We externally validated the recently suggested FSAC prediction model for hepatocellular carcinoma (HCC) in treatment-naïve Asian chronic hepatitis B patients starting potent antiviral therapy (AVT). The model reflects age, sex, presence of cirrhosis, and on-therapy changes in non-invasive fibrosis markers (NFMs) after 12 months of antiviral therapy, such as APRI and FIB-4. Our results highlighted better predictive performance for the FSAC model for HCC (Harrell’s c-index: 0.770) than the PAGE-B, modified PAGE-B, modified REACH-B, LSM-HCC, and CAMD models, which only use baseline parameters. A simplified version of FSAC score ), including only NFMs at 12 months, also showed a high c-index value (0.763). Our retrospective study suggests that the accurate measurement of intra-hepatic fibrotic burden during adequate AVT is necessary for predicting HCC development.n = 3026; median age, 50.0 years; male predominant (61.3%); cirrhosis in 1391 (46.0%) patients) receiving potent AVTs for >18 months between 2007 and 2018. During follow-up (median 64.0 months), HCC developed in 303 (10.0%) patients. Patients with low FIB-4 or APRI levels at 12 months showed significantly lower HCC risk than those with high NFM levels at 12 months . Cumulative 3-, 5-, and 8-year HCC probabilities were 0.0%, 0.3% and 1.2% in the low-risk group (FSAC ≤ 2); 2.1%, 5.2%, and 11.1% in the intermediate-risk group (FSAC 3−8); and 5.2%, 15.5%, and 29.8% in the high-risk group (FSAC ≥ 9) (both p < 0.001 between each adjacent pair). Harrell’s c-index value for FSAC score (0.770) was higher than those for PAGE-B (0.725), modified PAGE-B (0.738), modified REACH-B (0.737), LSM-HCC (0.734), and CAMD (0.742). Our study showed that the FSAC model, which incorporates on-therapy changes in NFMs, had better predictive performance than other models using only baseline parameters.Antiviral therapy (AVT) induces the regression of non-invasive fibrosis markers (NFMs) and reduces hepatocellular carcinoma (HCC) risk among chronic hepatitis B (CHB) patients. We externally validated the predictive performance of the FSAC prediction model for HCC using on-therapy NFM responses. Our multicenter study consecutively recruited treatment-naïve CHB patients ( Chronic hepatitis B virus (HBV) infection is a major public health problem affecting approximately more than 250 million people worldwide; it remains a leading cause of hepatocellular carcinoma (HCC), especially in endemic areas such as Korea ,2,3,4,5.Many models have been developed to help with predicting the risk of HCC development among HBV patients with or without AVT. Since the prognostic role of baseline serum HBV-DNA levels has been substantially attenuated in the present era of potent nucleos(t)ide analogues, models established within one decade  have adopted the presence of baseline cirrhosis rather than virological factors ,14,15. MHere, we aimed to externally validate the predictive performance of the newly developed FSAC model in comparison with other risk prediction models assessed at one time-point in an independent HBV cohort treated with ETV, TDF, or TAF.®  at the time of AVT initiation, as described in previous reports. Cirrhosis was histologically or clinically diagnosed as follows: (1) platelet count of <150/×103/μL and imaging findings suggestive of cirrhosis, including a blunted, nodular liver edge accompanied by splenomegaly (>12 cm) or (2) clinical signs of portal hypertension, such as gastroesophageal varices ) and were compared using Student’s The predictive performances of the risk scoring models for HCC development were assessed using Harrell’s C-index, time-dependent area under the receiver operating characteristic curve (TDAUC) at 3-, 5-, and 8-years from the index date and integrated area under the receiver operating characteristic curve (iAUC). Furthermore, lower values for the Akaike information criterion (AIC) were considered reflective of a better discriminatory ability for each model. Model performance was presented graphically using calibration plots, which compared the model prediction probability with the actual probability of HCC development. Discrimination and calibration were evaluated using the bootstrap method with re-sampling 1000 times.Statistical differences in parameters of predictive performance between FSAC and the other HCC-risk prediction models were evaluated using the bootstrap method with re-sampling 1000 times. If 95% CIs contained zero, there was deemed to be no significant difference in the parameters of predictive performance between the two models.http://cran.r-project.org/, accessed on 10 August 2021). Two-sided p-values < 0.05 were considered to indicate statistical significance.All statistical analyses were conducted using R package ; and higher median values of liver stiffness (14.3 vs. 7.7 kPa), FIB-4 (3.38 vs. 2.11), and APRI , compared to those without HCC   months, HCC developed in 303 (10.0%) patients (1.82 per 100 patient-years), and the cumulative probabilities of HCC development at 3-, 5-, and 8-years were 2.5%, 7.5%, and 15.3%, respectively. Patients who developed HCC were predominantly male (73.3% vs. 60.0%) and showed a significantly older age (median: 55.0 vs. 49.0 year); higher prevalence of cirrhosis (79.5% vs. 42.2%), diabetes mellitus (31.0% vs. 16.6%), hypertension (42.5% vs. 22.1%), and HBeAg negativity  . Likewisp = 0.001) and D , whose FIB-4 values at 12 months were ≥1.45, compared to those without , 952 (31.5%), 87 (2.9%), and 852 (28.2%) were classified into groups A, B, C, and D, respectively. Patients who developed HCC showed a higher prevalence of being classified into groups B  and D , whose APRI values at 12 months were ≥0.5, compared to those without , 733 (24.2%), 402 (13.3%), and 490 (16.2%) were classified into groups A, B, C, and D, respectively. Patients who developed HCC showed a higher prevalence of being classified into groups B . Subsequent multivariable analysis revealed that older age , male sex , presence of cirrhosis , higher liver stiffness values , hypertension , and lower platelet counts  were independently associated with an increased risk of HCC development , 0.769 (95% CI 0.745–0.791), 0.768 (95% CI 0.745–0.790), 0.768 (95% CI 0.743–0.789), and 0.769 (95% CI 0.744–0.791), respectively. The calibration plots for predicting 3-, 5-, and 8-year HCC development showed that the predicted probabilities were very close to the observed incidences .Harrell’s c-index values for PAGE-B, modified PAGE-B, modified REACH-B, LSM-HCC, and CAMD were 0.725 (95% CI 0.699–0.750), 0.738 (95% CI 0.712–0.764), 0.737 (95% CI 0.710–0.763), 0.734 (95% CI 0.706–0.762), and 0.742 (95% CI 0.715–0.768), respectively, whereas their iAUC values were 0.718 (95% CI 0.693–0.741), 0.722 (95% CI 0.701–0.744), 0.724 (95% CI 0.699–0.748), 0.731 (95% CI 0.705–0.755), and 0.743 (95% CI 0.717– 0.768), respectively . The FSAHarrell’s c-index and iAUC values of FSAC (2) model were 0.763 (95% CI 0.737–0.787) and 0.763 (95% CI 0.739–0.784), respectively .n = 1391, 46.0%) showed that the Harrell’s c-index and the iAUC value of the FSAC model were 0.668 (95% CI 0.633–0.701) and 0.661 (95% CI 0.627–0.694), respectively : scores 0–2); intermediate- (n = 1172 (38.2%): scores 3–8); and high-risk groups (n = 1009 (33.3%): scores 9–12), according to a previous study [p < 0.001, n = 0), 0.3% (n = 2), and 1.2% (n = 5) in the low-risk group; 2.1% (n = 23), 5.2% (n = 48), and 11.1% (n = 75) in the intermediate-risk group; and 5.2% (n = 49), 15.5% (n = 126), and 29.8% (n = 196) in the high-risk group, respectively (both p < 0.001 between each adjacent pair).Using FSAC score, we stratified patients into the three risk groups: low-  occurred during the median follow-up period of 64.0 months, allowing for highly acceptable statistical power. Second, among the low-risk group defined according to FSAC score (n = 845), only 6 patients developed HCC, with an annual incidence of 0.15%. Considering that current surveillance strategies to detect early-stage HCC may be cost-effective when annual risk exceeds at least 0.2% in patients without cirrhosis and 1.5% in patients with cirrhosis, the 27.9% of individuals in the low-risk group could likely avoid biannual abdominal ultrasonography-based HCC surveillance safely. Conversely, the intermediate- and high-risk groups, accounting for the remaining 72.1% of this study population, may require more delicate surveillance, given the suboptimal diagnostic sensitivity of abdomen ultrasonography and the overall poor prognosis of HCC detected at advanced stages. Thus, in order to achieve higher detection rates of HCC at early stages and greater cost-effectiveness, further studies on how to implement individualized surveillance strategies based on optimal visit intervals and the adoption of novel diagnostic modalities using radiology and/or serum biomarkers are warranted.Our study had several clinical implications. First of all, the prognostic performance of the FSAC model over other HCC-risk prediction models was reproduced. The large sample of >3000 patients with long-term follow-up enhanced the statistical reliability of the results. Moreover, a sufficient number of HCC cases ) which included only NFMs assessed at 12 months, also showed a Harrell’s c-index value similar to the FSAC model . The observations in this study are consistent with reports of excellent predictive performance for the CAGE-B and SAGE-B models that incorporate liver stiffness values on transient elastography after 5 years of AVT.n = 1102, 36.4%) [p < 0.001). Hence, further large-scale study with the serial follow-up of transient elastography during long-term AVT should be required.Although the predictive performance of FSAC model among our study population was acceptable, further research using novel biomarkers that can exclude the overestimation caused by active necro-inflammation before starting AVT  should be required in the near future , in orde, 36.4%) , we confOur study has several limitations. First, although liver stiffness values by transient elastography were available in all patients at the baseline, approximately two thirds of patients did not undergo transient elastography during long-term AVT, primarily because it is not reimbursed by the National Health Insurance Service in Korea. Since the predictive performances might vary depending on the sample size and HCC incidence, further studies are required in the setting of paired transient elastography tests. Second, when the HCC prediction models were assessed among a subgroup with cirrhosis, their prognostic performances were generally attenuated. This is most likely because the discriminatory power of the variables constituting the models  might become considerably attenuated in the relatively “homogenous” cirrhotic subgroup. Given that the majority of HCC rises in the setting of cirrhosis, especially among patients in Western countries, further studies concerning development of novel biomarkers should be required, allowing the optimized prognostication in a subgroup with cirrhosis.In this external validation study of a large-scale cohort, the FSAC model exhibited more robust predictive performance for HCC development, compared to other HCC-risk prediction models that use only baseline characteristics. For predicting HCC development, accurate measurement of fibrotic burden during long-term AVT is necessary.


