Neisseria gonorrhoeae is a global health concern with worrying trends of decreasing susceptibility to also the last-line extended-spectrum cephalosporin (ESC) ceftriaxone. A dramatic increase of reported gonorrhea cases has been observed in Sweden from 2016 and onward. The aim of the present study was to comprehensively investigate the genomic epidemiology of all cultured N. gonorrhoeae isolates in Sweden during 2016, in conjunction with phenotypic AMR and clinical and epidemiological data of patients. In total, 1279 isolates were examined. Etest and whole-genome sequencing (WGS) were performed, and epidemiological data obtained from the Public Health Agency of Sweden. Overall, 51.1%, 1.7%, and 1.3% resistance to ciprofloxacin, cefixime, and azithromycin, respectively, was found. No isolates were resistant to ceftriaxone, however, 9.3% of isolates showed a decreased susceptibility to ceftriaxone and 10.5% to cefixime. In total, 44 penA alleles were found of which six were mosaic (n = 92). Using the typing schemes of MLST, NG-MAST, and NG-STAR; 133, 422, and 280 sequence types, respectively, and 93 NG-STAR clonal complexes were found. The phylogenomic analysis revealed two main lineages (A and B) with lineage A divided into two main sublineages (A1 and A2). Resistance and decreased susceptibility to ESCs and azithromycin and associated AMR determinants, such as mosaic penA and mosaic mtrD, were predominantly found in sublineage A2. Resistance to cefixime and azithromycin was more prevalent among heterosexuals and MSM, respectively, and both were predominantly spread through domestic transmission. Continuous surveillance of the spread and evolution of N. gonorrhoeae, including phenotypic AMR testing and WGS, is essential for enhanced knowledge regarding the dynamic evolution of N. gonorrhoeae and gonorrhea epidemiology.The increasing transmission and antimicrobial resistance (AMR) in  Neisseria gonorrhoeae, the causative agent of gonorrhea, is a global public health concern with increasing transmission and development of antimicrobial resistance (AMR) (ce (AMR) . The incce (AMR) . This trce (AMR) . In 2020ce (AMR) . Nonethece (AMR) . Furtherce (AMR)  and the ce (AMR) .N. gonorrhoeae, there are many different molecular AMR determinants, for example, target mutations in: penA, encoding penicillin-binding protein 2 (PBP2), associated with resistance and decreased susceptibility to ESCs and penicillins; 23S rRNA associated with macrolide resistance; gyrA and parC, encoding subunits of DNA gyrase and DNA topoisomerase IV, respectively, associated with fluoroquinolone resistance; and 16S rRNA associated with spectinomycin resistance  result in decreased influx and increased efflux of antimicrobials, respectively. These mutations decrease the antimicrobial susceptibility further  is, due to its high resolution and accuracy, an ideal method for molecular epidemiology and enables collection of comprehensive data on gonococcal population characteristics and AMR determinants internationally  in Sweden in 2016, representing 72.0% of all reported gonorrhea cases (n = 1777) in the year, were included. When multiple isolates from the same gonorrhea episode were available, isolates from extragenital sites were prioritized. Accordingly, the examined gonococcal isolates were cultured from the urethra (n = 521), rectum (n = 291), pharynx (n = 150), or other/not reported (n = 65) in men and from cervix (n = 119), pharynx (n = 44), urethra (n = 34), rectum (n = 21), vaginal swabs (n = 14), or other/not reported (n = 20) in women. Multiple gonococcal infections of the same patient were considered separate episodes if the infections were > 3 weeks apart. AMR testing was conducted using Etest, according to the instructions of the manufacturer . Minimum inhibitory concentrations  of ceftriaxone, cefixime, ciprofloxacin and spectinomycin were interpreted according to the clinical breakpoints recommended by the European Committee on Antimicrobial Susceptibility Testing. For azithromycin, no clinical breakpoints are recommended and the epidemiological cut-off value of 1 mg/L (1 v11.0) was applied (isolates with azithromycin MIC > 1 mg/L are referred to as resistant hereafter). Additionally, isolates with MIC values of 0.064 to 0.125 mg/L for ceftriaxone and cefixime were interpreted to have a decreased susceptibility , and country of infection were extracted.2 was used to calculate significance and significance was set at p < 0.05.Statistical analysis was performed using logistic regression in IBM SPSS Statistics (version 25) with calculation of a 95% confidence interval and odds ratio (OR). Pearson’s χEthical approval for the study was obtained from the Swedish Ethical Review Authority .Genomic DNA was extracted using the QIASymphony DSP virus/pathogen kit  according to the manufacturer’s instructions, including RNase treatment and elution with Tris-HCl (pH 8), on the QIASymphony instrument (Qiagen). All DNA extractions were paired-end sequenced using Illumina HiSeq X platform . DNA extractions and sequencing libraries were quality controlled using Qubit BR/HS assays  and appropriate assays on the TapeStation according to manufacturer’s instructions .N. gonorrhoeae genomes (n = 1279) were sequenced with 50-100-fold coverage and initially quality controlled for contamination using Kraken (v1.1.1)  (N. gonorrhoeae multi-antigen sequence typing (NG-MAST)  (All (v1.1.1) . All rea(v1.1.1) ], geneti(v1.1.1) , N. gonoNG-STAR)  and N. gNG-MAST)  sequenceNG-MAST) . The NG-NG-MAST)  based onv3.14.1)  with theN. gonorrhoeae reference strain FA1090 (accession number LT591897) to obtain a multiple sequence alignment using multiple_mappings_to_bam pipeline3 with BWA-MEM  visualized using Microreact .All WGS sequence reads are available from the ENA (PRJEB47922).n = 21) except one (Västerbotten) were represented. Regarding origin of infection, 68.4% and 29.6% were reported as domestic (n = 875) and foreign (n = 379), respectively (n = 70), Germany (n = 32), Spain (n = 31), Denmark (n = 25), and Turkey (n = 18) (The study population included 252 women and 1027 men with median (mean) age of 24 (27.4) years and 30 (32.1) years, respectively . In totaectively . The mos(n = 18) . A highe4 and The phenotypic antimicrobial susceptibility of all gonococcal isolates by regional council is summarized in Figure 1n = 119) of all isolates (n = 1279) showed a decreased susceptibility. Cefixime resistance was found in 1.7% of isolates , and the highest resistance levels were in