comparison of nitrate utilization and nitrogen gas evolved with and without super plasticizer as an electron donor in 1.0 mmol/dm**^**3**^**of nitrate solutions and 1000 mg/dm**^**3**^**of activated sludge as the microorganism.** Run 2 was carried out with the electron donor and Run1 was carried out without the electron donor (blank). 2N~2~ is used for amount of N~2~ evolved in order to directly compare the mass balance with NO~3~^-^.Lines are shown to help identify the general trends shown by the data.Figure 2**Material balance of nitrogen species during Run 2 in the presence of super plasticizer as an electron donor, where the initial concentrations of nitrate and micro organism were 20 mmol/dm**^**3**^**and 1000 mg/dm**^**3**^**, respectively.** In series B (Runs 3--7), humic acids were used as electron donors with activated sludge as the microorganism. Ampule tests were carried out in Run 3 and Run 4 with changes in the initial NO~3~^--^ concentration, while vial tests were carried out in Run 5--7 to investigate temporal changes. The nitrogen balance in Run 3 and 4 (Figure [3](#Fig3){ref-type="fig"}) shows that the main reaction is the reduction of NO~3~^--^ to N~2~ in the case of the humic acids; as was found for the super plasticizers (Figure [2](#Fig2){ref-type="fig"}). Comparison of the NO~3~^--^ reduction rate in the presence of humic acid with super plasticizers (Figure [4](#Fig4){ref-type="fig"}) demonstrates that the amount of NO~3~^--^ reduced in any of Runs 5--7 was much lower than that of Run 2. Furthermore, there was no dependence of NO~3~^--^ reduction rate on the initial NO~3~^--^ concentration (Figure [4](#Fig4){ref-type="fig"}).Figure 3**Material balance of nitrogen species during glass ampoule experiments (Run 3 and 4) in the presence of humic acid.** Run3: Initial concentrations of NO~3~^--^, activated sludge and humic acid were 20 mmol/dm^3^, 1000 mg/dm^3^ and 100 mg/dm^3^ as carbon, respectively. Run4: Initial conditions = Run3, except for NO~3~^--^ = 50 mmol/dm^3^.Figure 4**Comparison of humic acid and super plasticizer as an electron donor from the viewpoint of nitrate utilization with activated sludge as microorganism.** Run 2 (shown as black circles and solid line) was carried out with super plasticizer and Run 3--7 (shown as dotted lines) were carried out with humic acids under the conditions shown in the Table [1](#Tab1){ref-type="table"}. Lines are shown to help identify the general trends shown by the data. In series C (Runs 8--11), tests were carried out using humic acids with *A. tolulyticus* as the microorganism. Run 8 was a blank test without humic acids and Runs 9--11 were with different concentrations of humic acids. There was no significant difference in the NO~3~^--^ reduction reaction and the growth of microorganisms between the blank (Run 8) and those with humic acids (Runs 9--11) (Figure [5](#Fig5){ref-type="fig"}). In each case, the concentration of microorganisms after eleven days decreased below one tenth of the initial value. By contrast, the activity of *A. tolulyticus* was confirmed in the experiments in series D (Runs 12 and 13) (Figure [6](#Fig6){ref-type="fig"}). In this series, glucose was used in Run 12 because it is easily decomposable in comparison with the humic acid used in Run 13. As shown in Figure [6](#Fig6){ref-type="fig"}, NO~3~^--^ was reduced within two days in the presence of glucose, significantly faster than in the presence of humic acids. This result indicates that *A. tolulyticus* can adequately reduce NO~3~^--^ in the presence of easily decomposable organics. It was therefore concluded that no significant denitrification using humic acids could be observed and might be due to a lack of decomposable organics initially introduced. This lack of initial decomposable organics could be a consequence of a dissolved portion of dead microorganisms acting as electron donors instead of the humic acids.Figure 5**Variation of nitrogen species and microorganism in the presence of humic acid as an electron donor (Run8-11).** Initial humic acid concentrations are 0 (small black circles and dotted line), 10 (black marks), 100 (gray marks) and 1000 (white marks) mg/dm^3^. Other initial conditions are the same: initial concentrations of NO~3~^--^ and microorganism (*Azoarcus tolulyticus*) were 50 mmol/dm^3^and 1000 mg/dm^3^, respectively. Lines are shown to help identify the general trends shown by the data.Figure 6**Comparison of nitrate utilization between glucose (Run 12) and humic acids (Run 13) as electron donors with*Azoarcus tolulyticus*as the microorganism.** Initial nitrate concentrations of nitrate, electron donors and microorganism are 50 mmol/dm^3^, 100 mg/dm^3^ and 1000 mg/dm^3^, respectively, by vial experiments. Lines are shown to help identify the general trends shown by the data. Figure [7](#Fig7){ref-type="fig"} shows the material balance of organic carbon in the presence of humic acid. Humic acids correspond to the soluble carbon component, because humic acids used for these experiments were sodium salts from Aldrich, and the microorganisms correspond to the solid carbon. Based on these assumptions, it is shown that carbon in the solid decreased and that carbon in the solution increased with time, indicating that some portion of the solid carbon dissolved. These results imply that soluble carbon produced from the debris of dead microorganisms is consumed by living microorganisms with NO~3~^--^ reduction occurring to some extent.Figure 7**Change in distribution of organic carbon in experiments using the humic acid as electron donors, where the concentrations of the humic acid were 0 (Run 8), 10 (Run9), 100 (Run10) and 1000 (Run11) mg/dm**^**3**^**.** In order to evaluate a consumption rate, the initial concentration of microorganisms should be low enough to avoid influence of the reuse of soluble organics from dead microorganisms. Thus, in additional set of experiments, series E (Runs 14 and 15), the initial concentration of microorganisms was set to 10 mg/dm^3^. In Run14 and 15, vial tests were carried out for 50 days to compare NO~3~^--^ reduction rate without and with the addition of humic acids, respectively (Figure [8](#Fig8){ref-type="fig"}). The amount of N~2~ gas produced was more in Run 15 than in Run 14 and suggests that the consumption rate of humic acids by microorganisms as electron donors might not be zero, but slower than that of other organic materials including organics from dead microorganisms.Figure 8**Comparison of evolved nitrogen gas between blank (Run 14) and humic acid (Run 15).** Two analytical data under each condition are shown in the bar graphs and their averages plotted by. *In-situ* experiments {#Sec9} --------------------- Results of the NO~3~^--^ reduction reaction using microorganisms from actual groundwater at Äspö HRL and an extract of the mixture of super plasticizers and cementitious materials are shown in Tables [4](#Tab4){ref-type="table"} and [5](#Tab5){ref-type="table"}. Table [4](#Tab4){ref-type="table"} shows concentrations of NO~3~^--^, NO~2~^--^ and NH~3~ and Table [5](#Tab5){ref-type="table"} shows data obtained from TNC and Q-PCR analyses. Samples were named S-1 to S-6 with regard to sampling times from 0 to 30 days (Table [5](#Tab5){ref-type="table"}). Sample S-1 is the solution sampled before the injection of NO~3~^--^ and super plasticizer solution. After the injection of NO~3~^--^ and organics, NO~3~^--^ concentration decreased and the number of microorganisms increased and so confirmed that the extract of super plasticizer acts as an electron donor for the NO~3~^--^ reducing bacteria from actual groundwater.Table 4**Concentrations of nitrogen compounds from*in-situ*experiments**Sample No.Sampling time (d)NO~3~^--^NO~2~^--^NH~3~mmol/dm^3^mmol/dm^3^mmol/dm^3^S-100.0000130.0000120.00200S-20.99.050.00560.00078S-33.99.050.00510.00027S-48.98.790.00430.00028S-5177.950.00160.00018S-6317.690.00440.00048Table 5**Analytical result of TNC and Q-PCR**Sample No.Time (d)TNC in solutionQ-PCR of NarG-DNAIn solutionOn biofilmcells/cm^3^σ (n=3)copies/gσ(n=4)copies/gσ(n=8 or 16)S-10130323.94E052.27E052.26E081.85E08S-20.9190543.63E051.17E051.95E087.70E07S-33.91,400997.98E053.12E053.93E083.53E08S-48.92,9001408.93E057.41E054.27E083.22E08S-5174,0001,3003.37E062.19E066.32E082.72E08S-6315,1007104.31E072.84E071.35E097.55E08 Discussion {#Sec10} ========== Reaction model outline {#Sec11} ---------------------- The reaction was modeled with reference to the anaerobic respiration process in ASM1 and ASM3C \[12.13\]. The ASM1 is the most fundamental model and ASM3C is constracuted with TOC-base (unit of both organics and microorganism is in TOC (g-C/dm^3^ or mol-C/dm^3^). Under anaerobic conditions where the electron acceptor is limited to NO~3~^--^, the growth rate of microorganisms with time, dxB/dt(mol-TOC/dm^3^/d), without considering their death, is modeled as (Henze et al. [@CR9];Duthy [@CR4]): where k3 is a rate constant (d^-1^), sS, sNO3, xB are concentrations of soluble organic materials (mol-TOC/dm^3^), NO~3~^--^ (mol-N/dm^3^) and microorganism (mol-C/dm^3^), respectively, and KsS and KsNO3 are half saturation concentration of organic materials (mol-TOC/dm^3^) and NO~3~^--^ (mol-N/dm^3^), respectively. Describing the death rate of microorganisms by bH·xB, where bH is the death rate constant (d^-1^), Equation (1) can be rewritten as: Soluble organics, sS, are consumed by the activity of bacteria and their rate of change is described by: where *x* is the specific growth rate given as the ratio of amount of organics to be transformed to the amount of consumed organics (mol/mol). Furthermore, the changing rate of NO~3~^--^ (electron acceptor) concentration and those of soluble organics (electron donor) can be related by where *z* \[(mol-N)/(mol-C)\] is the N/C ratio of the above pair of reactions. According to experimental studies of this work, when the above reactions are modeled, the following items should be considered: The growth rate and the NO~3~^--^ reduction rate depend on the consumption of organic materials. Among the organic materials examined in this study, humic acids were not easily consumed.By contrast, when no organic material exists or only hard-bio-decomposable organic materials exist, like humic acids, soluble organics produced from dead microorganisms can act as electron donors. In view of the results obtained in this study, organic materials can be listed in the order of ease of microorganism usage as: glucose \> plasticizer \> organic fraction produced from dead microorganisms \> humic acids 3)Biogenic reduction products of NO~3~^--^ are almost exclusively N~2~, but in some cases, depending on the species of microorganisms for example, products may include small amounts of NO~2~^--^ or NH~3~. In order to construct a reaction model considering these phenomena, the standard model, as adopted in the ASM, needs to be modified by the addition of reaction processes and parameters as described below. Organics should be categorized based on the degradability and reaction rate equation and parameters should be assigned to each category. From the viewpoint of geological disposal, important organics are super plasticizers, humic acids and also organics originating from dead microorganisms.In the ASM, production process of soluble organic materials from dead cell is described as hydration reaction of solid organics of dead cell. However, the results of this study suggest that it is also necessary to consider the direct production process of soluble organics from dead microorganisms. It is understood that this direct process may be important in the case of geological disposal environments where the existence of easy-biodegradable organic materials may be low.The NO~3~^--^ reduction reaction by microorganisms is known to follow the sequence of NO~3~^--^ → NO~2~^--^ → N~2~. In general, however, bio-modeling, including the ASM approach, adopts the direct process of N~2~ production from NO~3~^--^ reduction, neglecting any intermediate steps. Similarly, the modeling in this study adopts the direct process, given that NO~2~^--^ production was very low, even when actual groundwater was used (shown in Table [4](#Tab4){ref-type="table"}). Model parameters {#Sec12} ---------------- Before constructing a predictive model, data from the *in-situ* experiments were adapted for use in accordance with the following procedure: The groundwater flow experiment was conducted for thirty days, after which time bio-films had grown on the crushed rock surface. Therefore, the concentration of microorganisms was taken as the sum of those in the solution and in the bio-films.Concentration of microorganisms in the bio-films on TNC (total number of cells of microorganisms described in \"*in-situ* experiments\") base was calculated from the Q-PCR of solution and bio-films correlating with TNC of the solution. Bacteria concentrations from the sum of solution and bio-films on the TNC base were estimated from the sum of Q-PCR data of solution and bio-film, applying a factor obtained by correlating TNC and Q-PCR of the same solution.TNC was transformed to TOC because the model of this study uses microorganism concentrations in TOC base. Assuming that half of the decrease of TOC in the solution was used for the growth of bacteria (x = 0.5), a correlation factor was obtained by calculating the ratio of TOC decrease and microorganism increase throughout the experiment. The microorganism concentration of each time step was then transformed to TOC base using the above correlation factor.A ratio of NO~3~^--^ required to that of carbon used for bacteria growth was also calculated from the experimental data. The resulting adapted data used for the predictive model are shown in Table [6](#Tab6){ref-type="table"}.Table 6**Preparation of data analysis**SampleTime (d)TOC (gC/dm^3^)Count of microbe as TNC (cells/dm^3^)Count of microbe as TOCNO~3~^-^(g-N/dm^3^)In solution (TNC method)In total (correlated to TNC)In total (gTOC/dm^3^)S-10.00.00041.30E+082.42E+10\--S-20.90.1901.84E+082.09E+101.22E-030.125S-33.90.1941.15E+094.21E+102.46E-030.125S-48.90.1912.20E+094.58E+102.67E-030.122S-518.00.1852.86E+096.91E+104.03E-030.115S-631.00.1803.41E+091.68E+119.82E-030.113increase of amount−0.0141.22E+118.60E-03−0.012Parameters^\*,†^(1)(2)(3)*In-situ* test using extract of cement agent.^\*^TOC/Microbe correlation factor = −0.5×(1)÷(2) = 5.74E-14.^†^N/C = (3)÷(1) = 0.86≒1.0 (g-N)/(g-C). Model verification {#Sec13} ------------------ The reaction model was applied to the interpretation of *in-situ* experimental results. From an estimated N/C = 1.0 (Table [6](#Tab6){ref-type="table"}) and using k3 = 0.1 d^-1^ and bH = 0.005 d^-1^, parameters concerned with the growth rate of microorganisms, provided a good correlation with the prepared *in-situ* data (Figure [9](#Fig9){ref-type="fig"}). As indicated by the experiments, microorganisms grew very slowly using the extract of the mixture of super plasticizers and cementitious materials as an electron donor.Figure 9**Comparison between experiments and simulation results.** Data from the in-situ experiment using super plasticizer as an electron donor are plotted as symbols, while simulation results are shown as lines. Representative parameters to be used in Equation (3) are: k3 = 0.1 d^-1^, bH = 0.005 d^-1^ and N/C = 1 gC/gN. Table [7](#Tab7){ref-type="table"} shows values of k3 and bH obtained by this study compared with other values reported in the literature (Henze et al. [@CR9];Duthy [@CR4]). A typical value for k3, the maximum rate constant (corresponding to μ~H~) in the ASM, is 1.0 to 2.0 d^-1^. This is about ten times larger than the k3 = 0.1 d^-1^ found in this study. However, typical values of bH in the ASM is 0.05 to 0.1 d^-1^ and is about 2.5 to 10% of the maximum rate constant, which is compatible with the relation of bH and k3 (5%) found in this study. The k3 reported for the experiment using ethyl alcohol as an electron donor (Duthy [@CR4]) was also ten times larger than the k3 value found in this study.Table 7**Comparison of parameters between this study and previously reported values in the literature** (Henze et al. [@CR9];Duthy [@CR4])Source of datak3bHbH/k3Remarksd^-1^d^-1^%This study0.10.0055Obtained values calculated for extract from the mixture of super plasticizer and cementitious materialsHenze et al. ([@CR9])1.0 - 2.00.05 - 0.12.5 - 10Typical values shown as ASM\'s parameter for organics contained in municipal sewage.Duthy ([@CR4])2.4 - 3.10.0481.5 - 2.0Parameter obtained by one dimensional soil column using ethyl alcohol as organic materials From the *in-vitro* experiments, the extract of super plasticizers is a more bio-degradable organic material than humic acids, but less bio-degradable than easy-decomposable organic materials such as citric acid. The results shown in Table 7 support such an interpretation. Furthermore, the fact that bH was found to be low suggests that microorganisms originated from actual groundwater have relatively slow cycles of growth and death using hard-biodegradable organics instead of easy-biodegradable organics. Conclusions {#Sec14} =========== A series of *in-vitro* and *in-situ* experiments were conducted to investigate the NO~3~^--^ reduction reaction relevant to the geological disposal of NO~3~^--^ containing radioactive wastes. The NO~3~^--^ reduction reaction rate was found to be strongly affected by the degree of degradability of the existing organic materials. Data obtained from the *in-vitro* experiments, using activated sludge or microorganisms originating from an underground environment suggested that de-nitrofication rate using super plasticizer as electron donors were higher than humic acids. In the case of humic acids, the rate was found very low so that organics originated from dead microorganism could act as electron donors. The results were used to develop a NO~3~^--^ reduction reaction model. The model was verified by simulating data obtained from *in-situ* experiments using actual groundwater and microorganisms and provided a good correlation microorganism growth and organics consumption. Although obtained de-nitrification rate parameter for extracts from super plasticizer in the in-situ experiment was about one tenth of those obtained by the experiments using more decomposable organic compounds, nitrated reduction in near field of the repository might occur. **Competing interests** The authors declare that they have no competing interests. **Authors' contributions** KM, HM, KY, OK, KK and AH were members of a project team studying this issue. AH was a project leader and planned the principle scheme of the studies. Every in-vitro experiment was planned by KM and the plan was revised after discussing with OK and KK. Final conditions were confirmed by OK and AH. In-situ experiments were planned by OK and confirmed by HM. Data analyses and modeling studies were carried out by KM discussing with OK, KK, HM and AH. Parameter fitting for applying the bio-model to the in-situ experiments was carried out by KM, HM and KY using a computer program made by KM. All authors read and approved the final manuscript. This work was supported in part by the Ministry of Economy, Trade, and Industry through the program "Combined development of nitrate salt removal technology and an assessment system for the impact of nitrate on the co-locational disposal of TRU waste and HLW"(2007--2011). We also thank Dr. Pedersen of Microbial Analytics Sweden AB for suggestions on planning the on-site experiments at Äspö HRL.

Introduction ============ Aerosol transmission of *Mycobacterium tuberculosis* during active pulmonary disease results in exposure of a substantial proportion of the global population, although only a fraction of individuals develop clinical tuberculosis (de Jong *et al*., [@b11]). Based on priming of an antigen-specific immune response, it is estimated that two billion people have been infected by *M. tuberculosis*, of whom 5--10% will go on to develop disease (WHO report, updated annually). Epidemiology studies suggest that around 20% of individuals intensively exposed to *M. tuberculosis* show no evidence of a memory response, suggesting a level of innate resistance that can function prior to engagement of the adaptive immune system ([@b9]). We aimed to identify innate immunological mechanisms that underlie this diverse spectrum in clinical outcome ([@b2]). In light of the fact that natural killer (NK) cells can produce IFNγ ([@b34]), express cytolytic activity ([@b21]), and respond to conserved determinants of microbial pathogens through Toll-like and other innate immune receptors ([@b7]), we wished to investigate whether these cells could constitute one variable in the immune response to *M. tuberculosis*. Human NK cells display extensive phenotypic heterogeneity and plasticity within and between individuals. For instance, the level of CD56 surface expression discriminates two major subsets of NK cells whose frequencies in the blood vary significantly between individuals ([@b10]). Such variation could have functional immune consequences since CD56^bright^ cells are usually associated with cytokine production, and CD56^dim^ cells with natural cytotoxicity ([@b32]) although this dichotomy has been recently refined ([@b12]). Furthermore, each individual possesses a most likely unique repertoire of NK cells due to (i) the inherited set of genes and alleles coding for each different NK cell receptor, and (ii) the stochastic expression of these genes among NK cells from the same individual ([@b38]). Moreover, there is growing evidence that NK cell responses are tuned by a process that involves an interaction between Killer Immunoglobulin-like Receptors (KIR) on NK cells and host-specific MHC class I molecules ([@b6]). A consequence is that variations between individuals in the repertoire of KIR alleles expressed by NK cells can be associated with differences in responses to pathogen-associated signals and with resistance or susceptibility to different diseases ([@b31]; [@b20]; [@b19]). In the context of tuberculosis, a higher prevalence of KIR2DL3 among TB patients has been observed in two independent studies ([@b28]; [@b24]). In a murine model of tuberculosis, NK cells were found to be recruited to the lung and to produce IFNγ and perforin, although the absence of an infection phenotype following antibody-mediated depletion led the investigators to conclude that their functional role was redundant ([@b17]). In experiments using γ-chain^−/−^RAG^−/−^ mice in comparison with RAG^−/−^ immunodeficient mice, significant NK cell contribution was evident to the control of *M. tuberculosis* infection in the absence of T cell function ([@b14]), consistent with a model in which NK cells provide an alternative source of activities overlapping with those of other immune cells. However the extent to which NK cell contribution derived from mice studies can be extended to humans is notably limited by the inherent expression of independently evolved and structurally unrelated set of MHC class I-specific NK cell receptors belonging to the C-type lectin-like family as opposed to the immunoglobulin-like family in humans ([@b31]). In humans, several clinical studies have explored the potential association between peripheral blood NK cell counts and resistance or susceptibility to tuberculosis ([@b1]; [@b5]; [@b23]). A reduction in frequency and functionality of CD56^bright^ NK cells has been observed in patients with active tuberculosis and reciprocally, high blood levels were associated with protection in putative tuberculosis-resistant individuals. Variations in the number of NK cells in cord blood have also been suggested to influence the efficacy of BCG vaccination ([@b43]; van den Biggelaar *et al*., [@b39]). The present study provides the first evidence of the presence of NK cells in human granulomatous lesions showing that these cells are taking part in the immune response during pulmonary tuberculosis. The cytotoxicity against *M. tuberculosis*-infected cells has been addressed and the mechanisms characterized ([@b40]; [@b16]). However, direct IFNγ responses of peripheral blood NK cells to mycobacterial preparations focused only on attenuated strains, gamma-irradiated or heat-killed mycobacteria rather than fully virulent *M. tuberculosis* ([@b42]; [@b3]; [@b33]). Therefore, the consequence of a direct interaction between live *M. tuberculosis* and human NK cells is still unknown. Hence, we performed a systematic analysis of the responses of NK cells from various anonymous human blood donors, comparing cytokine response intensity to extracellular virulent *M. tuberculosis* H37Rv with the response to the attenuated *Mycobacterium bovis* BCG Pasteur strain. We observed that the major determinant of the NK cell response to mycobacteria is coming from the host and is independent of mycobacterial virulence. We describe an important variation of the cytokine response intensity between NK cells from different individuals and demonstrate a correlation with KIR gene content. Results ======= NK cells are recruited to the lungs during *M. tuberculosis* infection ---------------------------------------------------------------------- Tuberculosis is generally treated by chemotherapy. However, tuberculous patients suffering from multi-drug-resistant tuberculosis may undergo surgery as an adjunctive approach to reduce disease burden, which gives access to resected lung tissue. Based on NKp46, a single universal marker for mammalian NK cells ([@b41]), we used immunofluorescent microscopy to look for NK cells, screening sections from five formalin-fixed and paraffin embedded tuberculous lesions covering most of the different types of human granulomas as reviewed by [@b22]). We were able to detect numerous NK cells especially within inflammatory cell infiltrates in a sample representative of a necrotizing lesion ([Fig. 1](#fig01){ref-type="fig"}, panel b, c) and also within the well-vascularized fibrotic wall delimiting this granuloma ([Fig. 1](#fig01){ref-type="fig"}, panel a, d). The proximity of the NK cells to blood vessels was almost universal, suggesting recent extravasation. We observed a similar distribution within the consolidated area of a tuberculous pneumonia within the fibrotic surroundings of a large necrotizing granuloma (Fig. S1). In another sample, NK cells could be found infiltrating the epithelioid macrophage layer of a well-cuffed granuloma where liquefaction was evident, and within the chronic inflammatory area juxtaposed to it (Fig. S2). NKp46 signals were rarely observed in unaffected airway tissues and only few signals could be detected in the surroundings of a calcified granuloma or within its sclerotic rim (Fig. S3). These observations suggest that, during active tuberculosis, NK cells are recruited at the site of disease especially within highly inflamed granulomatous lesions. At this stage, NK cells can interact with infected cells and also extracellular mycobacteria released following lysis of infected cells by specific CD8 T cells or NK cells themselves. {#fig01} IFNγ production by NK cells in response to extracellular mycobacteria requires cytokine co-stimulation ------------------------------------------------------------------------------------------------------ We aimed to study the consequences of a direct interaction between NK cells and a virulent strain of *M. tuberculosis* and to determine whether mycobacterial virulence could affect this interaction. We therefore started screening for optimal time and conditions in which NK cells would respond to mycobacterial stimulation ([Fig. 2](#fig02){ref-type="fig"}). We cultivated purified human NK cells with or without single cell suspensions of *M. tuberculosis* H37Rv or *M. bovis* BCG (MOI 1:1) in the presence or absence of two common co-stimulatory cytokines for NK cell activity (i.e. IL-2 \[100 U ml^−1^) or IL-12p70 (1 ng ml^−1^)\]. We collected supernatants every 24 h for 3 days and measured release of IFNγ. In this experimental setting, cytokines or mycobacteria alone were not sufficient to independently trigger IFNγ production by NK cells. However, we observed progressive accumulation of IFNγ in culture supernatants from 24 h to 48 h that began to plateau after 72 h of contact with the mycobacteria and IL-2 or IL-12p70. In both cytokine environments, the attenuated BCG vaccine strain elicited a comparable response to virulent *M. tuberculosis* H37Rv. Although the plateau value varies between donors, this kinetic pattern of IFNγ production was found consistent across three independent experiments. {#fig02} IFNγ production by NK cells in response to extracellular mycobacteria is independent of mycobacterial virulence --------------------------------------------------------------------------------------------------------------- We subsequently compared the NK cell response from three anonymous donors that were isolated, cultivated for 72 h in the presence or in the absence of mycobacteria (MOI 1:1) and/or co-stimulatory cytokines, and analysed simultaneously. [Figure 3](#fig03){ref-type="fig"} illustrates the donor variability in the final amount of IFNγ released by NK cells following contact with mycobacteria, independently of mycobacterial strain. Indeed when looking at each donor individually, we confirmed that *M. tuberculosis* was able to trigger very similar cytokine response intensities as *M. bovis* BCG in both cytokine environments. Using intracellular antibody staining and polychromatic flow cytometry on another set of donors, we confirmed that IFNγ originated from NK cells ([Fig. 4](#fig04){ref-type="fig"}A). We also observed that the amount of IFNγ found in the supernatants after 72 h reflected the frequency of IFNγ positive NK cells 24 h post stimulation ([Fig.4](#fig04){ref-type="fig"}B). {#fig03} {#fig04} NK cell secretion profile after mycobacterial stimulation highlights substantial donor variability -------------------------------------------------------------------------------------------------- Since our previous observations suggested quantitative differences in the predisposition of NK cells from individual anonymous donors to respond to mycobacteria, we evaluated this variability in a larger donor sample size. Using standardized culture conditions, we recorded the cytokine response of purified NK cells from 52 independent donors after 72 h of contact with mycobacteria (MOI 1:1). Since neither the mycobacterial virulence nor the nature of the co-stimulatory cytokine influenced the NK cell response in the previous experiments, we arbitrarily chose to limit this screen to *M. bovis* BCG and IL-2 (100 U ml^−1^) as co-stimulatory cytokine. As a result of the lower threshold of detection of the bead fluorescent technology used in this experiment to measure cytokine production, we could now observe a slight but significant induction of IFNγ production by IL-2 alone but not with mycobacteria ([Fig.5](#fig05){ref-type="fig"}A). IFNγ production in response to co-stimulation with IL-2 and live mycobacteria extended over three orders of magnitude when comparing the different donor responses. In addition, since a previous study suggested that *M. bovis* BCG could trigger the production of TNFα by NK cells ([@b25]), we also measured the production of this cytokine in the same set of samples. As with IFNγ, we observed a slight but significant induction of TNFα secretion by IL-2 but also by mycobacteria alone and a synergistic effect of NK exposure to both mycobacteria and IL-2 ([Fig.5](#fig05){ref-type="fig"}B). TNFα production was also variable across the set of donors and there was a significant correlation between the ability of each independent donor to produce IFNγ and TNFα simultaneously (Spearman *r* 0.7426, *P* \< 0.0001). {#fig05} Mycobacteria preferentially trigger NK cell donor associated with KIR B haplotype --------------------------------------------------------------------------------- There is increasing evidence that different KIR/HLA genotypes influence NK cell potency and the threshold of their responsiveness ([@b18]; [@b6]). We therefore characterized the gene content of the KIR cluster for each of the 52 donors screened in the standardized assay. This characterization was performed by PCR using two independent sets of primers per gene as previously described ([@b27]) and summarized in [Table 1](#tbl1){ref-type="table"}. KIR haplotypes can be segregated in two groups (A and B) according to their gene content ([@b15]). In contrast to B haplotypes, which show higher genetic diversity, the haplotype A group is characterized by the absence of *KIR2DL5*, *KIR2DS1*, *KIR2DS2*, *KIR2DS3*, *KIR2DS5* and *KIR3DS1* and would express one single activating KIR, KIR2DS4. Interestingly, we found a significantly higher proportion of donors that were homozygous for the AA haplotypes within the group of low responders, i.e. below the median response ([Fig. 6](#fig06){ref-type="fig"}). Therefore, donors harbouring one or several activating receptors other than KIR2DS4 are significantly more represented in the group of high responders. When we looked for the activating KIR that could drive this association, we found that KIR2DS3 and KIR2DS5, two similar KIR with unidentified ligands, were significantly over-represented within the group of high responders \[χ^2^, d.f. (3.82, 1), *P* = 0.0253\]. {ref-type="table"} revealed a significant association between B haplotypes and high responders, defined as those above the median response of IFNγ.\ B. The activating receptors KIR2DS3 and KIR2DS5 were found to drive this association with a significant higher prevalence within the group of high responders (chi-squared test, *P* \< 0.05).](cmi0014-1734-f6){#fig06} ###### Analysis of the Leucocyte Receptor Complex (LRC) locus shows higher representation of KIR haplotypes B within high responders IFNγ 3DL3 2DL4 3DL2 2DL2 2DL3 2DL5 2DL1 3DL1 2DP1 2DS4 2DS1 2DS2 2DS3 2DS5 3DS1 Haplo ------------- -------- ------ ------ ------- ------- ------- ------- ------- ------- ------- ------- ------- ----------- ----------- ------- ----------- ------- Low E3 4.3 \+ \+ \+ − \+ \+ \+ \+ − \+ \+ − − \+ \+ B D3 4.4 \+ \+ \+ \+ − \+ \+ \+ \+ \+ − \+ \+ − − B H4 4.7 \+ \+ \+ − \+ − \+ \+ \+ \+ − − − − − A G4 5.8 \+ \+ \+ − \+ − \+ \+ \+ \+ − − − − − A S4 6.1 \+ \+ \+ − \+ − \+ \+ \+ \+ − − − − − A F4 8.4 \+ \+ \+ − \+ − \+ \+ \+ \+ − − − − − A M4 10.0 \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ − \+ \+ B T4 11.1 \+ \+ \+ − \+ − \+ \+ \+ \+ − − − − − A D\\4 13.5 \+ \+ \+ \+ \+ − \+ \+ \+ \+ − \+ − − − B L4 15.7 \+ \+ \+ − \+ − \+ \+ \+ \+ − − − − − A K3 15.8 \+ \+ \+ \+ \+ − \+ \+ \+ \+ − \+ − − − B E4 17.6 \+ \+ \+ − \+ − \+ \+ \+ \+ − − − − − A I4 20.0 \+ \+ \+ − \+ − \+ \+ \+ \+ − − − − − A C4 20.6 \+ \+ \+ − \+ − \+ \+ \+ \+ − − − − − A U3 24.8 \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ B R4 27.0 \+ \+ \+ − \+ \+ \+ − \+ − \+ \+ − \+ \+ B N4 29.8 \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ − \+ B G3 31.6 \+ \+ \+ − \+ − \+ \+ \+ \+ − − − − − A K4 33.2 \+ \+ \+ \+ \+ \+ \+ − \+ − \+ \+ − − − B J4 36.1 \+ \+ \+ − \+ − \+ \+ \+ \+ − − − − − A Q4 40.0 \+ \+ \+ \+ \+ − \+ \+ \+ \+ − \+ − − − B W3 48.9 \+ \+ \+ − \+ − \+ \+ \+ \+ − − − − − A J3 81.3 \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ − \+ \+ B A4 95.4 \+ \+ \+ − \+ − \+ \+ \+ \+ − − − − − A O3 115.4 \+ \+ \+ − \+ − \+ \+ \+ \+ − − − − − A I3 142.5 \+ \+ \+ − \+ \+ \+ \+ \+ \+ \+ − − \+ \+ B *Frequency* 100 100 100 34.62 96.15 34.62 100 92.31 96.15 92.31 30.77 38.46 **11.54** **23.08** 30.77 **53.85** High P3 148.9 \+ \+ \+ \+ \+ − \+ \+ \+ \+ − \+ − − − B F3 159.2 \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ − \+ B D4 160.2 \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ − \+ \+ − − B Z3 172.8 \+ \+ \+ \+ \+ − \+ \+ \+ \+ − \+ − − − B V3 207.0 \+ \+ \+ − \+ \+ \+ \+ \+ \+ − − − \+ \+ B H3 225.9 \+ \+ \+ − \+ − \+ \+ \+ \+ − − − − − A B4 237.8 \+ \+ \+ \+ \+ \+ \+ − \+ − \+ \+ \+ \+ \+ B Z2 256.3 \+ \+ \+ \+ − \+ − \+ − \+ \+ \+ − \+ \+ B E\\4 285.9 \+ \+ \+ \+ \+ − \+ \+ \+ \+ − \+ − − − B B\\4 345.3 \+ \+ \+ \+ − \+ \+ \+ \+ \+ − \+ \+ − − B B3 399.5 \+ \+ \+ − \+ \+ \+ \+ \+ \+ − − − \+ − B A3 427.0 \+ \+ \+ − \+ − \+ \+ \+ \+ − − − − − A X3 446.6 \+ \+ \+ − \+ \+ \+ \+ \+ \+ \+ − − \+ \+ B Y3 576.2 \+ \+ \+ − \+ \+ \+ \+ \+ \+ − − − \+ \+ B T3 608.8 \+ \+ \+ − \+ \+ \+ − \+ − \+ − − \+ \+ B S3 640.6 \+ \+ \+ − \+ \+ \+ \+ \+ \+ \+ − − \+ \+ B U4 1207.2 \+ \+ \+ \+ \+ − \+ \+ \+ \+ − \+ − − − B Q3 1581.0 \+ \+ \+ − \+ − \+ \+ \+ \+ − − − − − A M3 1770.7 \+ \+ \+ − \+ − \+ \+ \+ \+ − − − − − A C3 1793.6 \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ B P4 2662.4 \+ \+ \+ − \+ − \+ \+ \+ \+ − − − − − A O4 3320.4 \+ \+ \+ − \+ − \+ \+ \+ \+ − − − − − A L3 4416.6 \+ \+ \+ − \+ \+ \+ \+ \+ \+ \+ − − \+ \+ B C\\4 4528.6 \+ \+ \+ − − \+ \+ \+ \+ \+ − − \+ − \+ B R3 4930.4 \+ \+ \+ − \+ − \+ \+ \+ \+ − − − − − A N3 8259.7 \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ B *Frequency* 100 100 100 42.31 88.46 57.69 96.15 92.31 96.15 92.31 34.62 42.31 **26.92** **42.31** 46.15 **26.92** \* \* DNA was extracted from PBMCs and KIR genotyping established as previously described ([@b27]). The table summarizes the presence (+) or the absence (−) of each KIR gene within the LRC. NK cell donors were ranked (top to bottom) according to the amount of IFNγ measured after 72 h of contact with mycobacteria (MOI 1:1) in the presence of IL-2 (100 U ml^−1^). Using the median, two groups referred as low versus high responders were created for subsequent statistical analysis. Frequency for each gene or haplotype within each group has been calculated and significant changes are highlighted in bold (^\*^*P* \< 0.05). Discussion ========== Consideration of the potential contribution of NK cells is generally restricted to the early phase of mycobacterial infection, prior to engagement of adaptive immunity. Our demonstration of the presence of NK cells within mature granulomatous lesions is consistent with an involvement that extends into later stages of mycobacterial pathogenesis. Given their rapid turnover (\< 15 days) ([@b45]), the presence of NK cells in lesions suggests constant recruitment, which is reflected by the frequency of positive signals in the vicinity of blood vessels proximal to the lesions. Our observations suggest that NK cells are frequently recruited into inflamed tissues, patrolling the lesions where they could intercept infected cells migrating out of the granuloma as well as interact with extracellular mycobacteria released after lysis by cytotoxic cells. Therefore, variation in the host genetics that could affect NK cell responses would give rise to different levels of innate resistance to *M. tuberculosis* infection. We found that human peripheral blood NK cells have the potential to produce IFNγ when they encounter *M. bovis* BCG as well as *M. tuberculosis* in an appropriate cytokine milieu. This indicates that priming of NK cells by cytokines is sufficient to render NK cells responsive to mycobacteria. Release of IL-12 by macrophages and dendritic cells could therefore promote NK cell-mediated production of IFNγ during the early phase of infection, while IL-2 from T cells provides a potential stimulus for activation of NK cells at later stages. The fact that mycobacteria synergize with co-stimulatory cytokines to induce IFNγ production by NK cells is fully in line with previous reports showing the requirement of co-stimulatory cytokines following Toll-Like Receptor agonist stimulation ([@b7]; [@b35]). We did not perform an exhaustive screen of potential co-stimulatory cytokines but it is very likely that IL-15 among other cytokines may also synergize with the stimulation triggered by mycobacteria as observed for Pathogen-Associated Molecular Pattern recognition. Moreover, it has been shown that a cellular contact with APCs can modulate the cytokine response of NK cells that are recruited at the site of tuberculous pleurisy ([@b33]). Initial studies have described partial involvement of TLR2 and the natural cytotoxicity receptor NKp44 in recognition of *M. bovis* BCG ([@b13]; [@b25]). The fact that we observed similar induction of IFNγ production suggests that recognition of virulent *M. tuberculosis* and attenuated BCG vaccine by human NK cells are most likely controlled by the same set of pathogen receptors. In contrast, the level of IFNγ production by NK cells in response to mycobacterial stimulation varies over a 1000-fold between donors. Preferential expansion of particular NK cell subsets during viral infection has been shown to establish a long-lived protective memory response in mice ([@b36]), and it is attractive to speculate that mycobacterial infection or BCG vaccination could similarly establish an NK memory response. Having used anonymous blood donors, and therefore in the absence of medical records, we could not directly correlate NK cell responsiveness with BCG vaccination or previous *M. tuberculosis* infection for instance. However, using *M. tuberculosis* Protein Purified Derivative (PPD) to stimulate PBMCs from our panel of anonymous donors and IFNγ release assay as a read-out, we did not find any significant correlation between the NK cell responsiveness and memory immune response indicative of previous exposure to mycobacteria (Fig. S4). However, we cannot rule out the possibility of bystander amplification or modulation of NK cell activity by other infections ([@b26]). A major component of NK cell inter-individual variability is associated with the expression of specific HLA/KIR haplotypes revealed as a crucial determinant of NK cell responsiveness to tumour cell lines ([@b18]) and pathogen-associated signals ([@b19]). We have shown here that KIR B haplotypes, i.e. those harbouring multiple activating KIRs, and especially KIR2DS3 or KIR2DS5, correlates with a higher responsiveness to extracellular mycobacteria. These two receptors are similar to each other and together form the activating lineage III KIR ([@b29]). Unlike other activating KIR, KIR2DS3 and KIR2DS5 were not derived from a paired inhibitory receptor by recombination and their ligands still remain unknown ([@b8]). Our observations suggest a link between KIR genotype and the ability of the respective NK cell repertoire to react to mycobacteria. However, despite being statistically significant, the contingency test performed to study this association has limited statistical power. For instance, the use of the median as a cut-off to distinguish low from high responders has been performed arbitrarily in order to use all the collected data but other grouping would have led to different interpretations. Sample size calculation indicates that further study would require a much bigger sample size to fully attest the link between KIR haplotype and the cytokine response to *M. tuberculosis*. For instance, 2236 samples for each arm should be screened in order to detect a difference of 160 units of IFNγ on average between each haplotype group (5% significance level, 80% power). It also important to highlight the fact that KIR haplotype does not fully segregate low from high responders and some NK cell preparation from KIR A individuals showed high IFNγ response indicating that KIR haplotype is certainly not the only determinant of the cytokine response to *M. tuberculosis*. Still, this donor variability highlights a substantial potential impact on the pathogenesis of *M. tuberculosis* that has to be considered. One could argue that higher IFNγ production should confer a better protection against an intracellular pathogen through macrophage activation, although higher inflammation could also contribute to exacerbated tissue destruction that is required for transmission in tuberculosis. Three published studies have addressed the influence of KIR genotype in tuberculosis in Mexican, Lebanese and Iranian populations respectively ([@b28]; [@b24]; [@b37]). In the Lebanese study, KIR A haplotype was found more prevalent in the group of tuberculosis patients when compared with healthy controls (2.6:1 versus 1.5:1). However, no association could be found in the Iranian study. Therefore, the relations between NK cell repertoire, mycobacterial responsiveness and the protection or the sensitivity to tuberculosis need to be tested in various population settings. To conclude, our results contribute to the growing evidence that NK cell activities are regulated by a complex interplay between multiple stimulatory and inhibitory signals which generates extensive functional diversity in NK cell populations between individuals ([@b4]; [@b18]). Thus, with their ability to deliver a range of functions that complement various aspects of innate and adaptive immunity, NK cells may make an important contribution to diversity of the human immune response to tuberculosis. Efforts to identify correlates of immune susceptibility to tuberculosis and indicators of successful vaccination are therefore likely to be enhanced by evaluation of NK cell responses alongside measurement of T cell markers in immunological analyses. Experimental procedures ======================= Ethics statement ---------------- The tissues used in this study were collected between 2003 and 2006 as previously described ([@b22]) with written consent of the subjects, approval of the National Masan Tuberculosis Hospital IRB and an exemption by the US NIH, Office of Human Subjects Research. Blood samples, cells and cell cultures -------------------------------------- Fresh blood packs (Buffy coats and Cones) from healthy adult donors were purchased anonymously from National Blood Services, London, UK. Peripheral blood mononuclear cells (PBMCs) were prepared on a Ficoll-Paque density gradient (Amersham Biosciences AB, Uppsala, Sweden) by centrifugation (800 g, 30 min at room temperature), washed twice and frozen in RPMI 1640-FCS (5%)-DMSO (8.7%)-methyl-cellulose (0.1%). NK cells were selected from PBMCs by magnetic cell sorting using indirect NK isolation kit (Miltenyi Biotec, Auburn, CA, USA) according to manufacturer\'s recommendations. Average NK cell purity checked by flow cytometry (CD3^−^/CD16^+/−^/CD56^+/−^) was 97.51% ± 2.47 (standard deviation) across all donors. NK cells were cultured in complete RPMI 1640 medium, including 1 mM sodium pyruvate, and 1% heat-inactivated fetal calf serum. Recombinant human IL-2 and IL-12 were obtained from PeproTech EC. Culture and preparation of mycobacterial strains ------------------------------------------------ *Mycobacterium tuberculosis* H37Rv and *M. bovis* BCG Pasteur were grown at 37°C in Middlebrook 7H9 broth supplemented with ADC (Becton Dickinson, Sparks, USA). The strains were grown to mid-exponential growth phase and pelleted at room temperature. Single cell bacterial suspensions were then prepared as previously described ([@b30]). Briefly, the medium was discarded, bacteria were dispersed by shaking for 1 min with glass beads (3 mm diameter), and resuspended in PBS, pH 7.4. The remaining clumps were removed by centrifuging the supernatant for 10 min at 200 *g*. Bacteria were then plated on Middlebrook 7H11 agar plates supplemented with OADC (Becton Dickinson, Sparks, USA) to establish precise bacterial counts before and after freezing aliquots with glycerol (5% final v/v) and storage at −80°C. Cytokine production analysis ---------------------------- Cell culture supernatants were filtered using 0.2 μm 96-well filter plates (Corning) before detection of cytokines and chemokines using either ELISA kits (Peprotech) or combined cytokine singleplex assays (Invitrogen) on a Luminex^100^, χMAP^TM^ Technology. For intracellular cytokine detection, NK cells were stimulated for 24 h. Brefeldin A (BioLegend) was added during the last 6 h of culture before harvesting cells for antibody staining with anti-CD16-FITC (Miltenyi Biotec) and anti-CD56-PC7 (BD Bioscience), then fixed and permeabilized using BD Cytofix/Cytoperm™ buffer and stained with anti-IFNγ-PE (BD Biosciences). Cells were run on a BD Biosciences FACSCalibur flow cytometer and data analysed using FlowJo 7.6.4. Immunohistochemistry -------------------- Formalin fixed, paraffin embedded lung specimens obtained from lung resection surgery of TB patients with chronic MDR-TB were provided by the Tuberculosis Research Section of the Laboratory of Clinical Disease, NIAID, NIH, Bethesda, MD, directed by Dr Clifton E. Barry, III. Tissue sections (5 μm) were deparaffinized and rehydrated before high temperature antigen retrieval (citric acid 10 mM, pH 6) followed by a 20 min blocking step in PBS/0.15% BSA/0.1% Tween20 and further blocking with Fc Receptor Block solution (Innovex Biosciences) according to manufacturer\'s recommendations. Sections were then incubated overnight at 37°C with anti-human NKp46/NCR1 MAb (MAB 1850 & AF1850, R&D systems) (5 μg ml^−1^) followed by 2 h with Alexa Fluor® 594 chicken anti-mouse and donkey anti-goat IgG (Invitrogen) (20 μg ml^−1^) at room before mounting with DAPI (Vectashield). Sections were washed with PBS between each step. Bright-field and fluorescence image acquisition was performed using a Mirax MIDI Scan with an HXP120 lamp (Zeiss) and a HV-F22 camera (Hitachi). Annotated scaled images were converted to TIFF files using Mirax viewer software. Kirotyping ---------- DNA was extracted from 5·10^6^ PBMCs using QIAmp DNA mini kit (Qiagen). Oligonucleotide sequences and PCR amplifications were performed as previously described ([@b27]). Statistical analysis -------------------- Data analysis, correlation, paired *t*-tests, and contingency tests were performed using GraphPad Prism software. Without assuming a pre-defined distribution of the response tested, non-parametric statistical analysis has been used all across the study. Unless the direction of the association was expected prior to performing the assays, KIR2DS3/5 association study for instance, two-tailed statistical test were always performed. Statistical analysis of the KIR association study has been subjected to external review to the UCL statistical support services who performed sample size calculation. We would like to thank NIMR Histology Services and especially Dr Radma Mahmood for her technical support, Dr Helene Royo for fruitful briefing on fluorescence microscopy and Dr Andreas Wack for critical reading of the manuscript. We would also like to thank the patients and staff of NMTH for their kind participation in the research protocols at ITRC and NMTH. This work was entirely supported by the MRC core funds (U117581288). D.P. was holding a Career Development Fellowship from the MRC. The tissue collection, L.E.V. and S.E. were supported in part by the Intramural Research Program of the National Institute of Allergy and Infectious Disease, National Institutes of Health, USA, and in part by the Korean Ministry of Health, Welfare and Family. Supporting Information ====================== Additional Supporting Information may be found in the online version of this article: ###### NK cells presence in a tuberculous pneumonia sample. At the centre, H&E stain of a section from a tuberculous pneumonia sample resected from the lung of a tuberculous patient that was used for immunofluorescence microscopy assays. Insets from a representative immunostained serial section showing the presence of NK cells (NKp46^+^ in red) in various part of the lesion, within the consolidated area (top left), within lymphoid aggregates filling alveolar spaces (lower right) and at the border of a well-delimitated necrotic lesion (upper right). ###### NK cells presence in a consolidated and necrotizing tuberculous lesion. At the centre, H&E stain of a section from a tuberculous necrotizing granuloma that was used for the following immunofluorescence microscopy assays. Insets from a representative immunostained serial section showing the presence of NK cells (NKp46^+^ in red) starting notably to infiltrate the epithelioid macrophage layer delimiting a large necrotic lesion. ###### NK cells localization in a calcified tuberculous lesion. Top left, H&E stain of a section of a calcified granuloma that was used for the following immunofluorescence microscopy assays. Insets from a representative immunostained serial section reveal the presence of NK cells (NKp46^+^ in red) recently extravasated from a blood vessel (BV) (inset b), or within surrounding alveolar spaces (inset a, d) of a calcified granuloma. Few signals could be detected at the periphery and infiltrating the sclerotic rim (inset c). ###### PBMCs immune response intensity following exposure to *M. tuberculosis* antigens does not correlate with the ability of respective NK cells to respond to mycobacteria. Histogram comparing IFNγ production from (i) PBMC stimulated with Purified Protein Derivative (PPD) from *M. tuberculosis* (2 μg ml^−1^) for 24 h and (ii) NK cell preparation from the matching donor following exposure to *M. bovis* BCG for 72 h (MOI 1:1) in the presence of IL-2 (100 U ml^−1^). We observed substantial differences in the intensity of the immune response to PPD among the 52 donors (28 donors below 20 pg ml^−1^, 16 donors comprised between 20 and 100 pg ml^−1^ and 8 donors over 100 pg ml^−1^). However, there was no evident correlation between the memory response to PPD among PBMCs and the variable responsiveness of NK cell exposed to mycobacteria in the presence of co-stimulatory cytokine. [^1]: Re-use of this article is permitted in accordance with the Terms and Conditions set out at <http://wileyonlinelibrary.com/onlineopen#OnlineOpen_Terms>