Pseudomonas aeruginosa. The activity of the investigational combination cefepime-taniborbactam (FTB) and comparator agents was evaluated against clinical isolates of Enterobacterales from a 2018-2020 global surveillance study.Taniborbactam (formerly VNRX-5133) is a novel cyclic boronate-based broad-spectrum β-lactamase inhibitor with potent and selective direct inhibitory activity against both serine- and metallo-β-lactamases . Taniborbactam restores the activity of cefepime against many difficult to treat organisms, including cephalosporin- and carbapenem-resistant Enterobacterales and MICs of cefepime with taniborbactam fixed at 4 µg/mL and comparators were determined following CLSI M07-A11 guidelines against 10,543 Enterobacterales. Isolates were from community and hospital infections collected from 259 sites in 56 countries in 2018-2020. Resistant phenotypes were based on 2021 CLSI breakpoints. A set of 827 isolates with meropenem MIC ≥4 µg/mL (n=421) or with cefepime and/or ceftazidime MIC ≥2 µg/mL (n=406) was evaluated for the presence of MBLs, KPC, ESBLs, and OXA-48 group genes via PCR and sequencing. Forty-eight isolates with FTB MIC values of 16 µg/mL or greater were interrogated by WGS.50/90 values of 0.06/0.25 µg/mL and 99.5% inhibited at ≤8 µg/mL. FTB maintained activity against MBL-, KPC-, OXA-48 group, and ESBL-positive isolates . Isolates with elevated FTB MICs had IMP-type enzymes, variation in the cefepime target (penicillin binding protein 3), permeability defects in combination with acquired β-lactamases, and/or possible up-regulated efflux.Overall, 23.0% and 15.9% of isolates were nonsusceptible (NS) to cefepime and piperacillin-tazobactam (TZP), respectively (Table). FTB had potent activity against all Enterobacterales, with MICResults Tablein vitro activity of cefepime against Enterobacterales, including isolates nonsusceptible to recently-approved BL/BLI combinations and expressing serine and metallo-β-lactamases. This support the continued development of FTB as a potential new treatment option for challenging infections due to resistant Gram-negative pathogens.Taniborbactam significantly restored the Meredith Hackel, PhD MPH, IHMA (Employee)Pfizer, Inc. (Independent Contractor) Mark G G. Wise, PhD, IHMA (Employee)Pfizer, Inc. (Independent Contractor) Daniel F. Sahm, PhD, IHMA (Employee)Pfizer, Inc. (Independent Contractor)


Mycobacterium kansasii—termed rBCG-Mkan85B—which was used together with a booster immunization with plasmid DNA expressing the same M. kansasii Ag85B gene (DNA-Mkan85B). We reported that rBCG-Mkan85B/DNA-Mkan85B prime–boost immunization elicited various NTM strain-specific CD4+ and CD8+ T cells and induced Mycobacterium tuberculosis-specific immunity. In this study, to investigate the protective effect against M. kansasii infection, we challenged mice vaccinated with a rBCG-Mkan85B or rBCG-Mkan85B/DNA-Mkan85B prime–boost strategy with virulent M. kansasii. Although BCG and rBCG-Mkan85B immunization each suppressed the growth of M. kansasii in the mouse lungs, the rBCG-Mkan85B/DNA-Mkan85B prime–boost vaccination reduced the bacterial burden more significantly. Moreover, the rBCG-Mkan85B/DNA-Mkan85B prime–boost vaccination induced antigen-specific CD4+ and CD8+ T cells. Our data suggest that rBCG-Mkan85B/DNA-Mkan85B prime–boost vaccination effectively enhances antigen-specific T cells. Our novel rBCG could be a potential alternative to clinical BCG for preventing various NTM infections.The incidence of infections with nontuberculous mycobacteria (NTM) has been increasing worldwide. The emergence of multidrug-resistant NTM is a serious clinical concern, and a vaccine for NTM has not yet been developed. We previously developed a new recombinant Bacillus Calmette–Guérin (rBCG) vaccine encoding the antigen 85B (Ag85B) protein of  Mycobacterium tuberculosis complex and Mycobacterium leprae, such as Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium kansasii. Recently, the incidence of NTM infection has been increasing worldwide [Nontuberculous mycobacteria (NTM) are mycobacterium species other than the orldwide ,2,3. Altorldwide ,5,6,7,8.Since NTM pathogens are intracellular, cell-mediated immunity plays a major role in protecting against NTM. Previous reports have indicated that protective immunity against tuberculosis (TB) and NTM infection might overlap . EpidemiM. kansasii—termed rBCG-Mkan85B—which we used with a booster vaccination with plasmid DNA expressing the same M. kansasii Ag85B gene (DNA-Mkan85B) [M. tuberculosis and M. leprae [+ T cells and CD8+ T cells [d)-restricted epitopes that induced cross-reactive responses to M. tuberculosis and other related mycobacteria, including M. kansasii, in both BALB/c (H2d) mice and CB6F1 (H2b/d) mice [d-restricted peptide epitopes elicited polyfunctional CD8+ T cell responses that were also highly cross-reactive with those of other proteins in the Ag85 complex [M. kansasii, we challenged rBCG-Mkan85B/DNA-Mkan85B-prime–boost-vaccinated CB6F1 mice with M.kansasii and evaluated the protective effect and cellular immunity in this study.Previously, we developed a new recombinant BCG (rBCG) vaccine encoding the antigen 85B (Ag85B) protein of Mkan85B) . The Ag8Mkan85B) . The Ag8. leprae ,16. We r. leprae . We also T cells . Moreove/d) mice . The H2- complex . TherefoSpecific pathogen-free female CB6F1 mice, aged between 6 and 8 weeks, were purchased from Japan SLC Inc. . All animal studies were carried out under institutional guidelines approved by the Nihon University Animal Care and Use Committee (AP19MED029-3), the institutional committee for gene recombination experiments (2018MED21), and biorisk management and control (30-10-4). The institutional animal experimental guidelines are in accordance with the ILAR Guide. Mice were allowed free access to sterile water and standard mouse food, and their physiological conditions were assessed every few days.We used the previously prepared rBCG-Mkan85B strain and DNA-Mkan85B plasmid  in this BCG was cultured in Middlebrook 7H9 broth (Difco) supplemented with albumin dextrose complex (ADC) enrichment (Difco) and 0.05% Tween 80 at 37 °C. rBCG-Mkan85B was cultured in Middlebrook 7H9 broth (Difco) supplemented with ADC enrichment (Difco), 0.05% Tween 80, and 10 mg/mL kanamycin at 37 °C. Bacterial culture density was monitored by measuring the absorbance at 470 nm and 600 nm.6 CFU or 0.1 mg of bacilli intradermally (i.d.), and 100 µg of plasmid DNA in saline intramuscularly (i.m.) three times [Mice were immunized with the BCG vaccine or rBCG-Mkan85B at a concentration of 4 × 10ee times  . The bacteriological experiment was carried out under institutional guidelines approved by Nihon University biorisk management and control (30-10-4). M. kansasii was grown in Middlebrook 7H9 broth (Difco) supplemented with ADC enrichment (Difco) with 0.05% Tween 80 at 37 °C and harvested during the exponential growth stage. Infection of mice with M. kansasii was conducted intratracheally. Mice were anaesthetized with isoflurane and inoculated with 50 µL of a bacterial suspension containing 1 to 10 × 104 CFU of M. kansasii using a yellow pipet tip.At 6 weeks after infection, the mice were sacrificed, and the numbers of viable bacilli in their lungs were estimated. Lung tissues were homogenized with sterilized water. Tenfold serial dilutions of homogenate were inoculated onto duplicate 7H10-OADC agar plates (Difco) to determine bacterial loads. Colonies were counted after incubation for 3 weeks at 37 °C.Fractioned lung samples from each mouse were fixed with 10% neutral-buffered formalin and embedded in paraffin wax. The sections from these tissues were 4 mm thick and stained with hematoxylin and eosin (H&E) or with the Ziehl-Neelsen stain for acid-fast bacilli.+ epitope peptide (peptide-25: FQDAYNAAGGHNAVF) [M. kansasii. A seven-color flow cytometry panel was used to simultaneously analyze multiple cytokines at the single-cell level. The gating strategy used to identify cytokine-producing CD8+ and CD4+ T cells in mouse splenocytes is shown in Splenocytes and immune cells isolated from the lung were stimulated with a 9-mer Ag85B CD8 epitope peptide (YYQSGLSIV) (Pep8) , 15-mer GGHNAVF) , or tubeGGHNAVF) ,17. PPD All comparisons among recombinant and control groups and among immunization groups were conducted using one-way ANOVA tests with the Tukey-Kramer test using the JMP program (SAS Institute) and R version 4.0.3 (R core team 2020). Data are expressed as the mean ± SD.M. avium and M. abscessus [M.kansasii infection.According to previous epidemiological reports, BCG vaccination can reduce the risk of NTM infection in humans ,20,21. Ibscessus . TherefoM.kansasii strain for infection for another 6 weeks. The immunization schedule is shown in 4 (±1.5 × 104) pulmonary CFU per animal. Although BCG and rBCG-Mkan85B vaccination both reduced the pulmonary CFU  (p < 0.01) (The CB6F1 (H2b/d) mice were either left unimmunized or immunized with BCG or rBCG-Mkan85B for 6 weeks, followed by a nasal exposure to a virulent  < 0.01) , the eff < 0.01) A,D. Acid < 0.01) G,J. Alth < 0.01) B,C, the  < 0.01) H,I.M.kansasii infection. The CB6F1 mice were either left unimmunized or immunized with BCG or rBCG-Mkan85B/DNA-Mkan85B and then challenged with a virulent M.kansasii strain for another 6 weeks. The immunization schedule is shown in Next, we evaluated the efficacy of the rBCG-Mkan85B/DNA-Mkan85B prime–boost vaccination against To analyze the adaptive immune responses induced by BCG, rBCG-Mkan85B, and rBCG-Mkan85B/DNA-Mkan85B vaccination, we examined the induction of antigen-specific polyfunctional T cells using an intracellular cytokine staining (ICS) method.M. kansasii infection  [M. malmoense, M. marinum, or M. kansasii [M. avium and M. abscessus cross-reactive T cells in humans [+ T cell epitopes of the mycobacterial Ag85 complex elicited similar levels of cytokine production by polyfunctional T cells in mice vaccinated with an rBCG-Mkan85B/DNA-Mkan85B prime–boost strategy [Generally, NTM patients are considered to have been infected by environmental NTM, not by NTM from other patients. In other words, NTM do not cause airborne or droplet infections, unlike TB and SARS-CoV-2, the latter of which is responsible for coronavirus disease 2019 (COVID-19). However, increases in NTM incidence rates have been reported recently [ex (MAC) , M. malmn humans . Previoustrategy .M. kansasii. We showed that both BCG and rBCG-Mkan85B could protect the mice from virulent M. kansasii infection. However, contrary to our expectation, the efficacy of rBCG-Mkan85B vaccination was not superior to that of BCG vaccination. An ICS analysis revealed that M. kansasii antigen-specific polyfunctional CD4+ T cells were equally induced by BCG and rBCG-Mkan85B vaccination. However, similar to previous reports [M. kansasii peptides could not induce antigen-specific polyfunctional CD8+ T cells. Moreover, the present study demonstrated that rBCG-Mkan85B vaccination alone was not sufficient to induce M. kansasii Ag85B-specific polyfunctional CD8+ T cells. Therefore, we immunized mice with rBCG-Mkan85B and boosted them with DNA-Mkan85B three times. BCG and recombinant BCG prime–boost immunization were reported to be beneficial strategies for inducing antigen-specific CD8+ T cells [+ and CD4+ T cells. Regarding antigen-specific CD4+ T cells, several critical roles for eliminating mycobacteria have been reported [+ T cell counts have a high risk of developing NTM infection. In addition to that of CD4+ T cells, the importance of CD8+ T cells to protecting against mycobacterial infection was revealed by previous studies by other researchers. For instance, the airborne infection of mice [M. avium induced the activation of both CD4+ T cells and CD8+ T cells. However, the significance of CD8+ T cells in protection against NTM infection, especially M. avium infection, has been controversial. Although the depletion or genetic elimination of CD8+ T cells significantly decreases immunological responses to TB and increases susceptibility to M. tuberculosis [+ T cells was reported to be unrelated to M. avium infection [+ T cells occurred during experimental M. avium infection [+ T cells in M. kansasii infection has not been investigated thoroughly. In the present study, although BCG or rBCG-Mkan85B immunization could activate only M. kansasii-specific polyfunctional CD4+ T cells, rBCG-Mkan85B/DNA-Mkan85B prime-boost vaccination was able to elicit M. kansasii-specific polyfunctional CD8+ T cells. Nevertheless, the BCG- or rBCG-Mkan85B-immunized mice were shown to be protected against M. kansasii infection, and the rBCG-Mkan85B/DNA-Mkan85B prime-boost strategy drastically reduced the bacterial burden in the lungs of the mice. The results suggest a role for antigen-specific polyfunctional CD8+ T cells in protecting against M. kansasii infection.In the present study, we first challenged unvaccinated and BCG- or rBCG-Mkan85B-vaccinated mice with virulent  reports ,22,23,25 T cells ,17,24. Ireported ,26,27. I of mice , calves  of mice , and rherculosis ,31, the nfection . Gilbertnfection . On the + and CD8+ T cells [Since the end of 2019, COVID-19 has been a serious public health concern worldwide. Looking back on history, human beings have suffered from many kinds of infections. We have to continue to fight various pathogenic microorganisms and infectious diseases. Vaccines are the gold standard for protecting against infectious diseases. One of the aims of a vaccine is to induce effective neutralizing antibodies against the target pathogen. To protect against several pathogenic microbes, such as mycobacteria, the activation of cellular immunity is required in addition to the induction of humoral immunity. BCG has been used globally as a live attenuated vaccine against TB for a century. Various recombinant BCG strains have been designed, developed, and evaluated as novel vaccines against various infectious diseases. For instance, several rBCG strains expressing HIV antigens, such as HIV gag and env, have been reported to induce anti-HIV neutralizing antibodies and antigen-specific CD4 T cells ,33,34. W T cells ,36,37. MM. kansasii infection by inducing M. kansasii-specific polyfunctional CD4+ T cells. Furthermore, rBCG-Mkan85B/DNA-Mkan85B prime–boost vaccination induced antigen-specific CD4+ and CD8+ T cells and drastically reduced the CFU of M. kansasii in the lungs of vaccinated mice. Our data suggest that the combination of rBCG expressing Ag85B derived from M. kansasii with DNA that also expresses Ag85B derived from M. kansasii may be effective in enhancing antigen-specific T cells, resulting in more efficient control of M. kansasii infection.In conclusion, we demonstrated that BCG and rBCG-Mkan85B immunization protected CB6F1 mice from