Östergötland (4.0%), Stockholm (2.4%), and Skåne (2.3%) (n = 134) of isolates had a decreased susceptibility to cefixime. Eighty (6.3%) isolates had a decreased susceptibility to both ceftriaxone and cefixime. One of these 80 isolates was also resistant to azithromycin, i.e., a high-level azithromycin-resistant (MIC > 256 mg/L) strain cultured from a heterosexual male with a sexual contact in Thailand. Based on the WGS sequences, 20 (90.9%) of the cefixime-resistant isolates (n = 22) had mosaic penA alleles [penA-10.001 (n = 18) and penA-34.001 (n = 2)]  cefixime-resistant isolates did not contain any mosaic penA, however, these isolates harbored PBP2 A501V and P551S alterations (penA-13.001) as well as mtrR and porB1b mutations. Overall, 44 penA alleles were found among the 1279 isolates, of which the most common were penA-2.001 (n = 325), penA-2.002 (n = 232), penA-19.001 (n = 101), penA-44.001 (n = 98), and penA-14.001 (n = 94). Ninety-two isolates (7.2%) had mosaic penA alleles: penA-34.001 (n = 60), penA-10.001 (n = 23), penA-34.008 (n = 6), penA-72.001, penA-105.001, and penA-180.001 (n = 1 each) resulting in ceftriaxone MICs < 0.002-0.125 mg/L and cefixime MICs < 0.016-0.5 mg/L. The combination of mosaic penA with mtrR and/or porB1b mutations, which further decrease the ESC susceptibility, was found in 81 isolates, of which 49 (60.5%) isolates had decreased susceptibility to ceftriaxone and 17 (21.0%) and 60 (74.1%) isolates had resistance and decreased susceptibility to cefixime, respectively. PBP2 A501V/T, P551S and G542S alterations were found in 162 (12.7%), 56 (4.4%), and 45 (3.5%) isolates, respectively. The ceftriaxone and cefixime MIC range (mean) was 0.004-0.125 (0.035) mg/L and < 0.016-0.125 (0.040) mg/L for A501V/T, 0.004-0.064 (0.034) mg/L and < 0.016-0.064 (0.029) mg/L for P551S, and 0.004-0.032 (0.016) mg/L and < 0.016-0.032 (0.020) mg/L for G542S, respectively. The combination of PBP2 A501V/T and P551S alterations was present in 48 isolates (3.8%) with ceftriaxone and cefixime MIC range (mean) of 0.004-0.125 (0.07) mg/L and < 0.016-0.25 (0.07) mg/L, respectively. The combination of PBP2 A501T and G542S alterations was present in six isolates (0.5%) with ceftriaxone and cefixime MIC range (mean) of 0.016-0.064 (0.051) mg/L and 0.016-0.064 (0.045) mg/L, respectively.No resistance to ceftriaxone was found, however, 9.3% (e (2.3%) . FurthermtrR -35 A-deletion was found in all of them, two isolates had additionally a mosaic mtrD and the remaining three isolates had porB1b mutations. Mosaic mtrR was found in 1.1% (n = 14) of isolates, mosaic mtrC in 1.1% (n = 14), mosaic mtrD in 8.4% (n = 107), and mosaic mtrE in 1.0% (n = 13). The combination of mosaic alleles in all genes of the mtrRCDE operon was found in 1.0% (n = 13) of isolates with azithromycin MICs of 0.032-1.0 mg/L. One isolate harbored mosaic alleles in all genes except mtrE (azithromycin MIC = 1.0 mg/L). Notably, one isolate, which lacked known azithromycin resistance determinants, contained the previously described GC deletion in mtrC (Azithromycin resistance was found in 16 (1.3%) isolates, and the highest resistance levels were found in Dalarna (11.1%) and Stockholm (1.8%) . High-le in mtrC  (azithroparC was found in 451 (35.3%) isolates, all except one had the GyrA S91F mutation, consisting of the amino acid substitutions D86N (n = 137), S87I/N/R (n = 311), S88P (n = 27), and E91K (n = 10).Ciprofloxacin resistance was found in 653 (51.1%) isolates . All isorpsE genes.No resistance was found to spectinomycin, including no spectinomycin resistance determinants in the 16S rRNA or mtrR  and porB1b mutations  were found in 540 (42.2%) and 356 (27.8%) isolates, respectively.In total, n = 133), ST7363 (n = 125), ST1901 (n = 93), ST1588 (n = 89), and ST7359 (n = 67). Fifty-five MLST STs represented by single isolates were found. Using NG-MAST, 422 STs were identified and the five most prevalent STs were ST5441 (n = 89), ST5793 (n = 56), ST2992 (n = 41), ST11461 (n = 39), and ST387 (n = 35). In total, 260 NG-MAST STs were found in only one isolate. With NG-STAR, 280 STs were observed and the five most prevalent ones were ST442 (n = 133), ST55 (n = 63), ST158 (n = 63), ST231 (n = 61), and ST520 (n = 52). One hundred and forty-eight NG-STAR STs were represented by single isolates. Lastly, 92 NG-STAR CCs were found and 19 isolates were ungroupable. The five most common CCs were CC442 (n = 134), CC158 (n = 100), CC63 (n = 91), CC42 (n = 70), and CC390 (n = 69). Twenty-six NG-STAR CCs were represented by single isolates.Based on the WGS sequences, the most common MLST, NG-MAST, NG-STAR STs and NG-STAR CCs by sex and sexual orientation is summarized in penA types in sublineages A1 and A2 and linage B are summarized in n = 133) and ST1901 (n = 90); NG-MAST ST5441 (n = 89) and ST2992 (n = 41); NG-STAR ST442 (n = 133) and ST90 (n = 51); and NG-STAR CCs 442 (n = 134) and 63 (n = 91). The sexual orientation of the patients in A2 was homosexual male (57.9%), bisexual male (2.6%), heterosexual male (23.4%), women (14.5%), and not reported (1.7%) (penA (49/81) predominantly belonged to A2. The isolates with decreased susceptibility to ceftriaxone (n = 89) were cultured from MSM , heterosexual men , women  and not reported . The majority  of these infections were domestic and 73.0% (n = 65) were diagnosed in Stockholm. The most common molecular types among the isolates with decreased susceptibility to ceftriaxone were MLST ST1901 (n = 34), NG-MAST ST1407 (n = 25), NG-STAR ST90 (n = 33), and NG-STAR CC90 (n = 35). The 21 (95.5%) cefixime-resistant isolates in A2 were cultured from infections acquired heterosexually  or MSM (3/21). Furthermore, 14 isolates were from domestic and seven from foreign infections, in six different countries. The majority  were diagnosed in Stockholm, however, all cefixime-resistant isolates were from patients in larger metropolitan areas. The most common molecular types among the cefixime-resistant isolates were MLST ST7363 (n = 16), NG-MAST ST13876 (n = 9), NG-STAR ST232 (n = 10), and NG-STAR CC348 (n = 15).Phylogenomic analysis revealed two main lineages (A and B), of which one (A) was subdivided into two main sublineages (A1 and A2) (d (1.7%) . Isolaten = 11), or mtrR -35 A-deletion plus mosaic mtrD (n = 2). Sexual orientation of these patients was homosexual male (46.2%), heterosexual male (15.4%) and women (38.5%). These azithromycin-resistant isolates did not belong to any predominant ST nor did they cluster together in the