1. Introduction {#sec1-nutrients-09-00576} =============== A one-day workshop was convened in Marrakesh (Morocco) in October 2016 to discuss issues around iron interventions in Africa, with a focus on iron fortification. Iron deficiency (ID) is the most common nutritional disorder in the world and estimates have attributed about half the cases of anemia worldwide to iron deficiency \[[@B1-nutrients-09-00576]\]. Although the worldwide prevalence of anemia has slightly decreased in the past 20 years, the situation still raises important concerns in Africa, especially in the Central and Western parts of the continent \[[@B2-nutrients-09-00576]\]. Experts from nine African countries (Algeria, Côte d'Ivoire, Egypt, Ghana, Kenya, Malawi, Morocco, South Africa and The Gambia) from academia or governmental bodies contributed their scientific, clinical or public health expertise and their field experience to share knowledge and visions regarding the current situation and the most appropriate ways to address the specificities of ID in Africa. The objective of the workshop was to provide a landscape analysis of the needs and concerns facing African countries and to exchange knowledge on best practices that are currently in place or could be initiated in the different areas. The workshop covered three topics: (i)the prevalence and assessment of ID;(ii)the benefit/harm ratio of iron; and(iii)the national strategies to fight ID and their impact. This report summarizes the content of the presentations given by the authors and reflects the information, opinions and statements given by all the participants; it does not aim to be exhaustive. In this report, the words "anemia" and "iron deficiency" encompass anemia from all causes and iron deficiency from all causes, except when the wording "iron deficiency anemia" is used. 2. Iron Deficiency in Africa: Prevalence, Causes and Diagnosis Tools {#sec2-nutrients-09-00576} ==================================================================== The prevalence of anemia (from all etiologies) and of ID in the represented African countries is displayed in [Table 1](#nutrients-09-00576-t001){ref-type="table"}, as provided in the presentations and complemented by the participants throughout the workshop. Prof. Abkari presented anemia and ID prevalence data and its causes in Morocco. Further data about anemia prevalence in African young children and pregnant women can be found in WHO-based documents \[[@B2-nutrients-09-00576]\] and in the African Demographic and Health Surveys \[[@B3-nutrients-09-00576]\], and a selection of prevalence data is presented in [Table 1](#nutrients-09-00576-t001){ref-type="table"}. Although the diverse methodologies of survey prevent direct comparisons amongst countries, and despite numerous missing values, the prevalence of anemia from all causes and of iron deficiency (with or without anemia) appear elevated across the African continent, with South Africa consistently displaying the lowest figures. Anemia is widespread elsewhere, and affects more than 70% of young children and more than 45% of women in countries such as Côte d'Ivoire, The Gambia or Malawi. Iron deficiency often concerns more than half of young children except in Kenya, but less so in women, except in Egypt, as prevalence remains below 18%. Low dietary intakes of iron are a major cause of ID in all represented countries. Consumption of animal products, containing high amounts of bioavailable heme iron, is limited by cost and availability issues. In addition, African diets are usually not rich in vitamin C, which enhances iron absorption, and they often contain chelators which bind iron in the digestive tract and limit its absorption \[[@B19-nutrients-09-00576]\]. In Africa, foods from plant sources, such as cereal- or legume-based flours, are often rich in phytates, and many common foods or beverages may contain iron-binding phenolics. This is the case in Morocco where tea, rich in polyphenols, is the national drink consumed throughout the day \[[@B20-nutrients-09-00576]\], including by very young infants \[[@B21-nutrients-09-00576]\]. Although tea is consumed in other African countries, it is at a lower rate and its contributing role in ID is thought to be less than in Morocco. The nature of foods given to the child during complementary feeding appeared key to all participants. Indeed, iron stores at birth are usually adequate, because of maternal transfer; they drastically decrease between the 2nd and the 5th month of life. Complementary feeding practices are often detrimental to iron stores, especially when the child is switched early to iron-poor cow's milk, or suddenly given food from the shared family dish. In a growing number of situations, children may be introduced very early to unhealthy items (such as soft drinks) which do not help iron supplies and may pose threats to the acquisition of healthy eating habits. Besides diet, other reasons contribute to explain the high level of ID and anemia in African young children and women. Firstly, these age groups have increased requirements, linked to growth, pregnancy, lactation or menstrual blood losses. Secondly, idiopathic malabsorption, such as those due to celiac disease, can remain undiagnosed in many African populations, although they reach significant levels \[[@B22-nutrients-09-00576]\]. Thirdly, infections and parasitic infestations, leading to chronic blood losses, thus iron losses, are frequent in many African countries, although infectious agents may differ \[[@B23-nutrients-09-00576],[@B24-nutrients-09-00576]\]. Malaria is endemic in many areas of Sub-Saharan Africa, and many populations are also suffering from other parasites and chronic infections, e.g., cytomegalovirus or hepatitis. North African countries do not experience malaria and are less exposed to parasites \[[@B25-nutrients-09-00576]\], but they experience a high and possibly growing prevalence of *Helicobacter pylori*, especially in Algeria, but also in Egypt and Morocco \[[@B26-nutrients-09-00576]\]. A recent meta-analysis concluded on a plausible link between *Helicobacter pylori* infection and ID \[[@B27-nutrients-09-00576]\] but studies that have measured iron absorption in individuals with *Helicobacter pylori* infection have produced mixed results \[[@B28-nutrients-09-00576],[@B29-nutrients-09-00576],[@B30-nutrients-09-00576],[@B31-nutrients-09-00576]\], and the role of *Helicobacter pylori* in the etiology of ID in Africa remains uncertain. The African continent as a whole thus concentrates a high number of factors that increase the risk of ID. However, regions and even countries within the same African region, differ in the pattern of risk factors; for example, malaria is more prevalent in Sub-Saharan Africa than in the Maghreb; within Northern Africa, dietary habits are different between Morocco, where iron-chelating tea is highly consumed, and the neighbor Algeria where this drink is much less popular. These examples led the participants in the workshop to conclude that strategies to fight ID must consider regional and national specificities. Iron deficiency is most commonly assessed using hemoglobin (Hb) concentration. Limitations with this method include lack of specificity (all causes of anemia affect Hb concentration) and sensitivity as a drop in hemoglobin is a late manifestation of ID. ID can also be estimated using blood ferritin level, which assesses the size of body iron stores. However, ferritin is an acute phase reactant and it is elevated during infection or inflammation thus confounding the interpretation. Other biomarkers of iron include serum iron, serum transferrin, total iron binding capacity (TIBC) and unsaturated iron binding capacity, transferrin saturation, transferrin-ferritin index (TfR-F), soluble transferrin receptor (sTfR), zinc protoporphyrin, mean cell volume or mean cell hemoglobin concentration (MCHC). A summary of their meaning, practicalities, advantages and disadvantages is available elsewhere \[[@B32-nutrients-09-00576]\]. In addition, the setting of appropriate cut-off values for each one of these markers to identify ID in a consistent and comparable way is still a scientific challenge, as was illustrated during the workshop by Prof Phiri, who compared a wide range of ID markers in 381 severely anemic Malawian children (mean age: 20 months) \[[@B33-nutrients-09-00576]\]. The prevalence of ID according to each marker was assessed using internationally accepted WHO cut-off values and compared to the prevalence of ID estimated via bone marrow iron, considered as the "gold standard" measure, in spite of its invasiveness and subjectivity \[[@B34-nutrients-09-00576]\]. Sixty percent of children had malaria parasites and CRP was raised in 89% of them, indicating that inflammation was present in most children. ###### Hepcidin has been mentioned across the various sessions of the workshop. 1. This peptide acts as *the master regulator of iron* (^9^: it controls its dietary absorption, storage, and tissue distribution. 2. Hepcidin integrates signals from iron in serum, liver and bone marrow and from inflammation; it could thus act as a *biomarker reflecting iron status*, but its effective use as such still needs more research. 3. Hepcidin prevents iron absorption in inflammatory contexts and may blunt the efficacy of iron interventions in such contexts. Depending upon the different markers used, ID was detected from 1% (through TIBC) to 97.5% (through serum ferritin) of children, demonstrating a clear lack of consistency. When specificity and sensitivity were computed, a reasonably good performance was found for four markers: ferritin, sTfR, TfR-F index and MCHC. New cut off values were proposed for these markers, sometimes very different from conventional ones; TfR-F index and MCHC were thought to be the preferable tools to assess ID in such a population of severely anemic and infected young children. Many of the workshop participants confirmed challenges using currently recommended cut-off values and were keen to have more robust standardized guidelines. Similar studies would be welcomed in populations of different countries, ages and infectious or parasitic status, in order to know which markers and cut-offs would be the most appropriate. The need for a simple and reliable biomarker was strongly expressed, and several participants acknowledge that hepcidin, a protein which plays a central role in iron regulation, including absorption, recycling, and tissue distribution (see [Box 1](#nutrients-09-00576-box001){ref-type="boxed-text"} and \[[@B35-nutrients-09-00576]\]) could play this role. Indeed, there was a consensus that detecting individuals and populations who are really suffering from ID are of utmost importance: not only to clarify knowledge and statistics about prevalence rates, but most importantly to identify the right target populations for iron interventions. However, concerns were raised that the multiplication of markers and cut-off values would not ease the comparison across countries or the standardization of methods and would add even more complexity for policy makers. It was agreed that WHO guidelines should be followed for now, at least until evidence becomes available from current initiatives, such as the BRINDA project which aim at improving the interpretation of ID biomarkers in various contexts of infection burden; several methods are being proposed, including a regression model taking inflammation into account, instead of using ferritin cut-off value \[[@B36-nutrients-09-00576]\]. However, until such models are validated and accepted, serum ferritin remains the preferred marker, except where inflammation is prevalent, in which case the use of soluble transferrin receptor may be more appropriate \[[@B32-nutrients-09-00576]\]. ###### Highlights of session about prevalence and diagnosis of iron deficiency (ID). 1. Precise, reliable and comparable data on the prevalence of ID are lacking in many countries and population groups. Among other factors, this is due to biomarkers being frequently biased by inflammation. 2. ID is a worrying reality in young children and women, which does not seem to be currently decreasing. 3. ID-associated factors vary according to regions and countries, but inappropriate eating habits, and high burdens of infections and parasitic diseases are critical. **Research needs** 1. Initiate large epidemiological surveys on representative populations and with appropriate ID biomarkers including inflammatory markers. 2. Focus on post-partum anemia and ID, which are under-studied. 3. Improve ID biomarkers and their cut-offs and set clear interpretations according to context of use. 3. Benefits and Risks of Iron Interventions {#sec3-nutrients-09-00576} =========================================== As stated by one of the presenters during the workshop, "iron is probably the most widely used therapeutic in the world, but without a real test of its efficacy/risk balance". This paradox is raising a growing concern, particularly in settings with a high infection burden, which is addressed by several agencies or scientific bodies, such as the IUNS (International Union of Nutrition Societies) \[[@B37-nutrients-09-00576]\] so far without definitive and operational answers. As highlighted by Dr. Mwangi, an appropriate iron status is essential to enable each child to become a "five star" adult, fully developed both physically and cognitively. Iron is important at each developmental stage, from the fetal life, to infancy, childhood, then teen and adult ages and WHO has developed guidelines for iron supplementation in most of these groups \[[@B38-nutrients-09-00576],[@B39-nutrients-09-00576],[@B40-nutrients-09-00576]\]. One out of 10 maternal deaths can be attributed to iron deficiency anemia \[[@B41-nutrients-09-00576],[@B42-nutrients-09-00576]\]. According to the conclusion of a 2015 meta-analysis, preventive iron supplementation reduced maternal anemia at term by 70%, but data about maternal death, coming from two studies only, were inconclusive \[[@B43-nutrients-09-00576]\]. In an additional recent intervention trial in Kenya, anemia at birth affected 22% of women after iron supplementation vs. 50% of non-iron-supplemented women \[[@B44-nutrients-09-00576]\]. Ensuring an appropriate iron status post-partum is often neglected, while it is likely that low iron status are highly prevalent in this group, as 50--80% of women are anemic in low income countries \[[@B45-nutrients-09-00576]\]. Postpartum anemia is associated with an impaired quality of life, reduced cognitive abilities, emotional instability, and depression, which alter the interactions with the baby, with potentially negative impact on infant behavior and development \[[@B46-nutrients-09-00576]\]. Iron deficiency is rarely encountered in newborns, who obtain their iron stores via placental transfer from the mother's iron stores. Optimal maternal iron status is beneficial for the newborn as it increases the transfer of iron to the newborn and may increase birth weight. In the above quoted study, iron-supplementation of ID mothers led to a birth weight being increased by 234 g, compared to 149 g in non-ID mothers and fewer than 17 women needed to be supplemented to avoid one case of low birth weight. Infant iron stores may become depleted starting ages 4--6 months, as stores transferred during pregnancy are depleted at a time when requirements are high because of rapid growth and erythropoietic needs. Iron deficiency at this period and up to school age may irreversibly affect cognitive development and physical growth and provision of iron has shown positive effects in improving global cognitive scores, intelligence quotient and measures of attention and concentration. Iron supplementation also improved age-adjusted height and weight among all children; the strength of the conclusions, however, varies according to studies, ages and outcomes \[[@B47-nutrients-09-00576],[@B48-nutrients-09-00576]\]. Risks associated to iron interventions used to be limited to iron overload, with potential risks of tissue damage and oxidative stress \[[@B49-nutrients-09-00576]\]. These have long been known to occur in conjunction with genetic defects, such as beta-thalassemia or hereditary hemochromatosis, and/or in conditions which require regular blood transfusions \[[@B50-nutrients-09-00576]\]. From the early 2000s, concerns were raised about the effect of iron supplementation on increased susceptibility to infection, based on a potentially enhanced growth of pathogens from available iron. In 2006, the results of the Pemba study demonstrated that, in children living in malaria-endemic settings, iron and folate supplementation increased all-cause mortality and hospital admissions vs. a placebo control \[[@B51-nutrients-09-00576]\]. More concern is now given to the infectious context when implementing iron intervention, and recent systematic reviews have concluded that, overall, iron supplementation does not cause an excess of clinical malaria in children when proper malaria prevention and treatment is implemented \[[@B52-nutrients-09-00576]\], which is however often not possible in low-resource settings. Other infections could be affected by iron supplementation, as demonstrated by findings of an increased respiratory morbidity in iron-supplemented South African children \[[@B53-nutrients-09-00576]\], of diarrhea \[[@B54-nutrients-09-00576]\] or of increased infectious outbreaks in HIV positive Malawian children \[[@B55-nutrients-09-00576]\]. In a meta-analysis of trials in children aged 6--24 months, diarrhea, vomiting and fever were more prevalent in children receiving iron \[[@B1-nutrients-09-00576]\]. As early as 2002, a small, but significant increase in diarrhea had been reported in a systematic review of the effect of iron supplementation on incidence of infectious illness in children \[[@B56-nutrients-09-00576]\]. The mechanism of this effect may be explained by the fact that iron is a growth-limiting nutrient for many pathogenic gut bacteria, a topic developed during the workshop by Prof. Zimmermann. Indeed, pathogenic strains compete for unabsorbed dietary iron in the colon \[[@B57-nutrients-09-00576]\], whereas bifidobacteria and lactobacilli, which exert colonization resistance versus pathogens require little or no iron \[[@B58-nutrients-09-00576]\]. Clinical demonstration of these microbiological considerations has been provided in a six-month trial on anemic Ivorian schoolchildren receiving a biscuit containing 20 mg electrolytic iron four times a week or a placebo. Iron fortification caused a five-fold increase in enterobacteria, a five-fold decrease in lactobacilli and a five-fold increase in fecal calprotectin, an inflammatory marker \[[@B59-nutrients-09-00576]\]. These findings were confirmed in a controlled trial of multi-nutrient powder (MNP) in home fortification, in a younger population of Kenyan 6-month old children, whose microbiota was initially composed of 63% bifidobacteria but was also highly contaminated with pathogens. Iron supplementation significantly increased both the ratio of enterobacteria to bifidobacteria and enterobacteria to lactobacilli, increased inflammation and increased pathogenic *E. coli*. Diarrhea occurred in nearly 30% of children receiving iron, vs. in 8% of those who did not (non-significant difference) \[[@B60-nutrients-09-00576]\]. These findings raise safety concerns for African infants for whom increased dietary iron intake may enhance susceptibility to diarrhea and possibly, bacteremia and sepsis. In adults, and especially in pregnant or post-partum women, it would be plausible that iron supplementation could pose a potential risk of higher infections, for the same biological reasons as in children. It has indeed been observed that iron deficiency is associated with a reduced prevalence and density of *Plasmodium* parasites in placental blood; it is concerning that potential effects of iron interventions in increasing malaria could be more pronounced in pregnancy, when iron absorption is higher. However, no evidence of an enhanced *Plasmodium* infection has been so far demonstrated, including in a recent study on Kenyan women supplemented during pregnancy \[[@B61-nutrients-09-00576]\]. It should be pointed out that these women received preventive treatment for malaria from regular health services during the study. During the lively discussion following both presentations in this session, the overall feeling was that iron interventions for vulnerable populations such as infants and young women are still needed and that their benefits generally outweigh their risks. However, there was consensus that such interventions should be carried out with caution, especially in children with infectious risk. Safety of iron interventions can be further promoted by an appropriate control of worm infections, malaria and other infectious risks. Recently reported improvements in malaria control in Africa may raise some hope in this regards \[[@B62-nutrients-09-00576]\]. Discussion also focused on individualization of iron interventions, which should probably be preferred to population-wide programs, in order to target those who need and who are not at risk of unfavorable outcomes of iron intake. Individual decisions, taken at the clinic, by practitioner would enable targeted and probably safer approaches. Indeed, even though malaria is not present everywhere in Africa, many other conditions may exist that interfere with the benefit/risk ratio of iron interventions, such as high prevalence of HIV, such as in Malawi \[[@B55-nutrients-09-00576]\]. Other concerns include a high overall infectious risk in young children, usually not monitored or controlled, and the frequent inappropriate use of antibiotics, additionally perturbing the gut microbiota. In addition, the potential interaction of iron with other micronutrients, such as vitamin A, riboflavin and iodine, should be considered. ###### Highlights of session about benefits and risks of iron interventions. 1. *Clear benefits* regarding pregnancy, growth and development: -Decreased maternal anemia-Increased infant's birth weight-Improved cognitive capacity of children under 2 years 2. *Potential risks* may in high infectious context: -Enhanced pathogen growth (increased availability of iron in blood and unabsorbed iron in the gut).-Increased infectious risk in the absence of monitoring and treatment programs. 3. However, *benefits outweigh risks*, in particular when infection can be monitored and controlled. 4. *Caution should* remain, especially when infants and children are concerned and, if possible, interventions should be targeted to iron-deficient subjects. **Research needs** 1. Better assessments of the impact of iron intervention on growth outcomes and cognition in younger children. 2. Exploration of the harms of high/excessive iron status and associated outcomes, especially in pregnancy. 3. Investigations on the impact of iron on gut microbiota in various populations (e.g., pregnant women). 4. Strategies for Iron Intervention {#sec4-nutrients-09-00576} =================================== Because ID has long been recognized as a major global public health problem, international agencies, governments of concerned countries and non-governmental organizations have been working on potential solutions to control ID, as summarized by Prof. Prentice. These include:Increasing the dietary supply of iron-rich foods, among which animal-sourced ones offer highly bioavailable iron. This ideal solution is often difficult to implement for economic and practical reasons.Delaying cord clamping at birth is a simple but efficient means to increase the infant's body iron stores, which could be developed through appropriate education of health care professionals.Fortifying some foods within the usual diet can be done centrally on the whole supply of staple foods such as flour, without targeting population groups or individuals. Home fortification with MNPs or industrially fortified processed foods (e.g., biscuits, cereals, infant formulae) may allow some personalization and thus a better adaptation to individual's needs.Supplementing vulnerable groups is recommended by WHO for populations, living in settings where anemia prevalence is over 40%, including menstruating women \[[@B38-nutrients-09-00576]\], post-partum women \[[@B39-nutrients-09-00576]\] and children above six months \[[@B40-nutrients-09-00576]\]. According to WHO guidelines, supplementation is today most often recommended on a daily basis. Strategies currently implemented at the national level in African countries are diverse. Dr. M. Gbané detailed those existing in Côte d'Ivoire and participants shared their experience about practices in their respective countries. Fortification of staple foods (wheat and/or maize flour) exists in all represented countries but Algeria \[[@B63-nutrients-09-00576]\]. Iron compounds commonly used are either electrolytic iron (35 ppm in South Africa, or 45 ppm in Morocco), water soluble forms (60 ppm ferrous fumarate or sulfate in Côte d'Ivoire, Egypt, The Gambia and Ghana), or sodium-Ethylene Diamine Tetraacetic Acid chelated (EDTA) forms (5 to 50 ppm) in Kenya or Malawi \[[@B63-nutrients-09-00576]\]. The coverage is however often low, such as in Morocco, where only 35% of bakers using fortified flours in urban areas and in Côte d'Ivoire where the country coverage of fortified flour does not exceed 14%, due to political crises. Point of use fortification, using MicroNutrient Powders (MNP), is a more targeted approach, implemented in a few countries, such as Ghana or Kenya \[[@B64-nutrients-09-00576]\], but with a low adherence of populations after the first few months. Some iron-fortified infant and young children foods (formulae, growing up milk, cereals, etc.) exist in several countries (Algeria, Côte d'Ivoire, Egypt, etc.), but their use is usually not widespread. Policies for targeted supplementation of women or young children vary from one country to another. Malawi has no country-wide program, whereas Morocco, Kenya and Côte d'Ivoire have implemented intermittent supplementation for women, but none for children. Algeria targets women and at-risk newborns (premature, low birth weight and twins). The Gambia recommends daily supplementation of all pregnant women. Other public health policy measures are taken by countries in order to improve the efficacy of iron interventions via raising awareness and knowledge of health care professionals and populations. In Algeria, for example, the AAPNEM (Association Algérienne pour la Nutrition de l'Enfant et de la Mère) develops educational programs for mothers, but also for health care professionals and social workers. In Egypt, an awareness campaign is planned in 2017. The opinion of the participants was that none of these strategies have been completely effective, and trends from surveys on Hb levels and anemia prevalence from 1990 to 2010 appear to confirm this feeling; indeed, in several African countries, these figures have not improved and may even have worsened in a few cases \[[@B2-nutrients-09-00576]\]. Discussion showed that this could be due to a myriad of different reasons, more or less important according to countries and situations, among which participants identified: The difficulty of identifying and then reaching the target population groups and individuals.The lack of awareness, knowledge and understanding (especially regarding for home fortification) of caregivers, but also health professionals.The poor adherence to programs or prescriptions.The poor availability of iron-containing supplies (supplement, fortified staple foods, and fortified products), which may be missing at point of supply or purchase.The cost: Even for government-funded supplementation programs, which is not always the case, the subject should often pay a part of the cost. Iron-fortified products are often too expensive for the populations who would need them.The low bioavailability of some iron forms. Several countries, such as Morocco, are currently considering a change to the more bioavailable NaFeEDTA in their mandatory fortification programs.The infectious context: Infants and young children may be especially vulnerable to infection, both because they are exposed to pathogens and because their immune system is still immature. In addition, infection-induced hepcidin secretion limits iron absorption in contexts where the hygiene level is low \[[@B35-nutrients-09-00576]\]. The discussion also addressed the actions aimed at minimizing the risks linked to iron interventions. A first requirement should be to target only populations and individuals who really need iron. Screening for ID is thus seen as a mandatory step before implementing interventions, with a marked preference for not relying only on anemia prevalence. The second requirement should be a better control of the infectious and inflammatory risk in targeted populations: deworming, malaria prevention (mosquito nests) and treatment and increased water supply are needed to improve hygiene and sanitation and reduce pathogens, as promoted by UNICEF in its WASH (Water, Sanitation and Hygiene) programs. Education also appeared to all participants as a powerful lever to fight ID. Hygiene education and nutrition education are equally important in raising women's and mothers' awareness and understanding, enhancing their commitment and adoption of good practices. Education should stress the importance of iron for them and their children and promote exclusive breast feeding during the first months of life, appropriate complementary feeding and appropriate dietary diversification. Providing training to health care professionals is seen as critical to keep them aware of the risk of iron intervention in infected children and the need to screen for ID with appropriate markers and cut-offs. Professionals involved around delivery should know better about the importance of delayed cord clamping. Finally, health-care professionals should also be sensitized to the need of considering that, beyond young children, school children, post-partum women and child-bearing age women may also be at risk of ID. Another approach to cope with potential harms of iron interventions is to reduce the iron dose as low as possible by maximizing absorption. The amount of supplemental iron provided daily can be significantly reduced if efforts are made to increase its bioavailability by using well-absorbed forms of iron and enhancers of iron absorption. In a study on South African school-aged children with a low iron status, Prof Zimmermann's team demonstrated that providing only 2.5 mg of iron as NaFeEDTA together with ascorbic acid and exogenous phytase active at gut pH decreased the prevalence of ID by more than 75% vs. 35% in the control group \[[@B65-nutrients-09-00576]\]. Indeed, a lower iron dose would result in less unabsorbed iron entering the distal gut and would be expected to decrease the risk of iron-induced pathogen growth in the gut, and of associated infections. Another potential approach could be to co-fortify prebiotic components, such as galacto-oligosaccharides (GOS) with iron in MNPs, in order to support bifidobacteria in children's microbiota, thus maintaining colonization resistance and preventing or decreasing pathogen growth following iron supplementation. This has been tested by Prof. Zimmermann's group in 155 Kenyan infants aged 4--7 months, in a four-month long trial during which children were randomized into three groups, which received either no iron or 5 mg iron added or not to 7.5 g of GOS. ID prevalence decreased significantly in both iron-supplemented groups vs. in the no iron group. Furthermore, microbiota of children receiving iron + GOS had a more favorable profile (more *Lactobacilli*, more *Bifidobacterium longum*, less pathogenic *E. coli*, lower ratio of *Enterobacteriaceae* over *Lactobacilli* and *Bifidobacteria*) when compared to children receiving iron alone. This was associated with a lower rate of upper respiratory infections and diarrhea in children treated with iron + GOS, compared to both other groups. These data, still unpublished, were seen as extremely promising by the audience, although deserving confirmation in other studies and in other age ranges. ###### Highlights of session about iron intervention strategies. 1. Many African countries have governmental programs to fortify flour and to supplement pregnant women; supplementation of children is not systematic. 2. Coverage and efficacy of these actions are variable, but usually far from optimal, for many reasons (difficulty to reach the target population, cost and iron bioavailability of supplement/fortified foods, lack of awareness and compliance, etc.). 3. The risk of increased infections, especially in young children, acts as a bottleneck in areas with high infectious disease burden. 4. Improving availability of iron (chelated forms, absorption enhancers, etc.) would help to lower iron dose, thus to decrease harms, while keeping a similar efficacy. 5. Using prebiotics together with iron could lower risk of enteropathogen growth and infections in infants. **Research needs** 1. Demonstrate that increased clean water availability, washing practices and overall hygiene increase the safety and efficacy of iron intervention in young children. 2. Confirm and extend studies about prebiotics and determine which one(s) would be best adapted to an African context and to various ages and populations. 5. Fortified Food Products: Which Products for Which Target Group? {#sec5-nutrients-09-00576} ================================================================== The final discussion focused on practical recommendations that could be given regarding iron fortification in Africa. Regarding targets of such fortified food products, there was a consensus that children aged six months to three years are of primary concern. As this target is also the vulnerable to infections, it implies a careful monitoring of risks associated to iron fortification: knowledgeable parties should be involved, which means that education and training should be implemented together with fortification. Screening for ID and for infection should also be performed. Besides young children, other targets should be considered, namely school aged children, adolescents and women in the periconceptional period, as well as pregnant and post-partum women. Preferred iron forms could include NaFeEDTA, or ferrous fumarate or sulfate, because of their high bioavailability, along with ascorbic acid as an absorption enhancer; however, because it is sensitive to heat and oxidation, ascorbic acid requires some technical expertise to remain intact in food products. Iron bisglycinate is a well-absorbed form of iron, although potentially costly; other iron forms such as iron hydroxide adipate tartrate (IHAT) that are currently under investigation in Africa deserve future attention. The iron dose in a fortified food product should provide at a level equivalent to at least 20% of the dietary requirement, but is not intended to cover the entire daily iron supply: this low dose should lower the harms associated with iron. The food vehicle or matrix should be adapted to target- and country-relevant dietary habits, and should be available at an affordable cost. It should also be a food appreciated by the target groups (for children, cereal or biscuit, or porridge drinks for women, for example). Including prebiotics in iron-fortified products appears as a very promising route, especially in low hygiene environments, but this needs further research, both to confirm efficacy and to address practicalities (type of prebiotic, dose, technology and costs). It was clearly stressed that iron fortified food products should have a favorable nutritional profile in addition to iron content and should not introduce inappropriate dietary habits, i.e., should not favor overconsumption or unbalanced diets. Programs should ensure that the fortified products are eaten by those who need them most. 6. Conclusions {#sec6-nutrients-09-00576} ============== This workshop was an opportunity for experts from various backgrounds and countries to share their experience and questions around ID, which remains a public health problem on the African continent. It was clear to all participants that Africa is changing quickly and that there are reasons to be optimistic about its improving capacity to reduce ID. As a country/continent develops economically and gets better infrastructure, namely, in the sanitation, education and health domains, many infectious diseases will become easier to decrease or eliminate. The challenge would be to find the optimal route that avoids the "double nutrition burden" (i.e., coexistence of deficiencies and obesity), which means choosing smart interventions that fight disease and micronutrient deficiencies without predisposing the population to metabolic diseases. Among the routes offered to Africa to better and more efficiently address ID, the opportunities offered by developing partnerships among various stakeholders should be better exploited. These could include projects involving several or all entities among governments, international organizations and non-governmental associations, but also industries, including food industries. By joining different forces and expertise the issue of ID will be more efficiently tackled, in all its dimensions. This report contains inputs from the other workshop participants, listed here by alphabetical order: M. Bouchenak (DZ), K. Bouziane-Nedjadi (DZ), N. Chaoui (DZ), F. Derouiche (MO), E. Echoka (KE), L. El Ammari (MO), A. El Beleidy (EG), A.G. Konan (CI), I. Nassif (EG), A. Richmond (GH), C. Taljaard (ZA). A.P., M.Z., V.A.B. and R.B.S. wrote the first draft of the manuscript. The scientific material provided by M.W., K.P., A.A., M.G., A.P. and M.Z. during their presentations at the workshop were used to write the report. All presenters reviewed and approved the entire paper and gave additional inputs (additions and references), regarding the section they contributed most. The workshop "iron for Africa" has been sponsored by Danone. M.N.M., K.S.P., A.A., M.G., V.A.B., M.B.Z. and A.M.P.'s travel expenses have been covered by Danone. A.A., M.G. and V.A.B. have received fees from Danone for their participation to this workshop. R.B.S. is employed by Danone Nutricia Research. Hb Hemoglobin GOS Galacto-OligoSaccharides ID Iron Defiency IUNS International Union of Nutrition Societies MCHC Mean Cell Hemoglobin Concentration MNP MicroNutrient Powders sTfR Soluble Transferrin Receptor TfR-F Transferrin-ferritin Index TIBC Total Iron Binding Capacity nutrients-09-00576-t001_Table 1 ###### Prevalence (%) of anemia from all causes (A) and of iron deficiency (ID) in selected African countries. Age Group Algeria Egypt Côte d'Ivoire The Gambia Ghana Kenya Malawi \[[@B4-nutrients-09-00576]\] Morocco South Africa \[[@B5-nutrients-09-00576]\] ------------------------- --------------------------------- --------------------------------- --------------------------------- ---------------------------------- --------------------------------- ---------------------------------- ------------------------------------- --------- ------------------------------------------- ---------------------------------- --------------------------------- ---------------------------------- ---- ---- ---------------------------------- ---- ------ ---- Children below 5 64 \[[@B6-nutrients-09-00576]\] 51 \[[@B6-nutrients-09-00576]\] 27 \[[@B7-nutrients-09-00576]\] 64 \[[@B8-nutrients-09-00576]\] 75 \[[@B9-nutrients-09-00576]\] 51 \[[@B10-nutrients-09-00576]\] 73 \[[@B11-nutrients-09-00576]\] NA 66 \[[@B12-nutrients-09-00576]\] NA 46 \[[@B2-nutrients-09-00576]\] 21 \[[@B13-nutrients-09-00576]\] 73 NA 30 \[[@B14-nutrients-09-00576]\] NA 10.5 11 Child-bearing age women NA NA 39 \[[@B4-nutrients-09-00576]\] 51 \[[@B15-nutrients-09-00576]\] 54 \[[@B9-nutrients-09-00576]\] 17 \[[@B10-nutrients-09-00576]\] 60 \[[@B11-nutrients-09-00576]\] NA 42 \[[@B12-nutrients-09-00576]\] 16 \[[@B16-nutrients-09-00576]\] NA NA 46 NA 33 \[[@B17-nutrients-09-00576]\] NA 23 15 \* A: anemia; ID: Iron deficiency; NA: data not available. Data come from the most recent available sources. Data from representative surveys have been preferred (see references for date and scope of surveys). Anemia was identified by Hb blood levels below 110 mg/dL in children and pregnant women and 120 mg/dL in non-pregnant women \[[@B18-nutrients-09-00576]\]. [^1]: The authors wrote the report on the behalf of the workshop participants, listed in the acknowledgment section.