The Centers for Disease Control and Prevention (CDC) estimated direct healthcare costs of &271 million in 2018. CDC 2015 guidelines  recommended cephalosporin plus azithromycin for GC. We used real-world data to assess patterns of inappropriate Table 1) during the first uUGG episode . Generalized linear models were used for multivariate analysis.A retrospective cohort study of IBM MarketScan data  in patients ≥ 12 years old with uUGG. Eligible patients had an AB prescription ±5 days of uUGG diagnosis (index date) and continuous health-plan enrollment with ≥ 6 months’ baseline/≥ 12 months’ follow-up data. Patients with complicated urogenital GC were excluded. Patients were stratified by AB prescription , 77.1% had an IA/SO prescription (mostly due to IA AB class [~82.0%] and duration [24.0%]), while only 22.9% had an AP&OP prescription; uUGG episodes were more frequent with IA/SO (n=2386) than AP&OP (n=714) prescriptions during follow-up. Patients with IA/SO prescriptions had higher GC-related total adjusted costs per patient (PP) per index episode (&196) vs those with AP&OP prescriptions . Patients with IA/SO prescriptions also had higher GCrelated total adjusted costs PP during follow-up (&220) vs those with AP&OP prescriptions , mostly driven by higher outpatient ambulatory and emergency room (ER) adjusted costs with IA/SO  vs AP&OP prescriptions . ER visits PP at index and during follow-up were higher with IA/SO vs AP&OP prescriptions .Figure. GC-related costs per patient with uUGG, stratified by appropriateness of AB prescription*Table 2. GC-related HRU per patient with uUGG, stratified by AB prescriptionMost patients with uUGG were not prescribed treatments in accordance with CDC 2015 guidelines. High IA/SO AB prescriptions and associated healthcare costs suggest an unmet need for improved prescribing practices for uUGG in the US.Madison T. Preib, MPH, STATinMED Research  Fanny S. Mitrani-Gold, MPH, GlaxoSmithKline plc.  Ziyu Lan, MSc, STATinMED Research  Xiaoxi Sun, MA, STATinMED Research  Ashish V. Joshi, PhD, GlaxoSmithKline plc.


The predictive value of silver stained nucleolar organiser regions (AgNORs) was assessed in 229 patients with transitional cell bladder cancer followed up for over 10 years. The AgNORs were enumerated in pretreatment biopsy specimens. The AgNORs were related to clinical stage (T) (P = 0.0111), papillarity (P less than 0.0001), WHO grade (P less than 0.0001), DNA ploidy (P = 0.0010) and S-phase fraction (P less than 0.0001). Tumours presenting with pelvic lymph node involvement (P = 0.0085) or metastasis (P = 0.0780) at the time of diagnosis had more AgNORs than tumours confined to the bladder wall. Progression in T-, N- and M-categories (P = 0.0010-0.0030) was related to AgNORs and consequently they predicted bladder cancer related survival (P = 0.0005). The diploid tumours could be regrouped according to survival by AgNORs (P = 0.0001). In papillary tumours AgNORs predicted progression (P = 0.0110) and survival (P = 0.0038). In Ta-T1 tumours AgNORs predicted progression (P = 0.11) and survival (P = 0.0751) and also in T2-T3 tumours AgNORs contributed to survival significantly (P = 0.0039). The AgNORs subdivided WHO grade III tumours according to their ability to progress during the follow-up time (P = 0.0711). In a multivariate analysis AgNORs predicted progression independently in Ta-T1 category (P = 0.0165). AgNORs predicted recurrence free period like SPF (P = 0.0010). In conclusion, AgNORs are inferior to classic prognostic factors or DNA flow cytometric variables in muscle invasive bladder cancers whereas they have independent predictive value in superficial cancers.


The role of cholecystokinin (CCK) has been explored in pancreatic carcinogenesis following pancreatobiliary diversion (PBD), using the specific CCK receptor antagonist CR-1409. Male Wistar rats (n = 80) weighing 70-100 g were given weekly i.p. injections of azaserine (30 mg kg-1 week-1) for 3 consecutive weeks. One week later animals were randomised to receive either PBD or sham PBD and thereafter to receive s.c. injections of either saline or CR-1409 . Six months after operation surviving rats were killed as follows: sham + saline 20, PBD + saline 19, sham + CR-1409 14, PBD + CR-1409 11. Cardiac blood was taken for CCK assay and the pancreas was excised for wet weight measurement and quantitative estimation of atypical acinar cell foci (AACF), the precursor of carcinoma. PBD reduced median body weight (3-20% less than shams) but trebled the absolute and relative pancreatic weights (P < 0.001). CR-1409 blunted this adaptive response to PBD, reducing absolute pancreatic weight by 35% (P < 0.005). PBD quadrupled circulating CCK concentrations, regardless of the antagonist treatment. Acidophilic AACF occurred only in rats with PBD. CR-1409 markedly reduced the number of observed acidophilic AACF by 90% (P < 0.001) and the number of foci per pancreas by 93% (P < 0.001). Moreover, CR-1409 reduced the mean focal diameter of each lesion by 18% (P < 0.005), the mean focal volume by 58% (P < 0.05) and the percentage of pancreas occupied by acidophilic foci by 95% (P < 0.001). PBD enhances pancreatic carcinogenesis by causing hypercholecystokininaemia, and CR-1409 largely inhibits this enhancement.


Cytokines are small proteins that regulate immunity in vertebrate species. Marsupial and eutherian mammals last shared a common ancestor more than 180 million years ago, so it is not surprising that attempts to isolate many key marsupial cytokines using traditional laboratory techniques have been unsuccessful. This paucity of molecular data has led some authors to suggest that the marsupial immune system is 'primitive' and not on par with the sophisticated immune system of eutherian  mammals.IFN-γ, IL-2, IL-4, IL-6, IL-12 and IL-13, in the genome of the grey short-tailed opossum (Monodelphis domestica). Many of these genes were not predicted in the publicly available automated annotations.The sequencing of the first marsupial genome has allowed us to identify highly divergent immune genes. We used gene prediction methods that incorporate the identification of gene location using BLAST, SYNTENY + BLAST and HMMER to identify 23 key marsupial immune genes, including The power of this approach was demonstrated by the identification of orthologous cytokines between marsupials and eutherians that share only 30% identity at the amino acid level. Furthermore, the presence of key immunological genes suggests that marsupials do indeed possess a sophisticated immune system, whose function may parallel that of eutherian mammals. The marsupial and eutherian  lineages diverged approximately 180 million years ago. Marsupials are chiefly distinguished from other mammals by their unique reproductive strategies, with young born in an immature state with only the most rudimentary neurological and immunological systems , T Cell However, conventional experimental strategies using degenerate primers for reverse-transcriptase polymerase chain reaction (RT-PCR) and heterologous probes for screening genetic libraries have only identified the most phylogenetically conserved immune molecules, with cytokines proving particularly difficult to isolate . To datein vitro Mixed Lymphocyte Response -x(3)-L) is conserved . Inverted triangles indicate cysteine residues that are conserved across species. Dots represent identity to Monodelphis domestica sequence. Sequences used for alignment: Homo sapiens (Q9HBE4), Mus musculus (NP_068554.1), Gallus gallus (NP_001020006.1), Canis familiaris (NP_001003347.1), Sus scofa (Q76LU6), Bos taurus (Q76LU5).Click here for fileSyntenic region between human chromosome 4q27 and opossum chromosome 5, illustrating the gene cluster of interleukin 2 and 21. Transcriptional directions are indicated by arrows.Click here for fileIL-2Rγ amino acid sequences. Conserved cysteine residues are marked with an inverted triangle. Dots represent identity to Monodelphis domestica sequence. Completely conserved residues are shaded. Sequences used for alignment: Homo sapiens (NP_000197.1), Mus musculus (NP_038591.1), Gallus gallus (NP_989858.1), Rattus norvegicus (NP_543165.1), Canis familiaris (NP_001003201.1), Sus scrofa (NP_999248.1), Bos taurus (NP_776784.1).Click here for fileAlignment of IL-5 amino acid sequences. Dots represent identity to Monodelphis domestica sequence. Sequences used for alignment: Homo sapiens (NP_000870.1), Macaca mulatta (NP_001040598.1), Bos taurus (NP_776347.1), Canis familiaris (NP_001006951.1), Mus musculus (NP_034688.1), Macropus eugenii (AAD37462.1), Gallus gallus (NP_001007085.1).Click here for fileSyntenic region between human chromosome 5q23.3 and opossum chromosome 1, illustrating the gene cluster of interleukin 5, 4 and 13. Transcriptional directions are indicated by arrows.Click here for fileAlignment of IL-6 amino acid sequences. Residues involved in receptor binding in human IL-6 are denoted with diamonds. Cysteine residues conserved among all species are marked with an inverted triangle. PROSITE family motif is boxed. Dots represent identity to Monodelphis domestica sequence. Sequences used for alignment: Homo sapiens (NP_000591.1), Mus musculus (NP_112445.1), Oryctolagus cuniculus (Q9MZR1).Click here for fileAlignment of IL-12α amino acid sequences. Cysteine residues conserved among all species are marked with an inverted triangle. Dots represent identity to Monodelphis domestica sequence. Sequences used for alignment: Homo sapiens (NP_000873.2), Mus musculus (NP_032377.1), Gallus gallus (NP_998753.1), Rattus norvegicus (NP_445842.1), Ovis aries (NP_001009736.1) Canis familiaris (NP_001003293.1).Click here for fileAlignment of IL-10 amino acid sequences. Dots indicate identity to Monodelphis domestica sequence. Sequences used for alignment: Homo sapiens (NP_000563.1), Mus musculus (NP_034678.1), Gallus gallus (NP_001004414.1), Trichosurus vulpecular (AAD01799), Canis familiaris (NP_001003077.1), Sus scofa (Q29055), Cervus elaphus(P51746).Click here for fileAlignment of IL-19 amino acid sequences. Dots indicate identity to Monodelphis domestica sequence. Sequences used for alignment: Homo sapiens (NP_037503.2), Mus musculus (NP_001009940.1).Click here for fileAlignment of IL-20 amino acid sequences. Dots indicate identity to Monodelphis domestica sequence. Sequences used for alignment: Homo sapiens (NP_061194.2), Mus musculus (NP_067355.1), Tetraodon nigroviridis (AAP57416.1).Click here for fileAlignment of IL-24 amino acid sequences. Dots indicate identity to Monodelphis domestica sequence. Sequences used for alignment: Homo sapiens (NP_006841.1), Mus musculus (NP_444325.1), Rattus norvegicus (NP_579845.1), Tetraodon nigroviridis (AAP57418.1).Click here for fileAlignment of IL-26 amino acid sequences. Dots indicate identity to Monodelphis domestica sequence. Sequences used for alignment: Homo sapiens (NP_060872.1), Danio rerio (NP_001018635.1).Click here for fileAlignment of IL-22 amino acid sequences. Dots indicate identity to Monodelphis domestica sequence. Sequences used for alignment: Homo sapiens (NP_065386.1), Mus musculus (NP_058667.1), Sus scofa (AAX33671.1), Rattus norvegicus (ABF82262.1), Danio rerio (NP_001018628.1).Click here for fileNeighbour-Joining tree of IL-10 family ligand protein sequences rooted by midpoint. JTT amino acid substitution matrix was used and 500 bootstrap replicates performed. Branches supported by bootstrap values over 70 are in bold. Opossum sequences are marked by triangles. Sequences used for this analysis were Homo sapiens IL-10 (NP_000563.1), IL-19 (NP_715639.1), IL-20 (NP_061194.2), IL-22 (NP_065386.1), IL-24 (NP_006841.1), IL-26 (NP_060872.1); Mus musculus IL-10 (NP_034678.1), IL-19 (NP_001009940.1), IL-20 (NP_067355.1), IL-22 (NP_058667.1), IL-24 (NP_444325.1); Rattus norvegicus IL-24 (NP_579845.1); Sus scofa IL-10 (Q29055); Bos taurus IL-10 (P43480); Trichosurus vulpecular IL-10 (AAD01799); Gallus gallus IL-10 (NP_001004414.1); Cyprinus carpio IL-10 (BAC76885.1); Tetraodon nigroviridis IL-10 (CAD67786.1); Takifugu rubripes IL-10 (CAD62446.1) Danio rerio IL-26 (NP_001018635.1) and Monodelphis domestica. Sequences labels in the tree are abbreviated by the first letter from the genus with the first two letters from the specific name followed by the gene name.Click here for fileAlignment of CD4 amino acid sequences. Dots indicate identity to Monodelphis domestica sequence. Sequences used for alignment: Homo sapiens (NP_000607.1), Mus musculus (NP_038516.1), Macaca mulatta (BAA09671.1) Felis cattus (NP_001009250.1), Rattus norvegicus (NP_036837.1) Oncorhynchus mykiss (AAY42068.1).Click here for fileAlignment of CD8 amino acid sequences. Dots indicate identity to Monodelphis domestica sequence. Sequences used for alignment: Homo sapiens (NP_001759.3), Mus musculus (Q60965), Gallus gallus (NP_990566.1), Canis familiaris (NP_001002935.1), Sus scofa (NP_001001907.1), Rattus norvegicus (AAH88126.1).Click here for fileAlignment of IFNGR-2 amino acid sequences. Cysteine residues conserved among all species are marked with an inverted triangle. Dots indicate identity to Monodelphis domestica sequence. Sequences used for alignment: Homo sapiens (NP_005525.2), Mus musculus (NP_032364.1), Gallus gallus (NP_001008676.1).Click here for file