phylogenomic tree  also belonged to A2, and contained 23S rRNA gene mutations (n = 90), NG-MAST ST1407 (n = 33), NG-STAR ST90 (n = 51), and NG-STAR CC90 (n = 61).Resistance to ciprofloxacin was also prevalent (53.2%) in A2. The most common molecular types among the ciprofloxacin-resistant isolates in A2 were MLST ST1901 (n = 101) and ST1588 (n = 89); NG-MAST ST12001 (n = 21), ST9184 (n = 19), and ST18710 (n = 19); NG-STAR ST158 (n = 63) and ST426 (n = 22); and NG-STAR CC158 (n = 100) and CC309 (n = 48). The sexual orientation of the patients in A1 was homosexual male (32.8%), bisexual male (1.9%), heterosexual male (40.0%), women (22.5%), and not reported (2.8%) (penA). The remaining three  azithromycin-resistant isolates also belonged to A1 and appeared to represent the same gonococcal strain . All these isolates were also lacking 23S rRNA mutations and mosaic mtrD, but had mtrR -35 A-deletion plus porB1b mutations (azithromycin MICs of 2 mg/L). Furthermore, ciprofloxacin resistance was very common in A1 (90%). The most common molecular types among the ciprofloxacin-resistant isolates in A1 were MLST ST7363 (n = 101), NG-MAST ST12001 (n = 21), NG-STAR ST158 (n = 63), and NG-STAR CC158 (n = 100) (In lineage A1 (360 isolates), the most common molecular types were MLST ST7363 (d (2.8%) . A1 incln = 100) .n = 67) and ST1599 (n = 53), NG-MAST ST5793 (n = 56) and ST11461 (n = 39), NG-STAR ST55 (n = 63) and ST231 (n = 61), and NG-STAR CC42 (n = 69) and CC390 (n = 64). The sexual orientation of the patients was homosexual male (52.4%), bisexual male (0.8%), heterosexual male (22.8%), women (22.9%), and not reported (1.4%) (penA but no isolates comprised mosaic mtrD. The majority (93.3%) of isolates were ciprofloxacin susceptible.In lineage B (359 isolates), the most frequent molecular types were MLST ST7359 (d (1.4%) . Lineagen = 5) and epidemiological data  were diagnosed in metropolitan areas, especially in the capital city Stockholm. In 2016, resistance to ceftriaxone and cefixime remained low nationally; i.e., no resistance to ceftriaxone and 1.7% to cefixime. Resistance to azithromycin was also low (1.3%). The cefixime resistance was caused by penA-10.001 (n = 18), penA-34.001 (n = 2), or PBP2 A501V and P551S alterations in combination with mtrR and porB1b mutations (n = 2). The azithromycin resistance was caused by 23S rRNA A2059G (n = 1), 23S rRNA C2611T (n = 10) or mtrR -35 A-deletion plus mosaic mtrD (n = 2) or mtrR -35 A-deletion plus porB1b mutations (n = 3). None of the cefixime-resistant isolates was resistant to azithromycin. Prior to 2019, the Swedish gonorrhea guideline recommended ceftriaxone 500 mg, plus azithromycin 2 g if pharyngeal infection, for treatment of uncomplicated gonorrhea. However, from 2019, ceftriaxone 1 g monotherapy is the recommended treatment  and A1 contained the lowest proportion of MSM. Lineage B harbored most of the antimicrobial-susceptible isolates.Phylogenomic analysis showed that the isolates grouped into two main lineages as previously described , of whicpenA alleles in MLST ST1901 and ST7363 was originally documented in Japan  of the 21 Swedish regions, making preventions targeted on single sexual networks or risk group and in general gonorrhea control very challenging.The most prevalent MLST STs found in the present study have been described in previous studies. A national genomic Norwegian study described 21 STs with ≥ 10 isolates , all of in Japan  and thesin Japan , 2021. Iectively . The majd Europe . In SwedN. gonorrhoeae, NG-STAR CC  due to the lack of phylogenomic similarity of the isolates. NG-STAR with CC designation, which clusters based on AMR determinants and the closest alleles, appeared to correspond relatively well with the genome-based phylogeny and can also inform regarding resistance or decreased susceptibility to cefixime and ceftriaxone and presence of associated AMR determinants.The newly proposed classification method for -STAR CC , assigne-STAR CC , where iN. gonorrhoeae population in 2016 had a low prevalence of resistance to cefixime and no resistance to ceftriaxone. However, the limited cefixime resistance and especially the high level of decreased susceptibility to ESCs, predominantly domestically transmitted, remain worrisome and is a risk for development of resistance to ceftriaxone. The prevalence of azithromycin resistance was also low. Resistance and decreased susceptibility to ESCs and azithromycin and associated AMR determinants, such as mosaic penA and mosaic mtrD, were mainly found in the phylogenomic sublineage A2. Resistance to cefixime and azithromycin was more prevalent among heterosexuals and MSM, respectively, and both were predominantly spread through domestic transmission. Continuous surveillance of the spread and evolution of N. gonorrhoeae, including phenotypic AMR testing and WGS, is essential for enhanced knowledge regarding the dynamic evolution of N. gonorrhoeae and gonorrhea epidemiology.In conclusion, the Swedish https://www.ebi.ac.uk/ena, PRJEB47922.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The studies involving human participants were reviewed and approved by the Swedish Ethical Review Authority . Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.MU and RH designed, initiated, and coordinated the study. RH, DG, and LE were involved in the laboratory work, WGS, and/or bioinformatic analysis. A-KO, E-LE, and YL provided gonococcal isolates. IV and HF contributed with epidemiological data. RH, DG, and MU analyzed and interpreted all the data. RH and MU wrote the first draft of the manuscript. All authors read, commented, and approved the submitted manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Inflammatory osteolysis after total joint replacement (TJR) may cause implant failure, periprosthetic fractures, and be a severe threat to global public health. Our previous studies demonstrated that melatonin had a therapeutic effect on wear-particles induced osteolysis. Gut microbiota is closely related to bone homeostasis, and has been proven to be affected by melatonin. However, whether melatonin could play its anti-osteolysis effects through reprogramming gut microbiota remains elusive.Here, we demonstrated that melatonin could alleviate Ti-particles induced osteolysis, while this therapeutic effect was blocked by antibiotic cocktail treatment. Interestingly, transplantation of fecal microbiota from mice treated with melatonin reappeared the same beneficial effect. Analysis of the 16S rRNA revealed that melatonin could