Introduction {#s1} ============ Enterococci are official fecal indicator bacteria (FIB) in many countries for determining the bathing water quality and monitoring with the intention to protect swimmers from exposure to enteric pathogens ([@B1], [@B2]). An ideal FIB has a similar decay rate to enteric pathogens ([@B3]). However, the wide taxonomic range of microbes in the water relevant to the health of swimmers might have a different response toward environmental stress and growth factors such as pH values, solar radiation, salinity, predation, temperature and nutrients ([@B4]). Further, studies provide evidence that enterococci and enteric pathogens may persist longer in aquatic sediment and vegetation than in water ([@B4], [@B5]). In certain aquatic conditions, enterococci may grow in beach environments ([@B5], [@B6]) and enterococci originating from natural sources may pose unnecessary false positive alarms of a health risk. In contrast, the higher persistence of pathogens over FIB may jeopardize the health of beach users due to false negative alarms ([@B4]). Therefore, understanding the ecological interactions of different fecal indicators and pathogens with various biotic and abiotic factors found at bathing sites is important. In a warming climate, in which surface water temperatures are rising, the characterization and predictions of health risks to swimmers are more important than ever ([@B7]). In the brackish Baltic Sea region, pathogenic members of the *Vibrio* bacterial genus are a concern because their proliferation benefits from a rise in the sea surface temperature ([@B8]--[@B10]). In recent history, the summertime heat waves of 2014 and 2018 have shown that a rise in surface water temperatures together with water nutrients and favorable wind conditions can lead to extensive occurrences of *Vibrio* spp. in the coastal waters of the Baltic Sea ([@B11], [@B12]). Particularly in the northern parts of the Baltic Sea, a subsequent rise of *Vibrio* spp. infections, mainly wound and ear infections, have been noted ([@B11], [@B13]) and pose a significant health risks to certain groups of people, namely the elderly, immunocompromised patients and persons with open wounds ([@B14]). Several *Vibrio* species with pronounced pathogenic potential (including the non-O1/non-O139 serotype of *Vibrio cholerae* and *Vibrio vulnificus, Vibrio parahaemolyticus*, and *Vibrio alginolyticus*) are known to occur in the Baltic Sea region ([@B9]--[@B11], [@B13]), and notifiable *V. cholerae* infections in Finland have been associated with severe manifestations of illnesses such as septicemia ([@B11]). A rise in the bathing water temperature may have also indirect effects on microbial loads on beaches. High temperatures attract crowds to beaches for relief, especially if exceptional, extreme weather events occur ([@B7]). High temperatures may thus challenge beach management paradigms, and norovirus outbreaks have been associated with a sudden increase in the number of beach users ([@B15]). Furthermore, previous studies have detected viruses in surface water even when the FIB numbers were below the safe limit according to current monitoring protocols ([@B15], [@B16]). Such findings imply that FIB is insufficient for indicating the presence of enteric viruses in environmental waters; which justifies the need for introducing a virus indicator for fecal contamination to assess health risks of bathers ([@B3], [@B17]). Coliphages resemble human enteric viruses in their physical structure, composition, morphology, and survival characteristics in the environment and have been suggested as a potential virus indicator associated with human fecal contamination for monitoring bathing water ([@B3]). F-specific coliphage MS2 is one of the most commonly used virus indicators for water quality testing ([@B3]). Almost every year by the end of the bathing season, bathing sites on the coast of the eastern Gulf of Bothnia (Baltic Sea) have experienced problems with overly high enterococci counts, without a known contamination source. Instead of fecal contamination, excess growth of aquatic vegetation has been noted at these bathing sites, while our earlier study identified enterococci species of mostly *Enterococcus faecalis* ([@B18]). Additionally, a knowledge gap has been noted concerning the survival and growth strategies of *Vibrio* spp. and especially *V. cholerae* as an emerging etiological agent for vibriosis in the temperate coastal bathing sites of the Baltic Sea as a result of climate change ([@B19]). In addition, more knowledge is needed on the use of MS2 coliphage as a virus indicator for fecal contamination in coastal environments. This study evaluated the decay pattern of *E. faecalis*, one of the most dominant enterococci species in the human gut ([@B20]), in beach water, sediment and vegetation. The enterococci decay was compared with decay patterns of the pathogen *V. cholerae* and virus indicator MS2 coliphage in an experimental mesocosm simulating natural Nordic summer daylight conditions. Further, our objective was to investigate the role of aquatic vegetation in survival of *E. faecalis, V. cholerae* and MS2 coliphage in the beach environments. Materials and Methods {#s2} ===================== Mesocosm Preparation -------------------- Water, sediment, and vegetation were collected from an official bathing site located on the western coast of Finland (Gulf of Bothnia, the Baltic Sea) and transported immediately after collection to the laboratory in cool boxes. Approximately 80 liters (l) of coastal water was collected in plastic containers. Similarly, coastal sediment weighing about 30 kg was collected from the subtidal zone and mixed well. Further, aquatic vegetation (*M. sibiricum*) of about 3 kg in weight was manually picked with roots from the bathing site. As illustrated in [Figure S1](#SM1){ref-type="supplementary-material"}; two 50 l aquariums (Kuopion Akvaariovalaisin, Kuopio, Finland) were filled to form the study mesocosms with an approximately 4 cm layer of sediment and then by adding water (\~35 l) with a final volume ratio of sediment and water of 1:4 \[following Badgley et al. ([@B5])\]. About 2 kg (wet weight) of *M. sibiricum* was planted in one aquarium. The mesocosm without the vegetation was coded BS and the mesocosm with vegetation as BSM (BS, Baltic Sea; M, *M. sibiricum*). Mesocosms were exposed to identical artificial solar radiation simulating natural Finnish summer daylight conditions (19 h of daylight) using Sylvania Reptistar T8 full spectrum lamps (438 mm, ø 26 mm, 15 W, 6,500 K, UV-A 30%, UV-B 5%; Giant Valaisin, Kuopio, Finland). Both mesocosms were placed on a laboratory bench at ambient room temperature and covered to reduce evapotranspiration. The mesocosms were fitted with identical aquarium pumps (Ismatec ISM-1079B, VAC-115/230 Ecoline, Wertheim, Germany) without a filter to create a continuous mix of water and to avoid an anoxic environment. All the metallic equipment which came into contact with the samples, i.e., mechanical stirrers, scissors, blades and spoons were autoclaved prior to use. Aquariums and pumps were cleaned with a soap-water and 5 mg/l chlorine solution and then rinsed with tap water (3 times) and then with distilled water (3 times), prior to use. Both mesocosms were fitted with a minimum and maximum thermometer for measuring the temperature (°C). Spike Preparation ----------------- After settling the sediment (for about 30 min), suspensions of *E. faecalis, V. cholerae* and MS2 were spiked into the BS and BSM mesocosms. The *E. faecalis* 13V1235-1 strain was isolated from a coastal bathing site during a high enterococci count event \[([@B18]); sampling site E\]. The *V. cholerae* non-O1, non-O139 strain was isolated from a vibriosis patient in the 2014 heat wave on the western coast of Finland ([@B11]). Both strains were stored at −75°C or lower before use in this study. The strains of *E. faecalis* and *V. cholerae* were cultivated on tryptone soya agar medium (TSA) and a blood agar medium and incubated at 36°C for 48 and 24 h, respectively. Colony material was suspended in a phosphate buffer to reach an absorbance of 0.8 at 420 nm with the aim to spike about 3 × 10^8^ CFU of both *E. faecalis* and *V. cholerae* into the water of both mesocosms. The colony counts of *E. faecalis* and *V. cholerae* in the spikes were enumerated by spread plating on TSA and blood agar media, respectively. An MS2 coliphage (NCTC 12487) was produced using an *E. coli* host (ATCC 15597) following the principles presented in ISO 10705-1 (1995). Chloroform (1:10 v/v) was used to extract phages from the solution. The aqueous phase was centrifuged at 5,000 g for 20 min at 4° C and then filtered through a 0.45 μm filter (Acrodisc, Pall Corporation). The phage stock solution was stored at 4° C until use. The estimated initial number of the MS2 coliphage was the same as the *E. faecalis* and *V. cholerae*, being approximately 3 × 10^8^ PFU for each mesocosm. Mesocosm Monitoring ------------------- The physico-chemical and microbiological water quality was monitored for both mesocosms over time from duplicate samples. Natural background numbers of the microbial targets were enumerated as duplicates from the mesocosm water, sediment and vegetation before adding the spike. The sampling was continued after adding the spike (sampling for the initial numbers was conducted 30 min after the spike) and then at first twice a day (0.5, 1, 1.5, 2, 2.5, and 3 days), then every second day (4, 6, 8 and 10 days) and finally before ending the experiment on days 13, 20 and 27. A multi-parameter analyzer (Multi 3430; WTW GmbH, Weilheim, Germany) was used for measuring the pH, dissolved oxygen (mg/l) and conductivity (μS/cm) of the water samples. Further, the turbidity (NTU) was measured from subsamples in the laboratory at a wavelength of 860 nm with a Turb 555IR spectrophotometer (WTW GmbH&Co. KG, Weilheim, Germany) and the chloride concentration (mg/l) was monitored using the mercuric thiocyanate method as described in the manufacturers manual (Method 8113; HACH Lange GmbH, Duesseldorf, Germany). The workflow for each sampling event was as follows for both BS and BSM to avoid the unnecessary mixing of the mesocosm: (a) the temperature was recorded (b) water samples (\~50 ml) were collected with a peristaltic pump to measure physical characteristics (c) water samples (10--1,000 ml in duplicate) were collected with a peristaltic pump for microbial analysis, (d) vegetation samples (only from BSM; about 1 g in duplicate) were collected with the help of a sterile rod and scissors, and (e) sediment samples (about 3 g in duplicate) were collected with the help of a sterile 2 ml micro-centrifuge tube. For processing the vegetation and sediment samples, a phosphate buffer at a ratio of 1:10 was used and the mixture was vortexed for 2 min (1,100 rpm), settled for 30 s and then the eluent was transferred into a clean tube for microbial analysis \[the protocol modified from Whitman et al. ([@B21])\]. Target Enumeration ------------------ The culturable counts of intestinal enterococci including *E. faecalis* were enumerated using membrane filtration according to the international standard method ISO 7899-2:2000 as described by Tiwari et al. ([@B18]). In brief, after filtration of multiple sample volumes, the membranes (GN6, Pall Life Sciences, Michigan, USA) were incubated on a Slanetz & Bartley medium (S&B, Oxoid Ltd. Basingstoke, Hampshire, England) at 36 ± 2°C for 44 ± 4 h. A range of sample volumes were used to produce 10--100 presumptive colonies per membrane. After counting the presumptive enterococci, the membrane was transferred on a preheated bile aesculin azide medium (BEA, Scharlau, Barcelona, Spain) and incubated at 44 ± 0.5°C for 2 h. A black or brown color formation on bacterial colonies on the BEA medium confirmed the colony belonged to the group of intestinal enterococci. The enterococci counts in the water were presented as CFU/100 ml and in sediment and vegetation as CFU/100 mg (wet weight). The MS2 coliphage was measured using a culture-based method with a double agar layer (DAL) technique from 0.5 mL samples ([@B22]) using *E. coli* (ATCC 15597) as a host in two replicates. The MS2 counts in water were presented as PFU/100 ml and for the sediment and vegetation as PFU/100 mg (wet weight). For molecular analyses, a subsample of water (50--300 ml) was concentrated onto 0.45 micrometer-pore-size, 47-mm-diameter polycarbonate membranes (Nuclepore, Whatman) and 0.35 ml of each vegetation and sediment eluent was directly transferred into a micro-centrifuge tube. The membranes and tubes were stored at −75°C or lower prior to nucleic acid extraction. The total DNA and RNA were extracted from the samples using the AllPrep DNA/RNA Mini Kit (Qiagen GmbH) as previously described ([@B23]). The RNA was further purified using the Ambion TURBO DNA-free DNase kit (Life Technologies, Grand Island, NY), and then complementary DNA (cDNA) was synthesized using the random hexamer primed Superscript III system for RT-PCR (Life Technologies). The total RNA was stored at −75°C or lower, while the cDNA and the DNA extracts were stored at −20°C until use. The gene copy numbers of *Enterococcus* spp. and *Vibrio* spp. in the samples (including extraction and filtration blanks) were measured from cDNA and DNA extracts, and the *Vibrio cholerae* and Gram-negative bacteria gene copy numbers were measured from the DNA extract only. The qPCR assays were performed as previously described ([@B23]), by processing 8 μl of RNA in a cDNA synthesis (reverse transcription, RT). The primers and probes used in this study are listed in the [Table S1](#SM1){ref-type="supplementary-material"}. The targeted genes were 23S rRNA gene for *Enterococcus* spp. \[Entero1 assay; ([@B24])\] 16S rRNA gene for *Vibrio* spp. ([@B25]), and the *ompW* gene was used for the *V. cholerae* ([@B26], [@B27]). Total bacterial numbers in the BS and BSM waters were evaluated by using an assay targeted to 16S rRNA gene of all Gram -negative bacteria ([@B28]). The qPCR reactions were performed using a QuantStudio 6 real-time PCR system (Applied Biosystems) in 20 μl volume using the TaqMan Environmental PCR Master Mix (Life Technologies) for *Enterococcus* spp., *V. cholerae* and Gram- assays and the Power Sybr PCR Master Mix (Life Technologies) for a *Vibrio* spp. assay, all with primers and probes at final concentrations 0.2 μM (IDT Technologies, Inc). The cycling conditions included 95°C for 10 min of enzyme activation and pre-denaturation followed by 40 cycles at 95°C for 15 s of denaturation and at 60°C for 60 s of annealing, except for *Vibrio* spp., for which a 64°C annealing temperature was used and a melt curve analysis of the PCR amplicons was performed. Standard curves were generated using artificial gene fragments (gBlocks, IDT Technologies, Inc.) containing the sequences for each of the targeted genes. In qPCR, undiluted and 10 fold diluted cDNA and DNA samples were used as qPCR templates to detect PCR polymerase inhibition. For samples in which PCR inhibition was detected, qPCR data was generated using the results from diluted samples. Background signals, if detected in the filtration blanks, were subtracted from all the results to generate the final qPCR and RT-qPCR data per assay. The limit of detection (LOD) was set as 3 copies per reaction. The copy number per 100 ml of water and 100 mg of sediment and vegetation was calculated for those samples with values above the limit of quantification (LOQ) (i.e., as determined by the lowest value within the quantification range). The final qPCR, equivalent LOD (eLOD) and equivalent LOQ (eLOQ) values were calculated after taking into account the volume/mass of the processed sample, factors associated with the different processing steps of the RNA and DNA manipulations, and the dilutions used for each sample analyzed. Identification of Enterococci Colonies -------------------------------------- Representative enterococci colonies grown on an S&B medium were identified with partial 16S rRNA gene sequencing as done in our previous study ([@B18]). Enterococci colonies isolated before the spike were identified from all the substrates (water, sediment and vegetation). Further, the enterococci colonies isolated from water samples after 1, 2, and 10 days from the spike, sediment samples after 6 and 20 days and vegetation samples after 4 and 10 days were identified. From the selected samples, the nucleic acid extracts of all or at least 10 enterococci colonies grown were amplified with universal bacteria primers 8F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 787R (5′-CGACTACCAGGGTATCT AAT-3′) as described earlier by Ryu et al. ([@B29]). The quality of the sequences was checked and the consensus sequences from paired reads were made with the BioEdit, bioinformatics software ([@B30]). Finally, the enterococci species were identified with the NCBI Blast database ([@B31]). Decay Rate ---------- The decay characteristics of the targets were tested with GInaFit (Geeraerd and Van Impe Inactivation Model Fitting Tool) freeware add-in for Microsoft Excel 2010 ([@B32]). Models were selected based on the lowest root mean sum of the standard errors (RMSE), and the highest R^2^ value as done earlier by Kauppinen and Miettinen ([@B33]). The details of the microbial decay model equations are shown in [Supplementary Material](#SM1){ref-type="supplementary-material"} (Microbial decay and decay rate). GInaFit does not support modeling an erratic type of decay data. For that condition, the reduction equation was calculated with the following log-linear decay model equation \[as done by Badgley et al. ([@B5]) and Anderson et al. ([@B4])\]: L o g 10 N t N 0 = \- k t where k = the decay rate, N~t~ = the target count at time t days, N~0~ = the target count 0-day, t = the time (in days) when the target count is N~t~. Data Analyses ------------- All the data was log~10~ transformed and expressed as the log~10~ CFU/PFU/GC per 100 ml for the water and per 100 mg for the sediment and vegetation. Where applicable, mean of duplicate samples BS and BSM was used. The data analysis was done with SPSS (IBM SPSS Statistics for Windows, Version 25.0, IBM Corp., Armonk, NY). In this study, the significance was compared at a 95% confidence level, if not mentioned otherwise. The median numbers of the targets and water quality parameters between the two mesocosms were compared using Wilcoxon signed-rank test. The relation of culturable *E. faecalis* with other targets was calculated using Spearman\'s rank correlation test. The significance of the differences of the *k*-values was tested as done by Green et al. ([@B34]), by calculating the lower and upper confidence intervals with the following equation: Confidence interval (CI)  = k ± Standard Error (SE) \* α where the SE was obtained from GInaFiT tool and the α value was obtained from a Student\'s *t*-table on (n−2) degrees of freedom at a 95% confidence interval. The *k*-values were significantly different if the confidence intervals of two *k*-values did not overlap. Results {#s3} ======= Water Quality in Two Mesocosms ------------------------------ The effect of vegetation on the properties of water was calculated by comparing the physico-chemical properties and total numbers of Gram-negative bacteria in the water without vegetation and with vegetation (BS and BSM, respectively). [Table 1](#T1){ref-type="table"} shows the mean and median of the measured parameters of water in both mesocosms. The vegetation significantly reduced the median turbidity of the water in the BSM compared to the BS samples (*p*-value 0.008; [Table 1](#T1){ref-type="table"}). The mean oxygen and chloride concentrations were slightly higher in the water of vegetated BSM mesocosm water than in the BS water, but the difference was not statistically significant ([Table 1](#T1){ref-type="table"}). The water temperature, pH and electric conductivity were not affected by vegetation. The water temperature increased in both mesocosms during the course of experiment from about 19°C to 23°C. The numbers of Gram-negative bacteria measured as gene copies per 100 ml and indicating the total number of bacteria in water, were significantly higher in the BS compared to BSM (*p*-value 0.007; [Table 1](#T1){ref-type="table"}). ###### Physico-chemical parameters and the number of Gram-negative bacteria in the water of mesocosms without vegetation (BS) and with vegetation (BSM). **Parameters** **Mean (SD)** **Median** **Significance (Wilcoxon test; *p*-value)** ----------------------------------------------------- --------------- ------------- --------------------------------------------- ------- ------- Turbidity (NTU) 4.8 (3.6) 2.9 (2.7) 4.1 1.7 0.008 O~2~ (mg/l) 9.2 (0.6) 9.4 (0.6) 9.2 9.4 0.062 Chloride (mg/l) 2,200 (210) 2,000 (100) 2,220 2,000 0.073 Temperature (during sampling) (°C) 22.3 (1.7) 22.4 (1.4) 22.7 22.9 0.477 pH 7.7 (0.2) 7.6 (0.1) 7.7 7.6 0.450 Electric conductivity (μS/cm) 8,400 (240) 8,300 (140) 8,300 8,300 0.677 Gram-negative bacteria (log~10~ gene copies/100 ml) 7.7 (0.3) 7.2 (0.4) 7.8 7.5 0.007 *SD, standard deviation*. Target Detection and Decay -------------------------- The detection and decay rate of the targets was determined for each target microbe, as well as for the substrate from which the target was sampled, and also for both enumeration methods of enterococci (culture-based and molecular). Culturable enterococci were detected up to the end of the experiment in the water and sediment, but only up to the 20th day in the vegetation ([Figure 1](#F1){ref-type="fig"}). Further, when using molecular assays, the rRNA marker was more frequently detected than the rDNA marker ([Figures S2](#SM1){ref-type="supplementary-material"} and [S3](#SM1){ref-type="supplementary-material"}), indicating the higher 23S rRNA transcript copy numbers in the ribosomes of the viable *Enterococcus* spp. cells as compared to the copy numbers 23S rRNA gene in the *Enterococcus* spp. genome ([@B23]). However, culturable enterococci were detected more frequently than rRNA or rDNA *Enterococcus* markers, the result related to differences between the studied sample volumes and different limits of detection between the methods. *Vibrio* spp. genus-specific markers (rDNA, rRNA) were detected up to the end of the experiment in the water, sediment and vegetation ([Figure 2](#F2){ref-type="fig"} and [Figure S4](#SM1){ref-type="supplementary-material"}). The *V. cholerae* species-specific marker was less frequently detected than the genus-specific markers ([Figure S5](#SM1){ref-type="supplementary-material"}). Among the different substrates, *Vibrio* spp. was more frequently detected in the water than in the sediment or vegetation. MS2 coliphage was detected only by the 8th−10th days of the experiment in the water, and only during the 3 first days of the experiment in the sediment and vegetation ([Figure 3](#F3){ref-type="fig"}). {ref-type="table"}, [3](#T3){ref-type="table"}). BS---unvegetated mesocosm and BSM---vegetated mesocosm.](fpubh-07-00269-g0001){#F1} {ref-type="table"}, [3](#T3){ref-type="table"}). BS---unvegetated mesocosm and BSM---vegetated mesocosm.](fpubh-07-00269-g0002){#F2} {ref-type="table"}, [3](#T3){ref-type="table"}). BS---unvegetated mesocosm and BSM---vegetated mesocosm.](fpubh-07-00269-g0003){#F3} The decay characteristics of each target were calculated using the GInaFiT tool and with a classical log-linear model and are shown in [Tables 2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}, respectively. The culturable enterococci followed the biphasic or biphasic decay model with a shoulder with the first rapid decay phase before the delayed decay phase, in all the substrates (water, sediment and vegetation) ([Figure 1](#F1){ref-type="fig"}; [Table 2](#T2){ref-type="table"}). The decay rate (*k*-value) of the culturable enterococci in the first rapid decay phase was 7.0 and 9.7/day in water of vegetated (BSM) and unvegetated mesocosm (BS), respectively. The culturable enterococci numbers reached the background level after the 8th−13th days of the experiment. The decay rate of culturable enterococci in the sediments of the BS and BSM was 2.6 and 0.8 per day in the first phase and 0.3 and 0.1 per day in second phase of the biphasic decay, respectively ([Figure 4](#F4){ref-type="fig"}; [Table 2](#T2){ref-type="table"}). In the vegetation of BSM, the enterococci decay was log linear with the rate of 0.8 per day. The culturable enterococci had a significantly higher decay rate in water than in sediment and vegetation, as the upper and lower confidence interval of the k-value did not overlap (visual inspection; [Figure 4](#F4){ref-type="fig"}). ###### Log-linear and nonlinear GInaFiT decay model results for culturable enterococci and MS2 coliphages. **Mesocosm** **Substrate** **Log-Linear model and parameters** **Best fitted non-linear model and parameters** ---------------------------- --------------- ------------------------------------- ------------------------------------------------- ----- ------------ ------ ------ ------- ------------ ------------ ------------ ------ ----- **Culturable enterococci** BS W 1.9 13 5.7 −0.8 ± 0.2 0.60 1.2 B. S. −9.7 ± 4.0 −0.2 ± 0.1 1.42 ± 0.2 0.97 0.4 BSM W 1.9 10 5.9 −0.8 ± 0.2 0.70 1.1 B. S. −7.0 ± 1.7 −0.3 ± 0.1 1.13 ± 0.2 0.99 0.2 BS S 2.1 27 4.8 −0.3 ± 0.0 0.94 0.3 B. S. −2.6 ± 2.7 −0.3 ± 0.0 1.93 ± 0.7 0.96 0.3 BSM S 1.7 27 4.7 −0.3 ± 0.0 0.88 0.33 B. −0.8 ± 0.3 −0.2 ± 0.0 na 0.90 0.3 BSM V 3.2 10 5.5 −0.8 ± 0.1 0.96 0.29 -- -- -- -- -- -- **MS2 coliphage** BS W 0.2 8 4.5 −1.3 ± 0.1 0.97 0.57 -- -- -- -- -- -- BSM W ND na 4.6 −1.1 ± 0.1 0.98 0.2 -- -- -- -- -- -- BS S ND na 0.8 na na na -- -- -- \- -- -- BSM S ND na 0.7 na na na -- -- -- -- -- -- BSM V ND na 1.3 −0.5 ± 0.1 0.94 0.3 -- -- -- -- -- -- *BS, mesocosm without vegetation; BSM, mesocosm with vegetation; W, water; S, sediment; V, vegetation; N~nat~, background count before spike; N~0~, count at the zero hour (just after spike); t~bac~, time required for target counts to reach the background level; B.S., biphasic with shoulder; B, biphasic; D, 1--log reduction time; 4D, 4-log reduction time; R^2^, goodness of fit; RMSE, root mean sum of the standard errors; SE, standard error; SL, shoulder length; ND, not detected; na, not applicable*. ###### Decay rates (log-linear) of *Enterococcus* spp., *Vibrio* spp. and *V. cholerae* genetic markers. **Mesocosm** **Substrate** **log10(N~**nat**~) GC/100 ml or 100 mg** **t~**bac**~** **log~**10**~(N~**0**~) GC/100 ml or 100 mg** **log~**10**~(N~**min**~) GC/100 ml or 100 mg** **t~**min**~** **k~**min**~ (day^**−1**^)** ------------------------------------ --------------- ------------------------------------------- ---------------- ----------------------------------------------- ------------------------------------------------- ---------------- ------------------------------ ***Enterococcus*** **spp. (rDNA)** BS Water 2.88 10--13 5.96 3.48 10 −0.19 BSM Water 3.08 8--10 6.01 3.52 8 −0.19 BS Sediment ND \>0 2.96 na na na BSM Sediment ND \>0.5 1.81 na na na BSM Vegetation ND \>4 3.34 na 4 −0.03 ***Enterococcus*** **spp. (rRNA)** BS Water 4.57 13 7.55 3.17 20 −0.24 BSM Water 4.45 20 7.08 2.91 20 −0.22 BS Sediment 6.06 27 7.73 4.93 27 −0.08 BSM Sediment 6.57 6 7.09 5.00 27 −0.1 BSM Vegetation 6.73 13 8.31 5.61 27 −0.1 ***Vibrio*** **spp. (rDNA)** BS Water 5.42 2.5 6.71 3.84 8 −0.19 BSM Water 5.58 2 7.21 4.95 6 −0.38 BS Sediment 3.08 2 3.71 2.87 13 −0.15 BSM Sediment 3.49 3 4.73 2.98 13 −0.08 BSM Vegetation 3.82 \>27 4.55 4.16 4 −0.13 ***Vibrio*** **spp. (rRNA)** BS Water 4.4 \>27 9.25 5.97 8 −0.41 BSM Water 6.74 \>27 9.32 7.08 6 −0.36 BS Sediment 8.7 4 8.79 7.09 20 −0.13 BSM Sediment 8.94 3 8.81 6.87 8 −0.22 BSM Vegetation 9.46 3 9.52 8.98 20 −0.04 ***V. cholerae*** **(DNA)** BS Water ND \>27 5.65 2.37 8 −0.41 BSM Water ND \>27 5.88 2.85 6 −0.51 BS Sediment ND \>1 2.16 na na na BSM Sediment ND \>1 ND na na na BSM Vegetation ND \>27 2.44 2.07 3 −0.13 *BS, mesocosm without vegetation; BSM, mesocosm with vegetation; N~nat~, background count before spike; t~bac~, time required for target counts to reach the background level; k~min~, decay rate calculated with the equation Log~10~ (N~t~/N~0~) = --kt; where N~t~ was the lowest target count over the course of experiment after adding the spike and t was the respective day when the target count was the lowest; ND, not detected; na, not applicable. The k-value for the rDNA of Enterococcus spp. and V. cholerae in sediment and vegetation was not available due to their low detection frequency*. {#F4} The MS2 coliphage followed the log-linear decay model in water of both the mesocosms. The counts remained continuously higher in the BSM water than in BS water ([Figure 3](#F3){ref-type="fig"}). Further, a higher decay rate of the MS2 coliphage was noted in the BS water than in the BSM water, k-values were −1.3 and −1.1 per day, respectively, in the BS and BSM water ([Figure 4](#F4){ref-type="fig"}; [Table 2](#T2){ref-type="table"}). GInaFiT was not able to identify the decay model for the *Enterococcus* spp. genetic markers (rRNA, rDNA). However, from the visual inspection ([Figures S2](#SM1){ref-type="supplementary-material"} and [S3](#SM1){ref-type="supplementary-material"}), the biphasic decay of rRNA in water and sediment and an almost log linear pattern in vegetation was seen. The decay of the *Enterococcus* spp. genetic markers (rDNA, rRNA) was slower than the decay of culturable enterococci counts in all substrates. The markers exhibited a higher decay rate (k-value) in water (0.19--0.24/ day) than in sediment (0.08--0.1/ day) and vegetation (0.03--0.1/ day) ([Table 3](#T3){ref-type="table"}). It took 13 and 20 days after the spike in BS and BSM water, respectively, for *Enterococcus* spp. rRNA to reach the background level ([Figure S2](#SM1){ref-type="supplementary-material"}). Additionally, for genetic markers of *Vibrio* spp. (rDNA, rRNA) and *V. cholerae*, GInaFiT was not able to identify the decay model. On visual inspection, a biphasic decay pattern was seen as the gene copies dropped rapidly up to day 6--10 but later increased back nearly up to the spiked level ([Figure 2](#F2){ref-type="fig"}, [Figures S4](#SM1){ref-type="supplementary-material"} and [S5](#SM1){ref-type="supplementary-material"}). The log-linear decay rate of all *Vibrio* markers was lower in vegetation than in water or sediment with the exception of the rDNA marker in the sediment of the BSM water ([Table 3](#T3){ref-type="table"}). The Relationship of *Vibrio cholerae* and MS2 With Culturable Enterococci ------------------------------------------------------------------------- The relation between the enumerated numbers of culturable enterococci with other microbial targets in the water is shown in [Table 4](#T4){ref-type="table"}. Culturable enterococci had a strong positive correlation with *Enterococcus* spp. rDNA and rRNA markers. Overall, *Vibrio* spp. and *V. cholerae* markers had weak correlations with culturable enterococci. However, the correlation was strongly positive (ρ = 0.81--0.92) in the first 4 days after the spike. The culturable enterococci in the sediment and vegetation had a strong correlation with culturable enterococci in the water ([Table 4](#T4){ref-type="table"}). The correlation of the MS2 coliphage with culturable enterococci was strong in the first 6--8 days of the experiment (ρ \> 0.85). There was also strong relation between culturable enterococci counts and *Enterococcus* spp. rDNA and rRNA gene copy numbers in water (correlation coefficients ρ = 0.83--0.87, [Table 4](#T4){ref-type="table"}). ###### Comparing Spearman\'s rho correlation coefficient (ρ) between culturable enterococci with other microbial targets. **Targets** **Substrates** **27 days** **First 4 days** -------------------------------- -------------------------- ---------------- ---------------------------------------------------------------------------- ----------------------------------------- Culturable enterococci (Water) *Enterococcus* spp. rDNA Water 0.83[^\*\*^](#TN2){ref-type="table-fn"} 0.87[^\*\*^](#TN2){ref-type="table-fn"} *Enterococcus* spp. rRNA Water 0.85[^\*\*^](#TN2){ref-type="table-fn"} 0.86[^\*\*^](#TN2){ref-type="table-fn"} MS2 coliphage Water 0.87^[^\*\*^](#TN2){ref-type="table-fn"}[^\#^](#TN1){ref-type="table-fn"}^ 0.90[^\*\*^](#TN2){ref-type="table-fn"} *Vibrio* spp. rDNA Water 0.28[^\*^](#TN3){ref-type="table-fn"} 0.81[^\*\*^](#TN2){ref-type="table-fn"} *Vibrio* spp. rRNA Water 0.23 0.88[^\*\*^](#TN2){ref-type="table-fn"} *V. cholerae* DNA Water 0.33[^\*^](#TN3){ref-type="table-fn"} 0.92[^\*\*^](#TN2){ref-type="table-fn"} Culturable enterococci Sediment 0.86[^\*\*^](#TN2){ref-type="table-fn"} 0.73[^\*\*^](#TN2){ref-type="table-fn"} Culturable enterococci Vegetation 0.94[^\*\*^](#TN2){ref-type="table-fn"} 0.80[^\*\*^](#TN2){ref-type="table-fn"} The correlation was measured only until the MS2 coliphage was detected (up to 8--10 days). The correlation is significant at the 0.01 level (2-tailed). *The correlation is significant at the 0.05 level (2-tailed)*. Discussion {#s4} ========== This study identified biphasic with a shoulder, biphasic and log-linear decay patterns of culturable enterococci, *Vibrio* spp. and MS2 coliphage, respectively, in coastal bathing water. Culturable enterococci decayed rapidly in water for up to 2.5 days, before a slower decay phase. The identified slightly higher decay rate of culturable enterococci in this study than noted in earlier studies with coastal waters ([@B4], [@B35], [@B36]) could be due to differences in the experimental settings. While, our study used an environmental enterococci strain under 21 h of continuous artificial solar radiation each day at room temperature (21--23°C), Craig et al. ([@B35]) focused on the effect of the temperature, and did not mention the solar radiation, while Anderson et al. ([@B4]) used enterococci from dog feces, waste water and soil inoculum in their experiment on rooftop of a building with an open-air greenhouse, and Zhang et al. ([@B36]) spiked targets directly from treated sewage. The detected biphasic decay of *Enterococcus* spp. molecular markers (rDNA, rRNA) in water confirmed the recent findings by Ahmed et al. ([@B37]). Although the molecular marker decay seems to be slower than the decay of culturable enterococci counts, the strong correlation between the culturable counts and gene copy numbers generated using qPCR and RT-qPCR were found. This result indicates potential usability of molecular methods as alternatives to culture based methods for water quality monitoring. The biphasic decay of microbes could be due to the target strain heterogeneity; various strains might have different decay rates ([@B38]). In our study, this is not a probable explanation as a single *E. faecalis* strain was used as a spike, and all the identified isolates from the samples before the spike belonged to the same species. Thus, the cause of the initial rapid decay of enterococci could be due to a rapid inactivation after a sudden change of environmental conditions such as the solar radiation, nutrient availability, predation, salinity or water temperature, but could be also related to initial growth phase and high density of spiked bacterial cells ([@B39]). Similar decay is expected to take place after a fecal contamination incidence into bathing waters and must be taken into consideration when interpreting bacterial indicator results. Later on, the introduced bacteria might start to cope better with the prevailing environment, so after an adaption phase their decay rate might be lower as was noted in the present study. Additionally, *Vibrio* spp. and *Vibrio cholerae* genetic markers exhibited biphasic decay patterns. The good persistency of *Vibrio* spp. in water was as expected, since these bacteria are autochthonous to coastal waters ([@B8], [@B10], [@B40], [@B41]). The results presented herein correspond to one earlier study in which Zhang et al. ([@B36]) demonstrated the good persistence of *Vibrio* OTUs using high-throughput sequencing in a laboratory experiment. In the present study, the gene copies of *V. cholerae* dropped to a minimum by days 6--8 but then the numbers increased back nearly up to the spiked level. It is obvious that the current bathing water quality monitoring against fecal contamination ([@B1]) is not protecting bathers from *Vibrio* infection risks. In the present study, the observed lack of correlation between the genetic markers of *Vibrio* spp. and *V. cholerae* with culturable enterococci after the first 4 days highlights the deficiency of current monitoring practice. However, further studies are needed to investigate if *Vibrio* spp. counts could be enumerated from bathing waters using culture-based methods ([@B42]). It is possible that such counts could relate to counts of culturable enterococci better than the *Vibrio* marker gene copy numbers. The Nordic aquatic environment is rich in carbon and phosphorous and is favorable to the proliferation of *Vibrio* spp. ([@B9]). The pronounced proliferation of pathogenic *Vibrio* spp. in coastal waters is expected to cause greater public health concerns in future years due to global warming and the subsequent rise in sea surface temperatures ([@B8]--[@B10], [@B43]). *Vibrio* spp. have short generation times (\<10--30 min) at their optimum growth temperature (23--25°C) and can respond rapidly to changing environmental conditions ([@B10], [@B44]). Therefore, for health risk management purposes, early-warning modeling tools such as Vibrio Viewer \[<https://e3geoportal.ecdc.europa.eu/SitePages/Vibrio%20Map%20Viewer.aspx>; ([@B13])\] are useful. Further, the determination of *Vibrio* species from the bathing waters might become as a relevant effort to carry out, especially in cases of suspected waterborne infections. The log-linear decay pattern of the MS2 coliphage resembled the decay patterns reported in earlier laboratory studies on F-specific coliphages, adenoviruses, and polyomaviruses in coastal waters ([@B37], [@B45]). Further, Craig et al. ([@B35]) reported a relatively slower decay rate of somatic coliphages in coastal water mesocosms and found that the temperature affected the decay rate. MS2 is one of the most UV resistant phage and used commonly as a conservative virus surrogate ([@B46]). In the present study, vegetation might have sheltered MS2 as the decay rate was slower in the BSM water than in the BS water. In fact, MS2 counts correlated with the culturable enterococci in the first few days of the experiment, which makes sense in the mesocosm mimicking natural environment. The decay of culturable MS2 calls for further studies to investigate its survival mechanisms. More information is needed for comparison if similar behavior is seen with viral pathogens. In our study, bacterial targets persisted longer in sediment and vegetation than in water (except for *Vibrio* spp. rRNA). This better persistency detected herein has been reported also in several earlier studies in which the availability of nutrients, protection from UV radiation and predation, and lowered temperature have been identified as factors enhancing the survival ([@B4], [@B5], [@B17], [@B35], [@B36], [@B40], [@B47]). Further, the higher persistency of surface attached microbes in sediment or vegetation than in water may be due to the surface biofilm protecting microbes from external stress factors ([@B48]). Schets et al. ([@B49]) detected norovirus only in the sediment but not in the water at a bathing site during a bathing water outbreak. In our study herein, the poor detection frequency of the MS2 coliphage in sediment (12%) and vegetation (13%) could be due to the methodological limitations ([@B47]). Here, we used the same protocol for the separation of the MS2 coliphage and enterococci from sediment and vegetation, which was originally developed for enterococci ([@B21]). Vegetation can play further complex roles in aquatic systems and lead to changes in water turbidity, pH, temperature, and predator species ([@B50]), of which the turbidity change was noted in the present mesocosm study. Furthermore, in our study, the oxygen concentration was slightly higher in BSM than in BS water, potentially due to photosynthesis of vegetation. In general, due to the settling effect, vegetation in water may reduce suspended solid, leading to less turbid waters. However, due to the shedding effects, temperature and UV exposure may decrease enabling better microbial survival when vegetation is present. However, after growth phase, the decomposing vegetation might have an opposite effect on water quality. Such changes can have multiple effects on the decay rate of the studied targets. In our experiment, vegetation cover could have effect on enterococci decay as we observed a slightly higher decay rate of enterococci in BS water than in BSM water in the first phase of the biphasic decay pattern. Also the MS2 coliphage was detected in a shorter time (8 days) in BS water than in BSM water (10 days) and the vegetation seemed thus to reduce the decay rate in water. Consistent with our findings, also Badgley et al. ([@B5]) demonstrated that vegetation reduced the decay rate of enterococci in fresh water in a similar experimental setting. In contrast, however, Kleinhrinz et al. ([@B48]) demonstrated that vegetation increased the decay rate of *Escherichia coli, Salmonella*, and *Shigella*, and they argued that the targets rapidly attached to vegetation and thus recorded a lower count from the water column. In addition to the inactivation, the rapid reduction of microbial targets in the water could be due to sedimentation and attachment to the surface sediments ([@B35], [@B51]). The attachment of the targets to turbid suspended particles and their settling and deposition at the bottom of the mesocosm could thus have an effect on the initial rapid decay rate. In our study, culturable enterococci counts in the sediment and vegetation related strongly to culturable enterococci counts in the water. We did not notice the significant difference between the median value of enterococci counts in BS and BSM water. However, the total number of Gram-negative bacteria was higher in BS than in BSM water. These findings could partly be explained due to a partitioning effect. While the total numbers of bacterial targets were not different in the mesocosms, the BSM environment had three substrates (water, sediment, and vegetation), but the BS environment only had two (water and sediment). Thus, benthic sediment and aquatic vegetation could work as a sink and source of microbes like FIB and probable human pathogens ([@B6]). The FIB sink (due to sedimentation) in the benthic sediment and vegetation could contribute to the lower count of FIB in the water during regulatory monitoring of the microbial quality of bathing water, and their probable resuspension can complicate the interpretation of the results of water quality monitoring. The current microbial water quality monitoring protocol does not account for the microbial quality of the sediment and vegetation at bathing sites. The contamination and decay are simultaneous and continuous processes at each bathing site. Conclusion {#s5} ========== This study demonstrated the partitioning of pathogens and fecal indicators in coastal waters between water, sediment and vegetation. It also indicated that the decay of microbes was different in water than in sediment in presence or absence of vegetation. The current bathing water monitoring protocol does not account for the microbial content of sediment and vegetation at bathing sites and thus possibly underestimates the actual enterococci counts during the regulatory monitoring of water. Furthermore, enumerating only the enterococci does not seem enough for the prediction of free-living pathogens such as *Vibrio* spp. Finally, the different decay patterns observed between MS2 and enterococci emphasize the need for and importance of a viral indicator for assessing water quality more comprehensively. Data Availability Statement {#s6} =========================== All datasets generated for this study are included in the manuscript/[Supplementary Files](#s8){ref-type="sec"}. Author Contributions {#s7} ==================== All the authors participated in the project design, the execution of the experimental work, data analysis and manuscript writing. AT calculated the data and drafted the initial version of the manuscript. AK was incharge of the MS2 work. TP supervised the work. Conflict of Interest -------------------- The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors would like to express special thanks to all the people and institutions involved and helped contribute to the success of this work. Special thanks go to the site of the research for providing financial and technical help. We would like to sincerely thank the laboratory personnel of the Expert Microbiology Unit, at the National Institute for Health and Welfare, namely Tiina Heiskanen, Tarja Rahkonen and Iina Laaksonen. Further, special thanks also go to Anna-Maria Hokajärvi and Asko Vepsäläinen, for the support they provided during the process. The local health protection officer Maria Nylund from West Coast Environmental Unit is gratefully acknowledged for her help during the sample and material collection from the bathing site. In addition, the authors acknowledge Kaute Foundation for their personal grant to AT (grant number 20190366). Further, the scientific writing workshop organized by Young Water Professionals Finland of the International Water Association at Aalto University, in Helsinki and Professor Gustaf Olsson for his critical feedback on the manuscript are acknowledged. Supplementary Material {#s8} ====================== The Supplementary Material for this article can be found online at: <https://www.frontiersin.org/articles/10.3389/fpubh.2019.00269/full#supplementary-material> ###### Click here for additional data file. [^1]: Edited by: Nur A. Hasan, Center for Bioinformatics and Computational Biology, University of Maryland, United States [^2]: Reviewed by: Juan M. Gonzalez, Spanish National Research Council (CSIC), Spain; Durg Vijai Singh, Institute of Life Sciences (ILS), India; Siraje Arif Mahmud, University of Texas at Arlington, United States [^3]: This article was submitted to Environmental Health, a section of the journal Frontiers in Public Health