Animals were inoculated either with 10(6) or 10(7) MAT-LyLu cells, or with saline to serve as controls. Carcass weight in tumour-bearing (TB) animals decreased despite similar food and water intake in both groups. Absence of metastatic tumour cells from HL of all TB animals was confirmed by histological examination. Twenty-one days after inoculation, 31P MRS showed a 2.5-fold increase in [Pi]/[ATP] ratios in HL in vivo (P < 0.001) which was confirmed by 31P MRS of liver extracts in vitro (P < 0.005). Phosphodiester to ATP ratios were significantly increased (P < 0.05) in HL in vivo, but absolute PDE levels were similar in both groups. Phosphomonoester to ATP ratios did not change, although absolute phosphomonoester levels in HL were reduced by -41% (not significant). In HL extracts in vitro, sharp reductions in the levels of glucose-6-phosphate (P < 0.05), fructose-6-phosphate (P = 0.05), phosphocholine (P < 0.001), glycerophosphocholine (P < 0.001), and glycerophosphoethanolamine (P < 0.001) were observed. Electron microscopy revealed increased amounts and altered distribution of rough endoplasmic reticulum in HL. These findings show that experimental prostate cancer significantly affects hepatic phosphorylation status, phospholipid metabolism, and gluconeogenesis in the host animal, and demonstrate the value of combined MRS in vivo and in vitro in monitoring HL metabolism in cancer.


A longitudinal study of iron status markers (haemoglobin (Hb), serum (S-) iron, S-transferrin, transferrin saturation, S-ferritin) was performed in 31 chemotherapy treated patients with small cell lung cancer. At discovery, eight patients were anaemic (Hb less than 121 g l-1). Hb, S-iron and transferrin saturation were lower (P less than 0.01), and S-ferritin higher (P less than 0.01) than in healthy subjects. Chemotherapy induced an immediate fall in Hb (P less than 0.003), increase in S-iron (P less than 0.003) and transferrin saturation (P less than 0.001). Later in the disease a fall in S-transferrin (P less than 0.006) and an increase in S-ferritin (P less than 0.02) occurred. Thirty patients died during the 2 years observation. S-ferritin at discovery was correlated to performance status score  and to survival . Patients with S-ferritin less than or equal to 400 micrograms l-1 (n = 13) had longer survival than those with S-ferritin greater than 400 micrograms l-1 (n = 18) (P = 0.004).


Sequential exposure to these factors , however, resembling the order of secretion during liver embryogenesis, induced both glycogen-storage and cytokeratin (CK)18 expression. Additional exposure of the cells to trichostatin A (TSA) considerably improved endodermal differentiation, as evidenced by acquisition of an epithelial morphology, chronological expression of hepatic proteins, including hepatocyte-nuclear factor (HNF)-3β, alpha-fetoprotein (AFP), CK18, albumin (ALB), HNF1α, multidrug resistance-associated protein (MRP)2 and CCAAT-enhancer binding protein (C/EBP)α, and functional maturation, i.e. upregulated ALB secretion, urea production and inducible cytochrome P450 (CYP)-dependent activity.hMSC are able to undergo mesenchymal-to-epithelial transition. TSA is hereby essential to promote differentiation of hMSC towards functional hepatocyte-like cells. By utilizing in vitro models, pharmaceutical companies attempt to reduce clinical failure rates by accurately evaluating efficacy and safety much earlier in the drug discovery process , CD45-PE (DAKO), CD105-FITC , CD73-PE, CD90-PE, CD166-PE  and analyzed using a flow cytometer . Mesodermal differentiation was verified by means of different stainings (described below). To exclude spontaneous differentiation, hMSC were cultivated in regular MSCGM medium (Cambrex) and analyzed in parallel.3 cells/cm2 on 1 mg/ml collagen gel type I (BD) in basal medium supplemented with 2% (v/v) foetal bovine serum . Basal medium consisted of 60% (v/v) DMEM and 40% (v/v) MCDB-201  supplemented with 100 IE/ml penicillin , 100 μg/ml streptomycin, 1 mg/ml linoleic-acid bovine serum albumin, 0.1 mM L-ascorbic acid, 0.03 mM nicotinamide, 0.25 mM sodium pyruvate and 1.623 mM glutamine . At 100% confluence, cells were exposed to hepatogenic cytokines and growth factors, added either simultaneous as a cocktail ('referred to as 'cocktail-condition/-set-up/-exposure' or a derivative thereof)  or sequentially  . Differentiation media were changed every 3 days. From day 6 onwards, 1 μM TSA (Sigma) was added. The cocktail- and sequential-conditions, supplemented with 1 μM TSA will be further referred to as 'cocktail TSA-condition/-set-up/-exposure', and 'sequential TSA condition/-set-up/-exposure, respectively, or a derivative hereof.hMSC were plated at 21.5 10Cells were fixed with 10% (w/v) formalin  for 10 min at room temperature  or with methanol  for 2 min at -20°C (osteogenic differentiation). After fixation, adipocytes were identified as red-colored lipid droplets upon staining with Oil-red O (from Sigma) . MineralChondrocytic differentiation was determined using rabbit polyclonal anti-collagen II antibody .Cells were fixed either with ethanol (Merck) for 10 min at -20°C  or with 4% (w/v) paraformaldehyde  for 10 min at 4°C (nuclear and cytoplasmic markers). After fixation, liver-specific protein expression was analysed using primary antibodies against AFP (goat), (HNF3β (goat), HNF1α (rabbit), C/EBPα (rabbit), MRP2 , CK18  and ALB . HDAC inhibition was assessed using anti-acetyl histone H4 antibody . Respective secondary antibodies came from Jackson Immunoresearch, Cambridgeshire, UK. As a negative control, cells were incubated with appropriate gamma immunoglobulines (Jackson Immunoresearch) and immunostained under the same conditions. Cells were analysed using fluorescence microscopy with a Zeiss Axiovert scope.Cell morphology was analysed using phase-contrast light-microscopy (Nikon).ALB concentrations, secreted into the culture media, were analysed by ELISA .4Cl (Sigma), were measured colometrically in the culture media according to the manufacturer's instructions . Fresh culture media supplemented with 6 mM NH4Cl (Sigma) and 4 hours-cultured adult hepatocytes were used as negative and positive controls, respectively.Urea concentrations produced, after 24 hours-exposure of the cells to 6 mM NHEROD- and PROD-activities were assessed as previously described  with somTo evaluate the inducibility of CYP2B6 and CYP1A1/2, cells were, after 18 days of differentiation, exposed to PB  and MC , respectively. Media, supplemented with either PB or MC, were daily renewed. Fresh culture media and 4 hours-cultured adult hepatocytes were used as negative and positive controls, respectively.Results are expressed as mean ± SD. Statistical analyses were performed using Oneway Anova and Student's t-test. The significance level was set at 0.05.Albumin (ALB); alpha-fetoprotein (AFP); CCAAT-enhancer binding protein (C/EBP); cytochrome P450 (CYP); cytokeratin (CK); dimethylsulfoxide (DMSO); ethoxyresorufin-O-deethylase (EROD); fibroblast growth factor-4 (FGF-4); hepatocyte growth factor (HGF); hepatocyte nuclear factor (HNF); histone deacetylase inhibitor (HDAC-I); human adipose tissue-derived stromal cells (hADSC); human mesenchymal stem cells (hMSC); insulin-transferrin-sodium-selenite (ITS); lactate dehydrogenase (LDH); multidrug resistance-associated protein (MRP); multipotent adult progenitor cells (MAPC); methylcholantrene (MC); new chemical entities (NCEs); oncostatin M (OSM); pentoxyresorufin-O-dealkylase (PROD); Periodic-acid-Schiff (PAS) staining; phenobarbital (PB); trichostatin A (TSA).SS: Differentiation of expanded hMSC under hepatocyte-specific conditions. Analysis of multilineage  differentiation by means of staining, functionality assays, microscopic analysis. Preparation of the manuscript.TV, PP and MV: Optimization of the concentration of TSA and onset of exposure to TSA. Critical revision of the manuscript. ADB and IVR: Isolation, expansion and routine characterization of hMSC . Critical revision of the manuscript. VR: Head of department of Toxicology and promotor of the hereby associated PhD thesis. Microscopic analysis. Critical revision of the manuscript.All authors read and approved the final manuscript.3H] thymidine  incorporation into trichloroacetic acid precipitated-DNA  was measured using a scintillation counter . Graphs shown are representative for 3 independent experiments.DNA synthesis of sequentially (+/-1 μM TSA) and cocktail (+/-1 μM TSA)-exposed hMSC. hMSC, plated on 1 mg/ml collagen gel type I, were at 100% confluence treated with either the cocktail- or sequential-condition. From day 6 onwards, 1 μM TSA was added . Differentiation media were changed every 3 days. Samples for DNA synthesis were taken 2, 4, 6, 12 and 18 hours upon media change. [Methyl-Click here for fileCell viability analysis of sequentially (+/-1 μM TSA) and cocktail (+/-1 μM TSA)-exposed hMSC. hMSC, plated on 1 mg/ml collagen gel type I, were at 100% confluence treated with either the cocktail- or sequential-condition. From day 6 onwards, 1 μM TSA was added . LDH leakage into culture media was measured throughout culture time according to the Bergmeyer procedure  using a commercial kit . Graphs shown are representative for at least 3 independent experiments.Click here for fileCell death analysis of sequentially (+/-1 μM TSA) and cocktail (+/-1 μM TSA)-exposed hMSC. hMSC, plated on 1 mg/ml collagen gel type I, were at 100% confluence treated with either the cocktail- or sequential-condition. From day 6 onwards, 1 μM TSA was added . Differentiation media were changed every 3 days. Cells were, 12 hours upon media change, incubated with Alexa Fluor 488 annexin V (green fluorescent), propidiumiodide (red fluorescent) and the nuclear counterstain DAPI (blue fluorescent). The red, green, and both red and green-stained cells represent necrotic, apoptotic and death cells, respectively. 20 × 10 original magnification, phase contrast. Stainings shown are representative for at least 3 separate experiments.Click here for file