reverse dysbacteriosis triggered by osteolysis, and elevate the relative abundance of some short chain fatty acid (SCFA) producing bacteria. Moreover, butyrate was enriched by exogenous melatonin administration, while acetate and propionate did not show an evident difference. This was consistent with the results of the metagenomic approach (PICRUSt2) analysis, which revealed a general increase in the synthetic enzymes of butyrate. More importantly, direct supplementation of butyrate could also recapitulate the anti-osteolysis effect of melatonin. Further analysis identified that butyrate alleviated osteolysis via activating its receptor GPR109A, and thus to suppress the activation of NLRP3 inflammasome triggered by Ti-particles.Taken together, our results suggested that the benefits of melatonin mainly depend on the ability of modulating gut microbiota and regulating butyrate production.The online version contains supplementary material available at 10.1186/s12951-021-00915-3. As the essential treatment for end-stage arthritis, total joint replacement (TJR) could effectively relieve joint pain, stiffness, and improve joint function . However.  ** p < .01). Figure S3. Principle coordinate analysis (PCoA) plot between Ti-trans and MT-trans groups based on the Bray Curits distance. n = 6. Figure S4. Ti-particles induced osteolysis could be blocked by the treatment of either NLRP3 or caspase-1 inhibitor. (A) Representative view of calvarium from sham, Ti, MCC950, and Ac-YVAD-CMK groups via Micro-CT 3D reconstruction. (B-D) Quantification of bone erosion parameters (BV/TV) bone volume to tissue volume ratio, (BMD) bone mineral density, and total porosity. n=6. (E) Percentage of osteoclasts surface per bone surface . n = 5.(F) H&E and (G) TRAP staining of calvarial slices from each group. (H) H&E and (I) TRAP staining of calvarial slices from Sham, Ti and Butyrate group. Results are expressed as mean ± SEM (One-way ANOVA[post hoc:SNK] *** p < .001). Figure S5. Quantification of NLRP3, Caspase-1, IL-1β and GPR109A staining. (A) Quantification of NLRP3 immunohistochemical staining using integrated optical density/specimen area (IOD/Area). n=5. (B) Quantification of Caspase-1 immunofluorescence staining using mean density (integrated density/specimen area). n=5. (C) Quantification of IL-1β immunohistochemical staining using integrated optical density/specimen area (IOD/Area). n=5. (D) Quantification of GPR109A immunohistochemical staining. n=5. Results are expressed as mean ± SEM .


Anxiety Disorder (AD) is common in inpatient pediatric burn patients and likely related to pain/stress associated with acute care. This study ascertained if burn survivors reported higher anxiety levels based on sex, visibility of scars, or TBSA ≥ 50%.Burn-injured youth completed the Screen for Child Anxiety Related Disorders (SCARED) with parental consent. This 41 item self-report measures DSM-IV pediatric anxiety disorder symptoms: panic disorder (PD), separation anxiety (SA), generalized anxiety disorder (GAD), social phobia (SP) school phobia (SCP) and total anxiety (TA). The percentage of respondents above threshold for each disorder was calculated.112 survivors, mean age of 13, included boys (51%) & girls (49%). 83 reported visible scars. Females had higher percentages for TA (53%) vs. males (21%) (p < 0.001), PD (47%) vs. (7%) (p< 0.001), GAD (40%) vs. (16%) (p < 0.005), & SA (51%) vs. (21%) (p < 0.001). Youth with TBSA ≥ 50% (n=22) had higher precents for GAD (46%) vs. < 50% (24%) (p < 0.01). The visibly scarred had higher percent for GAD (38%) vs. hidden (7%) (p< .01).Female, visibly scarred, and patients with burns > 50% revealed increased AD symptoms. AD may be chronic, interfere with a child’s home & school function and lead to chronic distress, substance abuse, and isolation. Screening for anxiety in burn-injured youth is recommended.


The primary analysis of the STAT study demonstrated the feasibility, efficacy, and safety of using DTG/3TC as a first-line regimen in a test-and-treat setting through 24 weeks, with therapy adjustments for baseline resistance or hepatitis B virus (HBV) co-infection. Here we present secondary analyses through Week 48 of virologic outcomes in participants by baseline viral load (VL). 2, then antiretroviral therapy (ART) was potentially adjusted and participants remained on study. Efficacy analyses included proportion of participants with HIV-1 RNA < 50 c/mL regardless of ART regimen at Week 48, among all participants  and among participants with available HIV-1 RNA data at Week 48 . STAT is a single-arm study of treatment-naive adults with HIV-1 infection who initiated DTG/3TC ≤ 14 days after HIV-1 diagnosis without availability of screening/baseline laboratory results. If baseline testing indicated DTG or 3TC resistance, HBV co-infection, or creatinine clearance < 30 mL/min/1.73 mOf 131 enrolled, DTG/3TC treatment was adjusted in 10 participants, and of those with available data (n=7), all (100%) achieved HIV-1 RNA < 50 c/mL at Week 48. At Week 48, 82% (107/131) of all participants  and 97% (107/110) of those with available data  achieved HIV-1 RNA < 50 c/mL. Of participants with baseline VL ≥ 500,000 c/mL, 89% (17/19) achieved HIV-1 RNA < 50 c/mL at Week 48; the remaining 2 withdrew from study. Of participants with baseline VL ≥ 1,000,000 c/mL, 90% (9/10) achieved HIV-1 RNA < 50 c/mL at Week 48 (Table); the remaining participant withdrew consent. Of the 17 participants with baseline VL ≥ 500,000 c/mL with available data through Week 48, 76% (13/17) achieved virologic suppression by Week 24. One participant with baseline VL ≥ 500,000 c/mL switched from DTG/3TC before the Week 48 assessment. Of the 9 participants with baseline VL ≥ 1,000,000 c/mL with available data through Week 48, most participants  were suppressed by Week 24.Figure 1. Virologic outcomes at Week 48, overall and by baseline VL and CD4+ cell count: ITT-E missing = failure analysis.Figure 2. Virologic outcomes at Week 48, overall and by baseline VL and CD4+ cell count: observed analysis.Table. Viral Load by Study Visit Among Participants with Baseline HIV-1 RNA ≥1,000,000 c/mLThese data provide evidence for the efficacy and feasibility of using DTG/3TC as a first-line regimen in a test-and-treat setting, including among participants with very high baseline VL.Charlotte-Paige M. Rolle, MD MPH, Gilead Sciences Janssen Infectious Disease ViiV Healthcare  Tulika Singh, MD MS AAHIVS, Gilead ViiV  Moti Ramgopal, MD FIDSA, Abbvie Gilead Janssen Merck ViiV  Dushyantha Jayaweera, MD, mrcog(uk), face, Gilead (Research Grant or Support)Janssen (Research Grant or Support)viiv (Research Grant or Support) Peter Leone, MD, viiv healthcare (Employee) Jessica Matthews, BS, ViiV Healthcare (Employee) Michael Cupo, Ph.D., GlaxoSmithKline (Employee) Mark Underwood, PhD, GlaxoSmithKline (Shareholder)ViiV Healthcare (Employee) Kostas Angelis, PhD, GSK  Brian Wynne, MD, ViiV Healthcare  Deanna Merrill, PharmD, MBA, AAHIVP, GlaxoSmithKline (Shareholder)ViiV Healthcare (Employee) Christopher T. Nguyen, MD, ViiV Healthcare (Employee) Jean A. van Wyk, MB,ChB, GlaxoSmithKline (Shareholder)ViiV Healthcare (Employee) Andrew Zolopa, MD, GlaxoSmithKline (Shareholder)ViiV Healthcare (Employee)