EUS-guided biliary drainage has been widely attempted for failed endoscopic retrograde cholangiopancreatography.\[[@ref1][@ref2]\] For patients with surgically altered anatomy such as the Roux-en-Y procedure, EUS-guided hepaticojejunostomy (HJS) is performed. A covered self-expandable metal stent with a long length is normally used as an EUS-HGS stent to prevent stent migration,\[[@ref3][@ref4]\] but if this stent is used in EUS-HJS, collateral injury of the jejunal mucosa may occur.\[[@ref5]\] Therefore, a novel long plastic stent (Type IT stent; Gadelius Medical Co., Ltd., Tokyo, Japan) is used as an EUS-HJS stent \[[Figure 1](#F1){ref-type="fig"}\]. This plastic stent has a total length of 20 cm, an effective length of 15 cm, and 4 flanges. Among four flanges, two are at the distal and another at the proximal ends. The proximal end has a pigtail structure and the distal end is tapered. A disadvantage of the plastic stent is the risk of rupture during stent removal. This report describes balloon-assisted stent removal for a ruptured plastic stent using a balloon catheter (4 mm, REN biliary dilation catheter; KANEKA, Osaka, Japan), which top was 3 Fr. An 80-year-old male underwent total gastrectomy with the Roux-en-Y procedure due to gastric cancer 2 years earlier. During clinical follow-up, obstructive jaundice developed due to malignant peritonitis of recurrent gastric cancer. He underwent EUS-guided antegrade metal stent deployment combined with EUS-HJS using a Type IT stent \[[Figure 2](#F2){ref-type="fig"}\]. A Type IT stent was deployed form the common bile duct to the intestine. Clinical follow-up was performed using laboratory examination every 2 months. However, after 6 months, he was admitted because of cholangitis due to stent occlusion. EUS-HJS removal using a forceps biopsy device was attempted after safety guidewire placement, but the plastic stent was ruptured \[[Figure 3](#F3){ref-type="fig"}\]. To prevent stent migration into the biliary tract and rerupture, a 0.025-inch guidewire was inserted into the Type IT stent \[[Figure 4](#F4){ref-type="fig"}\]. Then, a fine-gauge balloon catheter was inserted into the Type IT stent over the guidewire \[[Figure 5](#F5){ref-type="fig"}\], and stent removal was successfully performed \[[Figure 6](#F6){ref-type="fig"}\]. Finally, the Type IT stent was deployed from the intrahepatic bile duct to the intestine \[[Video 1](#SD1){ref-type="supplementary-material"}\]. The fine-gauge balloon-assisted stent removal technique may be safe and useful for cases such as the present one. {#F1} {#F2} {#F3} {#F4} {#F5} {#F6} {#sec2-1} Declaration of patient consent {#sec3-1} ------------------------------ The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient has given his consent for his images and other clinical information to be reported in the journal. The patient understands that his name and initial will not be published, and due efforts will be made to conceal his identity, but anonymity cannot be guaranteed. Financial support and sponsorship {#sec3-2} --------------------------------- Nil. Conflicts of interest {#sec3-3} --------------------- There are no conflicts of interest. Video Available on: [www.eusjournal.com](www.eusjournal.com) ============================================================

Introduction ============ Transcatheter aortic valve implantation (TAVI) is an established treatment of severe aortic stenosis for elderly patients with high and intermediate surgical risk.[@suaa098-B1]^,^[@suaa098-B2] Ongoing studies aim to expand the indications for TAVI towards patients with lower risk, younger age, bicuspid anatomy, or even no symptoms. Intravascular haemolysis is found in 19--51% of patients with modern bi-leaflet mechanical prostheses and in 5% of patients with normally functioning bioprosthetic valves.[@suaa098-B3] High shear stress generated by turbulent flow across prosthesis can be caused by high-velocity jets due to prosthetic valve regurgitation or small prosthesis flow area.[@suaa098-B6] Transcatheter aortic valve implantation has so far higher incidence of paravalvular leakage (PVL) than surgical aortic valve replacement (SAVR) but better haemodynamic performance to SAVR concerning post-operative effective aortic valve area.[@suaa098-B7] Two groups have studied incidence and severity of haemolysis after TAVI so far[@suaa098-B8]^,^[@suaa098-B9] but no systematic study of intravascular haemolysis after TAVI with supra-annular self-expandable prosthesis has been published and the impact on long-term mortality is unknown. We believe this should be evaluated before extending the indications of TAVI to low-risk and younger patients with long expected survival. Methods ======= Study population ---------------- From April 2009 till January 2014, 102 consecutive patients with severe symptomatic aortic stenosis underwent TAVI in University Hospital Královské Vinohrady in Prague and agreed to participate in this study. All patients signed informed consent, data were prospectively entered into a dedicated anonymized database, the study design was academic without any industry sponsorship, in compliance with the Declaration of Helsinki, and protocol was approved by the local ethics committee. All patients were treated with supra-annular self-expandable CoreValve (Medtronic, Dublin, Ireland) prosthesis; sizes of 23, 26, 29, and 31 mm were available. Aortic regurgitation was semi-quantitatively evaluated by angiography at least 10 min post-implantation and graded according to Sellers;[@suaa098-B10]^,^[@suaa098-B11] grade ≥2 was considered as a positive finding. Eight patients did not survive till 1-year follow-up and were excluded from the analysis. The cause of death was two periprocedural complications; three cardiovascular; two non-cardiac; and one unknown. No excluded patient had clinical signs of haemolysis and only one excluded patient had moderate (or severe) aortic regurgitation post-TAVI. All remaining 94 patients represent our study cohort. Laboratory evaluation --------------------- Baseline blood samples were obtained 1 day before TAVI as part of the routine protocol and at 1-year (±2 months) follow-up. Analysis of haemoglobin, haptoglobin, lactate dehydrogenase (LDH), reticulocyte and schistocyte count, bilirubin, alanine aminotransferase, C-reactive protein (CRP) levels, and platelet count was performed at our standard hospital laboratory. We have used two definitions of haemolysis at 1-year follow-up: 1. According to Skoularigis criteria without schistocytes (Definition 1) to enable comparison with previous studies of haemolysis after TAVI.[@suaa098-B8]^,^[@suaa098-B9] 2. According to standard Skoularigis criteria (Definition 2) used in studies of haemolysis after SAVR.[@suaa098-B3] In short, Skoularigis criteria[@suaa098-B4] for haemolysis consider patients as having haemolysis when (i) serum LDH levels are over the upper limit of normal (in our laboratory 3.67 µkat/L) and (ii) any two of the following criteria are present: (a) haemoglobin level ˂13.8 g/dL for male patients and ˂12.4 g/dL for female patients; (b) haptoglobin level ˂0.5 g/L; (c) reticulocyte count ≥2%; and (d) presence of schistocytes in peripheral blood smear. The severity of haemolysis was assessed by serum LDH levels. Echocardiography and follow-up ------------------------------ Standard transthoracic echocardiography (TTE) was performed 1 day before TAVI and at hospital discharge. All images were analysed by experienced echocardiographer who was blind to haematologic laboratory parameters. Standard classification of aortic regurgitation as either none/trace, mild, moderate, or severe according to Valve Academic Research Consortium-2 criteria was used.[@suaa098-B12] Aortic valve area index (AVAI) was calculated post-TAVI, and the presence and severity of patient-prosthesis mismatch (PPM) was defined as follows: no PPM---AVAI \>0.85 cm^2^/m^2^; moderate PPM---AVAI ≥0.65 cm^2^/m^2^ but ˂0.85 cm^2^/m^2^; and severe PPM as AVAI ˂0.65 cm^2^/m^2^. All patients had clinical visit 1-year (±2 months) post-TAVI and all were offered long-term clinical follow-up at our centre, but this was not mandated by the protocol and some patients were managed locally. Mortality data were obtained from both our hospital database and the central database of The Institute of Health Information and Statistics of the Czech Republic. Statistics ---------- Continuous variables are presented in graphs and tables as mean and standard deviation. Categorical variables are reported as counts and frequencies. Testing of differences between groups was performed by the Student's *t*-test or Mann--Whitney U test. A *χ*^2^ test or Fisher's exact test was used to detect the difference between categorical variables. Laboratory markers of haemolysis (baseline and follow-up differences) were tested by a paired Student's test or Mann--Whitney U test. A log-rank test was used to compare survival curves. Results were considered statistically significant at a significance level of *P*-value \<0.05. All statistical analyses were performed in IBM SPSS Statistics version 26. Graphical analyses were performed in Sigmaplot version 14. Results ======= Intravascular haemolysis was present in 8 (9%) and 26 (28%) patients according to Definition 1 and Definition 2 of haemolysis, respectively. Baseline characteristics and procedural variables are described in *Table [1](#suaa098-T1){ref-type="table"}*, there were no significant differences between groups with and without haemolysis. No patient was treated with the CoreValve 23 mm prosthesis and no patient received two prostheses. An alternative approach was used in five patients, all from the subclavian artery. Sedation with local anaesthesia was our default approach to all patients with a transfemoral approach. ###### Baseline and procedural patient characteristics (*n* = 94) Haemolysis Definition 1 (Skoularigis without schistocytes) Haemolysis Definition 2 (standard Skoularigis) ----------------------------------------------------------- ------------------------------------------------------------ ------------------------------------------------ ------- -------------- ------------- ------- Age (years) 80.5 ± 7.1 78.3 ± 10 0.674 80.1 ± 7.3 80.8 ± 7.5 0.457 Men 43 (50) 6 (80) 0.163 34 (50) 15 (57.7) 0.332 Women 43 (50) 2 (20) 0.163 34 (50) 11 (42.3) 0.332 NYHA I + II 36 (41.9) 1 (12.5) 0.103 26 (38.2) 11 (43.2) 0.447 NYHA III + IV 50 (58.1) 7 (87.5) 0.103 42 (61.8) 15 (57.7) 0.447 Diabetes mellitus 44 (51.2) 6 (75) 0.179 38 (55.9) 12 (46.2) 0.269 Smoking 30 (34.9) 4 (50) 0.313 25 (36.8) 9 (34.6) 0.522 Hypertension 72 (83.7) 7 (87.5) 0.625 56 (82.4) 23 (88.5) 0.353 Sinus rhythm 59 (68.6) 6 (75) 0.528 47 (69.1) 18 (69.2) 0.6 GFR (mL/min/m^2^) 44.4 ± 20.3 45.9 ± 24.2 0.924 43.8 ± 20.1 46.5 ± 21.7 0.597 LV ejection fraction (%) 52.6 ± 12 56.8 ± 9.2 0.331 52.4 ± 11.6 54.4 ± 12.2 0.26 Mean aortic gradient (mmHg) 44 ± 14.4 51.5 ± 19 0.408 43.7 ± 14.4 47.1 ± 16.2 0.285 Aortic valve area (cm^2^) 0.75 ± 0.2 0.7 ± 0.1 0.715 0.7 ±  ± 0.2 0.7 ± 0.1 0.405 Pulmonary hypertension[^a^](#tblfn2){ref-type="table-fn"} 12 (14) 1 (12.5) 0.695 10 (14.7) 3 (11.5) 0.49 EuroSCORE I logistical (%) 20.6 ± 13.4 19.8 ± 15.1 0.73 19.8 ± 13.1 22.3 ± 14.7 0.486 Prosthesis size (mm)  26 45 (52.3) 6 (75) 0.445 38 (55.9) 13 (50) 0.715  29 39 (45.3) 2 (25) 0.445 29 (42.6) 12 (46.2) 0.715  31 2 (2.3) 0 (0) 0.445 1 (1.5) 1 (3.8) 0.715 Transfemoral approach 82 (95.3) 7 (87.5) 0.268 65 (95.6) 24 (92.3) 0.417 GFR, glomerular filtration rate; LV, left ventricle; NYHA, New York Heart Association; SD, standard deviation. Pulmonary artery systolic pressure over 50 mmHg. Laboratory parameters of haemolysis are summarized in *Table [2](#suaa098-T2){ref-type="table"}*. The difference between laboratory measurements at baseline and 1-year follow-up is shown in *Figure [1](#suaa098-F1){ref-type="fig"}* according to the presence or absence of haemolysis; we present data based on haemolysis Definition 2 but data based on haemolysis Definition 1 are similar. Only 20 (21%) patients had normal haemoglobin levels at baseline---per the definition above. Haemoglobin levels did not decline even in the groups with haemolysis---in fact, we observed slightly higher haemoglobin values in patients with haemolysis (*Figure [1](#suaa098-F1){ref-type="fig"}A*). Levels of LDH increased in both groups, but no patient had LDH levels above double the upper limit of normal value; in other words, all detected haemolyses were mild. Low levels of CRP at 1-year follow-up were demonstrated and prove that haptoglobin levels were not influenced by the acute-phase reaction. All other measured laboratory parameters did not change from baseline to 1-year follow-up. {#suaa098-F1} ###### Laboratory parameters at baseline and at 1-year follow-up Baseline 1 Year *P*-value -------------------------- -------------- -------------- ----------- Haemoglobin (g/dL) 12.03 ± 1.34 12.45 ± 1.38 0.034 Platelet count (10^9^/L) 192 ± 68 202 ± 71 0.177 Schistocyte count (%) 0.05 ± 0.06 0.06 ± 0.1 0.950 Retikulocyte count (%) 1.24 ± 0.49 1.31 ± 0.6 0.530 LDH (µkat/L) 3.12 ± 0.68 3.98 ± 0.82 \<0.001 ALT (µkat/L) 0.4 ± 0.23 0.43 ± 0.27 0.711 Bilirubin (µkat/L) 11.49 ± 6.98 10.76 ± 5.43 0.818 Haptoglobin (g/L) 1.45 ± 0.72 1.35 ± 0.78 0.441 CRP (mg/L) 7.7 ± 9.44 3.53 ± 2.81 0.004 Values are presented as mean ± SD. ALT, alanine aminotransferase; CRP, C-reactive protein; LDH, lactate dehydrogenase; SD, standard deviation. Post-implantation haemodynamic parameters are described in *Table [3](#suaa098-T3){ref-type="table"}*. Aortic regurgitation assessed by angiography in the operating room occurred in 12 (13%) patients and did not predict haemolysis. Moderate or severe aortic regurgitation (all paravalvular---no central regurgitation was detected) was diagnosed by TTE at discharge from hospital in 11 (12%) patients and was associated with a higher incidence of haemolysis---38% vs. 9% in patients with vs. without per Definition 1, *P* = 0.049 (similar results for Definition 2, see *Table [2](#suaa098-T2){ref-type="table"}*). Markedly higher incidence (approximately seven times) of haemolysis in patients with moderate or severe aortic regurgitation diagnosed by TTE at discharge is shown in *Figure [2](#suaa098-F2){ref-type="fig"}A*. We detected a trend towards higher mean prosthetic aortic valve gradient and smaller prosthetic aortic valve area in patients with haemolysis, but this finding did not reach statistical significance and the more appropriate AVAI values were very similar at 0.99 ± 0.34 vs. 0.92 ± 0.39, *P* = 0.408 in patients without vs. with Haemolysis Definition 1 (for all values see *Table [3](#suaa098-T3){ref-type="table"}*). Similarly, neither the severe PPM nor moderate or severe PPM was associated with the presence of haemolysis. *Figure [2](#suaa098-F2){ref-type="fig"}B* summarizes the frequency of moderate and severe form of PPM in our cohort and illustrates a similar incidence of haemolysis in these patients. {#suaa098-F2} {#suaa098-F3} ###### Post-implantation haemodynamic parameters according to the presence of haemolysis at 1-year follow-up Haemolysis Definition 1 (Skoularigis without schistocytes) Haemolysis Definition 2 (standard Skoularigis) ----------------------------------------- ------------------------------------------------------------ ------------------------------------------------ ------- ------------- ------------- ------- Moderate or severe AoReg by angiography 11 (12.8) 1 (14.3) 0.73 9 (13.2) 3 (11.5) 0.565 Echocardiography at discharge  Moderate or severe AoReg 8 (9.3) 3 (37.5) 0.049 5 (7.4) 6 (23.1) 0.044  Mean aortic gradient (mmHg) 7.7 ± 3.5 13.2 ± 9 0.081 7.6 ± 3.5 10 ± 6.3 0.191  Aortic valve area (cm^2^) 1.83 ± 0.61 1.67 ± 0.79 0.153 1.88 ± 0.64 1.64 ± 0.55 0.064  Aortic valve area index (cm^2^/m^2^) 0.99 ± 0.34 0.92 ± 0.39 0.408 1.01 ± 0.35 0.92 ± 0.31 0.228  Severe PPM 9 (11.6) 2 (33.3) 0.275 7 (11.3) 4 (17.3) 0.339  Moderate or severe PPM 34 (44.1) 4 (50) 0.518 27 (43.5) 11 (47.8) 0.456 AoReg, aortic regurgitation; PPM, patient-prosthesis mismatch; SD, standard deviation. All patients had mortality data at 6 years post-implantation (5 years post-evaluation for haemolysis) available. Overall, 67 (71%) patients died during 6 years of follow-up, 70% of cardiovascular and 30% of other causes. No patient expired due to haemolytic anaemia. Presence or absence of haemolysis had no impact on long-term patient survival, this is expressed as Kaplan--Meier survival curves in [*Take-home figure*](#suaa098-F3){ref-type="fig"}. The presence of moderate or severe aortic regurgitation post-TAVI was not associated with worse long-term prognosis in our population---mortality at 6 years was 72% in patients with trace or mild aortic regurgitation and 64% in patients with moderate or severe regurgitation, *P* = 0.46. Discussion ========== In this study, we provide the first data on intravascular haemolysis after implantation of supra-annular self-expandable transcatheter aortic prosthesis. The major findings of this study are the following: (i) incidence of intravascular haemolysis depends on the definition used, (ii) intravascular haemolysis is associated with turbulent blood flow and this is in the case of supra-annular self-expanding TAVI prosthesis associated with paravalvular aortic regurgitation but not with PPM, and (iii) intravascular haemolysis was mild in all diagnosed cases and did not have any prognostic impact till 6 years post-implantation. During the 1960s aortic valve replacement surgery was introduced with a mechanical prosthesis[@suaa098-B13] or bioprosthetic xenograft[@suaa098-B14] and the first report of haemolytic anaemia of mechanical origin followed shortly afterward,[@suaa098-B15] interestingly with authors correctly recognizing the role of turbulent blood flow. Since then, the incidence of clinical, symptomatic haemolysis decreased and became rare with reported rates under 1% with modern prosthetic valves.[@suaa098-B3]^,^[@suaa098-B4]^,^[@suaa098-B16]^,^[@suaa098-B17] However, mild subclinical haemolysis is commonly detected even with contemporary prostheses use, the reported incidence ranges from 18% to 51% and 5% to 10% in mechanical and biological prostheses, respectively. The wide range reflects the non-uniform diagnostic criteria used, this issue is covered in detail in two review articles.[@suaa098-B18]^,^[@suaa098-B19] Our results confirm this matter as a simple omission of one diagnostic parameter (schistocyte count) from standard Skoularigis criteria results in a three-times lower rate of haemolysis diagnosis (28% vs. 9%). Red blood cell survival analysis has been used in a small study and this approach might be more reliable in mild intravascular haemolysis detection and quantification.[@suaa098-B20] As far as the comparison of our results to published data: 1. There is only one report on haemolysis after surgical bioprosthetic valve in the aortic position. The study by Mecozzi *et al.*[@suaa098-B3] reported 3% incidence of haemolysis after stented surgical bioprosthesis in the aortic position. This is numerically clearly lower than our 28% incidence using the same Haemolysis Definition 2 criteria but should still be interpreted with caution as patient populations are quite different too; for example, age differs by 13 years. The majority of our patients (80%) had haemoglobin levels below the cut-off value per Skoularigis criteria even before TAVI. Hypothetically, replacing the absolute values of haemoglobin levels by drop of 1 g/dL or more from baseline would result in a dramatic reduction of haemolysis incidence to 9% in the old and high-risk TAVI patients. 2. There are two reports on haemolysis after TAVI and both have used our haemolysis Definition 1 criteria. Laflamme *et al.*[@suaa098-B8] have reported subclinical haemolysis in 15% of 122 patients following TAVI with mostly balloon-expandable prostheses; PVL had no impact on haemolysis but novel association between PPM and haemolysis was found. Ko *et al.*[@suaa098-B9] have reported subclinical haemolysis in 38% of 64 patients following TAVI with a mix of several prostheses used; moderate PVL and bicuspid aortic valve predicted haemolysis. These published data seem to report a numerically higher rate of haemolysis than our results (15--38% vs. 9%) but this comparison should be interpreted with caution as numbers are small. There seems to be a suggestion of a different predominant mechanism causing haemolysis: PPM for balloon-expandable TAVI prosthesis and PVL for supra-annular self-expandable TAVI prosthesis. Our results reflect a high rate (12%) of moderate or severe PVL after implantation of the first generation of a self-expandable prosthesis. This has improved considerably, recent data from a low-risk trial with 74% of patients treated with Evolut R prosthesis show moderate or severe PVL rate of 3.5% at 30 days post-implantation.[@suaa098-B7] Further improvement can be expected with Evolut PRO device use. On the other side, the rates of PVL might be higher in patients with bicuspid anatomy who will be more frequent in younger patients. Similarly to previously mentioned reports, all intravascular haemolysis was mild in severity in our study and no patient had severe symptomatic haemolysis. Levels of LDH can be falsely increased by other causes than haemolysis[@suaa098-B19] but mild elevations reliably rule-out severe haemolysis. As far as the clinical impact of subclinical haemolysis is concerned, Perek *et al.*[@suaa098-B21] reported a single-centre study with a possible negative impact of subclinical haemolysis on functional status at follow-up. Ko *et al*. found that mild haemolysis after TAVI was associated with an increased cardiovascular readmission rate at 1-year follow-up. We have not found any drop in haemoglobin levels even in patients with haemolysis at 1-year post-TAVI, but the development of anaemia in longer follow-up is possible. Turbulent blood flow does not only affect red blood cells but platelet activation might be linked to the same flow-induced mechanism as haemolysis.[@suaa098-B22] Our study provides the longest reported follow-up and did not find any impact of mild intravascular haemolysis (irrespective of the definition used) on patient mortality as a hard clinical endpoint. Limitations ----------- This study has important limitations. The small number of patients precludes multivariate analysis and does not allow us to draw any definitive conclusions. The intravascular haemolysis detection requires prospective laboratory testing of specific parameters that are not routinely clinically required, and this makes *post hoc* efforts to enlarge the number of patients by including other centres impossible. However, both our sample size and univariate data analysis are similar to most published literature on this topic. Selection bias due to eight excluded patients is unlikely but cannot be ruled out. All patients have received first-generation self-expandable TAVI device and our results should not be extrapolated to newer generations or different designs of valve prostheses. Mortality data reflect the high risk of enrolled patients and a small effect on mortality might be missed in our analysis. Conclusion ========== No severe symptomatic haemolysis was found after TAVI with self-expandable prosthesis. Mild subclinical intravascular haemolysis is present in 9--28% of patients depending on the definition of haemolysis. The presence of haemolysis is associated with moderate or severe aortic regurgitation but not with post-TAVI aortic valve area, this finding might be specific for the supra-annular self-expandable type of TAVI prosthesis. Subclinical haemolysis seems to be a benign condition with no detected impact on patient mortality at a 6-year follow-up. This topic warrants further study in younger low-risk patients and a more exact definition of intravascular haemolysis is needed. Funding ======= A.S., P.T., J.S., M.N., and V.K. were supported by the European Commission: Operational Programme: Research, Development, and Education, project INTERCARDIS, registration no. CZ.02.1.01/0.0/0.0/16_026/0008388. A.S., P.T., J.S., and V.K. were supported by Charles University Research Centre Program no. UNCE/MED/002. This paper was published as part of a supplement financially supported by the Cardiovascular Research Program of the Charles University 'Progres Q38'. **Conflict of interest:** P.T. reports consultant contract, lecture fees, and grant support from Medtronic. M.N. is an employee of Medtronic Czechia. V.K. reports consultant contract, lecture fees, and grant support from Medtronic and Abbott Vascular. All other authors have no potential conflict of interest.

Despite major advances in early detection and treatment, breast cancer remains the most frequently diagnosed cancer and the leading cause of cancer death in women worldwide. Strategies targeting the primary tumour have markedly improved in the past decade, but systemic treatments to prevent metastasis are less effective, and about 20%--30% of patients develop metastatic disease (metastatic breast cancer (MBC)). Several targeted agents have been introduced into chemotherapeutic regimens for MBC in recent years ([@bib3]; [@bib32]), and many others are in preclinical or early clinical stages of development, mostly in combination with cytotoxic chemotherapy ([@bib1]). However, once the disease metastasises to distant areas of the body, typically to the lung, bone, liver and central nervous system, it remains incurable, with a median survival between 2 and 4 years, depending on the subtype ([@bib12]; [@bib1]). Although representing only about 15% of all diagnosed breast cancer, the triple-negative subtype (TNBC), defined as negative for oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) expression, accounts for a disproportionate share of deaths, due to aggressive tumour behaviour, poor prognosis and lack of targeted therapies. TNBC generally occurs in younger women and is associated with a high risk of distant recurrence ([@bib24]). Thus, the as yet unmet need of effective agents for the management of metastatic TNBC (mTNBC) is fuelling the search for novel therapeutic targets ([@bib16]). A large body of evidence indicates that the insulin/insulin-like growth factor (IGF) pathway is strongly implicated in breast cancer ([@bib26]). The system consists of three ligands (IGF-1, IGF-2 and insulin); their cell surface receptors IGF-1 receptor (IGF-1R), IGF-2 receptor (IGF-2R, also known as the mannose 6-phosphate receptor M6P/IGF-2R) and two isoforms of the insulin receptor IR-A and IR-B, as well as hybrid IR/IGF-1Rs; six high-affinity binding proteins (IGFBP-1 to IGFBP-6) and their proteases ([@bib27]; [@bib22]). High IGF-1 plasma levels are a risk factor for breast cancer in premenopausal women ([@bib13]), and significantly higher serum IGF-1 levels have been found in breast cancer patients as compared with healthy controls ([@bib14]; [@bib17]). Loss of heterozigosity at the *IGF2* gene has long been observed as a frequent occurrence in human breast cancer samples ([@bib18]) and IGF-2 binding to IGF-1R and IR-A was found to facilitate activation of cell survival pathways ([@bib25]). The role of the IGF system in breast cancer has best been described in ER-positive tumours and cell lines, where it cooperates with the ER in promoting tumour growth and progression, while hampering the success of endocrine therapy ([@bib39]). Interestingly, IGF-1 has also been recently reported to promote proliferation and survival of TNBC cells ([@bib5]); in addition, a role has recently been described for IGF signalling in tumour metastasis and invasion ([@bib28]; [@bib37]; [@bib29]), increasing the appeal of the IGF system as a potential target for novel therapies directed at the ill-fated mTNBC subtype. Monoclonal antibodies and kinase inhibitors targeting IGF-1R have been the object of several clinical trials in different tumour types, but somehow the preclinical promise has failed to translate into therapeutic efficacy so far. This is possibly due to the redundancy and plasticity of the IGF system, whereby IR activation can compensate for IGF-1R disruption when this is specifically targeted ([@bib38]), especially when IGF-2 is the main stimulus acting on cancer cells. Most human cancers overexpress both IGF-1R and IR, leading to the formation of hybrid IGF-1R/IRs, and IGF-2 is a known ligand for all three receptors, displaying a far greater affinity for IR holoreceptors and IGF-1R/IR hybrids than IGF-1 ([@bib20]; [@bib2]). The role played by IGF-2 might be particularly relevant to therapeutic failure, as release of this factor from tumour cells can be driven by hypoxia-inducible factor-1 (HIF-1) as part of the adaptive response to reduced oxygen levels. Approximately 25--40% of all invasive breast cancer samples are hypoxic and express high levels of HIF-1*α*, the O~2~-labile HIF-1 *α*-subunit required for transcriptional activation ([@bib34]; [@bib35]). Thus, the well-known ability of HIF-1 to promote aggressive breast cancer disease might depend, at least in part, on the recruitment of IGF-2-dependent signalling; in addition, IGF-2 release by HIF-1 might account for the limited clinical success of IGF-1R-targeted agents. The present study was aimed at investigating the role played by IGF-2 in MDA-231 TNBC cell migration by emphasising its interactions with non-IGF-1R receptors. To this aim, we first verified that IGF-2-induced MDA-231 cell migration is associated with both IGF-1R and IR activation and that blocking IGF-1R with the selective catalytic inhibitor NVP-AEW541 ([@bib10]) was unable to suppress either cell migration or IR phosphorylation, whereas sequestering IGF-2 with the neutralising antibody MAB292 dramatically reduced both, and completely abrogated them in the presence of NVP-AEW541. In addition, we also observed that HIF-1*α* stabilisation reproduced, albeit on a lesser scale, the modifications observed in the presence of exogenous IGF-2 (i.e., increased cell migration and IGF-1R/IR phosphorylation), which could be prevented by adding the HIF-1 inhibitor topotecan and totally abolished by the topotecan/NVP-AEW541 combination. Overall, our data support the hypothesis that IR activation by IGF-2 may account for the failure of IGF-1R only-targeting agents to suppress TNBC cell migration *in vitro*, suggesting that it may also be involved in the disappointing clinical activity exhibited by such agents. Ligand-targeting compounds, or concomitant inhibition of the IGF system and HIF-1*α*, may prevent activation of compensatory signalling through IR or hybrid IR/IGF-1R and may provide an alternative or complementary strategy for the management of metastatic breast cancer in general and of TNBC in particular. Materials and methods ===================== Cell lines, reagents and drugs ------------------------------ Three human breast cancer cell lines were analysed: T47D and MCF-7 (ERα^+^, PR^+^, HER2^−^) and MDA-231 (ERα^−^, PR^−^, HER2^−^), obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) through the Tumour Microenvironment Laboratory (TML, SAIC-NCI Frederick, MD, USA), and authenticated by morphological inspection, growth curve analysis and short tandem repeat profiling, using the Promega PowerPlex 1.2 system (Promega, Madison, WI, USA) and the Applied Biosystems Genotyper 2.0 software (Applied Biosystems, Foster City, CA, USA) for analysis of the amplicons. An additional triple-negative cell line, MDA-468, was obtained from the same source and used to replicate some of the experiments for confirmation purposes. Cells were routinely cultured as monolayer at 37 °C and 5% CO~2~ in RPMI (or DMEM in the case of MDA468) medium supplemented with 5% fetal bovine serum (FBS, Euroclone, Milan, Italy) and 1% antibiotics (penicillin/streptomycin, Sigma Aldrich, Milan, Italy). NVP-AEW541 was kindly provided by Novartis Pharma (Basel, Switzerland): a DMSO stock solution (10 m[M]{.smallcaps}) was prepared and stored at −20 °C. MAB292, rhIGF-1 and rhIGF-2 were purchased from R&D Systems (Minneapolis, MN, USA): stock solutions were prepared following the manufacturer\'s instructions and stored at −20 °C. Topotecan (NSC 609699) was kindly provided by the TML (SAIC-NCI Frederick, MD, USA). Working dilutions of all drugs were prepared in culture media immediately before use. Quantitative RT-PCR ------------------- To analyse the components of the IGF and HIF systems at the transcript level, cells were seeded in dishes, allowed to grow for 24 h under normoxia (pO~2~ 21%) and subsequently incubated under hypoxia (pO~2~ 1%) for an additional 24 h. Transcription of HIF-1 target genes *(IGF2*, *VEGFA*, *PDK1)* was also assessed following treatment with a subtoxic concentration of topotecan (250 n[M]{.smallcaps}) during the 24 h of hypoxia. Total RNA was extracted following the manufacturer\'s instructions (RNeasy kit, Qiagen, Venlo, Netherlands) and quantitated (ND-1000, NanoDrop, Thermo Fisher Scientific, Waltham MA, USA); 250 ng (in 10 *μ*l) of mRNA were added to 15 *μ*l of RT solution (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems) and retro-transcribed following the optimised protocol. The cDNA thus obtained was used in a real time-PCR reaction: 2.5 ng (3.75 *μ*l) of cDNA were added with 21.25 *μ*l of a solution containing 2 × Master Mix (TaqMan or SYBR, Applied Biosystems), 3 *μ*[M]{.smallcaps} reverse and forward primers (final concentration 300 n[M]{.smallcaps}, custom made, Integrated DNA Technologies, Coralville, IA, USA), 20 × eukaryotic 18S rRNA endogenous control (VIC/TAMRA probe, Applied Biosystems) and H~2~O. The run was performed following the protocol provided by the manufacturer, and the results were analysed by the ABI7300 software (Applied Biosystems). Primer sequences are reported in [Supplementary Table S1](#sup1){ref-type="supplementary-material"}. Immunoblot analysis ------------------- To analyse the constitutive levels of the different components of the IGF and HIF systems, as well as the effects of growth factors and drugs on the IR/IGF-1R signalling pathway, cells were grown for 24 h and then treated with NVP-AEW541 (1 *μ*[M]{.smallcaps}) and/or MAB292 (5 *μ*g ml^−1^) for 24 h. Cells were exposed to rhIGF-1 or rhIGF-2 (50 ng ml^−1^) during the last 15 min before harvesting, following 6-h starvation in serum-free medium. The effects of hypoxia (1%) on the insulin/IGF system were assessed on cells grown for 24 h at 21% pO~2~ and shifted to hypoxia for an additional 24 h. NVP-AEW541 (1 *μ*[M]{.smallcaps}), MAB292 (5 *μ*g ml^−1^) and Topotecan (250 n[M]{.smallcaps}) were added, singly or in combination, during the 24 h of hypoxia. Cell lysates were prepared in RIPA buffer containing protease inhibitor cocktail (Sigma Aldrich); protein phosphatases were inhibited by adding phenylmethylsulfonylfluoride (1 m[M]{.smallcaps}) and sodium orthovanadate (Na~3~VO~4~, 1 m[M]{.smallcaps}) to the lysis buffer. Protein concentration was determined using assay kits based on the Bradford method (Bio-Rad Life Science, Hercules, CA, USA), and equivalent amounts of total cell lysates (100 *μ*g) were added with sample buffer 2 × (Laemmli, Sigma Aldrich), separated by 7% or 10% acrylamide SDS/PAGE under denaturing conditions and transferred onto PVDF membranes. Membranes were incubated overnight with the following primary antibodies: anti-phospho-IGF-1R*β* (Tyr1316), anti-IGF-1R*β*, anti-phospho-IR*β* (Tyr1361), anti-IR*β*, anti-phospho-Akt (Ser473) and anti-Akt (all from Cell Signalling Technology, Danvers, MA, USA); anti-IGF-2R (Abcam, Cambridge, UK); and anti HIF-1*α* (Novus Biologicals, Littleton, CO, USA). An anti-actin antibody (Sigma Aldrich) was used as a control. Membranes were then incubated with secondary anti-rabbit or anti-mouse antibody conjugated to horseradish peroxidase (Amersham, GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Immunoreactive bands were revealed by Enhanced Chemiluminescence Western Blotting Detection reagents (Amersham and Pierce) and visualised on Hyperfilm ECL (Amersham). ELISA assay ----------- The release of IGF-2 in culture media was evaluated under normoxic and hypoxic conditions. Cells were seeded onto six-well plates and allowed to grow for 24 h before starving and incubation at different oxygen levels (pO~2~ 21% or 1%). Supernatants were collected 24 h later and stored at −80 °C or immediately quantitated using a specific ELISA kit (Insight Genomics, Falls Church, VA, USA), according to the manufacturer\'s protocols. Flow cytometry -------------- Membrane expression of IGF-1R, IGF-2R and IR was evaluated in all cell lines. Cells were seeded in six-well plates and allowed to grow for 48 h; they were subsequently collected, counted and incubated for 1 h at 4 °C with specific conjugated antibodies (IGF-1R/PE, IGF-2R/FSC and IR/PE, R&D Systems) as well as IgG isotype control antibodies (R&D Systems). Red (PE) and green (FSC) fluorescence was then read using a Guava easyCyte (EMD Millipore, Billerica, MA, USA) flow cytometer. Background fluorescence, assessed in IgG isotype controls, was subtracted to the corresponding samples during analysis, and the percentage of fluorescent cells was calculated. Scratch wound-healing assay --------------------------- To evaluate the effect of the different compounds on migration of the three cell lines, cells were seeded at high density onto specific supports (*μ*-dish Culture Inserts, Idibi GmbH, Planegg/Martinsried, Germany) and allowed to grow for 24 h. The inserts were then removed, the medium was replaced with serum-free medium, with or without the different compounds, and the dishes were incubated in different conditions of oxygenation (pO~2~ 21% or 1%). Pictures of the wound scratch were taken at the time of removal (T0) and at regular time intervals for a total of 48 h. The experiments were repeated twice. Boyden chamber assay -------------------- To further assess the migration of MDA-231 cells, we also performed the Boyden chamber assay. Boyden chambers (Cell Biolabs Inc., San Diego, CA, USA) were pre-incubated for 30 min at 37 °C in 24-well plates containing 600 *μ*l of culture medium and FBS (1 : 1). Cells, previously starved for 24 h, were added on top of the chambers, treated with growth factors and/or drugs at different oxygen levels (pO~2~ 21% or 1%) and allowed to grow and migrate for 24 or 48 h. At the specified end points, the chambers were removed from the plates, fixed and stained in two steps (Eosin+AzurA/AzurB, 2 min each) and photographed. Analysis did not involve counting the number of migrating cells but only assessing visible differences between the treatments. The experiments were repeated twice. Results ======= *In vitro* assessment of IGF-stimulated migration and analysis of IGF system components in three human breast cancer cell lines ------------------------------------------------------------------------------------------------------------------------------- IGF-stimulated migration of MCF-7, T47D and MDA-231 cells was assessed using the scratch wound-healing and Boyden chamber assays. In the absence of stimulation, MCF-7 and T47D were unable to migrate, and even the addition of exogenous rhIGF-1 or -2 did not appreciably modify their behaviour; in contrast, MDA-231 cells, derived from a metastatic carcinoma, intrinsically exhibited some degree of migration, which was substantially increased following stimulation with rhIGFs ([Figure 1](#fig1){ref-type="fig"}). We then assessed the baseline levels of some major components of the IGF system, in order to evaluate their potential roles in the different migratory behaviour exhibited by the three cell lines. The results of PCR experiments ([Supplementary Figure S1](#sup1){ref-type="supplementary-material"}) indicate that IGF-2, IGF-1R and IGF-2R mRNAs are present in all cell lines, albeit at different levels: surprisingly, highly invasive MDA-231 showed lower levels of IGF-1R and higher levels of IGF-2R, as compared with both MCF-7 and T47D. IGF-1 mRNAs were undetectable in all cell lines and are not shown. Specific analysis of the two IR variants confirmed the results reported in the literature ([@bib23]): all the cell lines tested expressed both the A and B variants of the receptor, with a predominance of the former; more specifically, MDA-231 cells were found to possess the highest levels of IR-A mRNA. However, when considering protein levels, differences among cell lines were somewhat less evident: secreted IGF-2 in the extracellular medium was comparable in the three cell lines ([Supplementary Figure S2](#sup1){ref-type="supplementary-material"}, panel a), in spite of remarkable differences in transcript; IGF-1 expression was not investigated further, in view of the observed absence of transcript. Membrane expression of IGF-1R, as determined by flow cytometry, was consistent with transcript levels, whereas expression of IGF-2R and IR did not vary among cell lines ([Supplementary Figure S2](#sup1){ref-type="supplementary-material"}, panel b); however, it should be emphasised that IR-A and IR-B levels could not be determined separately, due to the lack of antibodies able to discriminate between the two isoforms. IGF-1R and IR activation following exposure to exogenous IGFs was assessed by western blotting, based on the levels of receptor phosphorylation, and is shown in [Figure 2](#fig2){ref-type="fig"} (densitometric analyses are reported in [Supplementary Figures S3, S4 and S5](#sup1){ref-type="supplementary-material"}). In unstimulated cells, both receptors exhibited a minimal degree of phosphorylation, which was markedly enhanced in response to the addition of rhIGFs to the culture medium. More specifically, as expected, IGF-1R was activated by both ligands in all three cell lines tested; in MDA-231, but not in the other cell lines, IR was also relevantly activated by rhIGF-2 and, to a lesser extent and rather more surprisingly, rhIGF-1. Exposure to the selective IGF-1R inhibitor NVP-AEW541 predictably abrogated IGF-1R phosphorylation in all cell lines but did not affect IR activation. The selective IGF-1R inhibitor NVP-AEW541 is only partially effective in inhibiting cell migration following exposure to rhIGFs ------------------------------------------------------------------------------------------------------------------------------- As shown in [Figure 3A and B](#fig3){ref-type="fig"}, exposure to NVP-AEW541 partially abrogated the migration potential of MDA-231 cells, as assessed by both the scratch wound healing and the Boyden chamber migration. As expected, the inhibitor appeared to be more effective in reducing rhIGF-1 than rhIGF-2-induced migration. The fact that migration was not completely abolished could be explained by the inability of NVP-AEW541 to prevent IR activation (shown in [Figure 2](#fig2){ref-type="fig"}) and to block the related downstream pathway, as indicated by the presence of some residual Akt phosphorylation following exposure to rhIGF-2 in the presence of NVP-AEW541 in MDA-231 cells, in spite of a near-complete block of IGF-1R phosphorylation ([Figure 4A](#fig4){ref-type="fig"}; densitometric analyses are reported in [Supplementary Figure S6](#sup1){ref-type="supplementary-material"}). Interestingly, NVP-AEW541 ehxibited some (slight) anti-migratory effect even on unstimulated cells, which might be explained by the presence of a low, baseline level of constitutive IGF-1R activation, as also suggested by the western blotting results shown in [Figure 4A](#fig4){ref-type="fig"}. Enhancing the effects of NVP-AEW541 on cell migration by inhibiting IGF-2 signalling with the specific monoclonal antibody MAB292 --------------------------------------------------------------------------------------------------------------------------------- To further demonstrate that IR activation by rhIGF-2 accounts, at least in part, for the lack of efficacy of NVP-AWE541 in inhibiting the migration of MDA-231 cells, receptor--ligand interaction was antagonised using a neutralising antibody (MAB292) that binds and sequesters IGF-2. As shown in [Figure 4A](#fig4){ref-type="fig"}, MAB292 caused a decrease in the phosphorylation of both IGF-1R and IR in MDA-231 cells following stimulation with rhIGF-2; furthermore, rhIGF-2-dependent IR phosphorylation, as well as downstream Akt phosphorylation, was totally abolished when NVP-AEW541 and MAB292 were added simultaneously to the culture medium. This last observation can probably be explained by the inability of the IGF-sequestering antibody to counteract the baseline constitutive activity of IGF-1R observed above. In agreement with the role played by IR in MDA-231 cell migration in response to rhIGF-2, results obtained in the scratch wound-healing ([Figure 4B](#fig4){ref-type="fig"}) and Boyden chamber assays ([Figure 4C](#fig4){ref-type="fig"}) indicate that, while IGF-1R inhibition alone was insufficient in preventing IGF-2-dependent migration, blocking the interaction of the growth factor with both signalling receptors using MAB292 showed some effect, which resulted in an almost complete abrogation of the migratory capacity of MDA-231 cells when NVP-AEW541 and MAB292 are added simultaneously. To confirm these findings on an additional TNBC cell line, the scratch wound-healing assay was also performed on MDA-468 cells stimulated with rhIGF-2, in the presence or absence of MAB292, NVP-AEW541 or both; the results are shown in [Supplementary Figure S7](#sup1){ref-type="supplementary-material"} and were similar to those obtained in MDA-231 cells. HIF-1*α* stabilisation decreases the anti-migratory effect of NVP-AEW541 ------------------------------------------------------------------------ Hypoxia has been shown to cause an increase in endogenous IGF-2 release, owing to transcriptional activation of the corresponding gene by HIF-1 ([@bib6]; [@bib30]). Thus, our study was extended to examine the mechanism of IGF-2-dependent migration, and its response to NVP-AEW541, in hypoxic MDA-231 cells. In agreement with literature data, secreted IGF-2 in the