PLoS Genetics, vol 2, issue 4, DOI:  10.1371/journal.pgen.0020053In http://www.ncbi.nlm.nih.gov) accession numbers for the sequence discussed in this paper are HVS1 (AY72095-AY721592)” should be “The GenBank (http://www.ncbi.nlm.nih.gov) accession numbers for the sequence discussed in this paper are HVS1 (AY720957-AY721592).”In the Supporting Information section, the sentence “The GenBank (


The expression of oestrogen receptor protein (ER) was examined in 151 cases of symptomatic or screening detected pure ductal carcinoma in situ (DCIS) of the breast by immunocytochemical assay (ERICA), in formalin-fixed paraffin-embedded tissue, with the monoclonal antibody H 222 (Abbott). Forty-eight tumours (31.8%) of cases were ER positive. Twenty-seven (17.9%) of cases showed high level ER expression and 21 (13.9%) of cases showed low level ER immunoreactivity. Significant associations of positive tumour ER immunoreactivity and non-comedo architecture chi 2 = 6.76; (d.f. = 1): P < 0.001, small cell size chi 2 = 4.49; (d.f. = 1): P = 0.034, higher S-phase fraction chi 2 = 4.71; (d.f. = 1): P = 0.03 and lack of c-erbB-2 protein overexpression chi 2 = 7.96; (d.f. = 1): P < 0.01 were identified. No significant associations of ER expression and patient age, histological grade of necrosis in DCIS, or DNA ploidy were found. ER expression is detectable in less than one third of symptomatic and screening detected cases of DCIS, implying that endocrine therapy of DCIS may be a more appropriate form of management for morphological subtypes of DCIS which show higher rates of oestrogen receptor expression, particularly those of non-comedo and small cell type.


Scientific Reports 10.1038/s41598-018-20137-2, published online 29 January 2018Correction to: This Article contains errors in References 8 and 9 which are incorrectly given as:et al. Colistin hetero-resistance in multidrug-resistant Acinetobacter baumannii clinical isolates from the Western Pacific region in the SENTRY antimicrobial surveillance programme. J Infection58, 138–144, 10.1016/j.jinf.2008.11.002 (2009).’‘Yau, W. et al. Synergistic killing of NDM-producing MDR Klebsiella pneumoniae by two ‘old’ antibiotics-polymyxin B and chloramphenicol. J Antimicrob Chemoth70, 2589–2597, 10.1093/jac/dkv135 (2015).’‘Rahim, N. A. 2.The correct references are listed below as refs


Microbacterium paludicola CC3 exhibits the capability to produce polysaccharide bioflocculants. Here, we report the whole-genome sequence of M. paludicola CC3, which may be helpful in understanding the genetic basis of the biosynthesis of polysaccharide bioflocculants as well as in promoting its production and application in industrial fields. Paenibacillus shenyangensis, Agrobacterium tumefaciens F2, and Paenibacillus wulumuqiensis  RSII platform using P6-C4 chemistry. The resulting sequencing reads with 320.4-fold coverage were then de novo assembled using Hierarchical Genome Assembly Process (HGAP) (http://www.ncbi.nlm.nih.gov/COG/), and the Gene Ontology (GO) Consortium (http://www.geneontology.org/). The metabolic pathways were predicted using the KEGG Automatic Annotation Server (KAAS) (http://www.genome.jp/tools/kaas/). rRNAs and tRNAs were identified using Barrnap 0.4.2 (http://www.vicbioinformatics.com/software.barrnap.shtml) and tRNAscan-SE version 2.0 (http://lowelab.ucsc.edu/tRNAscan-SE/), respectively. The clustered regularly interspaced short palindromic repeat (CRISPR) elements were detected using PILER-CR  , 9. Genes (HGAP) . FunctioPILER-CR .M. paludicola CC3. No plasmid sequences were detected. The chromosome was composed of 3,410,829 bp, with an average G+C content of 70.10%, which comprised 3,390 predicted genes, of which 3,209 were protein coding genes (CDSs), 32 were tRNA genes, 146 were rRNA genes, and 3 were microRNA genes. Pseudogenes and prophage genes were not identified, whereas 14 CRISPR candidates were detected in the genome of strain CC3. A series of genes encoding polysaccharide biosynthesis/modification proteins, such as mannose-1-phosphate guanylyltransferase .The sequence data for the genome of


AbstractPhlaeothripinae, Mystrothripslevissp. n. and Urothripslancangensissp. n., are described from China. Pentagonothripsantennalis Haga & Okajima and Plectrothripsbicolor Okajima are newly recorded in China.Two new species of fungivorous  Phlaeothripinae belong to Phlaeothrips lineage, which are usually taken from dead branches or leaf-litter and feed on fungal hyphae .All thrips specimens were extracted by using Tullgren funnels from leaf litter, and then sorted and preserved in 90% alcohol. Examined specimens were mounted into Canada balsam using the method outlined by Taxon classificationAnimaliaThysanopteraPhlaeothripidaehttp://zoobank.org/5399B364-0B5C-4B37-A2F5-896C751EF8A6. Holotype. Female aptera: CHINA, Guangdong: Guangzhou City, South China Botanical Garden (23Guangdong: Guan), in leaf litter of bamboo, 9.viii.2014 (Chao Zhao).Paratypes. 8 females 2 males, collected with holotype; 5 females 1 male, the same locality but collected on 20.xi.2015 (Chao Zhao).Female aptera . Distended body length 2030. Head length 190, width 185; eyes length 50; postocular setae length 75. Antennae length 470, segments I–VIII length (width) as follows: 49(43); 53(41); 68(32); 67(34); 65(34); 61(30); 54(24); 52(12). Pronotum median length 140, width across median part 310; length of major setae: pronotum anteromarginal setae 48, anteroangular setae 68, midlateral setae 90, posteroangular setae 85, epimeral setae 80. Metanotum median setae 40. Pelta length 100, width at base 170. Abdominal tergite IX S1 setae length 190, intermediate setae length 85, S2 length 190. Tube length 155, width at base 93, at apex 42; anal setae length 135.Male aptera . Distended body length 1620. Head length 160, width 155; eyes length 35; postocular setae length 70. Antennae length 375, segments I–VIII length (width) as follows: 34(36); 44(29); 50(32); 50(31); 55(28); 50(25); 45(20); 45(13). Pronotum median length 125, width across median part 260; length of major setae: pronotum anteromarginal setae 60, anteroangular setae 45, midlateral setae 73, posteroangular setae 66, epimeral setae 63. Metanotum median setae 30. Pelta length 65, width at base 110. Abdominal tergite IX setae S1 length 145, intermediate setae length 60, S2 length 45. Tube length 130, width at base 80, at apex 32; anal setae length 110.China (Guangdong).levis, is from the Latin adjective, meaning “smooth”, and refers to the dorsal surface of head and pronotum which are largely smooth. In contrast, most species of this genus are sculptured with distinct polygonal reticulation on head and pronotum.The specific epithet, Mystrothrips  to Japan  head and pronotum largely smooth ; (2) antennae uniformly brown (versus antennae segments I and II distinctly lighter than remaining segments in M.flavidus); (3) pelta semicircular with short lateral lobes ; (4) S1 and S2 setae on abdominal tergite IX much longer than tube (vs shorter than tube in M.flavidus).Of the seven species worldwide listed in the genus rothrips , two areto Japan . The newPageBreakTaxon classificationAnimaliaThysanopteraPhlaeothripidaeHaga & OkajimaPentagonothripsantennalis Haga & Okajima, 1979: 147.CHINA, Hunan: 2 females and 1 male, Zhuzhou City, Yanling County, Shennong Valley , in leaf litter, 16. ix. 2014 (Chao Zhao). Hubei: 1 male, Huanggang City, Yingshan County, Taohuachong , in leaf litter, 23.iv.2014 (Chao Zhao).Dorsal surface of body entirely reticulate; head longer than width, cheeks distinctly incut behind eyes; postocular setae well-developed with expanded at apex. Antennae 7-segmented, morphological segments VII and VIII fused with an incomplete suture, segments III and IV with two and three sense cones, respectively. Maxillary stylets short, wide apart. Pronotum with five pairs of well-developed major setae, strongly expanded at apex. Basantra absent. Fore tarsal tooth present in both sexes. Pelta transverse, shaped as a squashed ellipse, with distinctly polygonal reticulation. Sternite VIII with pair of stout or leaf-like posteromarginal setae submedially. Tube short than head. Pore plate of male absent.China ; Japan.Pentagonothrips, was originally established from Japan ; mesothoracic furcae closely fused together medially as well as metathoracic furcae joined together medially (but metathoracic furcae separated in Mystrothrips).The monobasic genus, om Japan . P.anteTaxon classificationAnimaliaThysanopteraPhlaeothripidaeOkajimaPlectrothripsbicolor Okajima, 1981: 313.CHINA, Guangdong: 1 female, Guangzhou City, Arboretum of South China Agricultural University , in leaf litter, 20.xi.2004 (Jun Wang); 1 male, Guangzhou City, Dafushan Forest Park , in leaf litter of Litchichinensis,17.iv.2016 (Chao Zhao).Body bicolored, yellow and brown. Head, thorax and tube brown, abdomen yellowish brown; all legs yellow; antennal segments II and III yellow, remaining segments brown. Head longer than broad, dorsal surface smooth except weakly sculptured posterolaterally. Antennal segments III and IV with two and three sense cones, respectively, segment VI with two sense cones. Maxillary stylets short, maxillary bridge developed and arched. Pronotum smooth, surrounded by stippled membrane with a distinct median longitudinal line. Metanotum with longitudinal striae medially. Mid tibia and hind tibia with one and two apical spur-like stout setae, respectively. Forewing parallel-sided with seven duplicated cilia. Pelta irregularly triangular with slender lateral lobes and a pair of campaniform sensilla. Abdominal tergites II–VII each with a pair of wing retaining setae; sternites V–VII with a pair of worm-like reticulate areas in both sexes; tergite IX S1 and S2 setae pointed, S1 setae longer than S2 but shorter than tube; tergite IX in male with a small median projection on posterior margin.China (Guangdong); Japan; Indonesia.P.bicolor, originally described from Japan and Indonesia . Holotype. Female aptera, CHINA, Yunnan province, Pu’er City, Lancang County, Nuozhadu Nature Reserve , 5.xi.2016 (Chao Zhao).Paratypes. 6 females, 3 males, collected with holotype.Female aptera . Body length 1400. Head length 180; maximum width 190. Pronotum length 110; median width 250; epimeral setae 20. Metathoracic epimeral setae 20. Abdominal tergite IX length 120, basal width 75, distal width 40. Tube length 130, basal width 22, apical width 25; anal setae 430. Antennal segments I–VIII length (width) as follows: 20(36), 28 (31), 37 (23), 39 (24), 45 (20), 40 (15), 47 (12).Male aptera. . Body length 1050. Head length 160; maximum width 160. Pronotum length 90; median width 185; epimeral setae 13. Metathoracic epimeral setae 13. Abdominal tergite IX length 105, basal width 55, distal width 40. Tube length 115, basal width 20, apical width 22; anal setae 370. Antennal segments I–VIII length (width) as follows: 22(33), 23 (31), 32 (19), 29 (22), 33 (20), 31(17), 39(13).China (Yunnan).The specific epithet is named after the type locality, Lancang County, Yunnan Province, China.Urothripstarai , particularly in the shape of antennae, but it can be differentiated from the latter by the following diagnostic characters: (1) head broadly rounded in front (vs slightly produced in U.tarai); (2) dorsal surfaces of head and pronotum largely sculptured with polygonal reticulation (vs head and pronotum distinctly tuberculate and without reticulation in U.tarai); (3) major body setae on head, pronotum, especially on abdominal tergites are stout, dilated and fan-shaped at apex ; (4) fore femora brown (while fore femora yellow in U.tarai).There are ten species recognized in this genus , of whicPageBreak