Due to antiretroviral treatment success, individuals with HIV are living longer. People aging with HIV  may be more likely to experience nutritional risk compared to their HIV-negative counterparts due to biopsychosocial factors. The DETERMINE checklist measure accounts for social and economic factors as well as aspects of the aging process that are not typically considered when examining nutritional risk and are important for PAWH. The current study examined nutritional risk and health-related quality of life (HRQoL) in PAWH using the DETERMINE checklist and PROMIS t-scores  through secondary analyses of 158 participants in the Strengthening Therapeutic Resources in Older patients agiNG with HIV (STRONG) study. DETERMINE nutritional risk scores (0-21) were separated into 4 groups . The sample was 55% male, 94% Black/African American and had a mean age=59 (SD=5.5). Most of the sample (74%) were at high or very high nutritional risk and low HRQoL t-score: physical M=43.7 (SD=9.5), and mental M=45.7 (SD=10.1). Mental and physical HRQoL were significantly (p<.001) associated with nutritional risk group as tested through linear regressions. Means were as follows: physical HRQoL low-risk M=53.4 (SD=10.6), moderate-risk M=47.4 (SD=8.9), high-risk M=43.5 (SD=8.1), very high-risk M=38.4 (SD=8.9); mental HRQoL low-risk M=54.0 (SD=8.9), moderate-risk M=49.1(SD=7.9), high-risk M=46.1(SD=9.5), and very high-risk M=39.5 (SD=9.7). These associations remained significant after controlling for age and sex. Higher nutritional risk as measured by the DETERMINE checklist in PAWH was associated with poorer physical and mental HRQoL.


In a Phase 3 trial, the Janssen COVID-19 vaccine, Ad26.COV2.S, showed robust efficacy against severe–critical COVID-19 in countries where different SARS-CoV-2 variants were circulating. We evaluated Ad26.COV2.S-elicited antibody neutralizing activity against variants of concern (VOC) B.1.1.7 (Alpha), B.1.351 (Beta), and B.1.617.2 (Delta) in sera from participants in clinical trials following a single dose of Ad26.COV2.S.10 viral particles [vp]) against VOC were assessed by wild-type virus neutralizing (wtVNA) and pseudovirion neutralization (psVNA) assays in sera from participants in Phase 1/2a and Phase 3 clinical trials, respectively. Geometric mean titers (GMTs) were determined at Days 29 and 71 after vaccination.Neutralizing activities of Ad26.COV2.S . Day 71 titers against the Delta variant were maintained for at least 8 months following a single dose of Ad26.COV2.S (5 x 1010 vp). In serum samples from Phase 3 participants (n = 8), psVNA titers against VOC were lower than the original strain at Day 71 after vaccination, with the lowest titers observed for the Beta variant . Smaller reductions in Nab titers for VOC were observed in the psVNA assay compared to wtVNA.In serum samples from Phase 1/2a participants (n = 6), at Day 29 after 1 dose of Ad26.COV2.S, wtVNA titers against VOC were lower than for the original strain (GMT = 573), with GMT = 65, 14, and 15 for Alpha, Beta, and Delta, respectively, representing 8.8-, 40.9-, and 37.7-fold decreases. By Day 71 after vaccination (n = 14), fold differences between the original strain (GMT = 375) and VOC  were smaller  than at Day 29, suggestive of B-cell maturation , B.1.351 (Beta), and B.1.617.2 (Delta) lineages in serum samples from participants who received Ad26.COV2.S. n = 6 samples at Day 29 and n = 14  samples at Day 71 after vaccination with a single dose of Ad26.COV2.S (5 x 10^10 vp dose level) were analyzed in wild-type virus neutralization assays against the SARS-CoV-2 Victoria strain , the B.1.1.7  the B.1.351 , and the B.1.617.2  lineages. Dots represent the IC50 (inhibitory concentration) titers per participant. Geometric mean titers (GMTs) and fold decrease in neutralizing activity between the original Victoria strain and each lineage are shown.Ad26.COV2.S-elicited serum neutralizing activity against VOC showed an overall decrease in titers relative to the original strain that was largest for the Beta variant, even though vaccine efficacy against severe–critical COVID-19 was maintained in countries where these variants were circulating versus in countries where they were not circulating. Over time, titers against variants increased, suggesting B-cell affinity maturation leading to increasing coverage of VOC.Mathieu Le Gars, n/a, Johnson & Johnson  Jerald Sadoff, MD, Johnson & Johnson  Mandy Jongeneelen, n/a, Johnson & Johnson  Dirk Heerwegh, n/a, Janssen Research and Development (Employee) Georgi Shukarev, MD, Janssen (Employee) Carla Truyers, n/a, Janssen Research and Development (Employee) Anne Marit de Groot, n/a, Johnson & Johnson (Employee) Gert Scheper, n/a, Johnson & Johnson  Jenny Hendriks, n/a, Johnson & Johnson  Boerries Brandenburg, n/a, Johnson & Johnson  Frank Struyf, n/a, Johnson & Johnson  Johan Van Hoof, n/a, Johnson & Johnson  Macaya Douoguih, MD, MPH, Janssen (Employee) Hanneke Schuitemaker, PhD, Johnson & Johnson