extracellular medium was significantly increased from 50.5±11.53 to 86.0±0.95 pg ml^−1^ (*P*\<0.05) following hypoxic incubation. Western blotting analysis indicates that both IGF-1R and IR were partially phosphorylated under hypoxia, even in the absence of exogenous growth factor stimulation, and that NVP-AEW541 decreased IGF-1R phosphorylation, while leaving IR phosphorylation virtually unaffected; in contrast, treatment with MAB292 reduced the phosphorylation of both receptors ([Figure 5A](#fig5){ref-type="fig"}; densitometric analyses are reported in [Supplementary Figure S8](#sup1){ref-type="supplementary-material"}). These observations correlate with the migratory behaviour of hypoxic MDA-231 cells, in that migration was almost completely blocked by MAB292, whereas NVP-AEW541 only caused partial inhibition ([Figure 5B and C](#fig5){ref-type="fig"}), similarly to what had been observed following exposure to exogenous rhIGF-2. HIF-1 inhibition decreases IGF-2-dependent signalling and enhances the effects of NVP-AEW541 on cell migration -------------------------------------------------------------------------------------------------------------- Given the ability of hypoxia to undermine the ability of selective IGF-1R inhibition to impair cell migration through HIF-1-dependent IGF-2 release, we finally evaluated the efficacy of the combination of NVP-AEW541 with HIF-1 inhibition in MDA-231 cells. To downregulate HIF-1 activity, we used topotecan, a DNA topoisomerase I poison that has been reported to inhibit the translation of HIF-1*α* mRNA ([@bib19]). The results of western blotting analysis confirm the effectiveness of topotecan as a HIF-1*α* inhibitor: as expected, and as shown in [Figures 5A](#fig5){ref-type="fig"} and [6A](#fig6){ref-type="fig"}, HIF-1*α* protein levels were consistently decreased following 24-h exposure to 250 n[M]{.smallcaps} of topotecan under hypoxia; this topotecan concentration was chosen based on a preliminary cytotoxicity assay, indicating that \<10% of the cells were killed by the drug under our experimental conditions. ([Supplementary Figure S9](#sup1){ref-type="supplementary-material"}, panel a). As a further corroboration of topotecan-induced HIF-1 inhibition, real time-PCR analysis showed that the mRNA levels of three HIF-1-regulated genes, namely, IGF-2, *VEGFA* and *PDK-1*, were restored to almost normoxic levels ([Supplementary Figure S9](#sup1){ref-type="supplementary-material"}, panel b) following topotecan treatment. It should be emphasised that, while topotecan alone caused a decrease in phosphorylation of both receptors, the topotecan/NVP-AEW541 combination achieved near-complete abrogation ([Figure 6A](#fig6){ref-type="fig"}; densitometric analyses are reported in [Supplementary Figure S10](#sup1){ref-type="supplementary-material"}). Accordingly, topotecan, both as a single agent and in combination with NVP-AEW541, was able to curtail the migration of MDA-231 cells in both assays ([Figure 6B and C](#fig6){ref-type="fig"}). Topotecan and the topotecan/NVP-AEW541 combination were observed to exert similar effects on hypoxia-induced migration of MDA-468 cells ([Supplementary Figure S11](#sup1){ref-type="supplementary-material"}), suggesting that our findings might be extended to other TNBC models. Discussion ========== The IGF axis and, more specifically, IGF-1R have long been considered as attractive targets for the development of novel anticancer therapies. Besides playing a crucial role in regulating cell growth, proliferation and differentiation, IGF-1R and its downstream signalling pathways have been implicated in the development of resistance to endocrine and targeted therapies ([@bib8]; [@bib15]). In addition, a role in tumour cell migration and metastatic potential and a direct involvement in the metastatic cascade in breast have recently been proposed ([@bib40]). Interestingly, IGF-dependent enhanced cellular proliferation has been documented in TNBCs, which can provide new targets for the so far limited therapeutic options of this sub-type that lacks expression of other druggable targets, such as ER or HER-2 ([@bib5]). In the present study, we have focused on the role of the IGF/insulin axis on cell migration and on the possibility to suppress this phenotype using NVP-AEW541, a selective IGF-1R inhibitor ([@bib10]), in MDA-231 cells, a human breast cancer cell line derived from a mTNBC. Two other cell lines, ER-positive MCF-7 and T47D, were also examined, but although both were shown to express IGF-1R and IR, neither displayed a migratory response to exogenous rhIGF-1 or rhIGF-2. Our results indicate that, while NVP-AEW541 effectively inhibits IGF-1R phosphorylation in response to both exogenous IGFs, only rhIGF-1-induced migration is significantly reduced by this compound, whereas the effect of rhIGF-2 on cell migration is remarkably resistant to this approach. Several different drug candidates targeting IGF-1R, including both anti-receptor antibodies and small-molecule receptor kinase inhibitors, have shown activity in model systems, and the former (e.g., figitumumab, ganitumumab, cixutumumab) have been the subject of intense clinical research. Although some Phase II studies of IGF-1R-specific antibodies showed activity with little toxicity, and there were reports of major responses, Phase III trial reports have documented a lack of efficacy, together with significant metabolic toxicity (chiefly hyperglycaemia; [@bib21]; [@bib8]; [@bib15]). Although systemic compensatory feedback mechanisms may account, at least in part, for the observed failure of agents specifically targeting IGF-1R in the clinical settings, our data indicate that compensatory mechanisms can also operate at the cellular level, significantly hindering the anti-migratory effect of NVP-AEW541 in our experimental setup. Our working hypothesis was that interaction of IGFs, especially IGF-2, with IR (which is not targeted by NVP-AEW541) might explain the failure of this compound to suppress breast cancer cell migration. MDA-231 cells do, in fact, express both IR-A and IR-B, and western blotting analysis indicates that NVP-AEW541 does not affect IGF-2-induced IR phosphorylation, nor is it able to completely abolish downstream signalling, as shown by residual activation of Akt. Rather more surprisingly, we also observed some degree of IR activation in cells stimulated with IGF-1, which was not prevented by NVP-AEW541 exposure; however, this finding could be due to the use of supraphysiological ligand concentrations, which might engage a significant fraction of IRs in spite of low affinity, while IGF-2 remains the most likely IR activator under physiological conditions. Alternatively, supraphysiological IGF-1 concentrations might cause indirect IR activation by increasing intracellular HIF-1*α* levels, as observed in experimental models derived from different tumour types ([@bib31]; [@bib33]; [@bib11]), and this in turn would cause the release of endogenous IGF-2. Dual IGF-1R/IR inhibition could prevail over this signal redundancy, but the role played by IR in cellular metabolism raises serious concerns regarding the safety of this approach in the clinic. The existence of two IR variants, the former of which (A) is preferentially expressed in tumours, whereas variant B is thought to have the major role in glucose metabolism, provides a window of opportunity for intervention specifically directed at tumours ([@bib4]); however, the fact that the two variants only differ in the presence/absence of exon 11 (coding for a fragment of 12 aa in the C-terminal portion of the *α* subunit of the receptor) makes the design of variant-selective inhibitors and antibodies particularly challenging. In order to simultaneously inhibit activation of both IGF-1R and IR by IGF-2, a different and probably more feasible strategy would be to block IGF-2 itself. IGF-2 has been reported to selectively activate IR-A in several tumour tissues, whereas its binding affinity for IR-B is negligible ([@bib7]; [@bib23]). Thus, sequestering IGF-2 would abrogate signalling through both IGF-1R and IR-A, as well as through their hybrid isoforms, all of which are frequently overexpressed in cancer cells, while sparing insulin-dependent signalling through IR-B, thereby reducing the risk of metabolic side-effects. MEDI-573 is the only antibody currently in clinical testing that exerts its effects by neutralising not only IGF-2 but also IGF-1, thus inhibiting IGF signalling through IGF-1R, IR and their hybrid receptors. The ongoing Phase I clinical trial has shown promising results and strongly suggests that MEDI-573 might achieve this result without inducing hyperglycaemia ([@bib9]). If confirmed, this observation would be consistent with better expectations of ligand- *vs* receptor-based targeting of the IGF axis. Our data obtained with a neutralising antibody specific for IGF-2 (MAB292) seem to support the involvement of IGF-2-induced IGF-1R/IR activation in the poor anti-migratory effect of NVP-AEW541. In MDA-231 cells exposed to IGF-2, MAB292 was able to prevent phosphorylation of both receptors; more significantly, stimulation of cell migration by rhIGF-2 was dramatically reduced by MAB292 and completely abolished when the antibody was combined with NVP-AEW541. IGF-2 neutralisation could be a particularly winning strategy, especially considering the fact that, at low oxygen levels that are frequently encountered within the tumour mass (pO~2~\<2.5%), HIF-1*α* stabilisation and subsequent dimerisation with HIF-1*β* leads to increased IGF-2 levels, as reported in the literature ([@bib6]; [@bib30]) and as observed both at the transcript and protein levels in the present experiments on MDA-231 cells. This HIF-1-dependent increase in IGF-2 release possibly explains why NVP-AEW541 fails to block MDA-231 cell migration, similarly to what has been observed in the presence of exogenous IGF-2 (although obviously to a lesser extent, considering the lower concentration of IGF-2 released under hypoxia as compared with the one administered exogenously). In support of the key role played by IGF-2 in obstructing the anti-migratory effect of NVP-AEW541 following HIF-1*α* stabilisation, addition of MAB292 is as effective in inhibiting the migration of hypoxic MDA-231 cells as it is following exposure to exogenous rhIGF-2. Based on this observation, we suggest that a novel strategy to overcome IR stimulation by IGF-2 following IGF-1R inhibition may be simultaneous HIF-1*α* inhibition. The discovery of HIF-1 (and later HIF-2) and its role in cancer cell proliferation and survival in conditions of reduced oxygenation (pO~2~\<2.5%) has led to a great interest in the development of HIF-1*α* and HIF-2*α* inhibitors. At present, the major problem in targeting HIFs is the lack of specific inhibitors: many chemotherapeutic drugs, in fact, have a complete or partial inhibitory activity on HIF-1*α*, but this is often accompanied by a number of off-target effects. Specific inhibitors of HIF-1*α* and/or HIF-2*α* are currently under validation ([@bib19]; [@bib36]). In our study, HIF-1*α* inhibition was achieved using topotecan, a topoisomerase I poison with major clinical applications in the management of ovarian and small cell lung cancer. Topotecan inhibits HIF-1*α* translation through a topoisomerase I-dependent but DNA damage-independent mechanism, suggesting that HIF-1*α* inhibition could be mechanistically distinct from cytotoxicity ([@bib19]). In our experiments, topotecan (used at subtoxic concentrations, so that HIF-1*α* inhibition could predominate over the cytotoxic effect of the drug) was confirmed to decrease HIF-1*α* protein levels, as well as the expression of different HIF-1 target genes (*IGF2*, *VEGFA* and *PDK1*). As a result of the decrease in IGF-2 release, phosphorylation of both IR and IGF-1R was reduced by topotecan treatment and nearly abrogated by concurrent treatment with topotecan and NVP-AEW541; importantly, the effect of NVP-AEW541 on migration of MDA-231 cells under hypoxia was restored to normoxic levels (or lower). Overall, we can conclude that combined inhibition of IGF-1R and HIF-1 appears to result in a synergistic effect on the migration of triple-negative MDA-231 breast cancer cells, and similar results obtained in MDA-468 cells, also triple-negative, raise the hope that our findings may be extended to this hard-to-treat breast cancer subtype. Thus, inhibiting the IGF and HIF systems during the early stages of breast cancer might provide a promising therapeutic option, most notably for TNBC, by preventing progression to MBC, besides reducing the contribution of these systems to the development of resistance against both cytotoxic and targeted agents. [Supplementary Information](#sup1){ref-type="supplementary-material"} accompanies this paper on British Journal of Cancer website (http://www.nature.com/bjc) This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. Supplementary Material {#sup1} ====================== ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. {#fig1} ![**Effect of NVP-AEW541 (1 *μ*[M]{.smallcaps} for 24 h) on total and phosphorylated IGF-1R and IR levels in MCF7, MDA-231 and T47D cells, in the absence of stimulation or after exposure to rhIGF-1 or rhIGF-2 (50 ng ml^−1^ in serum-free medium for 15 min: cells were grown in serum-free medium for the last 6 h of treatment before exposure to IGFs).** Actin levels were simultaneously assessed as an internal loading control.](bjc2014269f2){#fig2} ![**Effect of NVP-AEW541 (1 *μ*[M]{.smallcaps}) on the IGF-induced migration of MDA-231 cells, as assessed by the scratch wound-healing and Boyden chamber assays.** (**A**) Cells were grown in specific supports for 24 h, then the inserts were removed and the medium was replaced with serum-free medium containing NVP-AEW541 with or without rhIGF-1 or rhIGF2 (50 ng ml^−1^). Pictures were taken at the time of insert removal (T0) and at regular time intervals, up to 48 h. (**B**) Cells, previously starved for 24 h, were added on top of pre-incubated Boyden chambers and allowed to grow and migrate (in the presence of NVP-AEW541 with or without 50 ng ml^−1^ of rhIGF-1 or rhIGF-2) for 24 or 48 h: the cells able to migrate in the chamber were stained and photographed, and differences between the treatments were assessed by visual inspection.](bjc2014269f3){#fig3} ![**Inhibition of IGF-2 signalling by the specific monoclonal antibody MAB292 enhances the anti-migratory effect of NVP-AEW541.** (**A**) Comparative effects of MAB292 (5 *μ*g ml^−1^ for 24 h) and NVP-AEW541 (1 *μ*[M]{.smallcaps} for 24 h) on IGF-1R, IR and Akt phosphorylation in rhIGF-2-stimulated MDA-231 cells (50 ng ml^−1^ in serum-free medium for 15 min, following 6 h of starvation). Actin levels were simultaneously assessed as an internal control. (**B** and **C**) Comparative effects of MAB292 (5 *μ*g ml^−1^) and NVP-AEW541 (1 *μ*[M]{.smallcaps}) on rhIGF-2-induced migration of MDA-231 cells as assessed by the scratch wound-healing (**B**) and Boyden chamber (**C**) assays. The experiments were performed according to the same protocol as in [Figure 3A and B](#fig3){ref-type="fig"}.](bjc2014269f4){#fig4} ![**HIF-1*α* stabilisation decreases the anti-migratory effect of NVP-AEW541.** (**A**) Effect of HIF-1*α* stabilisation under hypoxic conditions (pO~2~ 1%) on total and phosphorylated IGF-1R and IR levels in MDA-231 cells, and effect of MAB292 (5 *μ*g ml^−1^ for 24 h) and NVP-AEW541 (1 *μ*[M]{.smallcaps} for 24 h) on IGF-R1 and IR phosphorylation. Cells were grown for 24 h under normoxic conditions (pO~2~ 21%) and subsequently incubated under hypoxia for 24 h, with or without MAB292 or NVP-AEW541. Normoxic controls were also included for comparison; actin levels were simultaneously assessed as an internal loading control. (**B** and **C**) Comparative effects of MAB292 (5 *μ*g ml^−1^) and NVP-AEW541 (1 *μ*[M]{.smallcaps}) in inhibiting the migration of unstimulated MDA231 cells, as assessed by the scratch wound-healing (**B**) and Boyden chamber (**C**) assays. Hypoxia was started at T0 following 24-h incubation under normoxic conditions; the experiments were performed according to the same protocol as in [Figure 3A and B](#fig3){ref-type="fig"}.](bjc2014269f5){#fig5} ![**HIF-1 inhibition decreases IGF-2-dependent signalling and enhances the anti-migratory effect of NVP-AEW541.** (**A**) Effect of topotecan (TPT; 250 n[M]{.smallcaps} for 24 h) on HIF-1*α* levels and on total and phosphorylated IGF-1R and IR in MDA-231 cells under hypoxic conditions (pO~2~ 1%), alone or in combination with NVP-AEW541 (1 *μ*[M]{.smallcaps} for 24 h), as assessed by western blotting. Cells were grown for 24 h under normoxic conditions (pO~2~ 21%) and subsequently incubated under hypoxia for 24 h, with or without TPT±NVP-AEW541. Normoxic controls were also included for comparison; actin levels were simultaneously assessed as an internal loading control. (**B** and **C**) Effect of TPT (250 n[M]{.smallcaps}), alone or in combination with NVP-AEW541 (1 *μ*[M]{.smallcaps}), on the migration of unstimulated MDA-231 cells, as assessed by the scratch wound-healing (**B**) and Boyden chamber (**C**) assays. Hypoxia was started at T0, following 24-h incubation under normoxic conditions; the experiments were performed according to the same protocol as in [Figure 3A and B](#fig3){ref-type="fig"}.](bjc2014269f6){#fig6}

{#sp1 .320}

Introduction {#ss1} ============ Diabetes is increasing in Japan to levels that are comparable with those of other countries, and a recent publication reported that the number of individuals 'strongly suspected of having diabetes' in Japan was approximately 8.9 million in 2007[^1^](#b1){ref-type="ref"}. Type 2 diabetes is characterized by impaired β‐cell function and insulin resistance. The increasing rate of diabetes in Japan probably reflects a complex interplay between genetic and environmental factors, including an increasingly Westernized diet, a more sedentary lifestyle and the 'thrifty' genotype characteristic of many Japanese people[^2^](#b2){ref-type="ref"}. Compared with other ethnic populations, Japanese patients with type 2 diabetes show markedly reduced basal and impaired early‐phase insulin secretion, but lower indices of insulin resistance[^3^](#b3){ref-type="ref"}. Accordingly, body mass index (BMI), which has a positive correlation with insulin resistance, is generally lower in Japanese type 2 diabetes patients, with a mean BMI of 24 kg/m^2^, compared with 27--30 kg/m^2^ and \>30 kg/m^2^ in European and US patients, respectively[^3^](#b3){ref-type="ref"}. In summary, these observations suggest that β‐cell failure might play a relatively greater part than insulin resistance in the pathophysiology of type 2 diabetes in Japanese people. This might be a result of loss of β‐cell mass or function. In Japan, sulfonylureas (SU) are widely used either as monotherapy or in combination with other oral antidiabetic drugs (OAD) to treat type 2 diabetes. In a cross‐sectional study of 17,000 Japanese type 2 diabetes patients, 72--78% on oral therapy were using SU[^4^](#b4){ref-type="ref"}. This is consistent with the known etiology of the disease in this population, where the key feature appears to be insufficient insulin secretion[^3^](#b3){ref-type="ref"}. The A Diabetes Outcome Progression Trial (ADOPT) study showed that treatment efficacy of glyburide (also known as glibenclamide) waned with successive treatment years[^5^](#b5){ref-type="ref"}. Whereas glyburide improved β‐cell function to almost normal levels within 6 months of initiation, the effect then decreased and β‐cell function declined to below baseline level. Inukai *et al.*[^6^](#b6){ref-type="ref"} reported that homeostasis model assessment of β‐cell function (HOMA‐B) gradually decreased over time after a transient improvement during 5‐year treatment with glibenclamide in Japanese type 2 diabetes patients. This paradoxical effect might result from increased β‐cell stress accelerating β‐cell apoptosis, as well as the natural decline in β‐cell function associated with disease progression[^7^](#b7){ref-type="ref"}. An alternative explanation is that desensitization to SU occurs, in which case, the state of decreased β‐cell function might be reversible[^7^](#b7){ref-type="ref"}. Among the newer treatments for type 2 diabetes are the incretin‐based therapies that include the glucagon‐like peptide‐1 receptor (GLP‐1R) agonists and dipeptidyl peptidase‐4 inhibitors (DPP‐4i), which exert their actions through potentiation of incretin receptor signaling. GLP‐1R agonists control blood glucose through regulation of islet function, principally with the stimulation of insulin and inhibition of glucagon secretion[^8^](#b8){ref-type="ref"}. Liraglutide (Novo Nordisk A/S, Bagsværd, Denmark) is a once‐daily, human GLP‐1R agonist. GLP‐1R agonists are glucose‐dependent insulin secretagogues, but their mechanism of action and target receptors on the β‐cell differ from SU. Response to physiological levels of GLP‐1 is reduced in type 2 diabetes patients; pharmacological levels of native GLP‐1 or GLP‐1 analog therapy can restore this response[^9^](#b9){ref-type="ref"}. In clinical trials, in predominantly Caucasian populations, treatment with GLP‐1R agonists is associated with sustained improvements in glycemic control, weight reduction and low hypoglycemia risk[^10^](#b10){ref-type="ref"}. In clinical trials in European and US populations, GLP‐1R agonists have shown favorable effects on several parameters of β‐cell function[^11^](#b11){ref-type="ref"}. In animal models, exposure to GLP‐1 is associated with an increase in β‐cell mass[^12^](#b12){ref-type="ref"}. In contrast to SU, it is therefore possible that GLP‐1R agonists might limit the progressive loss of β‐cell function. It is of clinical interest to determine whether the beneficial effects of liraglutide on β‐cell function evident in other populations could also provide clinical benefits in Japanese patients, and if these benefits could be sustained for longer periods than are achievable with SU. The results of glycemic control parameters, such as glycated hemoglobin \[HbA~1c~\], fasting plasma glucose (FPG), postprandial glucose (PPG) and seven‐point self‐monitored plasma glucose, as well as safety data, have been reported for two clinical trials with Japanese type 2 diabetes patients receiving liraglutide, either as monotherapy in one trial or added on to SU therapy in the other trial, for 24 weeks[^13,14^](#b13 b14){ref-type="ref"}. Here, we report the short‐term effect of liraglutide on β‐cell function in these trials. Materials and Methods {#ss2} ===================== Adult Japanese type 2 diabetes patients were screened and enrolled in one of two double‐blind, multicenter, randomized, parallel‐group clinical trials (trial A or B) if they were ≥20 years of age, with HbA~1c~≥7.4 to \<10.4% and BMI \<35[^13,14^](#b13 b14){ref-type="ref"}. Patients were to be on diet and OAD (trial A: ±OAD monotherapy -- biguanide, sulphonylamide, SU \[≤50% approved dose in Japan\], α‐glucosidase inhibitor, insulin secretagogue or insulin sensitizer, within approved Japanese dose ranges; trial B: SU monotherapy -- glibenclamide 1.25--10 mg/day, gliclazide 40--160 mg/day or glimepiride 1--6 mg/day). Patients with clinical conditions likely to interfere with the conduct of the trial were excluded. Trials were carried out in accordance with the Declaration of Helsinki[^15^](#b15){ref-type="ref"}, with informed consent of patients and approval of relevant ethics committees. In trial A (24 weeks; *n* = 411), patients were randomized (2:1) to once‐daily liraglutide (0.9 mg) or once‐ or twice‐daily glibenclamide (1.25--2.5 mg). In trial B (24 weeks; *n *=* *267), patients continued SU treatment (glibenclamide \[1.25--10 mg\], gliclazide \[40--160 mg\] or glimepiride \[1--6 mg\]), and were randomized to one of two daily doses of liraglutide (0.6 or 0.9 mg), or placebo. In trial A, a 4--6 week run‐in/screening period preceded a 2‐week dose‐escalation period followed by a 22‐week maintenance period. In trial B, a screening visit was followed by a start‐of‐treatment visit after 4 weeks, a 2‐week dose‐escalation period and a 22‐week treatment period. Patients in trial A were stratified by pretreatment therapy (±OAD) and, in trial B, according to type of SU. In both trials, liraglutide was initiated with 0.3 mg during week 1 and increased weekly (in 0.3 mg increments) to the final dose to minimize gastrointestinal side‐effects. Randomization lists were prepared by the contract research organization responsible for the study, Transcosmos Inc. (Tokyo, Japan). This organization also ensured that liraglutide was unidentifiable from placebo, blinded trial products and randomized patients, and informed the investigator and sponsor of randomization numbers. Dynamic allocation was used to guarantee a balanced allocation within strata of pretrial treatment. Randomization codes were maintained in sealed conditions until broken according to schedule. Liraglutide was given by subcutaneous injection in the abdomen using a prefilled pen once daily in the morning or evening in the upper arm, abdomen or thigh. Injections were to be given at the same time every day. The primary outcome measure in all trials was HbA~1c~ at the end of the trial (expressed by National Glycohemoglobin Standardization Program values). Secondary end‐points included seven‐point self‐measured PPG profiles, FPG, glucose homeostasis‐related parameters (fasting insulin, proinsulin, C‐peptide, glucagon, postprandial insulin and glucagon). These end‐points have been reported elsewhere. Secondary end‐points reported here include measures of HOMA‐B index, proinsulin:insulin ratio and proinsulin:C‐peptide ratio. A meal test (Japanese‐style breakfast) was also carried out at baseline and 24 weeks. For each individual patient, the content of the meal was identical at these time‐points. The meal test was standardized within each site, but differed across sites. Plasma glucose, insulin, glucagon, fasting proinsulin and C‐peptide were measured. Intact proinsulin concentrations were determined by enzyme‐linked immunosorbent assay, based on anti‐proinsulin monoclonal antibodies (IBL; Immuno‐Biological Laboratories, Hamburg, Germany). Human insulin does not cross‐react in this assay. Within a concentration range of 5--500 pmol/L, proinsulin Des 64--65 cross‐reacted with frequencies of 53--65%. All analyses were carried out by a central laboratory (Mitsubishi Kagaku BCL Inc., Tokyo, Japan), except the seven‐point plasma glucose profile, which was measured before and approximately 2 h after each meal and at bedtime by self‐monitoring using standardized glucose meters (Glutest Ace; Glutest PRO, Sanwa‐Kagaku, Nagoya, Japan; Glucocard Diameter or Glucocard Diameter α; Arkray KDK Corp., Kyoto, Japan) before the start of treatment and at study end. β‐cell function was assessed using the HOMA‐B index, where HOMA‐B =  360 × fasting insulin/(FPG − 63) and units of insulin and glucose were μU/mL and mg/dL, respectively. For insulin and glucagon (meal test), the area under the curve (AUC) was calculated using the trapezoidal rule. Trial A was carried out between December 2006 and November 2008, and trial B between November 2006 and October 2007. Statistical Analysis {#ss3} -------------------- Efficacy end‐point analyses included data from all patients who were randomized and received trial product with efficacy data. Safety analyses included all patients who received trial drugs. Primary and secondary end‐points were analyzed using an analysis of variance ([anova]{.smallcaps}) model, with treatment group and stratification factor as fixed effects and baseline value as a covariate. An ad hoc analysis, AUC~insulin 0--3 h~:AUC~glucose 0--3 h~, was carried out using [anova]{.smallcaps} model with trial (A or B) and treatment group (liraglutide or comparator) as fixed effects and corresponding baseline values as covariate. In trial A, sample size calculation was based on 1.2% common standard deviation (SD) for both treatments, and 0.0% true difference in HbA~1c~ at 80% power, a non‐inferiority margin for HbA~1c~ of 0.4% at a significance level of 2.5%. The sample size calculation in trial B was based on a mean difference of 0.6% in HbA~1c~ between 0.9 mg + SU and placebo + SU after 24 weeks, with a SD of 1.2 and 80% power. Results {#ss4} ======= Baseline demographics, patient characteristics and patient disposition are shown in [Table 1](#t1){ref-type="table-wrap"}. No baseline differences were noted between the trials. ######  Patient disposition and baseline characteristics by trial and by treatment group Trial A Trial B ------------------------------------------- ------------- ------------- ------------- ------------- ------------- Randomized to treatment 139 272 89 89 89 Not exposed 7 4 1 1 1 Completed 120 246 74 83 84 Included in the efficacy analysis 132 268 88 88 88 Age, years (mean \[SD\]) 58.5 (10.4) 58.2 (10.4) 58.6 (9.7) 59.1 (10.3) 61.3 (11.0) Male/female, *n*  86/46 183/85 57/31 53/35 59/29 BMI, kg/m^2^ (mean \[SD\]) 24.6 (3.8) 24.9 (3.7) 24.9 (4.0) 25.3 (3.6) 24.4 (3.4) HbA~1c~, %\* 9.18 (0.97) 9.32 (1.08) 8.85 (0.99) 9.00 (0.91) 8.61 (0.78) Duration of diabetes, years (mean \[SD\]) 8.5 (6.8) 8.1 (6.7) 10.1 (7.3) 9.3 (5.8) 11.6 (7.7) \*At baseline. The value for glycated hemoglobin (HbA~1c~; %) is estimated as a National Glycohemoglobin Standardization Program (NGSP) equivalent value (%) calculated by the formula HbA~1c~ (%) = HbA~1c~ according to the Japanese Diabetes Society (JDS) (%) + 0.4%, considering the relational expression of HbA~1c~ (JDS) (%) measured by the previous Japanese standard substance and measurement methods and HbA~1c~ (NGSP). BMI, body mass index; SD, standard deviation; SU, sulfonylureas. All liraglutide doses reduced mean HbA~1c~ relative to comparator. In trial A, HbA~1c~ was reduced by 0.50% points with liraglutide relative to glibenclamide, whereas in trial B, mean HbA~1c~ was 1.00 and 1.27% points lower than placebo in the 0.6 and 0.9 mg liraglutide treatment groups, respectively. Significant improvements in all other measured parameters of glycemia (FPG, PPG and self‐monitored plasma glucose) were also reported in each trial (data not shown). No major hypoglycemic events were reported, and liraglutide was well tolerated across both trials. In trial A, the overall rate of hypoglycemia (episodes/subject‐year of exposure) was significantly lower in liraglutide‐ than glibenclamide‐treated patients (0.8 vs 5.5; *P* \< 0.0001). In trial B, the number of all hypoglycemic episodes was higher in the 0.6 and 0.9 mg/day liraglutide + SU groups than in the placebo + SU monotherapy group (*P =* 0.0159 and *P* *=* 0.0085, respectively). Insulin levels in the 3‐h post‐breakfast period (AUC~insulin 0--3 h~) were higher with liraglutide than with the comparator in both trials ([Figure 1a](#f1){ref-type="fig"}). AUC~insulin 0--3 h~ was significantly higher at week 24 (last observation carried forward \[LOCF\]) in liraglutide‐treated groups than in the glibenclamide group (*P  =* 0.0165; trial A) or placebo + SU group (*P* \< 0.0001; trial B; [Figure 1a](#f1){ref-type="fig"}). {#f1} The AUC~insulin 0--3 h~:AUC~glucose 0--3 h~ ratio was increased from baseline values (0.06--0.09) by more than 40% with liraglutide (0.14--0.16) relative to comparators (0.09--0.11) in both trials. The estimated mean (95% confidence interval \[CI\]) treatment difference for AUC~insulin 0--3 h~:AUC~glucose 0--3 h~ after administration of 0.9 mg liraglutide vs comparator was 0.038 (0.028, 0.048). After administration of 0.6 or 0.9 mg liraglutide, the treatment difference (liraglutide − comparator) was 0.038 (0.029, 0.048). Fasting glucagon levels and AUC~glucagon 0--3 h~ at week 24 (LOCF) in the liraglutide group were significantly lower than in the glibenclamide group (trial A), but not different to the SU monotherapy groups (trial B; [Table 2](#t2){ref-type="table-wrap"} and [Figure 1b](#f1){ref-type="fig"}). ######  Analysis of the effect of liraglutide on glucose metabolism‐related parameters by trial and by treatment group   Trial A Trial B ----------------------------------------- -------------- ---------------------- -------------- --------------------- --------------------- Fasting insulin, μU/mL  End‐of‐study LS mean (SE) 6.93 (0.36) 7.16 (0.27) 6.93 (0.38) 7.29 (0.37) 7.14 (0.38)  Liraglutide--comparator, mean (95% CI)   0.24 (−0.53, 1.00)   0.36 (−0.53, 1.26) 0.21 (−0.68, 1.11)  *P*‐value for pairwise comparison   * P = *0.5413   NA NA Fasting glucagon, (pg/mL)  End‐of‐study LS mean (SE) 105.4 (2.6) 96.5 (1.9) 103.0 (3.5) 98.7 (3.4) 102.4 (3.5)  Liraglutide--comparator, mean (95% CI)   −8.9 (−14.5, −3.3)   −4.4 (−12.5, 3.8) −0.6 (−8.8, 7.5)  *P*‐value for pairwise comparison    *P *=* *0.002   NA NA Fasting proinsulin, pmol/L  End‐of‐study LS mean (SE) 10.32 (0.54) 6.04 (0.40) 10.15 (0.92) 9.15 (0.91) 8.47 (0.93)  Liraglutide--comparator, mean (95% CI)   −4.27 (−5.44, −3.11)   −0.99 (−3.16, 1.18) −1.67 (−3.84, 0.50)  *P*‐value for pairwise comparison   * P *\<* *0.0001   NA NA Fasting C‐peptide, ng/mL  End‐of‐study LS mean (SE) 2.44 (0.07) 2.55 (0.05) 2.45 (0.08) 2.76 (0.08) 2.77 (0.08)  Liraglutide--comparator mean (95% CI)   0.10 (−0.05, 0.25)   0.31 (0.11, 0.50) 0.32 (0.12, 0.51)  *P*‐value for pairwise comparison    *P =* 0.1740   *P =* 0.0021 *P =* 0.0016 CI, confidence interval; LS, least squares; NA, not available; SU, sulfonylureas. Fasting insulin was similar between liraglutide‐ and comparator‐treated patients ([Table 2](#t2){ref-type="table-wrap"}) in both trials. End‐of‐study fasting C‐peptide levels were significantly higher in liraglutide + SU‐treated patients than in those on placebo + SU in trial B (*P* = 0.0017), but there was no significant difference between liraglutide‐ and glibenclamide‐treated patients in trial A. The estimated mean of fasting proinsulin at trial end was significantly lower in liraglutide‐ than glibenclamide‐treated patients in trial A (*P* \< 0.0001), and was not significantly different for patients on liraglutide + SU and placebo + SU in trial B ([Table 2](#t2){ref-type="table-wrap"}). In trial B, the estimated treatment difference (\[liraglutide + SU\] − \[placebo + SU\]) in HOMA‐B index was significant for both doses of liraglutide (*P* = 0.047 and *P* = 0.0012 for 0.6 and 0.9 mg, respectively). No significant between‐treatment difference at the end of trial in HOMA‐B was observed in trial A (glibenclamide 34.9%; liraglutide 39.0%; *P* = 0.0997; [Table 3](#t3){ref-type="table-wrap"}). ######  Analysis of the effect of liraglutide on β‐cell function: related parameters by trial and by treatment group ** ** Trial A Trial B ---------------------------------------------- ---------------------- --------------- ---------------------- ---------------------- -------------- β‐cell function, HOMA‐B (%)  End‐of‐study LS mean (SE) 34.88 (2.31) 39.04 (1.75) 30.86 (5.26) 43.35 (5.13) 51.53 (5.30)  Treatment difference mean (95% CI) 4.15 (−0.80, 9.10) -- 12.49 (0.17, 24.81) 20.67 (8.22, 33.13)  *P*‐value for pairwise comparison *P* = 0.0997 -- *P *= 0.0470 *P *= 0.0012  *P*‐value for overall test -- *P* = 0.0050 Proinsulin:insulin ratio, (pmol/L)/(μU/mL)  End‐of‐study LS mean (SE) 1.79 (0.08) 0.99 (0.06) 1.86 (0.13) 1.29 (0.12) 1.17 (0.13)  Treatment difference mean (95% CI) −0.81 (−0.98, −0.63) -- −0.57 (−0.86, −0.27) −0.69 (−0.99, −0.40)  *P*‐value for pairwise comparison *P* \< 0.0001 -- *P* = 0.0002 *P* \< 0.0001  *P*‐value for overall test -- *P* \< 0.0001 Proinsulin:C‐peptide ratio, (pmol/L)/(ng/mL)  End‐of‐study LS mean (SE) 4.17 (0.19) 2.31 (0.14) 4.10 (0.26) 3.10 (0.26) 2.71 (0.26)  Treatment difference mean (95% CI) −1.85 (−2.26, −1.45) -- −0.99 (−1.60, −0.38) −1.38 (−1.20, −0.77)  *P*‐value for pairwise comparison *P* \< 0.0001 -- *P* = 0.0016 *P* \< 0.0001  *P*‐value for overall test -- *P* \< 0.0001 CI, confidence interval; HOMA‐B, homeostasis model assessment of β‐cell function; LS, least squares; SE, standard error; SU, sulfonylureas. Decreases in the proinsulin:insulin ratio from baseline (baseline of 1.79--2.15 across all groups) for liraglutide‐treated groups (decrease of 0.65--0.87 across all groups) were greater than in SU‐treated groups (decrease of 0.15--0.26), resulting in lower values for the liraglutide‐treated group than for the comparator or placebo‐treated group at week 24. Reduction in the proinsulin:C‐peptide ratio from baseline (baseline of 4.09--4.76 across groups) was also greater in liraglutide‐treated groups (decrease of 1.24--1.61) than in SU‐treated patients (decrease of 0.06--0.55). Discussion {#ss5} ========== The present report shows that 24 weeks' treatment with liraglutide provides a significant improvement in β‐cell function in Japanese type 2 diabetes patients. Additionally, liraglutide was associated with significantly greater improvements in key parameters of glycemia, namely HbA~1c~, FPG, PPG and seven‐point self‐monitored plasma glucose, than comparators, and these results have been reported elsewhere[^13,14^](#b13 b14){ref-type="ref"}. As previously described, liraglutide resulted in weight loss or no weight gain in Japanese patients. The overall improvement of glycemic control with 0.9 mg liraglutide seen in Japanese populations was not different to that observed with 1.2 mg liraglutide in non‐Japanese populations[^13,14,16^](#b13 b14 b16){ref-type="ref"}. Indirectly, these observations suggest that different degrees of SU insensitivity rather than β‐cell apoptosis are responsible for the failure of pretrial treatment in Japanese type 2 diabetes patients. Although SU and liraglutide have powerful insulin‐releasing effects on β‐cells, they exert their effect through separate, independent receptors[^17,18^](#b17 b18){ref-type="ref"}. GLP‐1R exist in the β‐cell plasma membrane, and receptor interaction leads to mobilization and exocytosis of insulin‐containing granules[^19^](#b19){ref-type="ref"}. This action is strictly glucose‐dependent. In contrast, SU stimulate insulin secretion by closing β‐cell adenosine‐5′‐triphosphate‐sensitive potassium channels (K~ATP~) through binding to SU receptor 1 (SUR1) and, according to recent evidence, by activating the cyclic adenosine monophosphate (cAMP) sensor exchange protein activated by cAMP -- with the exception of gliclazide -- also through direct binding[^17^](#b17){ref-type="ref"}. These events are not glucose‐dependent. The differences in β‐cell function reported here should, however, be considered in respect to the different levels of glycemic control achieved with liraglutide vs comparators. Recently, it has been shown that GLP‐1R agonists can improve impaired glucose metabolism in diabetic pancreatic β‐cells, resulting in an increase in adenosine‐5′‐triphosphate (ATP) production[^20^](#b20){ref-type="ref"}. As the closure of K~ATP~ channels by SU is ATP‐dependent[^21^](#b21){ref-type="ref"}, it seems likely that the combination of a GLP‐1R agonist and an SU would be more effective at stimulating insulin secretion than a GLP‐1R agonist alone. We have recently shown that active GLP‐1 levels after meal ingestion are extremely low in healthy Japanese subjects and Japanese patients with type 2 diabetes[^22^](#b22){ref-type="ref"}. It seems likely, therefore, that supplementation of GLP‐1R agonists that are resistant to degradation by DPP‐4 would be effective in enhancing the GLP‐1 effect. A significant improvement in β‐cell function with liraglutide was observed across the two trials. All indicators of β‐cell function were substantially ameliorated, showing that liraglutide positively affected the insulin response to glucose. A significant increase in postprandial insulin secretion (AUC~insulin 0--3 h~) compared with comparators was shown. In support of this observation, the postprandial insulin:postprandial glucose level ratio increased with liraglutide by \>40% across the two trials. Although insulin secretion by liraglutide from β‐cells is glucose‐dependent, insulin secretion diminishes despite the continued presence of liraglutide as glucose levels normalize[^23^](#b23){ref-type="ref"}. This could be expected to counter postprandial hyperglycemia, a feature of type 2 diabetes, even at an early stage, in Japanese type 2 diabetes patients. Liraglutide appears to have a positive impact on pancreatic glucoregulatory function, as shown by the trend to a reduction in fasting and postprandial glucagon levels. Trial A, in particular, showed significant glucagon reductions with liraglutide treatment compared with glibenclamide, and a similar trend was observed in trial B, despite not achieving significance. The absence of significance in trial B might be related to the long‐term use of SU, which are reported to increase prandial glucagon levels[^24^](#b24){ref-type="ref"}. Therefore, in patients receiving combination therapy with liraglutide and an SU, the observable effect on glucagon levels would be attenuated as a result of the opposing effects each agent has on glucagon secretion. In type 2 diabetes, normal suppression of glucagon after a meal is blunted, resulting in hyperglucagonemia and increased hepatic glucose production in many patients, thus exacerbating hyperglycemia[^25^](#b25){ref-type="ref"}. Therefore, counter‐regulatory responses affecting glucagon secretion are impaired in these patient groups, and larger, more focused studies using appropriately matched patient groups will be required to unambiguously determine the effect of liraglutide on glucagon secretion. Liraglutide promotes β‐cell preservation in animal studies, increasing β‐cell mass in rodents and inhibiting β‐cell apoptosis *in vitro*[^12,26^](#b12 b26){ref-type="ref"}. In our studies, liraglutide significantly increased HOMA‐B relative to both baseline and the comparators. In trial A, HOMA‐B measurements with 0.9 mg liraglutide and glibenclamide were significantly improved from baseline. These results were also in accordance with a previous monotherapy trial (LEAD‐3) carried out in a predominantly Caucasian population[^16^](#b16){ref-type="ref"}. Although liraglutide did not outperform glibenclamide regarding improvements in HOMA‐B, it is encouraging to observe that it can be as equally effective as such a potent and widely used insulin secretagogue[^27^](#b27){ref-type="ref"}. Consistent with this finding, Madsbad *et al.*[^28^](#b28){ref-type="ref"} reported a significant improvement from baseline in HOMA‐B with 0.75 mg liraglutide (23.6%), which was similar to that observed with glimepiride (1--4 mg, 24.6%) in 193 Caucasian type 2 diabetes patients. The magnitude of the liraglutide‐associated increase in HOMA‐B from baseline in the present study (in the order of 90--100%) was greater than that shown in other studies in mainly Caucasian study populations. In another study, treatment with liraglutide increased HOMA‐B relative to baseline by approximately 30%[^29,30^](#b29 b30){ref-type="ref"}. During liraglutide treatment, the fasting proinsulin:insulin ratio was decreased. This might suggest improved processing of insulin in the β‐cell, possibly as a result of a more appropriate pattern of insulinotropic action reducing overall β‐cell stress. An elevated proinsulin:insulin ratio is a principal feature of type 2 diabetes and pre‐diabetes[^31^](#b31){ref-type="ref"}, and shows β‐cell dysfunction[^32^](#b32){ref-type="ref"}. Hyperproinsulinemia might be caused by increased demand on the β‐cells (during hyperglycemia and as a consequence of insulin resistance), increasing the release of incompletely processed granules containing proinsulin. Alternatively, it is suggested that the increased proinsulin concentration might be a result of an intrinsic β‐cell defect in type 2 diabetes[^32^](#b32){ref-type="ref"}. The improvement in proinsulin:insulin ratio with liraglutide relative to SU therapy might reflect the different modes of action of the two agents. While both enhance insulin secretion from β‐cells, liraglutide's effect appears to be glucose‐dependent, and hence predominantly a postprandial effect, whereas that of the SU appears more or less continuous, leading to increased β‐cell stress and, potentially, an increased rate of β‐cell apoptosis[^33^](#b33){ref-type="ref"}. Taken together, the results from the present report suggest that treatment with liraglutide is at least as effective in Japanese patients with type 2 diabetes as in comparable populations. Despite the fact that the insulin secretory capacity of the β‐cells in Japanese type 2 diabetes patients might be substantially more impaired than in other populations, there appears to be sufficient β‐cell mass to preserve significant capacity for a glucose‐dependent insulin secretory response to liraglutide. This could indirectly show that the decreased insulin secretion seen in this population is more a consequence of impaired β‐cell function than lost β‐cell mass. The reported improvement in parameters of β‐cell function and glucoregulation provides scope for optimism that the therapeutic effects of liraglutide could be sustainable with long‐term therapy, possibly retarding the eventual decline in β‐cell secretory function that typifies SU therapy. This remains to be established in long‐term studies. The two trials used for this analysis were part of the development program for liraglutide in Japan, and were supported by Novo Nordisk Pharma Limited, Japan. We are grateful to Tomoyuki Nishida from Novo Nordisk Pharma Limited for assistance with statistical analysis, and Penny Butcher and Steve Banner at Watermeadow Medical, UK, for assistance with writing, which was funded by Novo Nordisk. Professor Yutaka Seino has served as a consultant/advisor and on speakers' bureaus for GlaxoSmithKline, Merck Global, Novartis, Novo Nordisk, Otsuka, Sanofi‐Aventis, Taisho and Takeda. Professor Kohei Kaku has received speaking honoraria/lecture fees from Astellas, AstraZeneca, Banyu, Daiichi‐Sankyo, Dainippon‐Sumitomo, Novartis, Novo Nordisk, Sanofi‐Aventis, Sanwa and Takeda, served as a consultant/advisor to Novo Nordisk, and received research funding from Astellas, Banyu, Daiichi‐Sankyo, Novo Nordisk, Sanofi‐Aventis and Takeda. Mads Frederik Rasmussen and Per Clauson are employees of and hold shares in Novo Nordisk.