Negative, cognitive and depressive symptoms, as well as physical comorbidities, have a great impact on the real-world functioning in patients with schizophrenia (SZ) . However, not all the studies have employed accurate psychometric instruments to assess these symptoms, nor have all these factors been studied simultaneously.The aim of the current study is to analyze the determinants of functionality in SZ measured by the Personal and Social Performance (PSP) scale, and considering not exclusively psychopathological and cognitive variables, but also aspects related to physical health and inflammation.Sample: 73 outpatients with SZ, duration of illness ≤10 years, under stable maintenance treatment .Clinical variables: PANSS, CGI-Severity, Clinical Assessment Interview of Negative Symptoms (CAINS) -Motivation/Pleasure (MAP) & Expression (EXP) domains-, Brief Negative Symptom Scale (BNSS), Calgary Depression Scale (CDS), MATRICS Consensus Cognitive Battery (MCCB), PSP.Biological variables: Glucose, cholesterol, LDL, HDL, triglycerides, TSH, prolactin, insulin, uric acid, alkaline phosphatase (APh), C-reactive protein (CRP), TNF-α, interleukin(IL)-6, IL-2, IL-1β, IL-1RA, homocysteine, HT (% hemolysis), lipid peroxidation (LPO), catalase.Pearson correlations were performed to select variables significantly related to PSP scores which were later included in stepwise multiple linear regression analyses. Age, sex, education, smoking, alcohol use, BMI, antipsychotic equivalent doses and other confounding factors were considered.Final model for PSP total score  identified that CGI-Severity (β= -0.279), PANSS-NM (negative Marder Factor) (β = -0.218), Asociality subscale of BNSS (β= -0.383) and IL-2 (β= -0.269) were significant predictors.Predicting variables included in regression models for specific PSP domains:- Self-care : PANSS-NM (β=0.458), Avolition subscale of BNSS (β=0.248), IL-2 (β=0.221), APh (β=0.201).- Useful activities : CGI-S (β=0.245), Avolition (β=0.557).- Social relationships : Asociality (β=0.578), PANSS-GP (β=0.276), CAINS-EXP (β=0.178).- Aggresive behaviour : PANSS-P (β=0.408), CDS (β=0.273).1.Negative symptoms are the most important determinants of a deficit in the real-world functioning in SZ, especially “asociality.”2.“Apathy” has a negative impact on self-care and useful activities domains.3.Proinflammatory cytokine IL-2 marks poor functionality.1. Harvey (2014). Assessing disability in schizophrenia: tools and contributors. J Clin Psychiatry.2. Strassnig et al. (2015). Determinants of different aspects of everyday outcome in schizophrenia: The roles of negative symptoms, cognition, and functional capacity. Schizophr Res.3. Menendez-Miranda et al. (2015). Predictive factors of functional capacity and real-world functioning in patients with schizophrenia. Eur Psychiatry.


Marinomonas fungiae strain AN44T was isolated from mucus of the coral Fungia echinata. Optimum growth occurs at 3 to 5% NaCl. The draft genome is 4.2 Mb, with 3,776 protein-coding genes. It harbors genes for the degradation of aromatic compounds, such as quinate, ferulate, p-coumarate, protocatechuate, and p-hydroxyphenylacetate. Marinomonas fungiae strain AN44T (JCM 18476T) is an aerobic, Gram-negative, motile, and rod-shaped bacterium belonging to the class Gammaproteobacteria and was isolated from mucus of the coral Fungia echinata from the Andaman Sea (N44T JCM 8476T is aman Sea .M. fungiae strain AN44T was generated at the DOE Joint Genome Institute (JGI) using the Illumina HiSeq 2000 platform (https://github.com/lh3/wgsim), and AllPaths-LG , respectively. The draft genome contains a total of 3,776 CDSs, 78 pseudogenes, 53 tRNAs, 16 rRNAs , 5 noncoding RNAs (ncRNAs), and 1 clustered regularly interspaced short palindromic repeat (CRISPR). Based on COG functional categories, the CDSs of the M. fungiae genome were distributed into categories of amino acid transport and metabolism (9.84%), carbohydrate transport and metabolism (5.92%), cell cycle control, cell division, and chromosome partitioning (1.08%), cell motility (3.51%), cell wall/membrane/envelope biogenesis (5.06%), chromatin structure and dynamics (0.09%), coenzyme transport and metabolism (5.47%), defense mechanisms (2.18%), energy production and conversion (6.61%), extracellular structures (0.44%), function unknown (5.41%), general function prediction only (7.31%), inorganic ion transport and metabolism (5.19%), intracellular trafficking, secretion, and vesicular transport (1.55%), lipid transport and metabolism (3.35%), mobilome prophages and transposons (1.08%), nucleotide transport and metabolism (2.47%), posttranslational modification, protein turnover, and chaperones (4.15%), RNA processing and modification (0.03%), replication, recombination, and repair (3.83%), secondary metabolite biosynthesis, transport, and catabolism (2.12%), signal transduction mechanisms (8.13%), transcription (7.97%), translation, ribosomal structure, and biogenesis (7.18%), and unassigned (28.88%).The genome was annotated using the JGI Microbial Genome Annotation Pipeline . Genes wp-coumarate, along with a single operonic gene cluster for central aromatic compound degradation via the protocatechuate meta-cleavage pathway, were found in the genome. Additionally, a single operonic gene cluster for the degradation of p-hydroxyphenylacetate was found.Further, putative genes or gene clusters for peripheral aromatic compound degradation pathways of quinate, ferulate, and LIQF00000000. The version described in this paper is the first version, LIQF01000000, and consists of sequences LIQF01000001 to LIQF01000036.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number


Nucl. Acids Res. (04 January 2016) 44 (D1): D447–D456. doi: 10.1093/nar/gkv1145.3Medizinisches Proteom Center (MPC), Ruhr-Universität Bochum, D-44801 Bochum, Germany.The authors wish to correct the affiliation of one of the authors. Gerhard Mayer's only affiliation should be 1European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SD, UK.He does NOT have a second affiliation at: The authors apologise to the readers for this error.