BRCA. In this study, we investigated the somatic mutation of DNA damage response genes in epithelial ovarian cancer patients using a multiple-gene panel with next-generation sequencing. In all, 69 serous, 39 endometrioid and 64 clear cell carcinoma patients were enrolled. Serous carcinoma patients (69.6%) had higher percentages of DDR gene mutations compared with patients with endometrioid (33.3%) and clear cell carcinoma (26.6%) . The percentages of DDR gene mutations in patients with recurrence  or cancer-related death  were higher than those without recurrence or living patients. In endometrioid carcinoma, patients with ≥2 DDR gene mutations had shorter PFS  and OS  than those with one mutation or none. In clear cell carcinoma, patients with ≥2 DDR gene mutations had significantly shorter PFS  and OS  than those with 1 DDR mutation or none. In the EOC patients, somatic DDR gene mutations were associated with advanced-stage tumor recurrence and tumor-related death. Type I EOC patients with DDR mutations had an unfavorable prognosis, especially for clear cell carcinoma.DNA damage response (DDR) is important for maintaining genomic integrity of the cell. Aberrant DDR pathways lead to accumulation of DNA damage, genomic instability and malignant transformations. Gene mutations have been proven to be associated with epithelial ovarian cancer, and the majority of the literature has focused on  Epithelial ovarian carcinoma (EOC) is a major cause of death in women worldwide, and patients are usually diagnosed at an advanced stage with a 5-year survival of less than 50% ,2,3,4. CBRCA mutations or homologous recombination deficiency (HRD) status is critical for selecting potential patients, but both positive and negative patients as defined by current HRD assays benefited from PARPi [Precision medicine is the current direction for cancer management depending on the specific genetic or molecular features of cancer. There are several subtypes of EOC—high-grade serous, clear cell, endometrioid, mucinous and low-grade serous—that could be viewed as distinct diseases for their differences in clinical course and pathological features ,7. To daom PARPi ,13,14,15BRCA 1/2 genes participating in HR and maintaining PARPi therapy for BRCA-mutated EOC is a good example of synthetic lethality [DNA damage response (DDR) is important for maintaining a cell’s genomic integrity, and the DDR pathway is composed of various molecules that detect DNA damage, activate cell-cycle checkpoints, trigger apoptosis, and coordinate DNA repair ,17,18. Sethality . Severalethality ,17. UndeThe study protocol was approved by the National Taiwan University Hospital Research Ethics Committee . Informed consent from all participants was obtained and the methods were performed in accordance with the guidelines and regulations. From December 2015 to October 2018, 172 women diagnosed with epithelial ovarian cancer who had received debulking surgery and adjuvant chemotherapy were enrolled. The cancerous tissue specimens collected during debulking surgery were immediately frozen in liquid nitrogen and stored at −70 °C. A portion of the tissue specimens were sent for pathological examinations to confirm the diagnosis and ensure tumorous tissue sufficient for the following experiments. Clinical data were obtained from medical records, including age, cancer stage, the findings during debulking surgery, treatment course and recurrence. Optimal debulking surgery was defined as a maximal residual tumor size <1 cm following surgery. The tumor grade based on International Union Against Cancer criteria, and cancer stage was based on International Federation of Gynecology and Obstetrics (FIGO) criteria . All patWe selected 60 genes involved in DNA damage response (DDR) for the gene panel , includi260/280 ratio.Genomic DNA was isolated using a QIAGEN Genomic DNA extraction kit according to the manufacturer’s instructions . The purity and concentration of the genomic DNA were checked by agarose gel electrophoresis and the ODThe genomic DNA was fragmented with Covaris fragmentation protocol . The size of the fragmented genomic DNA was checked by Agilent Bioanalyzer 2100  and NanoDrop spectrophotometer . The target gene library was generated with NimblGen capture kits . The samples were sequenced by Illumina MiSeq with paired-end reads of 300 nucleotides.The analysis algorithm was conducted according to our previous protocol . BrieflyThe sequence variants were classified according to the IARC variant classification . The patp value of less than 0.05.All statistical analyses were performed using the Statistical Package for Social Sciences software package  and R . One-way ANOVA was used to compare continuous variables and a chi-squared test was used for categorical variables. Survival curves were generated using the Kaplan–Meier method, and differences were calculated using the log-rank test. A multivariate Cox’s regression model was used to evaluate the prognostic factors for progression-free survival (PFS) and overall survival (OS). Statistical significance was set as a There were 172 EOC patients enrolled: 69 serous, 39 endometrioid and 64 clear cell carcinomas . There whttp://www.ncbi.nlm.nih.gov/snp, accessed on 28 September 2021) and bioinformatic analyses , MUTYH (6.4%) and BRCA2 (5.8%) for all patients. Serous carcinoma—TP53 (56.5%), BRCA2 (5.8%) and RAD51C (5.8%); endometrioid carcinoma—TP53 (15.4%), ATM (12.8%) and MSH2 (7.7%); clear cell carcinoma—MUTYH (9.4%), TP53 (4.7%), BRCA2 (3.1%) and ERCC8 (3.1%). The top three prevalent mutated subgroups of DDR genes were CCR (30.8%), HR (10.5%) and BER (7.0%) for all patients. Serous carcinoma—CCR (58.05%), HR (15.9%) and BER (5.8%); endometrioid carcinoma—CCR (23.1%), MMR (15.4%) and HR (7.7%); clear cell carcinoma—BER (9.4%), CCR (6.3%) and HR (6.3%). For detailed information, please refer to The pattern of prevalent mutated DDR genes was different among the histological subtypes . The prop = 0.048, chi-squared test). Endometrioid carcinoma (15.38%) had a higher percentage of MMR mutations than those of serous carcinoma (2.90%) and clear cell carcinoma (4.69%) . Low-grade tumors had a higher percentage of MMR mutations compared with high-grade tumors . Type II tumors had a higher percentage of DSBR mutations than type I tumors . Serous carcinoma (57.97%) had a higher percentage of CCR mutations than those of endometrioid carcinoma (23.08%) and clear cell carcinoma (6.25%) . Type II tumors had higher percentage of CCR mutations than those of type I tumors . The advanced-stage patients had a higher percentage of CCR mutations than the early-stage patients . The recurrent patients had a higher percentage of CCR mutations than those without recurrence . Patients who died of EOC had higher percentages of CCR mutations than living patients . Serous carcinoma (69.57%) had higher percentage of DDR mutations than those of endometrioid carcinoma (33.33%) and clear cell carcinoma (26.56%) . Type II tumors had a higher percentage of DDR mutations than type I tumors . The advanced stage patients had higher percentage of DDR mutations than the early-stage patients . Recurring patients had a higher percentage of DDR mutations than those without recurrence . Patients who died of EOC had a higher percentage of DDR mutations than