All relevant data are within the paper and its Supporting Information files. Introduction {#sec001} ============ There is a growing interest in the patterns and processes associated with geographical variation in population genetic structure across species' ranges since these often shift, expand and contract over time \[[@pone.0117113.ref001]--[@pone.0117113.ref004]\]. Historical and contemporary changes to population size and gene flow influence genetic diversity and population differentiation \[[@pone.0117113.ref003],[@pone.0117113.ref005]\]. These changes are particularly important in populations at geographical range limits, since these populations experience more rapid cycles of extinction, recolonization (with the associated founder events), severe population bottlenecks and asymmetric gene flow \[[@pone.0117113.ref003]\]. As a consequence, marginal populations tend to show greater than expected isolation by distance and have lower genetic diversity than populations located within the species' range \[[@pone.0117113.ref003]\]. They are therefore often of particular conservation interest \[[@pone.0117113.ref006],[@pone.0117113.ref007]\]. To complicate matters, it is increasingly recognized that isolated populations at the edge of species' distributions might not have dispersed, or become isolated, naturally but instead might have been assisted by humans. This has the potential to result in genetic admixture when animals are introduced from multiple source populations. As a consequence of human-mediated dispersal and resulting admixture, marginal populations might actually show higher genetic diversity than geographically more central populations \[[@pone.0117113.ref008],[@pone.0117113.ref009]\]. Therefore, it is important to establish the origin of marginal populations to be able to assign conservation priorities. This is well exemplified by the changing status of the pool frog (*Pelophylax lessonae*) in Britain. Initially considered to be present solely as a result of human introductions the native status of pool frogs was confirmed just in time to witness its extinction \[[@pone.0117113.ref010]\]. The species is now the focus of an active reintroduction program \[[@pone.0117113.ref011]\]. The common wall lizard (*Podarcis muralis*) exhibits a wide distribution across central and southern Europe. It also occurs in peripheral populations in Northern Europe where its status as a native species is debated. For example, while populations of wall lizards are known to be non-native in England \[[@pone.0117113.ref012]\] and parts of Germany \[[@pone.0117113.ref013]\], some isolated populations at the northern range limit in France, the Netherlands, and in Eastern Europe are of uncertain origin \[[@pone.0117113.ref014]\]. Of particular interest are populations on islands in the Golfe Normand-Breton, which were previously part of the French continental landmass and have been separated following climate and sea level changes about 7,000 BP \[[@pone.0117113.ref015],[@pone.0117113.ref016]\]. Jersey, the largest of Channel Islands (11,630ha) \[[@pone.0117113.ref017]\] and the Chausey archipelago (a group of islands, totaling 59ha) are now 25.5 and 17 km west of Normandy Coast, respectively \[[@pone.0117113.ref017],[@pone.0117113.ref018]\]. The presence and distribution of wall lizards on Jersey has been described by a number of authors \[[@pone.0117113.ref019]--[@pone.0117113.ref021]\] and it has been widely assumed that *P*. *muralis* is native to these islands. However, the species distribution on Jersey is noticeably patchy and restricted to old walls and ramparts on the north-eastern and eastern coast of the island \[[@pone.0117113.ref022]\], which suggests that they could have been introduced following the construction of the forts. Indeed, a population on the south east coastline of Jersey, cut off from the rest of the Island at high tide, is known to be a more recent introduction, although the origin of those animals is unknown \[[@pone.0117113.ref023]\]. The origin and genetic diversity of populations of *P*. *muralis* on the Channel Islands is of much interest as they are currently considered threatened and enjoy full protection status, despite that its present distribution is indicative of more recent introductions. Natural colonization of islands could have occurred from southern refugia, following climatic warming at the end of the Pleistocene and before the rising sea level, followed by separation from the mainland. Alternatively, colonization could have occurred subsequent to island isolation via rafting or the quarrying of granite. The aim of this study was to infer the origin of *P*. *muralis* populations on Jersey and Chausey Island and investigate the population genetic structure and diversity in relation to mainland populations. Based on our results we discuss conservation implications for these peripheral populations. Materials and Methods {#sec002} ===================== Study species {#sec003} ------------- The European wall lizard, *Podarcis muralis* (Laurenti, 1768) has a wide distribution in central and southern Europe \[[@pone.0117113.ref024]\] and shows a strong phylogeographic structure with several genetically and geographically distinct clades \[[@pone.0117113.ref025],[@pone.0117113.ref026]\]. This genetic structure is likely to have originated during isolation in southern glacial refugia in Italy on the Apennine Peninsula \[[@pone.0117113.ref025]\], the Balkans and on the Iberian Peninsula \[[@pone.0117113.ref024],[@pone.0117113.ref026]\]. The postglacial recolonization of western Europe expands to the northwest along the French coast of the English Channel, across southern Belgium and southernmost Netherlands towards south-western Germany \[[@pone.0117113.ref024]\]. Sampling, sequencing and genotyping {#sec004} ----------------------------------- We sampled 484 individuals from 21 populations between 2008 and 2013 (see [Table 1](#pone.0117113.t001){ref-type="table"} and [Fig. 1](#pone.0117113.g001){ref-type="fig"} in results section). We sampled lizards from all four locations on Jersey (St. Aubin Fort, Mont Orgueil Castle and Gorey, L'Etacquerel Fort and Fort Leicester, see Table C in [S1 File](#pone.0117113.s001){ref-type="supplementary-material"} for more information), from the Chausey archipelago (where the lizard is more widespread, see Table C in [S1 File](#pone.0117113.s001){ref-type="supplementary-material"} for more information) and from 19 populations in France (see Table C in [S1 File](#pone.0117113.s001){ref-type="supplementary-material"} for more information). We focused on mainland populations at the northwestern margin of the species distribution, i.e., close to the Channel Islands, but also included a number of populations in south-western France to compare the observed divergence between island populations with divergence across the entire western France lineage. {ref-type="table"}). (B) Parsimonious phylogenetic network reconstructed from 13 unique haplotypes sampled in our populations using a median-joining algorithm.](pone.0117113.g001){#pone.0117113.g001} 10.1371/journal.pone.0117113.t001 ###### Results from mtDNA and microsatellite analyses. {#pone.0117113.t001g} Region Population Code Latitude (^o^N) Longitude(^o^E) N~I~ [\*](#t001fn001){ref-type="table-fn"} N~H~ Haplotype[\*\*](#t001fn003){ref-type="table-fn"} A~R~ H~O~ (s.d) H~E~ (s.d) F~IS~ [~\*\*\*~](#t001fn003){ref-type="table-fn"} ---------------------- ----------------- ----------- ----------------- ----------------- -------------------------------------------- ------------ -------------------------------------------------- --------------- --------------- ------------ --------------------------------------------------- Jersey St. Aubin Fort AF 49.18712 -2.17103 15(15) 1 JER-H2(15) 2.12 0.237 0.409 **0.452** (0.071) (0.063) L'Etacquerel Fort EF 49.238267 -2.06698 17(17) 2 JER-H1(2) 2.14 0.313 0.401 **0.255** JER-H3(15) (0.077) (0.075) Fort Leicester LF 49.240243 -2.08162 14(14) 1 JER-H3(14) 2.86 0.375 0.532 **0.35** (0.104) (0.079) Mount Orgueil Castle OF 49.198904 -2.02013 34(34) 1 JER-H3(35) 2.71 0.403 0.552 **0.291** (0.1) (0.082) Chausey Archipelago Iles de Chausey CH 48.87425 -1.83016 31(34) 3 JER-H3(30) 3.21 0.547 0.613 0.144 CHA-H1(3) (0.104) (0.084) WFR-H5(1) France Cap Frehel CF 48.66451 -2.32066 12(11) 3 WFR-H1(6) 2.92 0.508 0.558 0.134 WFR-H6(3) (0.115) (0.101) WFR-H9(2) Chateau du Guildo CG 48.574464 -2.20691 25(5) 1 WFR-H5(5) 3.05 0.528 0.609 **0.155** (0.092) (0.091) Dinan DN 48.454352 -2.04734 25(5) 1 WFR-H5(5) 3.35 0.630 0.646 0.045 (0.049) (0.046) Sees SE 48.605425 0.172979 24(5) 1 WFR-H5(5) 2.46 0.451 0.480 0.085 (0.078) (0.078) Vitre VR 48.124012 -1.2144 20(5) 1 WFR-H5(5) 3.52 0.590 0.632 0.092 (0.079) (0.081) Josselin JO 47.953899 -2.54648 25(5) 2 WFR-H5(3) 3.63 0.589 0.634 0.091 WFR-H7(2) (0.086) (0.083) Pontchateau PC 47.436895 -2.08903 25(5) 1 WFR-H5(5) 3.76 0.513 (0.091) 0.550 (0.093) 0.088 Puybelliard PU 46.706436 -1.02946 22(5) 1 WFR-H5(5) 3.61 0.662 0.699 0.079 (0.079) (0.081) Pouzagues PZ 46.78435 -0.83917 25(5) 1 WFR-H5(5) 3.80 0.694 0.718 0.054 (0.076) (0.080) Saint Gervais GE 46.902738 -1.99874 25(5) 1 WFR-H5(5) 3.66 0.659 0.706 0.088 (0.085) (0.084) Bastide BA 42.939334 1.055994 25(5) 2 WFR-H3(2) 3.26 0.564 0.635 0.041 WFR-H8(3) (0.109) (0.095) Saint Michel MI 46.353210 -1.25172 25(5) 1 WFR-H5(5) 3.56 0.686 0.699 0.151 (0.086) (0.082) Saint Lizier LI 43.003259 1.138791 20(5) 2 WFR-H2(4) 3.8 0.704 0.708 0.031 WFR-H5(1) (0.085) (0.087) Saint Girons SG 42.982243 1.146273 25(5) 2 WFR-H2(4) 3.66 0.639 0.707 **0.12** WFR-H3(1) (0.108) (0.074) Nebias NE 42.896786 2.11586 25(5) 3 WFR-H5(3) 3.77 0.625 0.718 **0.15** WFR-H2(1) (0.079) (0.079) WFR-H4(1) Fontiers Cabardes FC 43.369587 2.248493 25(5) 3 WFR-H4(1) 3.45 0.610 0.650 0.087 WFR-H5(3) (0.105) (0.098) WFR-H2(1) \* Number of individuals used in microsatellite analysis and in parenthesis the number of individuals used in mtDNA analysis. \*\* Number of individuals sharing the same haplotype is shown in parenthesis \*\*\* Values in bold indicate significant deviation from Hardy-Weinberg equilibrium after correcting for multiple tests at the nominal level (5%), *p\>0*.*00024*. N~I~ (number of individuals), N~H~ (number of haplotypes), A~R~ (allelic richness), H~O~ (observed heterozygosity), H~E~ (expected heterozygosity) and F~IS~ (inbreeding coefficient). Ethics Information {#sec005} ------------------ Lizards were captured by noosing, and a small (ca 5mm) part of the tail was removed by inducing tail release with a pair of tweezers or, when the tail was regrown, using surgical scissors to provide tissue for genetic analysis. All lizards were released at the site of capture following sampling. The research was approved by the UK Home Office Ethical License PPL30/56 and all work and procedures during fieldwork were carried out under annual licenses and permits from the States of Jersey Government (Department of the Environment) and the French Government (Direction Régionale de l'Environnement, de l'Aménagement et du Logement). DNA extraction, sequencing and genotyping {#sec006} ----------------------------------------- We extracted genomic DNA from tail tissue preserved in ethanol (70--90%) with DNeasy 96 plate kit (Qiagen, Valencia, CA) following manufacturer's instructions (with overnight lysis). For the phylogenetic analysis we amplified a 656bp region of mitochondrion cytochrome b gene by polymerase chain reaction (PCR) using the primer pair LGlulk \[5′-AACCGCCTGTTGTCTTCAACTA-3′\] and Hpod \[3′-GGTGGAATGGGATTTTGTCTG-5′\] \[[@pone.0117113.ref012],[@pone.0117113.ref026]--[@pone.0117113.ref028]\]. Amplifications were carried out in a total volume of 15μl consisting of 7.5μl of MyTaq HS Mix (Bioline), 0.45μl (8pm) of each primer (Eurofins), 4.6μl PCR grade H~2~O and 2μl template DNA. PCR conditions were as follows: an initial denaturation step at 94^o^C for 1 min, followed by 35 cycles at 94^o^C for 1 min, 53^o^C for 45sec and 72^o^C for 1 min and a final extension step at 72^o^C for 10min. PCR products were purified using the MinElute 96 UF PCR Purification Kit (Qiagen, Valencia, CA). Sequencing reactions were carried out with BIGDye Terminator v3.1 Ready Reaction kit (Applied Biosystems, Warrington, UK) in both directions. Products were precipitated in isopropanol and analysed on an ABI 3130 automated capillary sequencer (Applied Biosystems, Warrington, UK). Mitochondrial DNA sequences from both directions were corrected by eye and aligned to obtain a consensus sequence. Accepted sequences were then aligned using MAFFT \[[@pone.0117113.ref029]\] implemented in [Geneious 6.1.7]{.smallcaps} \[[@pone.0117113.ref030]\] and trimmed into a uniform length of 656 base pairs (bp). We translated the sequenced *cyt-b* region to amino acid sequences, to verify that no premature stop codons disrupted the reading frame. Unique sequences were submitted to GenBank under the accession numbers KP118978-KP118990. To infer the genetic structure and diversity of our populations we genotyped 484 individuals at 10 polymorphic microsatellite loci; four described by Richard *et al*. \[[@pone.0117113.ref031]\] and six recently developed by Heathcote *et al*.\[[@pone.0117113.ref032]\] (Table A in [S1 File](#pone.0117113.s001){ref-type="supplementary-material"}). Multiplexed PCRs were carried out in a total volume of 11μl reaction mix containing 1μl of genomic DNA, 5μl of Qiagen MasterMix, 0.2μl of each primer (forward and reverse in equal concentrations) and 3.8μl (for multiplex 1 and 2) or 3.6μl (for multiplex 3) of PCR grade dH~2~O. PCR conditions were as follows: 15min of initialization step at 95^o^C, 26 cycles of 30sec at 94 ^o^C, 90sec at 57 ^o^C (for multiplex 1 and 2) or 55 ^o^C (for multiplex 3) and 1min at 72^o^C and a final extension step of 20min at 60^o^C. The 5'-end of each forward primer was labeled with a fluorescent dye either 6-FAM, HEX or NED. PCR products were run with an internal ladder (red ROX-500), on an ABI 3130 genetic analyser (Applied Biosystems Inc.) We scored alleles in [Geneious]{.smallcaps} 6.1.7 and any ambiguous peaks were repeated to confirm genotype. Phylogenetic analyses {#sec007} --------------------- We used the phylogenetic tree approach to assign haplotypes to known lineages by combining our sequences with 68 sequences (of varying lengths), obtained from GenBank, across the native distribution of the species (see Table B in [S1 File](#pone.0117113.s001){ref-type="supplementary-material"} \[[@pone.0117113.ref013],[@pone.0117113.ref025],[@pone.0117113.ref026],[@pone.0117113.ref033]--[@pone.0117113.ref036]\]). Three sequences belonging to *P*. *siculus* (AY185095) \[[@pone.0117113.ref037]\], *P*. *liolepis* (JQ403296) \[[@pone.0117113.ref038]\] and *P*. *melisellensis* (AY185097) \[[@pone.0117113.ref037]\] were used as outgroups in the phylogenetic analysis using Bayesian Inference (BI). We implemented BI analyses in [MrBayes]{.smallcaps} \[[@pone.0117113.ref039]\] under the GTR+G+I nucleotide substitution model as selected by the best-fit model applying the Akaike Information criterion (AIC) in [Mega 5.2 \[]{.smallcaps} [@pone.0117113.ref040] [\]]{.smallcaps}. The BI analysis was run with four chains of 1,000,000 generations and sampling every 100 trees. We discarded (burn-in-length) the first 10% of the trees after checking for convergence of the chains and the posterior probability branch support was estimated from the 50% majority-rule consensus tree. To investigate evolutionary relationships of our sequences, we constructed a parsimonious phylogenetic network using a median---joining algorithm in Network v.4.6.12 \[[@pone.0117113.ref041]\]. The method uses median vectors as a hypothetical ancestral sequence required to connect existing sequences within the network with maximum parsimony. Population genetics analyses {#sec008} ---------------------------- We checked the microsatellite data in M[icrochecker]{.smallcaps} V.2.2.3 \[[@pone.0117113.ref042]\] for null-alleles, large allele dropouts and scoring errors. Basic genetic diversity indices, observed and expected heterozygosities (H~O~, H~E~) were calculated with [Genalex]{.smallcaps} 6.5 \[[@pone.0117113.ref043]\] and allelic richness (A~R~) with F[stat]{.smallcaps} v.2.9.3 \[[@pone.0117113.ref044],[@pone.0117113.ref045]\]. Inbreeding coefficient (F~IS~) and deviations from Hardy-Weinberg equilibrium were also evaluated at the 0.05 nominal level for multiple tests using sequential Bonferroni corrections in F[stat]{.smallcaps} v.2.9.3 \[[@pone.0117113.ref044],[@pone.0117113.ref045]\]. We compared H~O~, H~E~, A~R~ in island versus mainland populations with a Welch Two Sample t-test and evaluated the correlation between expected heterozygosity and latitude with a Spearman's rank correlation test in R \[[@pone.0117113.ref046]\]. To infer population structure, we implemented a Bayesian analysis in [Structure]{.smallcaps} v.2.3.4 \[[@pone.0117113.ref047]\] using the admixture model \[[@pone.0117113.ref048]\]. The simulations were run with a burn-in of 100,000 iterations and a run length of 10^6^ iterations from K = 1 through 5. Runs for each K were replicated 10 times and the true K was determined according to the method described by Evanno *et al* \[[@pone.0117113.ref049]\] in the online software [Structure Harvester]{.smallcaps} v.0.6.93 \[[@pone.0117113.ref050]\]. We tested the level of genetic diversity within populations, among populations and among groups (as defined by the structure clustering analysis) by hierarchical analysis of molecular variance (AMOVA, \[[@pone.0117113.ref051]\]) in [Arlequin]{.smallcaps} 3.5.1.3 \[[@pone.0117113.ref052]\]. Population differentiation was assessed by calculating the F~ST~ values and visualized with a Principle Coordinate Analysis (PCoA) in [Genalex]{.smallcaps} 6.5 \[[@pone.0117113.ref043]\]. Results {#sec009} ======= Phylogeography {#sec010} -------------- Analysis of mtDNA sequences of 192 individuals revealed 13 unique haplotypes all nested within the Western France Clade ([Fig. 2](#pone.0117113.g002){ref-type="fig"}). The most common haplotype on the mainland (France) was WFR-H5, which was also present on Chausey (one individual) but not on Jersey ([Fig. 1A](#pone.0117113.g001){ref-type="fig"}). The parsimony network showed that WFR-H5 has a central position among French haplotypes and JER-H3 forms the centre of the cluster of Jersey and Chausey haplotypes, which are distinct from the rest of the mainland populations ([Fig. 1B](#pone.0117113.g001){ref-type="fig"}). {ref-type="supplementary-material"}.](pone.0117113.g002){#pone.0117113.g002} Population genetics {#sec011} ------------------- All 484 individuals were genotyped at 10 polymorphic loci, ranging from 10 to 56 alleles with mean number of 20.3 alleles per locus across all populations. Evidence of null alleles was observed in several loci but none were consistent across all populations, therefore we did not exclude them for further analysis (Table D in [S1 File](#pone.0117113.s001){ref-type="supplementary-material"}). Allelic richness, expected and observed heterozygosities ([Table 1](#pone.0117113.t001){ref-type="table"}) were all significantly lower (*p\<0*.*05*) in the island populations of Jersey and Chausey than in mainland France populations (Figure B in [S1 File](#pone.0117113.s001){ref-type="supplementary-material"}). There was a significant negative correlation (r = -0.84, *p \<0*.*05*) between latitude and expected heterozygosity ([Fig. 3](#pone.0117113.g003){ref-type="fig"}). {#pone.0117113.g003} The Bayesian clustering approach implemented in STRUCTURE suggested *K* = 3 best-fit the genetic data ([Fig. 4](#pone.0117113.g004){ref-type="fig"}, see also Figure A in [S1 File](#pone.0117113.s001){ref-type="supplementary-material"}). The Principle Coordinate Analysis (PCoA) based on F~ST~ values (see Table E in [S1 File](#pone.0117113.s001){ref-type="supplementary-material"}) between populations confirmed the results from STRUCTURE, identifying three clear groups corresponding to the samples from the Islands, North Western France and South Western France ([Fig. 5](#pone.0117113.g005){ref-type="fig"}). Analysis of Molecular Variance (AMOVA) revealed that 28% of the genetic variation was found among the three groups (clusters) and 50% was found within individuals ([Table 2](#pone.0117113.t002){ref-type="table"}). {ref-type="table"}.](pone.0117113.g004){#pone.0117113.g004} {#pone.0117113.g005} 10.1371/journal.pone.0117113.t002 ###### Hierarchical analysis of molecular variance (AMOVA) {#pone.0117113.t002g} Source of Variation d.f Sum of squares Variance components Percentage of variation Fixation indices *p* value ------------------------------------------ ----- ---------------- --------------------- ------------------------- ------------------ ----------- **Among groups** 2 686959.219 11893.80493 27.8 F~IS~ = 0.21047 \<0.05 **Among populations within groups** 18 342716.546 346.10698 8.09 F~SC~ = 0.11199 \<0.05 **Among individuals within populations** 460 1528192.078 577.644 13.5 F~CT~ = 0.27796 \<0.05 **Within individuals** 481 1042263.5 2166.86798 50.62 F~IT~ = 0.49377 \<0.05 **Total** 961 3600131.344 4280.42425 Discussion {#sec012} ========== Our data provides strong evidence that the wall lizard populations on the islands in the English Channel belong to a single origin. Furthermore, the analyses suggest that this mtDNA clade has been isolated from the mainland for a long period of time and should be considered native. The most parsimonious explanation for the origin of the common wall lizard on Jersey and Chausey Islands appears to be that the increasing sea levels 7000 BP isolated island populations from the mainland and from each other, resulting in independent population histories and hence divergence. It remains possible, however, that there is occasional gene flow between islands. For example, the presence of lizards on very small islets in the Chausey archipelago \[[@pone.0117113.ref018]\], which are unlikely to be large enough to sustain populations for thousands of years, might indicate that dispersal occasionally occurs between islands. In addition, the presence of the WFR-H5 haplotype on the island of Chausey, which is the most common haplotype on the mainland, might also provide evidence of occasional gene flow between mainland France and the islands. However, it could also be explained by retention of ancestral genetic variation or a more recent introduction. It is worth noting that a single isolated population on the coast of mainland France (Cap Frehel, CF; [Fig. 1](#pone.0117113.g001){ref-type="fig"}) also exhibits unique haplotypes, nevertheless it clusters with other mainland populations in all analyses. Anecdotal evidence suggested that human mediated dispersal might be the most likely explanation for one of the four current locations in Jersey, the population on St. Aubin Fort \[[@pone.0117113.ref023]\]. Although our mtDNA data revealed a different haplotype from other Jersey populations, the nucDNA clusters all Jersey populations together. This suggests that the source population was most likely animals from other Jersey populations and that the difference in haplotype represents a founder effect. Overall, these results confirm the suspected native status of Jersey and Chausey wall lizards. Thus, the lower genetic diversity of island populations compared to the mainland populations is expected given the lack of gene flow. This might have significant implication for the long-term persistence of the species on Jersey and Chausey Islands. However, since our data suggests that the species have been present on the islands for thousands of years it might have already been subject to a severe bottleneck that purged deleterious recessives \[[@pone.0117113.ref053]\]. The species might also have undergone a substantial reduction in abundance more recently. Historical references to the species on Chausey, dated in 1842 \[[@pone.0117113.ref054]\], and subsequently work recorded the species as very common \[[@pone.0117113.ref055]--[@pone.0117113.ref058]\]. Despite this, the current distribution of the species on Jersey is very restricted \[[@pone.0117113.ref023]\]. One partial explanation for this is that lizards on Jersey were part of a wider pet trade, with lizards being sent from Jersey to England as far back as 1761\[[@pone.0117113.ref021]\]. Indeed, by 1947 the pet trade in lizards had reached such proportions that the local government (States of Jersey) passed the Wildlife Protection (Jersey) Law 1947, which prohibited the buying, selling, exportation or killing of all reptiles and amphibians of Jersey, as a measure to control the increased trade for these animals as pets destined for England (however, none of the contemporary non-native populations in England originate from Jersey \[[@pone.0117113.ref012]\]). Not only might this explain the current patchy distribution of lizards on Jersey, it might also have contributed to their relatively low genetic variation. Geographically peripheral populations are often representatives of relatively widespread species within different political boundaries \[[@pone.0117113.ref059]\]. Their conservation value depends upon their genetic divergence from other conspecific populations because of the synergetic effects of isolation, genetic drift, and natural selection. Whether these range-edge populations merit the conservation effort that they are often subject to has been widely debated \[[@pone.0117113.ref006],[@pone.0117113.ref060],[@pone.0117113.ref061]\]. As this study clarified the native status of the wall lizard population on Jersey, it validates its current full protection status under the Conservation of Wildlife (Jersey) Law 2000 (as amended). The law prohibits the unlicensed taking, sale, keeping, injury and destruction of places for shelter (e.g. nest, dens or burrows) and disturbance of any resident animals. Given our results, it is important that Jersey conservation planners recognize the wall lizard's restricted distribution, vulnerability to future inbreeding depression, susceptibility to disease, predation and the island's ever-increasing urban development when developing species management strategies. For instance, should the granite walls and ramparts of historic fortresses where they are in highest abundance be developed or destroyed, the population's continued survival could be placed at risk. The lizard's long-term conservation status will depend upon increasing habitat connectivity, especially via coastline protection to connect their north-eastern and eastern coast populations on the island. Supporting Information {#sec013} ====================== ###### Table A, Details for the ten loci used in the study. Multiplexes one (1) and two (2) were developed by Heathcote *et al*. (2014) and multiplex three (3) was developed by Richard *et al*. (2012). Table B, List of sequence data used in the phylogenetic analysis. Information on sampling location, GenBank accession numbers and the reference study. Table C, Historical information on the island populations of the wall lizard. Table D, Table of null alleles per population per locus. Bold values indicated significant deviation from Hardy-Weinberg equilibrium (p\<0.05). Table E, Matrix of pairwise *F* ~*ST*~ values. Figure A, Plot of Delta K (ΔΚ). Calculated as in Evanno *et al*. (2005) from K = 2 to K = 4. Highest Delta K for *K* = 3. Figure B, Plots of genetic diversity indexes between island (group 1) and mainland populations (group 2). Genetic diversity is expressed as H~O~, H~E~ and A~R~. Differences in the mean numbers were compared with a Welch Two Sample t-test. (DOCX) ###### Click here for additional data file. We would like to thank Hannah MacGregor for assisting in the field. We also thank Tod Reeder and one anonymous reviewer for useful comments that improved the manuscript. [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: NC RG JG TU. Performed the experiments: SM NZ. Analyzed the data: SM. Contributed reagents/materials/analysis tools: TU RG JG. Wrote the paper: SM NC RG JG NZ GJW FA GMW TU. Sample and data collection: SM NC NZ GJW FA GMW TU. [^3]: ‡ SM and NC are joint first authors on this work.

INTRODUCTION {#s1} ============ Invasive cervical cancer (ICC) is the fourth most common malignancy among women worldwide, with an estimated 528,000 new cases and 266,000 deaths in 2012 and with 87% of cervical cancer deaths occurring in less developed regions \[[@R1]\]. Among Mexican women, ICC is the second most common neoplasia, just after breast cancer \[[@R2]\]. Infection with human papillomaviruses (HPVs) has been well documented as the main etiological factor for ICC since the viral genome is present in practically all cervical cancer tumors \[[@R3]\]. To date, 13 HPV genotypes have been defined as carcinogenic or high-risk (HR) viral types for cervical cancer \[[@R4]\]. The HPV viral oncoproteins E5, E6 and E7 can disrupt several host signaling pathways; for example, E6 can deregulate p53 and PDZ proteins and enhance the activation of cellular pathways such as PI3K, Wnt and Notch \[[@R5]\]. The Notch pathway is a highly conserved signaling system that plays a key role in cell differentiation, survival and proliferation \[[@R6]\]. In the canonical Notch pathway, a transmembrane Notch receptor (NOTCH 1--4) interacts with Delta-Serrate-Lag-type (Dll1, Dll3, Dll4, Jagged1 or Jagged2) ligands. This interaction triggers the sequential proteolytic cleavage of the Notch receptor, releasing the intracellular domain (NICD), which translocates to the nucleus and activates the transcription of target genes, including *Hes1, Hes5*, *Hey1, Cyclin D1* and *Myc* \[[@R7]--[@R9]\]. The participation of Notch signaling in cervical cancer remains controversial since both tumor suppressive \[[@R10]--[@R12]\] and oncogenic properties \[[@R13], [@R14]\] have been described. Talora *et al.* (2002) showed a lack of NOTCH1 expression in ICC samples and in cervical cell lines \[[@R11]\]. In contrast, Zagouras *et al.* (1995) and Yousif *et al.* (2015) found an increase in NOTCH1 expression throughout cervical cancer progression \[[@R14], [@R15]\]. Moreover, Jagged-1 and Delta-1 ligands have been reported as overexpressed in ICC and in cervical adenocarcinoma \[[@R16]\]. Several reports have suggested that NUMB is a negative regulator of NOTCH1 signaling \[[@R7]--[@R9]\]. The interaction of NUMB with NOTCH1 may result in increased NOTCH1 ubiquitination \[[@R17]\]. NUMB may also act as a scaffold for the E3 ligases Itch and Suppressor of Deltex Su(Dx) \[[@R7], [@R18], [@R19]\] and cooperates with α-adaptin (part of the endocytic AP2 complex), thereby promoting NOTCH1 endocytosis \[[@R8], [@R20]\]. In breast cancer, NUMB has been defined as a tumor suppressor protein \[[@R21]--[@R23]\]; nevertheless, its role in ICC is not clear. Chen *et al.* (2009) reported NUMB overexpression in cervical malignant lesions compared with normal epithelia, suggesting a role for NUMB in cervical cancer progression \[[@R24]\]. However, the relationship between NOTCH1 and NUMB in ICC is not clear. To understand the role of NOTCH1 and its negative regulator NUMB in cervical cancer, we investigated the expression and localization of NOTCH1 and NUMB in samples from 144 patients with cervical intraepithelial neoplasia (CIN) and ICC obtained from the Instituto Nacional de Cancerología-México from 2004 to 2017 using immunohistochemistry and determined their role as predictors of malignancy in ICC. RESULTS {#s2} ======= Characteristics of patients {#s2_1} --------------------------- We recruited cases with available paraffin-embedded samples from women diagnosed with CIN or ICC at the Instituto Nacional de Cancerologia, Mexico from April 2004 to January 2017. A total of 49 CIN and 95 ICC paraffin-embedded tissue samples and their clinical data were collected. The demographic and clinical characteristics of the patients are shown in Table [1](#T1){ref-type="table"}. Most patients were older than 30 years old in both groups, corresponding to 69.39% in the CIN group and 97.89% in the ICC group (*P* = 0.001). Smoking (10.20% vs. 11.57%) and alcohol consumption (0.00% vs. 4.21%) were not different among groups. A higher frequency of obesity was observed in CIN patients than in ICC patients (57.70% vs. 26.58%, *P* = 0.01). Additionally, hormone contraception usage was more common in the CIN group (58.62%) than in the ICC group (32.50%, *P* = 0.01). The number of previous sexual partners and the type of HPV were not different among groups. HPV types 16 and 18 were the most common in both groups (CIN vs. ICC: 63.15% vs. 51.06% for type 16; and 15.80% vs. 14.90% for type 18). ###### Demographic and clinical characteristics of patients (*n* = 144) with CIN and ICC treated at the Instituto Nacional de Cancerología-México from 2004 to 2017 Variable CIN (*n* = 49) ICC (*n* = 95) *P*-value ------------------------------------ ---------------- ---------------- ----------- ---------- ------------- Age  \<30 15 (30.61%) 2 (2.11%) **\<0.001**  ≥30 34 (69.39%) 93 (97.89%) Smoking status^a^  Smokers 5 (10.20%) 11 (11.57%) 1.00  Non-smokers 44 (89.80%) 84 (88.43%) Alcohol consumption^b^  Positive 0 (0.00%) 4 (4.21%) 0.36  Negative 49 (100%) 91 (95.79%) Body mass index, kg/m^2^  Normal (18.5--24.9) 4 (15.38%) 31 (39.24%) **0.01**  Overweigth (25--29.9) 7 (26.92%) 27 (34.18%)  Obesity (≥30) 15 (57.70%) 21 (26.58%)  Unknown^c^ 23 16 Hormone contraception usage  Yes 17 (58.62) 13 (32.50%)  No 12 (41.38) 27 (67.50%) 0.05  Unknown^c^ 20 55 Number of previous sexual partners  1 16 (38.10%) 22 (64.71%) 0.61  ≥2 26 (61.90%) 12 (35.29%)  Unknown^c^ 7 61 Human Papillomavirus (HPV)  16 12 (63.15%) 48 (51.06%)  18 3 (15.80%) 14 (14.90%) 0.49  Others^d^ 4 (21.05%) 32 (34.04%)  Unknown^c^ 30 1 e CIN: Cervical intraepithelial neoplasia. ICC: Invasive cervical cancer ^a^Smoking was defined as any tobacco consumption during their lifetime. ^b^Alcohol consumption was defined as any alcohol intake per week. ^c^Data not reported in the clinical files, and thus, this category was not included in the comparisons ^d^Other HPVs include types 6, 11, 31, 43, 42, 45 and 58. Bold: statistically significant. NOTCH1 and NUMB expression and localization in CIN and ICC {#s2_2} ---------------------------------------------------------- Normal cervical epithelium was used as positive control for NOTCH1 and NUMB expression in immunohistochemical analysis. Besides, confirmation of immunohistochemical results was done in a representative set of ICC samples with an alternative NOTCH1 antibody, obtaining 90% of concordance between antibodies, which confirms that the antibody used in this study is relievable ([Supplementary Figure 1](#SD1){ref-type="supplementary-material"} and [Supplementary Table 3](#SD1){ref-type="supplementary-material"}). Accordingly to the protein Atlas database, the immunostaining of NOTCH1 in normal squamous cells is moderate with a homogenous distribution in the cell \[[@R25]\], similar to what we observed in normal epithelium (Figure [1A](#F1){ref-type="fig"}). Intense NOTCH1 expression was more frequently observed in CIN samples (22.45%) than in ICC samples (3.16%, *P =* 0.001) (Table [2](#T2){ref-type="table"} and Figure [1A](#F1){ref-type="fig"}). NOTCH1 nuclear staining was more frequently observed in CIN samples than in ICC samples (77.55% vs. 15.79%). Additionally, in ICC samples, NOTCH1 protein expression was mainly observed in the cytoplasm (44.21%), while no cytoplasmic case was observed in CIN (*P =* 0.001) (Table [2](#T2){ref-type="table"} and Figure [1B](#F1){ref-type="fig"}). {#F1} ###### NOTCH1 protein expression intensity and cellular localization in samples of patients (*n* = 144) with CIN and ICC treated at the Instituto Nacional de Cancerologia-Mexico from 2004 to 2017 CIN (*n* = 49) ICC (*n* = 95) *P*-value -------------- ---------------- ---------------- ----------- ---------- -------- Intensity^a^  Negative 10 (20.41%) 26 (27.37%)  Weak 28 (57.14%) 66 (69.47%) 0.001  Intense 11 (22.45%) 3 (3.16%) Localization  Negative 10 (20.41%) 26 (27.37%)  Cytoplasm 0 (0.00%) 42 (44.21%) ˂0.001  Nucleus 38 (77.55%) 15 (15.79%)  Cyto/nuc^b^ 1 (2.04%) 12 (12.63%) CIN: Cervical intraepithelial neoplasia. ICC: Invasive cervical cancer. ^a^Intensity was categorized as weak = + and ++; and intense = +++ by immunohistochemistry. ^b^Presence of the protein in the cytoplasm and nucleus. Bold: statistically significant. NOTCH1 protein expression was evaluated by Western Blot in eight representatives ICC cases (Figure [2A](#F2){ref-type="fig"}). The densitometric analysis showed that immunostaining intensity correlates with protein expression (Figure [2B](#F2){ref-type="fig"}). Relative NOTCH1 protein expression is higher in intense immunostained cases (samples 1 and 6), than in weak cases (samples 2, 3 and 4 and 7). {#F2} For the NUMB protein, the normal epithelium showed weak cytoplasmic immunoreactivity (Figure [3A](#F3){ref-type="fig"}). Negative NUMB protein expression was more frequently observed in CIN samples than in ICC samples (65.31% vs. 42.11%, *P =* 0.014). Additionally, observations of the highest intensity for NUMB immunoreaction were more frequent in ICC than in CIN samples (0.00% in CIN vs. 6.31% in ICC) (Table [3](#T3){ref-type="table"} and Figure [3A](#F3){ref-type="fig"}). The NUMB protein, when present, was almost exclusively found in the nucleus in CIN samples (32.65% of nuclear NUMB in CIN vs. 2.04% of cytoplasmic NUMB), whereas in the ICC samples, its localization was heterogeneous, with a significant increase of NUMB expression in the cytoplasm (6.32% of nuclear NUMB in ICC vs. 35.79% of cytoplasmic NUMB) (Table [3](#T3){ref-type="table"} and Figure [3B](#F3){ref-type="fig"}). {#F3} ###### NUMB protein expression intensity and cellular localization in samples of patients (*n* = 144) with CIN and ICC treated at the Instituto Nacional de Cancerologia-México from 2004 to 2017 CIN (*n* = 49) ICC (*n* = 49) *P*-value -------------- ---------------- ---------------- ----------- ---------- -------- Intensity^a^  Negative 32 (65.31%) 40 (42.11%)  Weak 17 (34.69%) 49 (51.58%) 0.014  Intense 0 (0.00%) 6 (6.31%) Localization  Negative 32 (65.31%) 40 (42.11%)  Membrane 0 (0.00%) 2 (2.10%)  Cytoplasm 1 (2.04%) 34 (35.79%)  Nucleus 16 (32.65%) 6 (6.32%) ˂0.001  Mem/cyto^b^ 0 (0.00%) 8 (8.42%)  Cyto/nuc^c^ 0 (0.00%) 5 (5.26%) CIN: Cervical intraepithelial neoplasia. ICC: Invasive cervical cancer. ^a^Intensity was categorized as weak = + and ++; and intense = +++ by immunohistochemistry. ^b^Presence of the protein at the membrane and in the cytoplasm. ^c^Presence of the protein in the cytoplasm and nucleus. Bold: statistically significant. Relationship between NOTCH1 and NUMB expression {#s2_3} ----------------------------------------------- To determine if there was a relationship between the expression of NOTCH1 and NUMB (negative regulator of NOTCH1), we evaluated the expression, localization and positive tumoral area percentage of NOTCH1 and NUMB proteins in both CIN and ICC samples (Table [4](#T4){ref-type="table"}). In CIN samples, the most frequent condition was NOTCH1-positive and NUMB-negative expression (44.90% vs. 26.32% for the same condition in ICC). In contrast, in ICC samples, the most frequent condition was NOTCH1-positive and NUMB-positive expression (34.69% in CIN vs. 46.32% in ICC). We also evaluated NOTCH1 and NUMB localization and we found that the most frequent combination was nuclear NOTCH1 and nuclear NUMB in CIN samples (75.51% vs. 16.84% in ICC). The most frequent condition observed in ICC samples was cytoplasmic NOTCH1 and nuclear NUMB (22.44% in CIN vs. 47.36% for the same condition in ICC). The distribution of frequencies for both expression and localization among the CIN and ICC groups were statistically significant (*P* = 0.014 for expression; and *P* = 0.001 for localization) (Table [4](#T4){ref-type="table"}). Additionally, we evaluated the correlation between the distribution of NOTCH1 and NUMB throughout serial histological sections and found to be statistically significant in ICC but not in CIN (*r =* 0.226, *P =* 0.116 in CIN vs. *r =* 0.306, *P* = 0.002 in ICC) ([Supplementary Figures 2 and 3](#SD1){ref-type="supplementary-material"}). ###### Demographic and clinical characteristics of patients (*n* = 144) with CIN and ICC treated at the Instituto Nacional de Cancerología-México from 2004 to 2017 Variable CIN (*n* = 49) ICC (*n* = 49) *P*-value ------------------------------------------------ ---------------- ---------------- ----------- ---------- ----------- Protein expression  Negative^a^ NOTCH1 and negative^a^ NUMB 10 (20.41%) 15 (15.79%) **0.014**  Negative^a^ NOTCH1 and positive^b^ NUMB 0 (0.00%) 11 (11.58%)  Positive^b^ NOTCH1 and positive^b^ NUMB 17 (34.69%) 44 46.32%  Positive^b^ NOTCH1 and negative^a^ NUMB 22 (44.90%) 25 (26.32%) Protein localization  Nuclear^c^ NOTCH1 and nuclear^c^ NUMB 37 (75.51%) 16 (16.84%) **0.001**  Nuclear^c^ NOTCH1 and cytoplasmic^d^ NUMB 1 (2.04%) 0 (0.00%)  Cytoplasmic^d^ NOTCH1 and nuclear^c^ NUMB 11 (22.44%) 45 (47.36%)  Cytoplasmic^d^ NOTCH1 and cytoplasmic^d^ NUMB 0 (0.00%) 34 (35.78%) CIN: Cervical intraepithelial neoplasia. ICC: Invasive cervical cancer. ^a^The negative condition was defined as an absence of immunostaining in the sample. ^b^The positive condition was defined as the presence of any immunostaining in the sample. ^c^Nuclear expression was defined as any positive nuclear immunostaining. ^d^Cytoplasmic expression was defined as any positive immunostaining outside of the cell nucleus. Bold: statistically significant. Association of NOTCH1 expression with malignancy status {#s2_4} ------------------------------------------------------- Multivariable-adjusted analysis for the association between nuclear NOTCH1 expression and malignancy status showed a negative and significant association (β = --2.836, 95% CI = --3.694, --1.978, *P =* 0.001) (Table [5](#T5){ref-type="table"} and [Supplementary Table 1](#SD1){ref-type="supplementary-material"}). This association was consistent through sensitivity analyses, including age (β = --3.122, 95% CI = --4.180, --2,063, *P =* 0.001) and contraceptive consumption (β = --3.790, 95% CI = --5.459, --2.120, *P =* 0.001) as covariates ([Supplementary Table 1](#SD1){ref-type="supplementary-material"}). Moreover, the negative association of nuclear NOTCH1 with malignancy persisted even after adjustment for cytoplasmic NUMB expression (β = --3.428, 95% confidence interval \[95% CI\] = --5.127, 1.728, *P =* 0.001) (Table [5](#T5){ref-type="table"}). ###### Multivariable-adjusted model for the association between nuclear NOTCH1 expression and ICC diagnosis in patients with cervical cancer treated at the Instituto Nacional de Cancerologia-Mexico from 2004 to 2017 (*n* = 95) Variable β (95% CI) *P*-value ---------------------- --------- ---------------------- ----------- NOTCH1 expression^a^ --3.428 (--5.27, --1.728) **0.001** Age 0.092 (0.015, 0.168) **0.018** HC 0.973 (**--**0.595, 2.541) 0.223 NUMB expression^b^ 2.074 (**--**0.358, 4.506) 0.094 ^a^Nuclear NOTCH1 expression was defined as any positive nuclear immunostaining at the cell nucleus. ^b^NUMB expression was defined as any positive immunostaining at the cell cytoplasm. Age was included as a continuous variable. HC: Hormonal contraception use. β = Estimate for the association between NOTCH1 expression and patient characteristics. 95% CI = 95% confidence interval. Bold: statistically significant. Association of NUMB expression with ICC {#s2_5} --------------------------------------- We also explored the association between NUMB expression and malignancy status through sensitivity analyses (Table [6](#T6){ref-type="table"} and [Supplementary Table 2](#SD1){ref-type="supplementary-material"}). In the univariable regression model, cytoplasmic NUMB expression was associated with cervical malignancy (β = 3.28, 95% CI = 5.310, 1.262, *P* = 0.001), and this association was persistent in the multivariable model adjusted by age (β = 3.487, 95% CI = 5.548, 1.427, *P =* 0.001) and contraceptive consumption (β = 2.946, 95% CI = 0.809, 5.082, *P* = 0.007) as covariates. Remarkably, when the model was adjusted by nuclear NOTCH1 expression, the significance of the association was lost (β = 2.074, 95% CI = --0.358, 4.506, *P =* 0.094) (Table [6](#T6){ref-type="table"}). ###### Multivariable-adjusted model for the association between cytoplasmic NUMB expression and malignancy in patients with cervical cancer treated at the instituto nacional de cancerologia-mexico from 2004 to 2017 (*n*= 95) Variable β (95% CI) ---------------------- --------- ------------------ NUMB expression^a^ 2.074 (0.358, 4.506) Age 0.092 (0.015, 0.168) HC --0.973 (--2.541, 0.595) NOTCH1 expression^b^ --3.428 (--5.525, 1.728) ^a^NUMB expression was defined as any positive immunostaining in the cell cytoplasm ^b^NOTCH1 expression was defined as any positive immunostaining in the cell nucleus. Age was included as a continuous variable. HC: Hormonal contraception use. β = Estimate for the association between NUMB expression and patient characteristics. 95% CI = 95% confidence interval. Bold: statistically significant. Effect of NOTCH1 expression on prognosis {#s2_6} ---------------------------------------- To determine the potential effect of NOTCH1 on cervical cancer prognosis, we explored the association of NOTCH1 expression with overall survival (OS) in patients with malignant lesions. Patients with cytoplasmic NOTCH1 expression showed a longer OS than those with nuclear NOTCH1 expression, but it was only a borderline association (*P* = 0.08) (Figure [4](#F4){ref-type="fig"}). {#F4} DISCUSSION {#s3} ========== In the present study, we showed that ICC samples exhibited lower NOTCH1 expression than CIN samples and that this differential expression is also related to higher NUMB expression. We also showed that nuclear NOTCH1 expression is negatively associated with malignancy independent of known risk factors for ICC, including age and use of hormonal contraceptives as well as NUMB expression. In contrast, the association of NUMB with malignancy was not independent; it relied on NOTCH1 expression. Based on these findings, we conclude that the loss of nuclear NOTCH1 might be a key factor involved in cervical carcinogenesis. To our knowledge, this is the first study to clarify the expression and localization of NOTCH1 and NUMB in cervical cancer. It is well known that a persistent infection with high-risk HPV is an etiological factor for cervical cancer \[[@R26]\]. All our samples were positive for HPV infection according to molecular examination, and the main HPV types were 16 and 18, as expected for the Mexican population, with no differences between groups \[[@R27]\]. The HPV type was not related to the expression of either NOTCH1 or NUMB. Concomitant factors for HPV infection and CIN development include: age, menarche, parity, age of first intercourse, number of sexual partners, use of hormonal contraceptives, body mass index, smoking and alcohol consumption \[[@R28]\]. Accordingly, we found that women in the cancer group were older than those in the CIN group. This finding is expected since ICC has a peak age incidence around the fourth decade of life \[[@R29]\]. Alterations in NOTCH signaling have been associated with tumorigenesis, but its activity is dissimilar among cancer types \[[@R22], [@R30]--[@R32]\]. In ICC, the role of the NOTCH pathway is controversial \[[@R10], [@R12], [@R13]\]. Some authors have proposed that NOTCH1 expression levels are stage-specific: 1) in early lesions, NOTCH1 expression is upregulated; and 2) in malignant lesions, NOTCH1 expression is downregulated \[[@R33]\]. In our samples, the distribution of positive cases was not different between groups, but NOTCH1 immunostaining was weaker in malignant lesions, suggesting a reduction of the expression of NOTCH1 protein. In order to confirm the obtained results, NOTCH1 protein expression was evaluated by Western blot in ICC available cases, demonstrating coincidence with the immunohistochemical analysis which supports the loss of NOTCH1 expression in ICC. Moreover, NOTCH1 localization has been used as a marker of activation \[[@R34]\], since signaling transduction relies on NICD nuclear translocation. In our samples, the loss of nuclear immunoreactivity in ICC samples, could be related to inactivation of the canonical pathway. This could be confirmed by the analysis of the expression of NOTCH1 target genes, such as those belonging to the *Hes* or *Hey* family \[[@R7], [@R9]\]. Chen *et al.* (2009) showed that NUMB expression increased during cervical carcinogenesis \[[@R24]\]. Increased NUMB expression has also been reported in oral squamous cell carcinoma (OSCC) \[[@R31]\], hepatocellular cancer (HCC) \[[@R35]\] and endometrial cancer \[[@R36]\]. In contrast, other studies have suggested NUMB as a tumor suppressor protein \[[@R37]\]. NUMB downregulation has been reported in breast cancer and non-small-cell lung carcinomas (NSCLCs), probably due to extensive degradation \[[@R22], [@R38]\]. We found an increase in NUMB expression in ICC compared to CIN, and this overexpression of NUMB correlates with a decrease in NOTCH1 expression. Even more, we found that premalignant lesions tended to express NUMB in the nucleus which is upregulated in cervical cancer with a cytoplasmic localization. Besides, cytoplasmic NUMB expression was associated with a decrease in nuclear NOTCH1 expression in cervical cancer samples, suggesting the potential regulation of NUMB over NOTCH1, as previously suggested \[[@R17], [@R39]\]. In the correlation analysis, we expected that tissue areas with NUMB positive expression would correlate with negative NOTCH1 areas. In CIN samples, we did not find a correlation, but we found low positive correlation between the expressing areas of NOTCH1 and NUMB in ICC reflecting that both proteins are present in the cytoplasm in the transformed tissue. This could suggest, that a cytoplasmic NUMB isoform expressed in ICC, could avoid NICD nuclear translocation and thus, inhibit its activity \[[@R19], [@R40]\]. As previously mentioned, the differences in NUMB localization in our groups (CIN vs. ICC) might be due to different isoform expression. *NUMB* mRNA can be alternatively spliced, giving rise to at least six isoforms of the protein with differences in the size of the phosphotyrosine-binding domain (PTB) and the proline-rich region (PRR) \[[@R41]\]. However, analysis of the NUMB isoforms is beyond the aims of our study since there are no isoform-specific NUMB antibodies available. The expression of another suppressor as p63 \[[@R33], [@R47]\]. It is worth to mention that the protein Atlas database report a moderate NOTCH1 immunoexpression in cervical cancer, with a heterogeneous localization in the cell \[[@R25]\]; this is opposite to our results since we found a diminished NOTCH1 expression in cervical cancer. We are not able to distinguish the membranal protein since our antibody is against the intracellular domain. On the other hand, our results of NUMB expression agree with those reported in the Protein Atlas since in both cases the expression is strong with a cytoplasmic localization. NOTCH1 expression has been proposed as a poor prognostic factor for many types of cancer, such as breast, gastric and lung cancer \[[@R42]--[@R44]\]. In this study, we showed that nuclear NOTCH1 expression was negatively correlated with malignancy status, this means that loss of nuclear NOTCH1 expression, the presumably active protein, might favor neoplastic progression from precursor cervical lesions (CIN) to cancer (ICC). Similar findings were recently observed in a study of small cell lung cancer, in which high NOTCH1 expression was an independent favorable prognostic factor \[[@R45]\]. Besides, we found that patients with ICC and cytoplasmic NOTCH1 expression tended to exhibit longer OS compared with those with nuclear NOTCH1 expression. Our findings related to OS are limited by the low number of ICC samples used for this analysis. Poor survival of patients with nuclear NOTCH1 and 3 expressions has already been described in non-small cell lung cancer and cervical carcinoma \[[@R44], [@R46]\]. The data showed here, support the results of Talora *et al.