With limited and low-genetic barrier drugs used for the prevention of mother-to-child transmission (PMTCT) of HIV in sub-Saharan Africa, vertically transmitted HIV-1 drug-resistance (HIVDR) is concerning and might prompt optimal pediatric strategies.The aim of this study was to ascertain HIVDR and viral-tropism in majority and minority populations among Cameroonian vertically infected children.A comparative analysis among 18 HIV-infected children (7 from PMTCT-exposed mothers and 11 from mothers without PMTCT-exposure) was performed. HIVDR and HIV-1 co-receptor usage was evaluated by analyzing sequences obtained by both Sanger sequencing and ultra-deep 454-pyrosequencing (UDPS), set at 1% threshold.10 copies/mL, and 526 (282–645) cells/mm3, respectively. All children had wild-type viruses through both Sanger sequencing and UDPS, except for 1 PMTCT-exposed infant harboring minority K103N (8.31%), born to a mother exposed to AZT+3TC+NVP. X4-tropic viruses were found in 5 of 15 (33.3%) children (including 2 cases detected only by UDPS). Rate of X4-tropic viruses was 0% (0/6) below 5 years , and became relatively high above 5 years  versus CD4 >15% ; similarly for CD4 ≤200 (3/4 [75%]) versus CD4 >200  age, viremia, and CD4 count were 6 (4–10) years, 5.5 (4.9–6.0) logNGS has the ability of excluding NRTI- and NNRTI-mutations as minority species in all but 1 children, thus supporting the safe use of these drug-classes in those without such mutations, henceforth sparing ritonavir-boosted protease inhibitors or integrase inhibitors for the few remaining cases. In children under five years, X4-tropic variants would be rare, suggesting vertical-transmission with CCR5-tropic viruses and possible maraviroc usage at younger ages. From these observations, we postulated that minority DRMs in HAART-naïve children might grow-up through selective drug-pressure and populate plasma in a short-frame, herein justifying the rapidly emerging DRMs we observed at failure. Although not yet clinically endorsed, pediatric minority DRMs might be more concerning in the context of PMTCT, henceforth underscoring an unmet clinical need.,11 Coupled to previous knowledge on the detection of DRMs by next-generation sequencing (NGS),–14 we thus hypothesized that using NGS to assess DRMs in vertically infected HAART-naïve children would contribute in designing long-term HAART strategies for SSA-children.As the footprint of long-term HAART depends largely on the effectiveness of first-line drugs in sustaining viral suppression, establishing adequacy between pediatric HAART and DR-mutations (DRMs) would be clinically relevant. As HAART would be reaching 1.5 million children by 2020, as high as 20% virological failure (VF) is expected, favored by high-viremia and poor adherence in children.,16 Without optimal strategies, VF would quickly overcome HAART success, maintaining children vulnerable.Current pediatric HAART-regimens consist of lamivudine (3TC), abacavir (ABC), or zidovudine (AZT), associated to ritonavir-boosted lopinavir (LPV/r) or NVP. LPV/r is recommended to overcome PMTCT-resulting non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance, whereas NVP matches with postnatal prophylaxis.,8 Although not yet approved for under 16 years, the CCR5 antagonist—maraviroc—might represent a suitable antiretroviral alternative for children, pending proof-of-concept towards relevant pediatric clinical trials. Particularly, there are limited evidence on the potential effectiveness of maraviroc for SSA-children in PMTCT, initial-HAART and/or following treatment-failure.–21 With rising concerns of minority variants on response to several classes of antiretrovirals, a genuine delineation of HIV-1 tropism, considering both minority and majority quasi-species,,23 could rationalize maraviroc suitability for pediatric HAART-policies in SSA.Moreover, pediatric HAART options are limited in SSA, urging the quest for a wider therapeutic portfolio.Based on these assumptions, we aimed to ascertain DRMs and HIV-1 co-receptor usage, in majority and minority viral populations, from children according to maternal PMTCT-exposure in a resource-limited setting (RLS).22.1A comparative study was conducted in 2015 among 18 HIV-1 vertically infected Cameroonian children, all HAART-naïve, stratified according to maternal antiretroviral exposure during pregnancy: control-group  versus case-group (7 children from mothers exposed to reverse transcriptase inhibitors [RTIs]). For each child, a plasma sample was collected to perform both Sanger- and 454 ultra-deep pyrosequencing (UDPS).2.2 Briefly, viral RNA was extracted from plasma using QIAamp Viral RNA minikit , following manufacturer's instructions. PR/RT-containing region was then reverse-transcribed and amplified using SuperScript One-Step for long templates reverse transcriptase polymerase chain reaction (RT-PCR) of Invitrogen kit , with an eventual second-round seminested PCR. Direct sequencing was then performed using 7 overlapping primers.Protease (PR)/RT Sanger sequencing was performed as previously described. Briefly, viral RNA containing the V3-loop region was reverse-transcribed and amplified using an RT/Taq mix, with an eventual second-round seminested PCR. Direct sequencing was then performed using 4 overlapping primers.V3 loop Sanger sequencing was performed as previously described.2.32O DNase RNase free, 1 μL forward primer (10 μmol/L), 1 μL reverse primer (10 μmol/L), 1 μL RNase Out (40 U/μL Invitrogen) and 1.2 μL RT/TAQ, for a final volume of 50 μL. RT-PCR conditions were the following: 1 cycle 50°C, 30 minutes; 1 cycle 94°C, 2 minutes; 40 cycles ; a final extension 68°C, 10 minutes. Forward and reverse primers were respectively 5’GACAGGCTAATTTTTTAGGG3’  and 5’GATAAATTTGATATGTCCATTG3’ . Nested-mid PCR was then performed with the Fast Start HiFi PCR system  using 5 pairs of barcoded-modified forward and reverse primers for each amplicon , 8 μL MgSO4 (5 mmol/L), 2.8 μL H2.4Ten microliters viral RNA were reverse transcribed with 1-step RT-PCR system using forward  and reverse  primers, under the following conditions: 1 cycle 50°C, 30 minutes; 1 cycle 94°C, 2 minutes; 35cycles ; a final extension 68°C, 10 minutes. A nested mid-PCR was then performed with the Fast Start HiFi PCR system  as previously described.2.5PR/RT PCR products  and V3 loop (one fragment of 367 bps) were purified using Agencourt AMPure PCR purification beads  and quantified with Quant-iT PicoGreen double-stranded DNA assay kit  on a GloMax multidetection system . Phylogenetic analyses excluded any possible sample contamination (data not shown).Pooled purified PCR products were clonally amplified by emulsion PCR and pyro-sequenced on the 454 GS junior platform  as previously described.2.6The entire PR (amino acid position: 1–99), RT (1–251) and the entire V3 loop (1–35) sequences obtained after 454-pyrosequencing were de-multiplexed and then quantified using the SFF tool Roche. Using a home-made Perl script and SHORAH package 0.5.1, sequences were filtered and corrected for homopolymeric region-associated errors and aligned against HIV-1 consensus B. Final alignments were manually checked for insertion or deletion in homopolymeric regions that could result in a frame shift. Nucleotidic/aminoacidic variants were evaluated and quantified by a home-made pearl script, and sequences were considered reliable when showed an intra-patient frequency ≥1% in both forward and reverse strands.2.7http://hivdb.stanford.edu/pages/download/resistanceMutations_handout.pdf) and geno2pheno.v2.5 (http://coreceptor.geno2pheno.org/), respectively. Using a quantitative interpretation, viruses were considered CXCR4-tropic (X4-variants) by UDPS when ≥2% viral species had a false-positive rate (FPR) ≤3.5%, or by Sanger sequencing when FPR was ≤10%, describing the probability of classifying an R5-virus falsely as an X4-variant.PR/RT DRMs and HIV-1 co-receptor usage were interpreted using Stanford HIVdb list  available at 2.9HIV-1 DRMs and coreceptor usage were compared between the two PMTCT-groups. Coreceptor results by Sanger sequencing and UDPS were considered concordant if viral-tropism was identical from both sequencing technologies. Viral-tropism was explored according to age and CD4 count.P values <.05 were considered statistically significant.All statistical analyses were performed using the statistical open source environment R.v.3.1.1. 2.10Ref. N°034/NEC/SE), proxy-informed consent was provided, unique identifiers were used for privacy and confidentiality, and a material transfer agreement was established.Ethical clearance was obtained from the Cameroon National Ethics Committee  cells/mm3, respectively, without any significant difference between the 2 groups (data not shown). In the control, neither children nor their mothers had any antiretroviral exposure. Antiretroviral history of children belonging to the case-group, considered at higher risk of HIVDR, is described in Table Overall, median (interquartile range [IQR]) age, viremia, and CD4 count were 6 (4–10) years, 5.5 (4.9–6.0) log3.2HIV-1 subtyping revealed 50% CRF02_AG (9/18), 33.3% F (6/18), 11.1% CRF01_AE (2/18), and 5.6% CRF11.cpx (1/18).3.3PR/RT sequences were successfully obtained both through Sanger sequencing and UDPS for 17/18 children. The median UDPS coverage was of 1642 (IQR: 1269–5193) reads. In the entire covered PR/RT regions, the 2 sequencing technologies showed total concordance in variants detection, and all UDPS variants with frequencies <20% were not detected by Sanger sequencing , an accessory polymorphism weakly selected under etravirine (ETR) and rilpivirine (RPV), was found in a child aged 8 years from the control group.By using UDPS, 1 (aged 1 year) of 7 children 14.3%) from the case-group harbored viruses with K103N , a nonpolymorphic mutation causing high-level resistance to NVP and efavirenz (EFV). This infant was born from an RTI-treated mother (AZT + 3TC + NVP). Thus, Sanger sequencing and UDPS were performed also for the mother (ID-18613). UDPS revealed a virus harboring 2 major DRMs: L74 V at minority-level (2.5%), causing high- and intermediate-level resistance respectively to didanosine and to ABC; Y181C at population-level (96.7%), causing high- and intermediate-level resistance respectively to NVP and to EFV, ETR, and RPV , a polymorphic accessory mutation selected under EFV, in a child aged 6 years Table .Other variants, found even at RTI-associated drug resistance positions, were with minimal or no effect on drug susceptibility or virological response. Of note, in either group, no major DRMs to ritonavir-boosted protease inhibitors (PI/r) were found by both Sanger sequencing and UDPS.3.4V3 loop sequencing was successful by both Sanger sequencing and UDPS for 15 of 18 children and the mother ID-18613, with an overall viral-tropism concordance of 87.5% 14/16) between Sanger sequencing and UDPS (Table /16 betweX4-tropic viruses were found in 5 of 15 (33.3%) children (including 2 cases detected only by UDPS), all aged above 5 years. Specifically, in 1 child (ID-11621) UDPS provided an added value in tropism-determination compared to Sanger sequencing. Indeed, a clinically relevant quantity of minority X4-tropic variants (frequency: 3.9%) was detected by UDPS in this child . In another child (ID-10196), despite an R5-tropism (FPR = 79.7%) determined by Sanger sequencing, a discordant tropism was observed through UDPS with a high percentage of X4-tropic variants , because of insertions detected only at minority levels.P = .040). As expected, X4-tropic viruses were higher with CD4 ≤15% (4/9 [44.4%]) versus CD4 >15% ; similarly for CD4 ≤200 (3/4 [75%]) versus CD4 >200 . No statistical difference was found in X4-variants between the 2 PMTCT-groups: 2 of 7 (28.6%) case group versus 3 of 8 (37.5%) control group, P = 1.000.Of relevance, the rate of X4-tropic viruses was 0% (0/6) among children under 5 years , and became significantly higher as from 5 years and above , known to be associated with resistance to NNRTIs used both for PMTCT and first-line HAART in SSA, was found in a PMTCT-exposed infant, thus suggesting NNRTI-sparing regimens for such children.,30,34 Discrepancy in DRMs between mother and infant would be due to sample collection later after delivery (at the moment of infant HIV diagnosis), with possible selection following prophylaxis/breastfeeding; as previously reported in similar RLS . This infant (aged 1 year), compared to the median age of the study population (6 years), suggests that circulating DRMs might have fade-up with increasing age.,33 NNRTI mutations (E138A and V179D), found in children without PMTCT-exposure, are known as polymorphisms with little or no effect on drug susceptibility or virological response. The ability of NGS in excluding minority RTI-mutations  supports the safe use of NNRTIs/NRTIs in those without such mutations, thus sparing from inappropriate switch to PI/r- or integrase inhibitor-containing regimens.,17,33–35In this high CRF02_AG-infected population,,37 as well as a baseline FPR <60 as previously demonstrated.,39 Further investigations might help in establishing novel public health strategies for an eventual usage of maraviroc in children.,40 As current PMTCT-practice might not be an independent factor for viral-tropism , CCR5-antagonist (maraviroc) could be a useful therapeutic weapon for pediatric HAART.,18,40Coreceptor usage in these children provides a clue for clinical application. Indeed, X4-variants appeared to be associated with older ages and lower CD4 cells, suggesting limited vertical transmission by CXCR4-tropic viruses, and later appearance of X4-variants with chronicity, immunological impairment,,39 Interestingly, by detecting minority insertions associated with a complete discrepant result on Sanger sequencing, UDPS appears very useful in validating tropism determination for non-B subtypes.Of the two children showing discordant results between the two sequencing techniques, the added value of UDPS in detecting X4-tropic minority variants is in accordance with previous reports.,22,41Therefore, UDPS might provide additional information in detecting DRMs and viral-tropism, confirming the added value of this technology for both clinical diagnostics and management of non-B HIV-infected children.,43In spite of this added value of UDPS, implementing NGS is more challenging in RLS , suggesting the need for simpler and affordable approaches integrating minority variants (point-of-care or pragmatic sequencing).,13,44–46 This study therefore provides relevant data to be used as base for further/enlarged studies.A potential study limitation could be the relatively small sample size, which makes the study probability relatively large. Also, in the PMTCT-exposed group, only 3 of 7 were exposed to triple ART, calling for subsequent investigations with scale-up of option B+. Moreover, HIV-1 variants were investigated only in plasma compartment, suggesting the need for exploring HIV variability in several compartments  and the impact on treatment and monitoring strategies in SSA.In a nutshell, NGS could help in identifying PMTCT-exposed children harboring minority NNRTI-DRMs, therefore serving for a timely switch of treatment and limiting failure rate. NGS also reveals a possible absence of X4-variants among children below 5 years, thus suggesting possible public health approaches using maraviroc. These preliminary evidences, generated on a small sample of mainly CRF02_AG-infected individuals, merit further investigations for improved pediatric-HAART strategies in RLS.Conceptualization: A. Nanfack, C-F. Perno, C. Fokunang, C. Tangimpundu, D. Armenia, D. Takou, E. Temgoua, F. Ceccherini-Silberstein, G. Cappelli, J. Fokam, M-M. Santoro, P. Koki, V. Colizzi.Data curation: D. Armenia, F. Ceccherini-Silberstein, J. Fokam, L. Carioti, M. Bellocchi, M-M. Santoro.Formal analysis: D. Armenia, F. Ceccherini-Silberstein, J. Fokam, L. Carioti, M. Bellocchi, M-M. Santoro.Funding acquisition: C-F. Perno, J. Fokam, V. Colizzi.Investigation: A. Ndjolo, A. Nanfack, C-F. Perno, C. Fokunang, C. Tangimpundu, D. Takou, E. Temgoua, F. Ceccherini-Silberstein, G. Cappelli, J. Fokam, J. Torimiro, M-M. Santoro, P. Koki, V. Colizzi.Methodology: D. Armenia, D. Takou, F. Continenza, F. Ceccherini-Silberstein, J. Fokam, L. Carioti, M. Bellocchi, M-M. Santoro.Project administration: A. Ndjolo, C-F. Perno, C. Fokunang, F. Ceccherini-Silberstein, G. Cappelli, J. Fokam, J. Torimiro, P. Koki, V. Colizzi.Resources: A. Ndjolo, C-F. Perno, J. Fokam, V. Colizzi.Software: D. Armenia, D. Takou, F. Continenza.Supervision: A. Ndjolo, C-F. Perno, C. Fokunang, F. Ceccherini-Silberstein, G. Cappelli, M-M. Santoro, P. Koki, V. Colizzi.Validation: A. Ndjolo, C-F. Perno, C. Fokunang, C. Tangimpundu, D. Armenia, E. Temgoua, F. Continenza, F. Ceccherini-Silberstein, G. Cappelli, J. Fokam, J. Torimiro, L. Carioti, M. Bellocchi, M-M. Santoro, P. Koki, V. Colizzi.Visualization: A. Nanfack, C-F. Perno, C. Tangimpundu, D. Takou, E. Temgoua, F. Continenza, J. Fokam, M-M. Santoro.Writing – original draft: C-F. Perno, F. Ceccherini-Silberstein, J. Fokam, M-M. Santoro.Writing – review & editing: A. Ndjolo, A. Nanfack, C. Fokunang, C. Tangimpundu, D. Armenia, D. Takou, E. Temgoua, F. Continenza, G. Cappelli, J. Torimiro, L. Carioti, M. Bellocchi, P. Koki, V. Colizzi.We are appreciative to our institutional staff that participated locally in the enrolment and in sample processing. We thank Domenico Di Carlo for statistical analyses.