living patients .We evaluated the correlations between the mutation of DDR genes, the clinicopathologic parameters and outcome of the EOC patients. As shown in p = 0.0072, log-rank test, p = 0.022, log-rank test, p = 0.56, log-rank test, p = 0.47, log-rank test, p = 0.0035, log-rank test, p = 0.015, log-rank test, p = 0.0056, log-rank test, p = 0.0046, log-rank test, EOC patients without DDR gene mutation had longer progression-free survival (PFS) (p = 0.011), 1 DDR gene mutation (HR: 1.71 (1.12–2.60), p = 0.013), endometrioid carcinoma (HR: 0.17 (0.08–0.37), p < 0.001), type II tumor (HR: 2.69 (1.81–4.00), p < 0.001), advanced-stage carcinoma (HR: 5.29 (3.16–8.85), p < 0.001), high-grade tumor (HR: 5.57 (2.26–13.70), p < 0.001) and optimal debulking surgery (HR: 0.28 (0.18–0.41), p < 0.001) were significant in the univariate Cox regression model (p = 0.001) and optimal debulking surgery (HR: 0.51 (0.32–0.80), p = 0.004) were important prognostic factors in the multivariate analysis. Cancer-related death with TLS gene mutation (HR: 33.76 (3.95–289.00), p = 0.001), 1 DDR gene mutation (HR: 1.96 (1.20–3.20), p = 0.007), endometrioid carcinoma (HR: 0.12 (0.04–0.38), p < 0.001), type II tumor (HR: 1.88 (1.19–2.96), p = 0.007), advanced-stage carcinoma (HR: 6.84 (3.28–14.25), p < 0.001), high-grade tumor (HR: 17.97 (2.50–129.29), p = 0.004) and optimal debulking surgery (HR: 0.26 (0.16–0.41), p < 0.001) were significant in the univariate Cox regression model. Type II tumor (HR: 0.35 (0.20–0.60), p < 0.001), TLS gene mutation (HR: 9.57 (1.08–84.83), p = 0.042), advanced-stage carcinoma (HR: 4.82 (2.09–11.09), p < 0.001) and optimal debulking surgery (HR: 0.38 (0.22–0.64), p < 0.001) were important prognostic factors in the multivariate analysis.Tumor recurrence with CCR gene mutation (HR: 1.68 (1.12–2.50), on model . AdvanceATM, BRCA1/2, BRIP1, MLH1, MSH2, MSH6, PALB2, RAD51C and RAD51D [BRCA test alone. The percentage of BRCA 1/2 somatic mutation in serous carcinoma was 7.2, which was compatible with the 6–7% in previous studies [BRCA HR somatic mutation of our study was more than 10% in serous and endometrioid carcinomas, and the MMR somatic mutation was around 15% in endometrioid carcinomas, which was compatible with the previous study [Our study showed that nearly half of the epithelial ovarian cancer (EOC) patients had DNA damage response (DDR) gene mutations with varied proportions of histological subtypes. Two-thirds of serous adenocarcinoma patients, one-third of endometrioid adenocarcinoma patients and one-fourth of clear cell carcinoma patients had DDR gene mutations. Our DDR gene panel consisted of the genes involved in single-strand break repair, double-strand break repair and cell cycle regulation, including the genes recommended by National Comprehensive Cancer Network (NCCN) guidelines as cost-effective tools for assessing the lifetime risk of EOC, such as d RAD51D . The maj studies ,35,36,37us study .ARID1A, PIK3CA, KRAS and PPP2R1A) might be related to chromatin remodeling, cell proliferation, cell cycle checkpointing and cytoskeletal organization [ARID1A, PIK3CA, PPP2R1A or TP53 in ovarian clear cell carcinoma did not correlate well with the prognosis [ARID1B, ARID3A, CREBBP, CSMD3, CTNNB1, LPHN3, LRP1B, MAGEE1, MLH1, MLL3, MUC4, PIK3R1, PTEN and TP53 [Our study showed that ovarian clear cell carcinoma patients with DDR gene mutations had an unfavorable survival prognosis. Those who had somatic DDR mutations were significantly associated with advanced-stage carcinomas, tumor recurrence and tumor-related death. The trend was different in histological subtypes as serous carcinomas or type II tumors with DDR mutation showed a better survival trend. Non-serous or type I EOC patients with DDR mutations had a poor prognosis, especially in clear cell carcinoma. Ovarian clear cell carcinoma is an aggressive drug-resistant subtype of EOC in association with endometriosis and glycogen accumulation. It accounts for about 5–13% of all EOCs in Western populations, but up to 20–25% in East Asia, including Taiwan [nization ,47,48,49rognosis . Other iand TP53 ,46,48,49BRCA gene tests or companion HRD assays are currently suggested for PARPi, but there are unmet problems that need to be resolved [BRCA (tBRCA) mutations, including germline (gBRCA) or somatic (sBRCA), derived the greatest benefit from PARPi maintenance therapy [BRCA mutation, and another 6–7% patients had an sBRCA mutation with a negative gBRCA test [BRCA mutated patients tested negative for tBRCA [BRCA HR gene mutations were usually pooled together to interpret the association with clinical outcomes in previous studies because of their relatively low prevalence [BRCA somatic mutations  derived benefit from olaparib in study 19 [BRCA HR gene mutations , but the sensitivity in discriminating a rucaparib response was only 11% [BRCA wild type EOC patients still benefitted from PARPi, which indicated that a BRCA test by itself was inadequate for selecting EOC patients for PARPi [BRCA HR genes could be used to predict a PARPi response, especially in non-serous EOC patients.Our DDR gene panel could provide a scientific rationale for patient selection in future clinical trials that target DNA damage repair response pathways, especially in clear cell carcinoma. resolved ,14,15,20resolved ,50,51. G therapy ,13,14,15RCA test ,35,36,37or tBRCA ,53,54. Tevalence ,56,57. Tstudy 19 . In ARIEonly 11% . HoweverATM, BRCA1/2, BRIP1, MLH1, MSH2, MSH6, PALB2, RAD51C, RAD51D and STK11 to assess the lifetime risk of EOC [BRCA gene, there is a difference among laboratories in the VUS reporting rate (3–50%), detection protocols and management strategies [There were limitations to our study. First, germline gene mutations were not investigated. These not only inform the patients but also identify family members of the possible risk of malignancy ,53,54. Tk of EOC , but howk of EOC ,61,62. Tk of EOC . Even inrategies . FurtherOur study found that nearly half of the EOC patients had DDR gene mutations of varying proportions in the histological subtypes. Patients with somatic DDR mutations were significantly associated with advanced-stage carcinoma, tumor recurrence and tumor-related death. Type I EOC patients with DDR mutations had an unfavorable prognosis, especially for clear cell carcinoma. A broad multiple-gene DDR panel would provide not only comprehensive information of gene mutations but also a rationale for a future study of a novel therapy target for DNA damage response pathways.