* (2012) that NOTCH1 is downregulated in later stages of cervical carcinogenesis. Thus, NOTCH1 seems to have a suppressive function in ICC. Moreover, Sun *et al.* (2009) found a similar behavior for NOTCH1, demonstrating its suppressive role through the induction of We acknowledge that the present analysis has several limitations, including that this is a retrospective study based mainly in paraffin embedded tissue; therefore, we lack enough fresh biological material to perform immunofluorescence for co-localization confirmation. Moreover, since our Institution is a cancer reference center, we could collect only a relatively low number of samples in the CIN group. Still, the consistency of the results from our sensitivity analysis suggests that the sample size was not a limitation. Additionally, we obtained a limited number of ICC samples exhibiting nuclear NOTCH1 expression. Nonetheless, the present study is distinctively unique due to the analysis of NOTCH1 and NUMB expression and localization in CIN and ICC samples. In conclusion, nuclear NOTCH1 is highly expressed in premalignant lesions, while the lack of nuclear NOTCH1 is an independent predictor of malignancy. Additionally, the association of NUMB with malignancy is dependent on NOTCH1 expression. We propose that the loss of nuclear NOTCH1 may contribute to cervical carcinogenesis. These results point to target the NOTCH pathway as a therapeutic in cervical cancer. MATERIALS AND METHODS {#s4} ===================== Tissue specimens {#s4_1} ---------------- A total of 144 biopsy tissue samples were collected from January 2004 to December 2016 in the Pathology Department of the Instituto Nacional de Cancerología in México City: 95 ICC samples (11 adenocarcinomas and 84 squamous cell carcinomas) and 49 cervical premalignant lesions (including 29 low-grade squamous intraepithelial lesions: CIN I and II; and 20 high-grade lesions: CIN III and *in situ* carcinoma). Hematoxylin and eosin staining confirmed histopathological diagnoses. Clinical and pathological parameters were collected from the medical files. This project was approved by the Institutional Review Board (INCAN/Of.CEI577/15). Antibodies {#s4_2} ---------- The antibodies used for immunohistochemical staining were polyclonal antibodies against the C-terminus of human NUMB isoforms (p65/p66 and p71/p72) (Santa Cruz Biotechnology Inc. Dallas, Texas. sc-15590) (1:30) and activated NOTCH1 (against the cleaved intracellular fragment, NICD) (Millipore, Merck, New Jersey, USA. 07-1231) (1:50). The NOTCH1 C-20 polyclonal antibody (Santa Cruz Biotechnology Inc. Dallas, Texas, sc-6014) (1:50) was used for immunostaining validation. Immunohistochemical assays {#s4_3} -------------------------- Immunohistochemical staining was performed in serial sections of paraffin-embedded tissues. The slides were incubated at 60° C for 1 h, dewaxed in xylene and rehydrated in alcohol. Antigen retrieval was performed by boiling the tissues for 1 min in 0.1 mol/L citrate buffer (pH = 6) at 80° C, incubating for 30 min in a water bath, and cooling down for 5 min. Subsequently, the slides were incubated for 20 min in 0.3% H~2~O~2~ blocking buffer (EnVisio System-HRP, Dako, California, USA). Slides were incubated with the corresponding antibody overnight at 4° C in a wet chamber and later washed with phosphate-buffered saline (PBS pH 7.4) prior to incubation with anti-mouse secondary antibody (DakoCytomation EnVisio System-HRP, California, USA) for 30 min. Positive staining was detected with 3,3′-diaminobenzidin, and then, the slides were counterstained with Mayer's hematoxylin (Merck, New Jersey, USA). Finally, the slides were preserved with rapid mounting media (Merck) and covered with a glass coverslip. Normal cervical epithelium was included as a control, accordingly to the protein Atlas database (<https://www.proteinatlas.org/>) that showed positive immunoreaction for NOTCH1 and NUMB proteins. Primary antibodies were replaced with PBS for the negative controls. Evaluation of immunohistochemical staining {#s4_4} ------------------------------------------ The immunohistochemical evaluation was performed by a senior pathologist and an experienced examiner of the Instituto Nacional de Cancerología-México in a double-blind fashion. A Nikon ECLIPSE E200 optical microscope with a 10x eyepiece and 10x and 40x objective lens was used. The staining intensity was defined as: weak (including + and ++), when the immunoreaction was visible only with a 40x objective; and intense (+++), when staining was visible even using a 10x objective. Localization was categorized as membranal, cytoplasmic, nuclear, or combinations among them. Percentage of positivity was assessed by quartiles. Technique validation was performed using an alternative NOTCH1 antibody in a representative set of samples. Western blotting {#s4_5} ---------------- NOTCH1-antibody specificity was assessed by Western Blot analysis in 22 random paraffin embedded tissues. Protein extraction was performed using the Qproteome FFPE Tissue Kit (Quiagen, Hilden, Germany). Briefly, 5 serial sections from the same block were cut with a thickness of up to 15 μm and placed in a 1.5 ml collection tube. For deparaffinization, 1 ml xylene was added into the tube and vortexed vigorously for 10 s and incubated for 10 min at room temperature (15--25° C), 100 μl of extraction buffer was added and heated. After centrifugation, the supernatant was transfered into a new tube. Total protein quantification was determined with bicinchoninic acid. After protein electrophoresis, proteins were transferred to a nitrocellulose membrane and tested with NOTCH1 (Millipore, Merck, New Jersey, USA, 07-1231) and GAPDH antibodies (Santa Cruz Biotechnology, Dallas, Texas). Clarity kit (Bio Rad, California, USA) was used for chemiluminescent protein detection. Densitometric analysis was performed using ImageJ software (Image Processing and analysis in Java). HPV detection {#s4_6} ------------- HPV typing was performed as previously described \[[@R27]\]. Primers from the L1 region were used (MY09/MY11/HMB01 and L1C1/L1C2.1/L1C2.2). DNA from HeLa and CaSki cells was used as a positive control. Products were analyzed by electrophoresis on 2% agarose gels stained with ethidium bromide. PCR products were directly sequenced with the BigDye Terminator v3-1 Cycle Sequencing Kit (Applied Biosystems). HPV sequences were aligned using BLAST software (<http://www.ncbi.nlm.nih.gov>, NCBI GenBank). Statistical analysis {#s4_7} -------------------- We evaluated the different distributions of NOTCH1 and NUMB expression, intensity and localization among groups (CIN vs. ICC) using the chi-squared test. We also explored correlations between NOTCH1 and NUMB expression in histological sections using Pearson coefficients. Multivariate analysis was conducted to determine the association between NOTCH1/NUMB expression and cancer status. For the association between NOTCH1 and NUMB in malignancy, we included the following confounders: age (continuous) and hormone contraception (categorical). We used three sets of models: unadjusted; adjusted for age; and adjusted for age and hormone contraception. We used the Akaike information criterion (AIC) to evaluate goodness-of-fit. The effect of nuclear NOTCH1 expression on OS was observed using the Kaplan and Meier method, and the log-rank test was used to compare groups. All analyses were performed using R software (R Project for Statistical Computing, Wien). SUPPLEMENTARY MATERIALS FIGURES AND TABLES {#s5} ========================================== **Author contributions** Immunohistochemical staining: Elenaé Vázquez-Ulloa, Ana Clara Ramos-Cruz and Alma Chávez-Blanco. Immunohistochemical data interpretation: Alejandro Avilés-Salas and Elenaé Vázquez-Ulloa. Database analysis: Elenaé Vázquez-Ulloa, Ana Clara Ramos-Cruz and Diddier-Prada, Marcela Lizano. Statistical analysis: Adriana Contreras-Paredes and Diddier-Prada. Sample collection: Adriana Contreras-Paredes and Alma Chávez-Blanco. Study design: Adriana Contreras-Paredes and Marcela Lizano. Manuscript preparation: Elenaé Vázquez-Ulloa, Diddier-Prada, Luis A Herrera-Montalvo, Adriana Contreras-Paredes and Marcela Lizano. This work is part of the Ph.D. thesis of María Alicia Elenaé Vázquez-Ulloa from the Programa de Maestría y Doctorado en Ciencias Bioquímicas at the Universidad Nacional Autónoma de México, who thanks CONACyT México for scholarship CVU 347097. We thank Sofía Ochoa MSc. for advising on the statistical analysis and Dr. Marika Sjöqvist for valuable comments regarding the manuscript. **CONFLICTS OF INTEREST** The authors declare that there are no conflicts of interest. **FUNDING** This work was partially supported by CONACyT CB-2010-01-151493; Instituto Nacional de Cancerología, México; and a scholarship to María Alicia Elenaé Vázquez-Ulloa CONACyT 347097. ICC : Invasive cervical cancer CIN : Cervical intraepithelial neoplasia HPV : Human papilloma virus HR : High risk NICD : Notch intracellular domain OS : Overall survival OSCC : Oral squamous cell carcinoma HCC : Hepatocellular carcinoma PTB : Phosphotyrosine-binding domain PRR : Proline-rich region NSCLC : Non-small cell lung carcinoma

Introduction {#Sec1} ============ M*ycobacterium leprae* is the main causative agent of leprosy, a disease that affects the skin, nerves, and mucosa of the upper respiratory tract in humans^[@CR1]^. A second, distantly related leprosy bacillus, *Mycobacterium lepromatosis*, was recently discovered in humans and red squirrels (*Sciurus vulgaris*)^[@CR2]^. Leprosy is curable with multidrug therapy (MDT), but remains a public health problem in South America, Africa, South and Southeast Asia, and Micronesia, where over 200,000 new leprosy cases are reported each year^[@CR3]^. MDT, comprising rifampicin, dapsone, and clofazimine, has been used intensively since the 1980s and a few second-line drugs, ofloxacin, minocycline, and clarithromycin, are sometimes employed as therapeutic agents^[@CR4]^. The emergence of drug-resistant (DR) and multidrug-resistant (MDR) *M. leprae* is increasingly reported^[@CR5]--[@CR12]^. For dapsone, rifampicin and ofloxacin, the resistance mechanism has been attributed to missense mutations in the drug resistance determining regions (DRDR) of the *folP1*, *rpoB*, and *gyrA* genes, respectively. *M. leprae* is an obligate intracellular pathogen that has never been cultured axenically but can infect wild or experimental animals. The nine-banded armadillo (*Dasypus novemcinctus*) or the mouse footpad (MFP) can be used to produce bacilli, but both methods are cumbersome and time-consuming^[@CR13]^. The genome of *M. leprae* is the smallest among mycobacteria (3.3 Mb) with 1614 genes encoding proteins and a remarkable 1300 pseudogenes^[@CR14]^. Such reductive evolution is a hallmark of bacteria that have changed their lifestyle from free-living to strictly host-associated^[@CR15]^. Due to its 14-day generation time and the absence of horizontal gene transfer, the genome of *M. leprae* is highly conserved, with \<300 single-nucleotide polymorphisms (SNPs) observed between distantly related strains, and only a few SNPs between close relatives^[@CR5],[@CR16]--[@CR18]^. Four SNP types (branches 1--4) and 16 SNP subtypes (A--P) were defined by surveying 78 informative SNPs and six single-base insertion/deletions (InDels)^[@CR16],[@CR19]^. Genotyping a large panel of *M. leprae* strains revealed strong geographical associations and suggested possible routes of dissemination of leprosy^[@CR16]^ whereas, a recent phylogenetic analysis of 16 whole-genome sequences of modern and ancient *M. leprae* strains, implicated the 3K subtype (branch 0) as the most ancestral^[@CR17]^. Leprosy seems to have appeared during the Iron Age (1200--600 BC) and the date of the most recent common ancestor of *M. leprae* was estimated to be from 2543 BC to 36 AD, based on whole-genome sequence analysis^[@CR17]^. Similarly, the earliest accepted written record of leprosy is from 600 BC^[@CR20]^, and the earliest osteological evidence dates from around 300 BC^[@CR21]--[@CR25]^. The oldest genomic evidence of leprosy is for samples from 80 to 240 AD in Central Asia^[@CR26]^. In this study, we develop and apply methods to isolate and purify *M. leprae* DNA that enable whole genome sequences to be obtained directly from human biopsy material, thus removing the necessity for passage through animals. This approach was successfully used to generate 120 new *M. leprae* genome sequences from drug-susceptible and DR strains from around the world, thereby enabling detailed phylogenetic and phylogeographic comparisons to be performed, new mutations associated with antimicrobial resistance to be detected, and the likely origin of leprosy to be proposed. Results {#Sec2} ======= Isolating *M. leprae* DNA from human skin biopsies {#Sec3} -------------------------------------------------- Genome sequencing has become routine practice in microbiology^[@CR27]^, especially for micro-organisms that can be readily isolated, which is not the case of the leprosy bacillus. For decades, the sole source of *M. leprae* DNA suitable for genomics was from bacteria isolated 12 months after infection of armadillos or mice. Recently, we have developed and optimized methods that enable *M. leprae* DNA to be extracted directly from fresh or formalin-fixed skin biopsies from leprosy patients^[@CR28]^. These methods include enrichment of *M. leprae* DNA by array capture^[@CR17]^ but this is less practical for large population-based investigations. The DNA extraction method used in this study was applied directly to punch biopsies from clinically well-characterized patients of known bacillary index (BI) and exploits the fact that *M. leprae* resides intracellularly. Host cells are first disrupted and their DNA degraded, leaving the bacilli intact. The bacilli are then lysed and their DNA extracted and used for library preparation. This approach was applied to 106 biopsies whose BI ranged from 0 (no bacilli visible) to 6 (\>1000 bacilli per microscopic field) thereby enabling a relationship between BI and sequencing efficiency to be established (Fig. [1](#Fig1){ref-type="fig"}). As expected, there was a direct correlation between genome coverage and the BI but, surprisingly, successful coverage could even be achieved with some specimens whose BI was as low as 1+.Fig. 1Correlation between bacillary index and successful sequencing. The content of *M. leprae* DNA in sequencing libraries derived from human skin biopsies was determined and found to be proportional to the bacillary index (not available for all samples). Empty circles are samples that were not included in the study due to insufficient genome coverage. Sample count and sequencing success rates are given at the top of each category Genome analysis of patient and animal cohort {#Sec4} -------------------------------------------- We analyzed a total of 154 *M. leprae* genomes from 25 countries (Fig. [2](#Fig2){ref-type="fig"}, Supplementary Data [1](#MOESM4){ref-type="media"}), of which 120 were newly sequenced and 34 were previously published (Supplementary Data [1](#MOESM4){ref-type="media"}). The cohort comprised 147 human samples, 6 from red squirrels and 1 from an armadillo that were all naturally infected. Genome sequences were obtained directly from 109 human samples, 30 from bacilli passaged in mice, and 8 from armadillos. Thirty of these strains were from patients who had relapsed or not responded to MDT the remainder (124) were from supposedly drug-susceptible strains (87 were from confirmed primary cases, while disease history was unknown for the others).Fig. 2Geographic distribution of the *M. leprae* samples used in this study. World map shows the number of registered cases of leprosy per 10,000 population (prevalence rates) in 2015 as reported by the World Health Organization (<http://apps.who.int/neglected_diseases/ntddata/leprosy/leprosy.html>). Blue numbers indicate ancient *M. leprae* strains A total of 3053 SNPs and 219 InDels (excluding tandem repeats) was found (Supplementary Data [2](#MOESM5){ref-type="media"}). The average SNP difference among the 154 genomes was 114. We found a total of 988 non-synonymous alleles (0.62 per protein-coding gene, or 0.61 per kb of protein-coding genes) and 530 synonymous SNPs (0.33 per protein-coding gene or 0.33 per kb of protein-coding genes), and 1763 mutations in intergenic regions and pseudogenes (1.07 mutations per kb of intergenic regions and pseudogenes). The SNP density for each gene is given in Supplementary Data [2](#MOESM5){ref-type="media"}. Of the 219 InDels, 58 (27%) were in protein-coding genes. Phylogeny of *M. leprae* {#Sec5} ------------------------ Phylogenetic analysis using both maximum parsimony (MP) and Bayesian inference resulted in consistent tree topologies and revealed distinct lineages and sublineages of *M. leprae* (Fig. [3](#Fig3){ref-type="fig"}). Strains belonging to the same SNP subtypes^[@CR16]^ clustered within single branches, with the exception of SNP subtype 3K, which is represented by a newly discovered ancestral lineage, termed here 3K-1, and the ancestral lineage referred to earlier as branch 0^[@CR17]^ and termed here 3K-0 (Fig. [3a](#Fig3){ref-type="fig"}). All strains from the two most ancestral lineages, 3K-0 and 3K-1, originated from Japan (8), China (1), Korea (1), the Marshall Islands (1), and New Caledonia (1), in agreement with earlier genotyping studies of hundreds of *M. leprae* strains which confirmed the predominance of the 3K genotype in East Asia, notably in Japan, China, and Korea^[@CR16],[@CR29]--[@CR31]^Fig. 3Phylogeny of *M. leprae*. **a** Maximum parsimony tree of 154 genomes of *M. leprae*. The tree is drawn to scale, with branch lengths representing number of substitutions. *M. lepromatosis* was used as outgroup. Bootstrap values (500 replicates) are shown next to the branches. Dots indicate protein-changing mutations in the corresponding gene as given in Table [1](#Tab1){ref-type="table"}. **b** Bayesian phylogenetic tree of 146 genomes of *M. leprae* calculated with BEAST 2.4.4. Hypermutated samples with mutations in the *nth* gene were excluded from the analysis. The tree is drawn to scale, with branch lengths representing years of age. Samples were binned according to geographic origin as given in the legend. Posterior probabilities for each node are shown in gray. Location probabilities of nodes were inferred by the Discrete Phylogeny model. *M. leprae* in East Africa showed higher diversity with subtypes 2E, 2F, and 2H representing distinct lineages (Fig. [3a](#Fig3){ref-type="fig"}). The geographic distribution of those lineages corroborates the findings of earlier studies reporting the presence of SNP type 2 in Medieval Europe, the Middle East and East Africa^[@CR16]^. Two Ethiopian isolates, belonging to the 2F subtype, clustered closely with medieval European strains dating from the 11th to 12th century (Fig. [3](#Fig3){ref-type="fig"}), which supports the hypothesis that the ancient Greek and Roman routes^[@CR32]^ connecting Europe, the Middle East, East Africa, and South Asia^[@CR16],[@CR33]--[@CR35]^ contributed to the dissemination of SNP type 2 *M. leprae*. West Africa, on the other hand, harbors exclusively SNP type 4, suggesting that overland migration between East and West Africa was limited. SNP subtypes 4N, 4O, and 4P, albeit sharing the same ancestor, do not form a monophyletic clade as previously hypothesized^[@CR16]^. Rather, the 4O and 4P subtypes cluster together in a branch distinct from 4N (Fig. [3](#Fig3){ref-type="fig"}). Brazil, as expected, contains a great diversity of several *M. leprae* lineages, with the SNP type 4 and SNP subtype 3I being the most prevalent^[@CR36]^. The 3I genotype was common in medieval Europe^[@CR17],[@CR21],[@CR37],[@CR38]^, and is still present in red squirrels in the United Kingdom^[@CR2]^. The modern Brazilian strain Br2016-45 branched between two medieval strains from Europe (Fig. [3](#Fig3){ref-type="fig"}), making it the most ancestral contemporary 3I strain in the Americas to date. The broad diversity of 3I genotypes from Brazil probably derives from multiple introductions from Europe. On the other hand, the strains circulating in the Southern USA and associated with zoonosis from the nine-banded armadillo, I-30, NHDP-55 and NHDP-63^[@CR18]^, originated much more recently (Fig. [3b](#Fig3){ref-type="fig"}), in agreement with the rapid expansion and spread of the armadillo population since its introduction to this region about 150 years ago^[@CR39]^. Good representation of most *M. leprae* lineages enabled identification of lineage-specific markers. A set of 235 SNPs and 25 InDels were specific to single lineages or groups of related lineages (Supplementary Data [2](#MOESM5){ref-type="media"}), of which 73 non-synonymous SNPs and 5 InDels were within protein-coding genes. These new lineage-specific markers can be used for future genotyping schemes. Dating analysis {#Sec6} --------------- Dating analysis of *M. leprae* (Fig. [3b](#Fig3){ref-type="fig"} and Supplementary Figure [1](#MOESM1){ref-type="media"}) was done using BEAST v2.4.4^[@CR40]^ and the results were very similar to those obtained from a ten-fold smaller number of contemporary isolates^[@CR17]^. The most recent common ancestor (TMRCA) of all *M. leprae* strains was estimated to be 3699 years old (95% Highest Posterior Density (HPD) 2731--4838 ya) and the substitution rate was 7.8 × 10^−9^ per site per year. Overlapping results were obtained when using different models (Supplementary Note and Supplementary Table [1](#MOESM1){ref-type="media"}), indicating that the dataset was robust and sufficiently informative. A striking observation is the relative youth of the SNP type 1 lineage and its association with South Asia (Fig. [3b](#Fig3){ref-type="fig"}). Earlier studies revealed a predominance of SNP subtype 1D in India and Nepal, followed by 1C, 1A^[@CR16]^, and 2E, 2G, and 2H^[@CR33]--[@CR35]^. SNP type 1 predominates in Thailand^[@CR41]^, Bangladesh, Indonesia, and Philippines^[@CR16]^. The current phylogeography of *M. leprae* implies that humans brought leprosy to South Asia from other parts of the continent. Hypermutated *M. leprae* strains {#Sec7} -------------------------------- Eight *M. leprae* strains (85054, S15, Amami, Zensho-4, Zensho-5, Zensho-9, Br14-3, and Br2016-15), belonging to five different SNP subtypes, had unusually long branches in the MP tree (Fig. [3a](#Fig3){ref-type="fig"}) because they contained on average 92 more SNPs than the other strains but approximately the same number of InDels. Comparative analysis revealed one unique feature linking the observed "hypermutated" strains, namely deleterious mutations in the endonuclease III gene *nth* (*ML2301*) due to frameshifts and premature stop codons (Table [1](#Tab1){ref-type="table"}).Table 1Mutations in genes associated with drug resistanceSample*folP1* ML0224*rpoB* ML1891c*gyrA* ML0006*gyrB* ML0005*fadD9* ML0484c*ribD* ML1340*ethA* ML0065*pks4* ML1229*nth* ML2301c2188---2007\.....G62D^1^\...2188---2014\.....G62D\...85054**P55LS456L**G52E..W878\*D256N..R197\*2DDS**P55R**\...L396PS58R\...LRC-1A..I851TV214G\.....Airaku-3**T53I**..N304fs\....Amami**P55L**\....R236C..L145fsARLP_08.H200Y\...\....ARLP_10\....Y927DG61C\...ARLP_30..G1115R\...\...ARLP_52..G1115R\...\...Bn8-46**P55R**\...\.....Bn8-52**P55L**N145H\...W1108fs\...Br14-1**P55RS456M**..G148fs\....Br14-2**P55RS456M**..G148fs\....Br14-3**P55LH451YA91V**.Q107\*G94D..E173\*G448DT433IBr14-4**P55RS456L**..L396PI56T.Y171NBr14-5**P55RS456L**..A919EC222W\...Br2016-15**P55L**.V731IT503IG796SA63T.M14IN142fsBr2016-16\....L998fsK267fs\...Br2016-18..S307L\...\...Br2016-21\...\....I932fs.Kusatsu-6**P55L**T433I\....K477fs**D441Y**Ml10-93**P55R**\...R73fsS58R\...Ml10-98\.....A10fs\...Ml2-10**T53R**\...\.....Ml6-50**T53R**\...\.....Ml6-55**T53R**\...\.....Ng14-35**P55R**.S307L.A594TD34del\...S15**T53I**G432S..D466NQ117\*D63NT334IG146fs**H451**DD323NS9**T53I**R791Q\...S58N\...Thai-237..I851T\...\...US57..G362E\...\...Zensho-2**P55L**\...Y562fsG94D\...Zensho-4**T53I**P51S**A91V**D464NR314CD77NA25TQ1719\*L163fs**S456L**Zensho-5**P55LS456L**\...G204CG390A.N142fsP583LZensho-9**P55LH451Y**..A973TP150LP383L.E122\*G681SIn bold, substitutions or residues known to confer drug resistance in *M. leprae*; \* premature stop codon, fs frameshift, dot (.) wild type. RpoB numbering is based on *M. leprae*, *E. coli* numbering in brackets: 51 (126), 52 (127), 171 (246), 200 (275), 432 (507), 433 (508), 441 (516), 448 (523), 451 (526), 456 (531), 681 (756), 791 (866). ^1^Coverage below the threshold, both "2188" isolates come from the same patient but after an interval of 7 years^[@CR28]^ Drug resistance {#Sec8} --------------- DR-associated SNPs were detected in the DRDR in 24 strains for *folP1*, 11 strains for *rpoB*, and 2 strains for *gyrA* (in bold in Table [1](#Tab1){ref-type="table"}). Previously described mutations were identified in *folP1* at codons 53 (*n* = 7) and 55 (*n* = 17), except in one isolate (Bn8-52), which had mutations at codons 55 and 145. Eleven strains had known mutations that confer rifampicin resistance in their *rpoB*-DRDR, while two strains (Kutatsu-6 and S15) harbor one additional mutation, and one (Br14-3) has two additional mutations in the DRDR (Table [1](#Tab1){ref-type="table"}). One of these additional mutations (G432S) does not confer rifampicin resistance to recombinant *Mycobacterium smegmatis*^[@CR42]^ whereas no information is available for the remaining three (T433I, G448D, and T508I) except that the G448A substitution does confer rifampicin resistance in *M. tuberculosis*^[@CR43]^. Also, 5 of the 11 rifampicin-resistant strains had additional missense mutations in *rpoB* (85054, Br14-4, Zensho-4, Zensho-5, and Zensho-9) while 2 strains (ARLP_08 and S9) presented non-synonymous SNPs outside the DRDR (Fig. [4](#Fig4){ref-type="fig"}). Compensatory mutations in *rpoA* and *rpoC*, encoding the alpha and beta-prime subunits of RNA polymerase, can occur in rifampicin-resistant *M. tuberculosis*^[@CR44]^. We found one non-synonymous SNP in *rpoA*, substitution T187P in the rifampicin-resistant strain Br14-5, and seven non-synonymous SNPs in *rpoC* (Supplementary Data [2](#MOESM5){ref-type="media"}), of which two occurred in the drug-resistant strains S15 (A258T) and Zensho-4 (H1133Y).Fig. 4Mutations of *M. leprae* genes associated with antimicrobial resistance. Triangles point to the location of the mutation in the protein. Black triangles indicate known resistance-conferring mutations identified in this study that are situated in the drug resistance determining regions (DRDR): D dapsone, Q quinolone, R rifampicin. Orange border means the mutation was found to be homoplasic. Triangle size reflects the number of isolates from this study harboring the mutation, ranging from 1 to 17. Frameshifts and premature stop codons are in turquoise. Substitutions predicted to have an impact on the biological function of the protein^[@CR75]^ are in bold. Proteins are drawn to scale Two strains had known quinolone resistance mutations in the DRDR of *gyrA* and six harbored different single mutations elsewhere in the gene. Three isolates had a missense mutation in *gyrB*, including two within the DRDR (Table [1](#Tab1){ref-type="table"}). Five strains harbor deleterious mutations in the *ethA* gene, encoding a mono-oxygenase that activates thioamide prodrugs in *M. tuberculosis*^[@CR45],[@CR46]^. Interestingly, in addition to *ethA* and *nth*, three genes (*fadD9*, *ribD*, *pks4*) were mutated almost exclusively in MDR strains occurring 18, 19, and 4 times, respectively (Table [1](#Tab1){ref-type="table"}). Retracing the emergence of drug resistance in leprosy patients {#Sec9} -------------------------------------------------------------- Prior to the introduction of MDT in the 1980s, patients were treated with dapsone or other antimicrobials as monotherapies of varying duration^[@CR7],[@CR8]^. Since genomics uncovered new mutations that are associated with antimicrobial resistance in other bacteria, such as those in *ethA* and *gyrB*, this prompted us to try and retrieve the clinical records of six patients whose strains displayed resistance to three or more drugs (dapsone, rifampicin, quinolones, and thioamides). Four of these extensively drug-resistant (XDR) strains were from multibacillary patients in Japan who had received a succession of monotherapies in the pre-MDT era and our genome analysis enabled the chronology of resistance emergence to be retraced. This is illustrated in Fig. [5](#Fig5){ref-type="fig"}, and sadly exemplified by the strain from patient Zensho-4 who was diagnosed in 1963 and first treated with protionamide followed by thiambutosin, both of which show cross-resistance and likely require activation by the EthA mono-oxidase that acquired the A25T missense mutation^[@CR47]^; then treatment began with dapsone leading to emergence of the T53I mutation in *folP1*, followed by rifapentine that selected the S456L mutation in *rpoB*, and continued with ofloxacin, to which resistance arose from the A91V mutation in *gyrA* and D464N in *gyrB*. Molecular drug susceptibility testing was performed in 1998 and the patient finally cured by a regimen comprising clofazimine, minocycline, chloramphenicol and levofloxacin/sparfloxacin. The fifth XDR strain was from a newly diagnosed Brazilian case (Br2016-15) with no history of treatment for leprosy, confirming the ongoing transmission of primary antimicrobial resistance, while details of the sixth case could not be recovered.Fig. 5Timeline of the leprosy treatment and emergence of drug resistance in the XDR strains. Mutated genes conferring resistance to the corresponding drugs are shown in red. Arrows span from the onset of disease to the end of treatment. Horizontal lines show the period when a drug was given. Dotted lines mean irregular treatment. CAM chloramphenicol, CLO clofazimine, DDS dapsone, DPT thiambutosine (diphenylthiourea), ETO ethionamide, INH isoniazid, KAN kanamycin, LVX levofloxacin, MIN minocycline, OFX ofloxacin, PTO protionamide, RIF rifampicin, SMP sulfamethoxypyridazine, SPX sparfloxacin, STR streptomycin, TZA thiozamin Genes under positive selection {#Sec10} ------------------------------ We also identified genes containing an unusually high number of polymorphisms, multiple alleles, and homoplasies (Supplementary Figure [2](#MOESM1){ref-type="media"}), which could be indicative of positive selection^[@CR48]^. Strikingly, the distribution of these polymorphic sites around the genome was not random as they were often clustered, especially proximal to either side of the origin of replication (Supplementary Figure [3](#MOESM1){ref-type="media"}). Protein-changing mutations were found in 540 genes, with an average of 1.77 mutations per gene (STD 2.12). Table [2](#Tab2){ref-type="table"} contains a ranking of genes with at least five non-synonymous mutations or regions with one or more homoplasy (excluding VNTRs). The most polymorphic gene by far was *ML0411*^[@CR49]^ encoding the serine-rich antigen, a member of the immunogenic, surface-exposed PPE protein family. Two other known T-cell antigens whose genes display variability are Lsr2 and EsxA (Table [2](#Tab2){ref-type="table"}). Other than *nth*, three other polymorphic genes (*ML1040c*, *ML1750c*, and *ML1512c*) code for proteins that appear to function in nucleic acid or cyclic nucleotide metabolism (Table [2](#Tab2){ref-type="table"}).Table 2Highly polymorphic genes and genomic regions of *M. leprae*Gene or regionDescriptionNon-synonymous mutations (multi-allele loci)Synonymous mutationsHomoplasy*ML0411*Serine-rich antigen32 (4)14*ML1040c*Putative ATP-dependent helicase19 (1)00*fadD9*Probable fatty-acid-CoA ligase1611*ML1750c*Putative nucleotide cyclase1710*ML1512c*Putative ribonuclease J1700*ribD*Bifunctional enzyme riboflavin biosynthesis protein1701*rpoB*DNA-directed RNA polymerase (beta chain)13 (1)13*ML1753c*Probable transcriptional regulatory protein900*gyrA*DNA gyrase (subunit A)881*ML1300*Conserved hypothetical protein800*nth*Endonuclease III701*ctpC*Metal cation-transporting *P*-type ATPase C720*rpoC*DNA-directed RNA polymerase (beta chain)750*ML2687c*Probable conserved transmembrane protein700*ML1052c*Conserved hypothetical protein710*ML0009*Hypothetical protein610*ML0283*Cation-efflux transporter component610*ML2700*Conserved transmembrane protein670*mfd*Transcription-repair coupling factor610*ppsC*Phenolphthiocerol and DIM synthesis610*ethA*Activates the pro-drug ethionamide500*trpE*Biosynthesis of tryptophan (at the first step)510*pknB*Transmembrane serine/threonine-protein kinase530*fas*Fatty acid synthase510*esxA*Early secretory antigenic target201*lsr2*Dominant T-cell antigen and stimulates lymphoproliferation.413*folP1*Dihydropteroate synthase5 (2)12*ML1752c*Conserved hypothetical protein211*ppsA*Phenolphthiocerol and DIM synthesis431*ML0803*Two-component sensor kinase2 (1)00*ML0237*Conserved hypothetical protein (pseudogene)NA11*ML0010c-ppiA*Intergenic regionNA31 Discussion {#Sec11} ========== Here we have optimized and applied highly sensitive procedures to extract *M. leprae* DNA directly from human skin biopsies that is suitable for whole-genome sequencing. The resultant genome sequences were analyzed phylogenetically and used to retrace the origin of the leprosy bacillus, and to identify polymorphisms that had been positively selected during evolution. Such polymorphisms might reflect pressure from the human immune system, from MDT or other forces. It is striking that the ancestral lineages of *M. leprae* predominate in East Asia, although we should keep in mind that Central Asia has been understudied, so it would be interesting to sequence more samples spanning the East--West axis of Asia, including the Middle East, where the 3K genotype is also present^[@CR16]^. Nevertheless, given the current data on the distribution of the 3K subtype we can deduce that the ancestor of *M. leprae* originated within Eurasia, probably in the Far East. Endonuclease III (Nth) and the formamidopyrimidine and endonuclease VIII family (Fpg/Nei) of DNA glycosylases are central to the base excision repair pathway in bacteria^[@CR50]^. Mycobacterial genomes usually contain a single *nth* and two *fpg/nei* genes but *M. leprae* has lost both *fpg/nei* orthologues and retained the *nth* gene. Nth, Fpg, and Nei may have overlapping functions and, in enteric bacteria, mutator phenotypes were observed when *nth* was inactivated in combination with the *fpg* and *nei* genes^[@CR51]--[@CR53]^. In *M. smegmatis*, deletion of *nth* and both the *nei* homologs resulted in elevated spontaneous mutation frequencies and increased sensitivity to oxidative stress^[@CR54]^. Therefore, in the absence of Nei, inactivation of *nth* in *M. leprae* should lead to increased sequence variability, which is consistent with our results. Strikingly, all *nth* mutants were also drug-resistant so Nth loss likely favors emergence of drug resistance, and *nth* mutations might serve as a surrogate marker for potential drug resistance and treatment failure. A link between a higher mutation rate and drug resistance was observed in strains of *M. tuberculosis* (which has *nth* and two *fpg/nei* genes) belonging to lineage 2, but the molecular basis for this is unknown^[@CR55]^. For a pathogen with an extremely reduced genome such as *M. leprae*, a hypermutator phenotype could be detrimental and ultimately lethal. Drug resistance is alarming for leprosy control. There is growing evidence for primary quinolone resistance in strains of *M. leprae* from patients who have never been treated with quinolones for leprosy but may have received this drug for other infections^[@CR56]^. Five new GyrA mutations were identified in this study, but their effect on FQ resistance remains to be determined. Since two of them arose independently in the GyrA intein, which is removed by protein splicing, they may not impact quinolone activity (Fig. [4](#Fig4){ref-type="fig"}). Three non-synonymous mutations were found in *gyrB* (Table [2](#Tab2){ref-type="table"}) and experimental evidence exists for two of them conferring quinolone resistance in in vitro assays or in *M. tuberculosis*^[@CR57],[@CR58]^. To our knowledge, this is the first report of *M. leprae* clinical isolates harboring mutations in *gyrB*. Thus, despite their apparent rarity, mutations in *gyrB* should be systematically assessed in drug resistance screening. A range of known and new mutations was detected in the DRDR and elsewhere in *rpoB* (Fig. [4](#Fig4){ref-type="fig"}; Table [1](#Tab1){ref-type="table"}). Some of these might have a compensatory role in restoring fitness, that is known to be reduced to various degrees in rifampicin-resistant mutants of *M. tuberculosis*^[@CR59],[@CR60]^. Similarly, compensatory mutations in *rpoA* and *rpoC* can occur in rifampicin-resistant *M. tuberculosis*^[@CR44]^. The *rpoA* substitution T187P in the rifampicin-resistant *M. leprae* strain Br14-5 was shown to be compensatory in *M. tuberculosis*^[@CR44]^. Rifampicin-resistant isolates of *M. tuberculosis* harbor more mutations in *rpoC* compared to rifampicin-susceptible isolates^[@CR44],[@CR61]^. In our case, we observed no clear correlation between rifampicin resistance and mutations in *rpoC*, which occurred in two resistant and two wild-type strains. Arguably the most intriguing finding of the present investigation was the remarkably high frequency of mutations in the *fadD9* and *ribD* genes and in 19/23 cases these occur in strains that have at least one mutation that is associated with resistance to a leprosy drug (Fig. [4](#Fig4){ref-type="fig"}; Table [1](#Tab1){ref-type="table"}). Functional information for *fadD9* is scarce and the mutations found are predicted to either abolish protein production (8/16) or to cause detrimental amino acid changes (Fig. [4](#Fig4){ref-type="fig"}). In the case of *ribD*, 14 different missense mutations were found in a group of 17 variant alleles, indicating that this is likely an essential function. From studies with *M. tuberculosis* it is known that *ribD* encodes an alternative dihydrofolate reductase, with relatively low activity compared to that conferred by the bona fide dihydrofolate reductase gene, *dfrA*^[@CR62]^. In clinical isolates of *M. tuberculosis*, a promoter mutation causes overexpression of *ribD* that is associated with resistance to the old drug, *para*-amino salicylic acid (PAS), and to certain DHFR inhibitors^[@CR63]^. This suggests that the mutations detected in the *M. leprae ribD* gene may also confer resistance to PAS and support for this is provided by the fact that vadrine (2-pyridyl-(4)-1,3,4-oxydiazolone-(5)-*p*-aminosalicylate) was used as a drug to treat leprosy before dapsone became widely available^[@CR64]^. It is thus possible that the *ribD* mutations we report here arose nearly 60 years ago following treatment with vadrine or another PAS derivative. Our discovery of these mutations and those in *fadD9* should encourage further experimentation in order to establish their true role and contribution to antimicrobial resistance, especially to clofazimine. Methods {#Sec12} ======= Sample collection {#Sec13} ----------------- Samples were taken from leprosy patients as punch biopsies of skin (preserved in 70% ethanol or formalin-fixed and paraffin-embedded (FFPE)), which is standard diagnostic procedure for leprosy, or from mouse foot-pads. Details about the samples used in this study are given in Supplementary Data [1](#MOESM4){ref-type="media"} and below. Origin of S15: Strain S15 corresponds to strain 92041^[@CR65]^, which was isolated from a lepromatous leprosy patient originally from Martinique. The origin of S15 was erroneously attributed to New Caledonia in Monot et al.^[@CR16]^, and the error was subsequently propagated in several publications^[@CR2],[@CR17],[@CR66],[@CR67]^. Origin of the case Ng14-3: We mention in the Results that West Africa harbors only SNP-type 4 strains. Strain Ng14-35 (subtype 1D) is an exception, but the patient (a native of Nigeria) developed leprosy during a prolonged stay in Libya. LRC-1A, an unidentified strain from Japan: A sample obtained from the Leprosy Research Center (LRC) in Tokyo, initially labeled as Airaku-2, did not contain the mutations in *folP1* and *rpoB* previously detected in Airaku-2 by PCR sequencing^[@CR7]^. Therefore, we renamed this sample to LRC-1A (standing for SNP subtype 1A sample from LRC). DNA extraction and library preparation {#Sec14} -------------------------------------- DNA was extracted from 101 human skin biopsies with known BI using a customized in-house protocol combining host tissue digestion and the QIAmp microbiome kit for host DNA depletion, strong bacterial cell lysis and silica-based purification. Punch biopsies (6 mm) in 70% ethanol were first rehydrated in Hank's balanced solution prior to mincing with scissors. Cells were detached from the tissue by 30 min incubation at 37 °C with a mixture of 0.5 U of collagenase and dispase, followed by incubation at 56 °C with 10 mg/ml of trypsin until complete digestion. Free cells were then suspended in 1 ml of phosphate-buffered saline (PBS) and DNA was extracted using the QIAmp DNA microbiome extraction kit according to the manufacturer's recommendations. Each run of extraction included a batch of five to nine samples and one blank control (500 μl of Hank's balanced solution). The presence of *M. leprae* was assessed by PCR using RLEP primers^[@CR2]^ prior to library preparation. Libraries were prepared from 50 µl of extracted DNA using the Kapa Hyperprep kit as described previously^[@CR2],[@CR5]^. DNA from FFPE samples was extracted using the truXTRACTM FFPE DNA kit (Covaris) as described previously^[@CR28]^. Libraries prepared from the extracted DNA were used directly for shotgun sequencing. *M. leprae* DNA extraction quality was assessed from the percentage of *M. leprae* DNA present in the library inferred by alignment to the reference genome sequence, with a minimum threshold set at 1%. This threshold was chosen because it yields an average genome coverage of at least 5× per sample in a multiplexed run of 10 samples on one HiSeq 2500 lane (yielding around 20 million reads per sample and 100 bases per read). Library enrichment {#Sec15} ------------------ Libraries with low *M. leprae* content underwent enrichment using whole-genome tiling arrays as described previously^[@CR17]^. Briefly, Illumina libraries were hybridized onto custom Agilent SureSelect Capture Arrays containing ca. one million DNA probes (60 bp) spanning the entire *M. leprae* genome (tiled every 4 bp), followed by elution and PCR amplification. Sequencing {#Sec16} ---------- Sequencing was performed on Illumina Hi-Seq 2000, Hi-Seq 2500, or Mi-Seq instruments. Sequence processing {#Sec17} ------------------- We took precautions in analyzing the data to avoid false-positive SNP calls. All raw reads were adapter- and quality-trimmed with Trimmomatic v0.33^[@CR68]^. The quality settings were "SLIDINGWINDOW:5:15 MINLEN:40". Paired-end (PE) data were additionally processed with SeqPrep (<https://github.com/jstjohn/SeqPrep>) to merge overlapping pairs. This increases the accuracy of sequence in the overlapping area, avoids problems in estimating coverage and creates longer reads, which facilitates InDel calling. Duplicate reads were omitted from downstream analyses. This is especially important for libraries with insufficient *M. leprae* DNA fragments, which is not uncommon for low BI samples or samples that are difficult to process, like FFPE samples. In these cases, library enrichment with array-capture, or very deep sequencing often produce a high number of duplicate reads (DNA fragments that were sequenced multiple times, seemingly increasing the overall genome coverage), with each read having dozens or even hundreds of copies. Such reads will amplify possible artefacts and sequence errors, resulting in false SNP calls. Sequence analysis {#Sec18} ----------------- Preprocessed reads were mapped onto the *M. leprae* TN reference genome (GenBank AL450380.1) with Bowtie2 v2.2.5^[@CR69]^. We filtered out all reads with mapping quality below 8 and omitted repetitive regions in the reference sequence. We also omitted ribosomal RNA (rRNA) genes because alignments in these regions tend to be error prone. This is because rRNA genes are highly conserved in bacteria, so sequences from other species could map to the *M. leprae* reference sequence. This usually happens when the content of *M. leprae* DNA in a sequencing library is scarce and is even more pronounced when libraries with low *M. leprae* content undergo array-capture. However, because lineage-specific mutations were previously observed in the *M. leprae rrs* gene^[@CR30]^, we manually checked the alignments corresponding to the rRNA genes and added the curated results to Supplementary Data [2](#MOESM5){ref-type="media"}. SNP calling was done using VarScan v2.3.9^[@CR70]^. To avoid false-positive SNP calls the following cutoffs were applied: minimum overall coverage of five non-duplicated reads, minimum of three non-duplicated reads supporting the SNP, mapping quality score \>8, base quality score \>15, and a SNP frequency above 80%. InDel calling was done using Platypus v0.8.1^[@CR71]^ followed by manual curation. Completed genome sequences of *M. leprae* Br4923 and *Mycobacterium lepromatosis* (GenBank JRPY00000000.1) were aligned against the *M. leprae* TN reference using LAST^[@CR72]^ using the default parameters for the former and the gamma-centroid option for the latter. Mixed samples {#Sec19} ------------- A large number of missing values, especially in lineage-specific loci, points to the presence of more than one strain in a sequencing library. Although not thoroughly tested, in our opinion mixed data sets are mostly due to technical problems or contamination because in some cases we were able to identify the problematic strains. The possible presence of multiple *M. leprae* strains in single skin lesions was not tested in this study, but we expect it to be extremely low. Overall, a few mixed data sets were detected and some were removed from this study, except for samples that we deemed important and describe below. Nevertheless, results were not biased because loci with mixed alleles were treated as missing values. Zensho-4 seems to contain a fraction of another strain (possibly around 40%) that is closely related to it. Only a few loci had mixed alleles, and these include the A91V substitution in *gyrA* (supported by 62% of reads) and the D464N substitution in *gyrB* (supported by 41% of reads). The latter was attributed to Zensho-4 for simplicity. Similarly, Zensho-5 seems to contain around 30% of Zensho-4. This is the main reason why we could not detect SNPs specific only to Zensho-5 (Fig. [3a](#Fig3){ref-type="fig"}), since such SNPs would be "diluted" with wild-type alleles from Zensho-4 and could not pass the SNP "purity" threshold. We included these two samples in this study because they are multi-drug-resistant and belong to the SNP-type 3K-0. Furthermore, mutations in genes conferring drug resistance from this study match with those from earlier reports of these samples, confirming their identity^[@CR73]^. Thai-311 contains \<20% of an unidentified 3K-0 strain that belongs to the Kyoto-1/Zensho-5 cluster of strains (Fig. [3a](#Fig3){ref-type="fig"}). SNP calling was not significantly affected. Finally, sample Ye2-3 contained around 25% of an unidentified strain belonging to SNP-type 4. Because we only have few samples from Yemen, we decided to keep Ye2-3 in this study. Phylogeny and dating analysis {#Sec20} ----------------------------- Concatenated SNP alignments were used for the analyses. MP trees were constructed in MEGA6^[@CR74]^ using 500 bootstrap replicates. Sites with missing data were partially deleted (80% coverage cutoff), resulting in 3046 variable sites used for the tree calculation. The Subtree-Pruning-Regrafting algorithm was used as the MP search method. Dating analysis and discrete phylogeography were done using BEAST2 v2.4.4^[@CR40]^. Details are given in the Supplementary Note. Data availability {#Sec21} ----------------- Sequence data are available from the NCBI Sequence Read Archive (SRA) under accession number SRP072827. Accession numbers for all samples used in this study are given in the Supplementary Data [1](#MOESM4){ref-type="media"}. Other relevant data supporting the findings of the study are available in this published article and its Supplementary Information files, or from the corresponding author upon request. Electronic supplementary material ================================= {#Sec22} Supplementary Information Peer Review File Description of Additional Supplementary Files Supplementary Data 1 Supplementary Data 2 **Electronic supplementary material** **Supplementary Information** accompanies this paper at 10.1038/s41467-017-02576-z. **Publisher\'s note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. We thank the Genomic Technologies Facility at the University of Lausanne for Illumina sequencing and technical support and all the patients and clinical staff who participated in the study. This work was supported by the Fondation Raoul Follereau, the Swiss National Science Foundation grant IZRJZ3_164174, the Swiss Cooperation and Development Center (CODEV), the Heiser Program of the New York Community Trust for Research in Leprosy (grant numbers P15-000827 and P16-000976), and grants CNPq 428964/2016-8 and CAPES PROAMAZONIA 3288/2013. S.T.C., P.S., C.A., and A.B. designed the study. C.A., P.S., S.G., A.N.B.F., and P.B. processed the samples, extracted DNA, and prepared sequencing libraries. A.B. and C.L. processed the data. A.B. analyzed the data and prepared figures and tables. A.B., C.A., and S.T.C. interpreted the results and wrote the manuscript with input from other authors. S.G., Y.M., M.N., K.B., C.G.S., M.B.S., R.C.B., M.A.C.F., F.B.F., J.G.B., J.A.C.N., S.B.-S., A.L., A.R.A.-S., Y.A.-Q., A.S.A., G.B., L.V.C., F.S., C.R.J., M.Ko., A.F., S.O.S., M.G., O.K., M.M.A.S., G.O.P., P.N.S., E.N.S., M.O.M., P.S.R., I.M.F.D.B., J.S.S., A.A., M.M., and M.Ka. participated in identification of leprosy cases, patient management, sample collection and preparation, and microscopy. Competing interests {#FPar1} =================== The authors declare no competing financial interests.