Halopteris filicina (Grateloup) Kützing, Dictyota dichotoma (Hudson) J. V. Lamouroux, Posidonia oceanica (L.) Delile and Flabellia petiolata (Turra) Nizamuddin from the Adriatic Sea (single point collection). VOCs were investigated by headspace solid-phase microextraction (HS-SPME) and analysed by gas chromatography and mass spectrometry (GC-MS/FID). H. filicina headspace contained dimethyl sulfide , C8-compounds ), benzaldehyde , alkane C17, dictyopterene D and C , tribromomethane (V), 1-iodopentane, others. F. petiolata headspace was characterized by DMS (22.2%), 6-methylhept-5-en-2-one (9.5%), C17 (9.1%), II (6.5%), compounds I-V. DMS (59.3%), C15 (14.5%), C17 (7.2%) and C19 (6.3%) dominated in P. oceanica headspace. Sesquiterpenes were found in D. dichotoma, predominantly germacrene D (28.3%) followed by other cadinenyl (abundant), muurolenyl and amorphenyl structures. Determined VOCs may be significant for chemosystematics and chemical communications in marine ecosystem.Performed phytochemical study contributes to the knowledge of volatile organic compounds (VOCs) of  The research on the algae VOCs has been continued by different teams cyclohepta-1,4-diene). Proposed biogenesis of dictyopterenes starts from (3S)-1,cis-5-undecadien-3-ol and (3S)-1,cis-5,cis-8-undecatrien-3-ol by dehydration and cyclization.Carotenoid cleavage dioxygenases (CCDs) catalyze oxidative cleavage of carotenoids, resulting in the production of norisoprenoids—apocarotenoids  via glycso occur . The bre2-unit, which can potentially occur via either the β-oxidative pathway or non-oxidatively [Benzenoid and phenylpropanoid volatile compounds, primarily derived from phenylalanine require shortening of the carbon skeleton side chain by a Cdatively .P. oceanica followed by F. petiolata and H. filicina indicating those plants as source of sulfur compounds in marine ecosystem. Their headspace contained individually variety of C8-compounds (e.g. fucoserratene), benzaldehyde, alkanes C15, C17 and C19, dictyopterene D and C, others. Sesquiterpenes were found in D. dichotoma, predominantly germacrene D indicating similarity to terrestrial aromatic plants. Identified VOCs contain different types of organic compounds that may be significant for chemosystematics and ecology .Considering limited data available on the chemical composition of marine plants from the Adriatic Sea, the present research is contribution toward their better chemical characterization. Significant differences were found among the headspace VOCs from 3 seaweeds and 1 seagrass. High abundance of DMS was found in


In t test)  .S1 Fign = 8 biological replicates per genotype. (B) Percent of male only Spen KD larvae floating. (C) Percent of female only Spen KD larvae floating. (D) FB-specific Spen KD  with different insertion site as Spen KD in Fig 1A compared to KD control (dcg>iw). (E) Genetic background controls (iSpen/+ and iw/+) for (D). (F) As the Spen hairpin insertion site appears to result in a lean phenotype, KD animals were normalized to their genetic background. (G) As in (A), three additional independent Spen hairpin constructs (dcg>iSpen) tested in different density solutions and compared to KD control (dcg>iw). (H) Genetic background controls (iSpen/+’s and iw/+) for (G). P value obtained by ANOVA. *P < 0.05, ** P < 0.01, ***P < 0.001, **** P < 0.0001. Error bars represent SEM.(A) Percent of floating larvae in different density solutions. FB-specific Spen KD  as in Fig 1A with additional dcg/+ background control. Fifty larvae per genotype per experimental replicate, (TIF)Click here for additional data file.


Klebsiella pneumoniae infections. Herein, we report the draft genome sequences of two colistin-resistant K. pneumoniae isolates (BA41763 and B6753). The sequence data indicate that BA41763 and B6753 contain genomes of ~5.9 and 5.7 Mb in size with several plasmids.Resistance to colistin is a major threat that limits therapeutic choices for treating carbapenem-resistant  Klebsiella pneumoniae (CRKP) infections are difficult to manage, with very few remaining treatment options, such as colistin and tigecycline  with 49× and 23× coverage, respectively. Assembled genome sequences were annotated in PATRIC, the bacterial bioinformatics database and analysis resource (http://www.patricbrc.org) (http://rast.nmpdr.org/) (http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html).To further understand the molecular mechanism behind colistin resistance, we determined their whole-genome shotgun sequences in Ion Torrent PGM using 400-bp chemistry. https://www.patricbrc.org). Similarly for B6753, 5,754,328 bp with 6,248 CDS were identified. Also identified were 13 rRNAs, 72 tRNAs with 34 and 82 ARDB and CARD antimicrobial resistance genes and 72 and 181 virulence factors from the VFDB and Victors database, respectively.The genome of BA41763 had 5,944,266 bp with 6,678 coding sequences (CDS), 10 rRNAs, and 66 tRNAs. The annotation revealed 32 and 85 ARDB and CARD antimicrobial resistance genes, respectively. In addition, 74 and 183 virulence factors from VFDB and the Victors database, respectively, were identified (https://cge.cbs.dtu.dk//services/MLST/) (https://cge.cbs.dtu.dk//services/ResFinder/) (rmtf and aacA4), beta-lactam , fluoroquinolones lb-cr, oqxA, qnrB66), fosfomycin (fosA), macrolide (mph(A)), rifampin (ARR-2), and sulfonamide (sul2) resistance genes. Similarly, for B6753, we found aminoglycoside -Vla), beta-lactam , fluoroquinolone (aac(6′)lb-cr), fosfomycin (fosA), macrolide (mph(E), msr(E)), phenicol (catB3), sulfonamide (sul1), tetracycline (tet(D), and trimethoprim . However, plasmid-mediated colistin resistance genes mcr-1 and mcr-2 were absent. B6753 had a mutation in the mgrB gene with a premature stop codon resulting in a truncated protein of 27 amino acids, but there were no mutations in the mgrB gene of BA41763.Next-generation sequencing (NGS) also revealed that BA41763 and B6753 were of sequence types (STs) ST147 and ST14, respectively, as analyzed by the MLST 1.8 tool (https://cge.cbs.dtu.dk//services/PlasmidFinder/) (PlasmidFinder 1.3 (Finder/)  revealedLZYN00000000 and MEBR00000000, respectively. The versions described in this paper are the first versions, LZYN01000000 and MEBR01000000.This whole-genome shotgun project of both the isolates BA41763 and B6753 has been deposited at DDBJ/ENA/GenBank under the accession numbers


EditorThe following manuscript has been retracted from our November – December, 2016 issue. It has been retracted on a request from the authors as they forgot to properly acknowledge the source of their data. The authors are grateful to those who pointed out this mistake. - Retraction in: Pak J Med Sci 2016;32(6):1500-1505. doi: https://doi.org/10.12669/pjms.326.11460Link: http://pjms.com.pk/index.php/pjms/article/view/11460/4800Effective role of lady health workers in immunization of children in Pakistan1, Azka Naeem2, Unaiza Shahid3, Wajiha Noor Syed4, Urva Khan5, Nayyar Misal Zaidi6Saira AfzalRetracted on February 7, 2017


A 24-year-old male from Wangdue Phodrang district, Bhutan, presented with a history of unexplained intermittent low-grade fever and cough for 8 months. He also complained of weight loss, abdominal discomfort, and one episode of hemoptysis and convulsion. He was a cow herder by profession. A computed tomography scan showed hepatosplenomegaly  with mesenteric lymphadenopathy. Biochemical evaluation showed elevated alkaline phosphatase 1096 IU/L), lactate dehydrogenase 330 IU/L), and C-reactive protein (40.8 mg/dL) and reverse albumin/globulin ratio. Complete blood count showed pancytopenia confirmed by peripheral smear. In addition, left shift of neutrophils and giant platelets were observed. Erythrocyte sedimentation rate (26 mm/h) was increased. Prothrombin time (20 s) and activated partial thromboplastin time (51 s) were prolonged. The bone marrow aspirate smear revealed intracellular and extracellular Leishman-Donovan bodies . In view096 IU/L,30 IU/L,


C. elegans cell divisions that produce an apoptotic daughter cell exhibit Daughter Cell Size Asymmetry (DCSA), producing a larger surviving daughter cell and a smaller daughter cell fated to die. Genetic screens for mutants with defects in apoptosis identified several genes that are also required for the ability of these divisions to produce daughter cells that differ in size. One of these genes, ham-1, encodes a putative transcription factor that regulates a subset of the asymmetric cell divisions that produce an apoptotic daughter cell. In a survey of C. elegans divisions, we found that ham-1 mutations affect primarily anterior/posterior divisions that produce a small anterior daughter cell. The affected divisions include those that generate an apoptotic cell as well as those that generate two surviving cells. Our findings suggest that HAM-1 primarily promotes DCSA in a certain class of asymmetric divisions. Caenorhabditis elegans somatic development is essentially invariant. Almost all of the somatic divisions are asymmetric, generating two daughter cells that differ in fate  [4p::gfp] .LG II. ynIs25 [flp-12p::gfp] [2p::gfp] , 19.LG III. nIs107[tbh-1p::gfp + lin-15(+)] [rdvIs1 [egl-17p::mCherry:his-24 + egl-17p::myristolated mCherry + pRF4] [n-15(+)] , rdvIs1  + pRF4] .LG IV. ham-1(gm279) [ ham-1gm29 [7].LG V. zuIs178 [-119(+)] .LGX. gmIs81 [7p::gfp] .ltIs44[pie-1p-mCherry::PH(PLC1delta1) + unc-119(+)]  [lqIs80[scmp::gfp::CaaX] [Unmapped: -119(+)] , leIs270-119(+)] , lqIs80[p::CaaX] .kyEx581[ocr-4p::gfp + lin-15(+)] , Ex  [Extra-chromosomal arrays: ::dsRed] .C. elegans promoter. The A/PVM, SDQR/L, A/PQR and URXR/L neurons were detected using the gmIs81 reporter. The SMB, OLQ, ASK, MC and RIC neurons were detected using the reporters flp-12p::gfp, ocr-4p::gfp, srbc-66p::gfp, ceh-19p::gfp and tbh-1p::gfp, respectively. When an extra neuron was detected in a ham-1 mutant, its position was in close proximity to the normal position of the single neuron found in wild-type animals. Missing neurons were only scored when using integrated transgenes, since extra-chromosomal arrays can be lost during cell divisions. Statistical analysis was performed using the two-sample Z-test for proportions.All neurons were detected with transcriptional reporters that express fluorescent proteins under control of the indicated lqIs80[scmp::gfp::CaaX]. V5.pa lineage analysis was performed by compiling and reordering images of rdvIs1[egl-17p::myr mcherry + egl-17p::H2B::mcherry] L2 larvae. The mcherry markers are upregulated in all cells of the V5.pa lineage. V5.paa daughter cells size measurements were performed at the 3- and early 4-cell stages, before V5.paap and V5.paaa migrations occurred. T.pp and V5.pa neuroblast daughter cell sizes measurements were performed as previously described for the Q neuroblasts' daughters , which labels the cells of the P3-8 lineage. Ratios are indicated in the text ± standard deviation.T.p lineage analysis was performed in early L2 larvae using aughters , 12. TheFor embryonic neuroblasts, automated lineaging was performed as described . After nFor each genotype and ACD, the measured daughter cells size ratio was plotted on graphs where horizontal dotted lines indicate 1:1 ratios, and horizontal grey bars indicate the median of each ratio distribution. Statistical analysis was performed using the Mann-Whitney U-test.ham-1 mutants produce abnormal numbers of neurons in specific lineages . The cells boundaries are indicated by dotted lines and the name of each cell is indicated. Right panels are schematic representations of the corresponding cells. Black arrows describe cell displacements. Colored arrows indicate the direction of neurite extensions. Upon V5.pa division, the anterior daughter V5.paa moves to a more dorsal position above its sister V5.pap. The V5.paa neuroblast divides asymmetrically before V5.pap to produce the smaller anterior daughter V5.paaa, which will become the PDE neuron, and the larger posterior daughter V5.paap. The V5.pap glioblast's division produces daughter cells of equal size, the anterior daughter V5.papa, which will become the PDE socket cell, and the posterior daughter V5.papp, which will become the PDE sheath cell. Before the V5.paap division, both V5.paap and V5.paaa migrate anteriorly and ventrally, respectively. V5.paap then divides to generate the larger anterior daughter V5.paapa which will become the PVD neuron, and the smaller posterior daughter V5.paapp, which dies. After completion of all cell divisions, PDE and PVD neurons extend their neurites. Abbreviations: so: socket cell; sh: sheath cell. Scale bars: 10 μm.(A) Schematic diagram of the V5.pa lineage, which produces two neurons (PDE and PVD), two neuron-associated support cells (PDE socket cell and PDE sheath cell) and one apoptotic cell (V5.paapp). V5.paa and V5.paap daughters are unequal in size, whereas V5.pap division is symmetric in size. (B) Description of the cell division sequence and cell movements that occur during posterior deirid development. Left panels are representative fluorescent labeling of the left V5.pa lineage at all stages of development by (TIF)Click here for additional data file.