INTRODUCTION {#s1} ============ Bladder cancer (BC) is the 9th most common cancer worldwide, and the 4th most common cancer in more developed world \[[@R1]--[@R3]\]. In the last years, BC risk has been linked to the smoking, to the occupational exposures \[[@R4]\], to the use of drugs like cyclophosphamide or chlornaphazine \[[@R5]\], to the environmental pollutants and the arsenicals \[[@R6], [@R7]\], and to the chronic inflammation with the related up-regulation of some pro-inflammatory proteins like interleukin (IL)-6, tumor necrosis factor (TNF) and C-reactive protein (CRP) \[[@R8]\]. Moreover, a strong correlation between altered expression of various clock genes (PER1 and PER2) and common tumor markers (TP53, PTEN and PAI-1) evidenced that a disturbed function in the cellular clock system can clearly represent an another mechanism of BC progression \[[@R9]\]. In particular, many studies reported experimental evidences about the role of the environmental chemicals in BC carcinogenesis. In fact, the increased COX-2 expression and the activation of the mitogen activated protein kinase pathway in BC cells exposed to monomethylarsonous acid, a metabolite of inorganic arsenic, resulted a strong mechanism for BC carcinogenesis \[[@R10]\]. Moreover, it has been demonstrated in mice and rats the BC carcinogenicity of both 3-amino-1-methyl-5H-pyrido\[4,3-b\]indole, that is formed in cooked meat and fish \[[@R11]\], and pellets containing crude tryptophan pyrolysate and its derivates \[[@R12]\]. On the other hand, the exposure to high concentrations of diesel engine emissions, belonging to the sub-category of pollutants named as vehicle emissions, as well as insecticides, fungicides and pesticides resulted to contribute to carcinogenesis \[[@R13]--[@R15]\]. Our research group works in Campania, a region of Italy, in which BC incidence is very high with 75.3 cases per 100000 inhabitants, in comparison to only 19 cases per 100000 inhabitants in Europe in 2012 \[[@R3]\]. In the last decades, large areas of the Naples and Caserta provinces have been extensively contaminated by the widespread burial and open air dumping and incineration of industrial toxic waste \[[@R16]\]. Recently, the serum levels of arsenic measured in living subjects of five municipalities in the Naples province resulted to be higher than expected compared to national average \[[@R16]\]. Since the etiology of BC development is multi-factorial, only its early detection can reduce mortality. Hence, the development of new non-invasive biomarkers would benefit patients. Some reviews in literature report a summary of studies conducted through different omics approaches aimed to evaluate the transcriptome, the miRNome, the metabolome and the long non coding RNAs profiling involved in BC \[[@R16]--[@R21]\]. However, any detailed information is reported until now about what genes/proteins modulated by environmental exposure or by only arsenicals are involved in BC development or progression, how they are correlated, and what metabolic pathways are affected. Therefore, our aim was to select the genes/proteins modulated by environmental exposure (comprising arsenicals, pollutants, smoking, insecticides, fungicides and pesticides) or by only arsenicals in BC starting from Comparative Toxicogenomics Database (CTD), and to correlate them by a computational network approach. Through this method, we identified: i) the specific HUB nodes, that are nodes with a large degree and have connections with many other nodes, ii) the HUB-HUB interaction sub-network between HUB nodes that can be considered as specific of the correlation between BC and environment, and, hence, of the BC development due to environmental exposure, iii) the correlations between HUB nodes with circadian genes and miRNAs, iv) effect of HUB nodes on survival outcome of BC patients and their mutational status in this cancer. Therefore, further experimental studies could be focused on these HUB nodes to verify their utility as new diagnostic and/or prognostic biomarkers as well as potential targets for chemoprevention approach. RESULTS AND DISCUSSION {#s2} ====================== Selection of environmental chemicals implicated in BC and the related modulated proteins {#s2_1} ---------------------------------------------------------------------------------------- To create an interaction network between proteins modulated by environmental exposure in BC, we followed a protocol reported in Figure [1](#F1){ref-type="fig"}. Firstly we extracted the list of environmental chemicals implicated in BC and of the related proteins modulated by them using Comparative Toxicogenomics Database (CTD) \[[@R22]\]. The selected chemicals were grouped in the four following categories: arsenicals, smoking, pollutants and others, comprising insecticides, fungicides and pesticides, by considering also the studies already reported in literature \[[@R10]--[@R15]\] ([Supplementary Table 1](#SD1){ref-type="supplementary-material"}). About the identified proteins, we found 51 proteins modulated by arsenicals, 40 by smoking molecules, 94 by pollutants and 73 by other chemicals (Figure [2](#F2){ref-type="fig"} and [Supplementary Table 2](#SD1){ref-type="supplementary-material"}). A molecular pathway analysis was conducted on all the proteins belonging to these four categories ([Supplementary Table 3](#SD2){ref-type="supplementary-material"}), and the molecular pathways, that were common or specific between the four categories, were extracted ([Supplementary Table 4](#SD2){ref-type="supplementary-material"}). In details, we can evidence the presence of some common pathways, that are strictly correlated to the cancer, among which: i) TNF and NOD-like receptor signaling pathway were common between arsenicals and smoking sub-groups; ii) Rap1, estrogen, prolactin, TGF-beta and VEGF signaling pathways were common between arsenicals, pollutants and "others" sub-groups; iii) hematopoietic cell lineage and Cytokine-cytokine receptor interaction molecular pathway were common between smoking and "others" sub-groups; iv) chemical carcinogenesis was common between smoking, pollutants and "others" sub-groups, and v) drug metabolism - cytochrome P450 was common between smoking and pollutants sub-groups. However, it is important to underline that, although different categories of chemicals are able to regulate similar pathways even if through different proteins along the same pathway, they can generate distinct molecular/cellular outputs. {#F1} {#F2} Moreover, we evaluated also the pathways that are specific for each chemical category, and found for example: i) regulation of actin cytoskeleton, Wnt and Chemokine signaling pathway for arsenicals; ii) drug metabolism - other enzymes and metabolism of xenobiotics by cytochrome P450 for pollutants; iii) AK-STAT signaling pathway for "others" sub-group. Any specific molecular pathway for smoking was evidenced ([Supplementary Table 4](#SD2){ref-type="supplementary-material"}). Moreover, a detailed analysis of the proteins modulated by only one chemical sub-category showed that FBXW8 was modulated only by arsenicals, ANXA2R only by smoking chemicals, and nineteen proteins (ASXL2, RALGPS1, STAG2, SMC1B, SMC1A, TYMP, SLC12A7, ARID1A, TACC3, APOBEC3B, MLL2, LOXL4, CALMHM1, NRSN1, EOMES, KMT2A, CHD6, VWA3A, KDM6A) only by pollutants (Figure [2](#F2){ref-type="fig"} and [Supplementary Table 2](#SD2){ref-type="supplementary-material"}). In details, FBXW8 is a member of the F-box protein family that acts in phosphorylation-dependent ubiquitination whereas ANXA2R may act as a receptor for annexin A2 that plays a role in the regulation of cellular growth and in signal transduction pathways. Regarding the nineteen proteins modulated by the pollutants, only STAG2, SMC1A and SMC1B resulted to be involved in the enriched molecular pathway named cell cycle. However, through the Venn diagram analysis, we verified also the presence of twenty-two proteins that are modulated in BC by all four chemical sub-groups: CDH1, KRAS, IGFBP3, GSTM1, IGF1, TP53, IGFBP5, GSTP1, PTGS2, TNF, GPX1, CXCL8, CDKN2A, SOD2, EGFR, NQO1, MYC, CDKN1A, MT2A, BIRC3, FAS and ESR1 (Figure [2](#F2){ref-type="fig"}). These proteins are involved in the following molecular pathways: p53 signaling, MAPK signaling, Apoptosis, ErbB signaling, Cell cycle and Glutathione metabolism ([Supplementary Table 5](#SD1){ref-type="supplementary-material"}). Since our principal aim was to identify a sub-network of genes/proteins modulated by environmental exposure in BC, we decided to focus our attention on the proteins modulated by all four chemical sub-groups and to create the general network of interaction between BC and environment. Identification and analysis of HUB nodes in "BC and environment" network {#s2_2} ------------------------------------------------------------------------ On the basis of the human molecular interactome and of the list of the twenty-two proteins, reported above, the "BC and environment" interaction network was created and analyzed. In detail, we mapped our twenty-two proteins on the human molecular interactome (INTACT) \[[@R23]\], extracted their related interaction network (named as "BC and environment") composed by 1839 nodes and 2376 interactions and analyzed it by the related topological properties, reported in the Methods section, in order to understand the position and the role of these proteins present as nodes in the network ([Supplementary Figure 1](#SD1){ref-type="supplementary-material"}) \[[@R24]\]. In this way, we selected HUB nodes, which are the nodes with the strongest coordination role by considering a consensus of four out of six measures of centrality and topology (see Methods section). The following fifteen HUB nodes were identified: KRAS, IGFBP3, GSTM1, TP53, GSTP1, TNF, CXCL8, CDKN2A, SOD2, EGFR, NQO1, MYC, CDKN1A, FAS, and ESR1. They resulted to be up-regulated in BC by microarray studies with the exception of GSTP1 and ESR1 \[[@R25]--[@R31]\], and be involved in specific molecular functions and pathways ([Supplementary Tables 5](#SD1){ref-type="supplementary-material"} and [6](#SD1){ref-type="supplementary-material"}). We can speculate that the most part of identified HUB nodes is involved in molecular pathways correlated to cancer development and do not seem specific for BC. However, among these nodes, it is important to evidence the presence of GSTP1 and ESR1, about which few information is reported in BC. In general, GSTP1 catalyzes the conjugation of many hydrophobic and electrophilic compounds with reduced glutathione. It is reported that its polymorphisms are among the genetic determinants related to lead-induced inflammatory response and may modulate the response to epithelial oxidative changes caused by air pollutant exposure in lung \[[@R32]\]. Hence, it could be interesting and useful to study how GSTP1 is involved in BC development after environmental chemical exposure. In regard to ESR1, it is an estrogen receptor involved in sexual development and reproductive function. Considering that some environmental chemicals such as endosulfan and dieldrin are endocrine disruptors that can cause negative effects on the endocrine functions by miming the action of steroid hormones due to their structure similar to these last ones, we can think in future to investigate in more detail how ESR1 can be affected by these chemicals and through what mechanisms it can be involved in BC carcinogenesis. About the obtained network a detailed analysis of the statistical centrality and topological measures has permitted to evidence its effectiveness and its robustness. In fact, it is important to underline that our network has a centralization value of 0.423, a network density of 0.001, a heterogeneity value of 9.899 and characteristic path length of 3.330. Overall these data evidenced that: i) the network effectiveness is elevated with nodes that are highly correlated between them; ii) the network is of small world type characterized by short path lengths \[[@R33]\]. Moreover, the plot of the node degree distribution showed a decreasing trend demonstrating that our network had scale free property indicating that it follows the role that "riches get richer" ([Supplementary Figure 2](#SD1){ref-type="supplementary-material"}) \[[@R34]--[@R37]\]. On the other hand the clustering coefficient graph showed a decreasing trend highlighting the tendency of our network to contain HUB nodes \[[@R37]\]. However, considering that the betweenness centrality is a measure to obtain inferences on the importance of inter-connected proteins on the basis of load placed on the given node in the network, the increasing trend of the betweenness centrality in our network demonstrated that the following five HUB nodes had the maximum load: i) TP53 (tumor protein p53), a DNA binding tumor suppressor protein, ii) MYC (c-MYC), a multifunctional and nuclear phosphoprotein that plays a role in cell cycle progression, apoptosis and cellular transformation, iii) EGFR (Epidermal Growth Factor Receptor), a cell surface receptor, iv) ESR1 (estrogen receptor), a nuclear hormone receptor and v) CDKN1A (Cyclin Dependent Kinase Inhibitor 1A) which functions as a regulator of cell cycle progression at G1. Then, to study if in the network there were clusters and/or modules characterized by groups of nodes correlated between them, a cluster analysis was performed considering as statistically significant only the clusters with p-values lower than 0.001. In this way four clusters with density values ranging from 0.004 to 0.170 were selected ([Supplementary Figure 7](#SD1){ref-type="supplementary-material"}). They comprised639, 345, 68 and 25 proteins/nodes, respectively, and five HUB nodes. In detail, EGFR was in cluster 1, MYC in cluster 2, ESR1 in cluster 3, and GSTP1 and NQO1 in cluster 4. These clusters were named as EGFR cluster, MYC cluster, ESR1 cluster and GSTP1-NQO1 cluster, respectively. A functional analysis performed on the proteins present in the four clusters evidenced that each cluster comprised proteins involved in different metabolic pathways in comparison to those present in the other clusters. In fact, the only common pathways between at least two clusters were: i) "cell cycle and Epstein-Barr virus infection" in the case of EGFR and MYC clusters, ii) "RNA transport" in the case of ESR1 and MYC clusters, and iii) "proteoglycans in cancer" in the case of EGFR and ESR1 clusters ([Supplementary Table 7](#SD2){ref-type="supplementary-material"} and [Supplementary Figure 4](#SD1){ref-type="supplementary-material"}). Overall the cluster analysis demonstrated that our "BC and environment" network comprised specific functional sub-networks in which some HUB nodes play crucial roles. Then, to select the sub-network of HUB nodes that were more correlated between them and to define the related HUB -- HUB interaction sub-network, we extracted the interactions between HUB nodes in "BC and environment" network. In this way, it was possible to evidence that: i) four HUB nodes exhibited direct HUB--HUB interactions in the network (EGFR-IGFBP3 and TP53-CDKN1A) and ii) the other HUB nodes were linked between them through only one node (Figure [3](#F3){ref-type="fig"}). This finding suggested the strict functional relationship between these HUB nodes and how the HUB -- HUB sub-network could represent a panel of proteins on which we can focus further studies in order to verify the possibility to use them as specific of the involvement of the environmental chemicals in BC initiation. {#F3} Moreover, the mutational status of the identified HUB nodes was evaluated. This analysis showed that: i) eight HUB nodes (ESR1, FAS, GSTM1, IFGBP3, KRAS, SOD2 and TNF) did not show mutations in BC, ii) CXCL8, NQO1 and MYC had one missense mutation, iii) GSTP1 had two missense mutations, iv) CDKN1A and EGFR had three missense mutations, v) CDKN2A had four missense mutations and vi) TP53 had thirty-eight missense mutations ([Supplementary Table 8](#SD2){ref-type="supplementary-material"}). Moreover, the co-occurrence of three mutations, i.e. T384S in EGFR and P151H in TP53, D136H in CDKN1A and R273S in TP53, and G146V in GSTP1 and G245S in TP53, resulted as statistically significant (p-value \<0.001) suggesting their importance in BC initiation. Then, to highlight in greater detail the involvement of these HUB nodes in BC development, we searched if they correlated with clock genes already reported to be altered in BC \[[@R9]\], and if they can be target of miRNAs reported to be implicated in BC \[[@R38]\]. In general, the circadian clock system comprises both negative and positive regulators, based on an auto-regulatory transcriptional and translational feedback program. In this context, PER and CRY proteins bind to the promoter region of BMAL1 and CLOCK, that are two transcription factors, and are capable to reduce the transcription of many genes during ambient light exposure \[[@R9]\]. In particular, the basic helix-loop-helix (bHLH)/PAS domain transcription factor plays a crucial role in the controlling the biological clock that controls the circadian rhythms. Also the urinary system is regulated from the circadian rhythms. In fact, during day and night both urine excretion and extrusion are actively regulated by several internal factors and hormones. Such circadian variations led us to postulate that similar to other organs, the perturbation of the clockwork may contribute to the dysregulation that develops during BC development \[[@R9]\]. Since clock genes are able to modify the gene regulation, they may interact with the transcription of oncogenes and/or tumour suppressor-genes. In fact, a recent paper reported the close correlation between altered expression of various clock genes and common tumor markers in BC evidencing how a disturbed function in the cellular clock may be an important additional mechanism contributing to cancer progression \[[@R9]\]. Hence, we searched if there were correlations between the proteins codified from circadian genes (PER1, PER2, PER3, CRY1, CRY2, BMAL1, CLOCK, ANTI-CSNK1α1L, CSNK1α and CSNK1ε) and our HUB nodes. As visible in [Supplementary Figure 5](#SD1){ref-type="supplementary-material"}, there was a direct correlation between TP53 (HUB node) and CSNK1ε (circadian node) and other circadian nodes correlated with the other HUB nodes through one or two nodes. This demonstrated the strict relationship between circadian rhythms and HUB nodes and confirmed how our HUB nodes can have an important role in BC development in according to the other previous analysis. The list of the miRNAs implicated in BC by microarray studies and verified experimentally by RT-PCR, and the list of the genes targeted from these miRNAs was extracted by mirNET database ([Supplementary Table 9](#SD1){ref-type="supplementary-material"}) \[[@R38]\]. In this way, it was possible to select only the miRNAs able to target our HUB nodes. In detail, our analysis evidenced that thirteen miRNAs (hsa-mir-7-5p, hsa-mir-17-5p, hsa-mir-26a-5p, hsa-mir-30a-3p, hsa-mir-30c-5p, hsa-mir-30e-5p, hsa-mir-101-3p, hsa-mir-125b-5p, hsa-mir-133b, hsa-mir-199a-3p, hsa-mir-520b, hsa-mir-639 and hsa-mir-644a) correlated with seven HUB nodes (EGFR, SOD2, MYC, KRAS, ESR1, CDKN1A and TP53) (Figure [4](#F4){ref-type="fig"}). {#F4} In addition, starting from these data, to understand if our HUB nodes can have a clinical validity and utility, we decided to perform a set of bioinformatics analyses on available gene expression datasets BC (TCGA-BLCA). The purpose was to assess if there was an association between the expression in BC of our HUB nodes and patients survival. Our analysis evidenced that high expression of EGFR, TP53, MYC, GSTP1, NQO1 and KRAS as well as the association of high co-expression of TP53-EGFR, MYC-EGFR, KRAS-EGFR, NQO1-EGFR, TP53-MYC had a negative effect on the survival (Table [1](#T1){ref-type="table"} and [Supplementary Figure 6](#SD1){ref-type="supplementary-material"}). ###### Overall survival related to high expression/high co-expression of HUB nodes in the "BC and environment" and "BC and arsenicals" networks by SynTarget online tool using public TCGA_BLCA dataset (Bladder Urothelial Carcinoma)^31^ Nodes Suvival effect p-Value -------------------------------- ---------------- ---------- **BC and environment network** EGFR Negative 0.00124 TP53 Negative 0.00898 MYC Negative 0.0276 GSTP1 Negative 0.0478 NQO1 Negative 0.0379 KRAS Negative 0.017 TP53-EGFR Negative 0.000301 MYC-EGFR Negative 5.23e-06 KRAS-EGFR Negative 0.000591 NQO1-EGFR Negative 0.000134 TP53-MYC Negative 0.00835 **BC and arsenicals network** KRAS Negative 0.017 ERCC4 Negative 0.00424 ERCC4-KRAS Negative 0.0385 The p-values lower than 0.05 were considered as statistically significant. Identification and analysis of HUB nodes in "BC and arsenicals" network {#s2_3} ----------------------------------------------------------------------- Since BC incidence is very high in the Campania region in Italy \[[@R3]\] and the SEBIOREC study demonstrated an higher level of arsenic in the serum of subjects living in the province of Napoli compared to national average \[[@R16]\], a particular attention was directed to establish an interaction between arsenicals and fifty-one proteins known to be modulated by them (Figure [2](#F2){ref-type="fig"}). In this study we followed the same protocol used in the case of "BC and environment" network (Figure [1](#F1){ref-type="fig"}). As reported in the first Results paragraph, the functional analysis on these proteins evidenced that they were involved in a set of molecular pathways ([Supplementary Table 3](#SD2){ref-type="supplementary-material"}). These fifty-one proteins were mapped on the human molecular interactome and the related interaction network named "BC and arsenicals" network was extracted and analyzed by different topological properties, as reported above for the "BC and environment" interaction network ([Supplementary Figure 7](#SD1){ref-type="supplementary-material"}) \[[@R24]\]. This network comprised 353 nodes (proteins) and 378 interactions (edges), and the following nine HUB nodes: PSMB2, TNF, BIRC3, FANCA, KRAS, CCNE1, ERCC4, PABPC1, and PRSS3. It is important to evidence that two HUB nodes, TNF and KRAS, were in common with the "BC and environment" interaction network whereas the others are uniquely regulated by arsenicals. Notably, all the HUB nodes, at exception of BIRC3 and FANCA, were already resulted to be up-regulated in BC by microarray studies \[[@R25]--[@R31], [@R39]--[@R43]\], and be involved in specific molecular functions ([Supplementary Table 10](#SD1){ref-type="supplementary-material"}). However, among the proteins already studied in BC, PSMB2 is a very interesting node; in fact, it is hypermethylated in BC and was identified as indicator of adverse health effects associated with arsenic exposure \[[@R44]\]. On the other hand, very few information is reported about BIRC3 and FANCA in BC. In particular, BIRC3 inhibits apoptosis by binding to tumor necrosis factor receptor-associated factors, and is dysregulated in some cancers, and FANCA is a DNA repair protein that may operate in a post-replication repair or a cell cycle checkpoint function. Notably, it could be interesting to study in future how these two proteins can be affected by arsenicals and through what mechanisms they can be involved in BC carcinogenesis. A detailed analysis of the obtained network showed that it had a good centralization equal to 0.402 and a network density value of 0.006. The characteristic path length of 3.752 confirmed that also this network followed the small-world rule \[[@R32]\] as we evidenced for "BC and environment" network. Moreover, we can underline also that: i) the decreasing trend of the node degree distribution plot indicated that our network had scale free property with the occurrences of modules ([Supplementary Figure 8A](#SD1){ref-type="supplementary-material"}) \[[@R26]--[@R29]\], ii) the decreasing trend of the clustering coefficient graph showed the tendency of our network to contain HUB nodes ([Supplementary Figure 8B](#SD1){ref-type="supplementary-material"}) \[[@R43]\], and iii) the increasing trend of the betweenness centrality demonstrated the presence of a HUB node with maximum load like PABPC1 (Polyadenylate-Binding Protein 1) that is involved in cytoplasmic regulatory processes of mRNA metabolism such as pre-mRNA ([Supplementary Figure 8C](#SD1){ref-type="supplementary-material"}). Then, to study if in the "BC and arsenicals" network there were modules characterized by groups of nodes correlated between them, a cluster analysis was performed as in the case of "BC and environment" network. This showed the presence of two clusters (with significant P-values lower than 0.005) comprising 15 and 10 proteins/nodes with density values ranging from 0.608 to 0.644 and three HUB nodes ([Supplementary Figure 9](#SD1){ref-type="supplementary-material"}). In detail, CCNE1 was in cluster 1 whereas TNF and BIRC3 were in cluster 2. However, cluster 1 comprised also CDKN1A, a HUB node related to "BC and environmental" network. The proteins present in cluster 1 resulted to be involved in specific metabolic pathways such as PI3K-Akt signaling pathway, p53 signaling pathway and cell cycle, whereas those in cluster 2 in Apoptosis, NF-kappa B signaling pathway, TNF signaling pathway and Adipocytokine signaling pathway. However, the only common pathway between two clusters is "Pathways in cancer" ([Supplementary Table 11](#SD1){ref-type="supplementary-material"} and [Supplementary Figure 10](#SD1){ref-type="supplementary-material"}). As in the case of "BC and environment" network, cluster analysis highlighted that the "BC and arsenicals" network comprised two sub-networks in which the identified HUB nodes played important functional roles. Moreover, to select the HUB -- HUB interaction sub-network in "BC and arsenicals" network, we focused on the interactions between HUB nodes, and evidenced that two HUB nodes (TNF-BIRC3) exhibited direct HUB--HUB interactions in network whereas the other HUB nodes were linked among them through one or two nodes (Figure [5](#F5){ref-type="fig"}). Moreover, three HUB nodes identified for the arsenicals (ERCC4, PABPC1 and CCNE1) resulted to interact directly with three HUB nodes evidenced in the "BC and environment" network (ERCC4-EGFR, PABPC1-ESR1, and CCNE1-CDKN1A) suggesting the strict correlation between "BC and arsenicals" and "BC and environment" networks. {#F5} However the analysis of HUB-HUB interaction sub-network in "BC and arsenicals" network evidenced how, with the exception of TNF and KRAS being in common between two networks, the remaining seven HUB nodes (PSMB2, BIRC3, FANCA, CCNE1, ERCC4, PABPC1, and PRSS3) can be considered as specific of the arsenicals involvement in BC development. The analysis of the mutational status of all the HUB nodes, showed that: i) six HUB nodes (CCNE1, KRAS, PABPC1, PRSS3, PSMB2 and TNF) had not mutations in BC, ii) BIRC3, ERCC4 and FANCA had one, two and four missense mutations, respectively ([Supplementary Table 12](#SD1){ref-type="supplementary-material"}). No co-occurrence mutations resulted statistically significant. Also in the case of "BC and arsenicals network", we evaluated if there was a correlation between our HUB nodes and the clock genes resulted to be altered in BC \[[@R9]\]. As visible in [Supplementary Figure 11](#SD1){ref-type="supplementary-material"}, there was no direct correlation between HUB nodes and circadian nodes but the circadian nodes correlated with the HUB nodes through one or two nodes. This suggested that the relationship between circadian rhythms and HUB nodes in "BC and arsenicals" network was weaker in comparison to what observed for the "BC and environment" network. Then, we evaluated if our HUB nodes can be targets of miRNAs, resulted already as implicated in BC using the same protocol reported above. This analysis evidenced that seven miRNAs (hsa-mir-7-5p, hsa-mir-17-5p, hsa-mir-26a-5p, hsa-mir-30a-3p, hsa-mir-125b-5p, hsa-mir-520b and hsa-mir-646) correlated with four HUB nodes (FANCA, CCNE1, KRAS and PABPC1) (Figure [6](#F6){ref-type="fig"} and [Supplementary Table 13](#SD1){ref-type="supplementary-material"}). {#F6} Among these miRNAs, only hsa-mir-646 did not correlate with the HUB nodes of "BC and environmental" network. Hence, it could be specific of the involvement of arsenicals in BC development. Finally, we evaluated if there was an association between the expression of our HUB nodes in BC and patients survival as in the case of "BC and environmental" network. Our analysis showed that only the high expression of ERCC4 and KRAS and their high co-expression (ERCC4-KRAS) resulted to have a negative effect on the survival in BC patients ([Supplementary Figure 12](#SD1){ref-type="supplementary-material"} and Table [1](#T1){ref-type="table"}). CONCLUSIONS {#s3} =========== No many papers were already published about the direct linking between the environmental or arsenic exposure and the related modulated proteins in BC. For example, it has been shown that arsenic exposure is positively associated with PRSS3 promoter methylation levels in BC \[[@R45]\] whereas the effect of NQO1 Pro187Ser polymorphism resulted to be more important in never smokers because no consistent results were obtained about tobacco-related BC risk \[[@R46]\]. Therefore, aim of this work was to highlight the correlation between genes and proteins that are modulated by environmental exposure or only by the arsenicals by network approaches, and to identify the related HUB nodes that can be considered as specific of the BC development due to environmental or arsenicals exposure, and on which can be useful to focus further experimental studies in order to verify their utility as new diagnostic and/or prognostic biomarkers or targets. In summary, in our study we reported the creation of two networks named as "BC and environment" and "BC and arsenicals", defined a set of HUB nodes and the related HUB-HUB interactions, and evidenced that these HUB nodes showed significant mutations implicated in BC and correlated with genes involved in circadian rhythms. In addition, we identified: i) a sub-network of interactions between miRNAs and genes that is specific of the correlation between BC and environment or arsenicals, and ii) the genes correlated to negative effect on the survival in BC patients. On the basis of our results, we can underline that many identified HUB nodes in "BC and environment" and "BC and arsenicals" networks are proteins already known as to be involved in molecular pathways correlated to the development of cancer and BC. However, we evidenced also the presence of other proteins for which molecular interactions in BC were unknown. For example in "BC and environment" network we found the estrogen receptor, ESR1, that can be object of further investigations because some environmental chemicals are endocrine disruptors that can mimic its structure. Hence, this finding can cause problems in the endocrine system and could represent an initiation point for BC. Moreover, in the case of "BC and arsenicals" network, we identified two HUB nodes, BIRC3 and FANCA for which the role in BC is unknown. Therefore, it can be useful to study if and how these two proteins can be affected by arsenicals and if there are specific mechanisms through which they can contribute to BC carcinogenesis. Certainly these hypothesis may be verified by experimental studies but could represent starting points for the identification of new markers for BC. Finally, it is important to underline that all our data fall within the SPES project (<http://spes.campaniatrasparente.it/>), in which our group is involved, that is an exposure study in susceptible population. Its aim is to evaluate the effects on human health of different sources of contamination. Through the analysis of the spatial distribution of the sources of contamination and the concentration values of exposure, genetic susceptibility, immune and oxidative biomarkers in biological fluids of youth living groups in the "Land of Fires" municipalities, it will be possible to identify areas that have the same potential index of risk. Therefore, the SPES project is collecting sera from healthy donors/patients of susceptible population to contamination, and, hence, the network studies in this work can provide knowledge useful for further hypothesis-driven experimental studies and targets discovery in BC. In fact, the identification of chemicals-regulated proteins could help to search for specific markers in selected populations. Therefore, considering the results of this study, we could think to evaluate: i) the expression of TP53 and EGFR, and their mutations (T384S of EGFR and P151H of TP53) in sera of susceptible individuals to the environmental chemicals exposure to verify if they can be used as markers for BC and ii) the expression of ERCC4, KRAS and of hsa-mir-646 in individuals exposed to contamination of drinking water with arsenicals to understand if and what among these three molecules can be used as markers for BC initiation. MATERIALS AND METHODS {#s4} ===================== Network analysis {#s4_1} ---------------- Comparative Toxicogenomics Database (CTD) was used to extract the list of environmental chemicals implicated in BC and the related chemical--protein interactions \[[@R22]\]. Through the Cytoscape software platform for the visualization of complex networks and their integration (<http://www.cytoscape.org/>), a network related to the interactions between the proteins modulated by the selected environmental chemicals was constructed using as reference the human molecular interactome (INTACT) \[[@R23]\] where all interactions are derived from literature curation or direct user submissions and are freely available. Some statistical analyses were performed the following three measures of centrality: i) the degree that indicates the number of interactions of a particular node with other nodes in the network; ii) the betweenness centrality that evaluates the importance of a node in the network and how the other interactions in the network are controlled by this node \[[@R47]\]; and iii) the closeness centrality of a node that is calculated as the sum of the length of the shortest paths between the node and all other nodes in the graph and ranges from 0 to 1 \[[@R48]\]. Then, we evaluated also other topological analyses like average characteristic path length, network density, and centralization \[[@R48]--[@R50]\]. The characteristic path length is calculated by finding the shortest path between all pairs of nodes, adding them up, and then dividing by the total number of pairs. This indicates the number of steps that takes to get from one member of the network to another. The density of a network is defined as a ratio of the number of edges to the number of possible edges \[[@R48]\] whereas the centralization produces rankings, which seek to identify the most important nodes in a network model ranging from 1 to 0 \[[@R49]\]. Finally, we performed a cluster analysis by means of Cluster-One \[[@R50]\] that is the task of grouping a set of objects in such a way that objects in the same group (called a cluster) are more similar to each other than to those in other groups (clusters) \[[@R51]\]. Functional and Pathway Analyses were performed by DAVID program \[[@R52]\]. miRNA evaluations {#s4_2} ----------------- The miRNAs able to target the HUB nodes were selected by MirNet tool \[[@R53]\]. In details, we performed the following protocol: firstly, we extracted the list of miRNAs involved in BC; secondly, starting from this miRNA list we extracted all their targets; finally we selected only the miRNAs that targeted our HUB nodes. Then, using the list of our HUB nodes and the related miRNAs, an interaction network between selected miRNAs and HUB nodes was constructed by the Cytoscape package. Survival gene effect analysis {#s4_3} ----------------------------- Bionformatic analyses were performed by SynTarget online tool able to test the synergetic effect of genes on survival outcome in cancer (<http://www.bioprofiling.de>) using public TCGA_BLCA dataset (Bladder Urothelial Carcinoma) \[[@R53]\]. In details, PPISURV tool was used to test if our genes can be used as biomarker of cancer survival. Gene mutation analysis {#s4_4} ---------------------- Mutation analysis was conducted by cBioPortal for Cancer Genomics tool able to analyze cancer genomics data in order to test the presence of mutations on genes involved in cancer (<http://cbioportal.org>) \[[@R54], [@R55]\]. SUPPLEMENTARY MATERIALS FIGURES AND TABLES {#s5} ========================================== **Author contributions** Alfredo Budillon, Gennaro Ciliberto, Susan Costantini and Maurizio Montella conceived this study; Andrea Polo and Susan Costantini performed the experiments; Anna Crispo, Pellegrino Cerino, Luca Falzone, Saverio Candido, Aldo Giudice, Giuseppina De Petro collaborated to the discussion of the results; All the Authors approved the final version of the manuscript. **CONFLICTS OF INTEREST** The authors declare no conflicts of interest. **FUNDING** This study was partially supported by Italian Ministry of Health to Istituto Nazionale Tumori di Napoli (progetto 5x1000 2016-2017). Andrea Polo is supported by a fellowship from the SPES project (<http://spes.campaniatrasparente.it/>)