Correction to: BMC Pharmacol Toxicol 20, 5 (2019)https://doi.org/10.1186/s40360-018-0280-8After publication of the original article , an erroThe incorrect sentence is:P < 0.0001).SIB peak plasma concentrations were 207.1 mg/L for SPC and 12.6 mg/L for SM tablets. All pharmacokinetic parameters differed significantly between formulations (The correct sentence is:P < 0.0001).SIB peak plasma concentrations were 207.1 ng/L for SPC and 12.6 ng/L for SM tablets. All pharmacokinetic parameters differed significantly between formulations (The original article has been corrected.


Breast cancer is the most common cancer in women . Triple-First, we generated a cisplatin (DDP)-resistant TNBC cell line, MDA-MB-231/DDP, which is 5-fold more resistant to cisplatin than its parental line MDA-MB-231  Fig. a. IntereP = 0.005). Consistently, in both validation cohort of 128 samples . Likewise, low-SNORD33 group showed significantly reduced overall survival (OS) compared with high-SNORD33 group , validation group  and combined cohorts   patients who received first-line platinum-based chemotherapy Fig. f. In theles Fig. a, low-SN38) Fig.  revealedROC test was used to evaluate the value of SNORD33 in predicting PFS in mTNBC patients treated with platinum-based regimens in the combined cohort. SNORD33 signature showed the strongest predictive value (AUC = 0.652) for PFS, compared with other single clinicopathological risk factors , suggesting better performance of SNORD33 signature as a surrogate predictor for platinum-based chemotherapy outcome Fig. m. Based P = 0.023) than high-SNORD33 group  [RPL13A (host gene), DICER enzyme (an endoribonuclease), or SNORD32a (homologue to SNORD33 modifying 18S rRNA during cell proliferation) in MDA-MB-231 cells  . It part(RPL13A) . Does SNlls Fig. a. The insis Fig. b. We obssis Fig. b. We thenes Fig. c. Down-rnes Fig. . ConsistTogether, we identified plasma SNORD33 signature as a predictor for the sensitivity of platinum-based chemotherapy in mTNBC patients. Furthermore, we showed that MeCP2 could convey SNORD33 associated platinum resistance. Our findings may have immediate translational relevance for precision platinum-based chemotherapy.Additional file 1. Methods.Additional file 2: Supplementary Figure 1. Aberrantly expressing RNA in 231/DDP cells. Supplementary Figure 2. SNORD33 knockdown increases proliferation and decreases apoptosis of TNBC cells. Supplementary Figure 3. Reduced SNORD33 level is correlated with poor prognosis of mTNBC patients who received first-line platinum-based chemotherapy. Supplementary Figure 4. Increased cell viability was observed in cisplatin treated cell lines with SNORD33 knockdown. Supplementary Figure 5. Reduced SNORD33 level correlates with poor prognosis of non-small cell lung cancer (NSCLC) patients who received first-line platinum-based chemotherapy. Supplementary Figure 6. MeCP2 is a candidate protein binding with SNORD33. Supplementary Figure 7. Down-regulation of MeCP2 partially rescues SNORD33 knockdown increased cell colony formation. Supplementary Figure 8. Down-regulation of MeCP2 rescues SNORD33 knockdown decreased cell apoptosis and induced alteration of apoptotic markers.Additional file 3: Supplementary Table 1. Baseline characteristics in mTNBC patients received first-line platinum-containing regimens. Supplementary Table 2. Univariate and multivariate analysis of prognostic factors associated with progression-free survival and overall survival in the combined cohort. Supplementary Table 3. Baseline characteristics in mTNBC patients received first-line non-platinum-containing regimens. Supplementary Table 4. Baseline characteristics in lung adenocarcinoma patients received first-line platinum-containing regimens. Supplementary Table 5. Univariate and multivariate analysis of prognostic factors associated with progression-free survival in lung adenocarcinoma patients.Additional file 4.


CMV infection is common post-kidney transplant (KT). Valganciclovir (VGC) prophylaxis (Px) has lessened CMV infection among high-risk (CMV D+/R-) KT recipients (KTRs), but VGC can induce neutropenia. We quantified the burden of CMV infection among CMV D+/R- KTRs and healthcare resources required to manage these patients (pts).Retrospective study of pts undergoing KT between Jan 2014-Dec 2018. Study and control groups (gps) were CMV D+/R- and R+ KTRs, respectively. Standard post-KT immunosuppression was tacrolimus and mycophenolate mofetil (MMF). D+/R- and R+ KTRs received VGC Px (900 mg/day) for 6 and 3 months (mos), respectively.Clinical characteristics did not differ between D+/R- (n=131) and R+ (n=140) pts. Median VGC Px duration was longer for D+/R- . Within the first 6 mos post KT, a higher proportion of D+/R- KTRs received ≥1-course of granulocyte-stimulating factor (G-CSF) . VGC Px was stopped prematurely/intermittently in 20% and 10% of D+/R- and R+, respectively, due to neutropenia (p=0.02); corresponding data for stopping MMF for ≥1 mos were 32% and 21% (p=.05). 50% of D+/R- pts received < 3 mos Px. Leukopenia prompted hospitalization in 3% of D+/R- vs 0% of R+ pts (p=.05). CMV infections did not differ between gps ; however, VGC-resistant CMV was higher in D+/R- gp . Between 6-12 mos post-KT, D+/R- KTRs had higher rates of CMV infection , VGC resistance , hospitalization due to CMV , MD intervention , and infectious disease (ID) referral . 57% of CMV resistance was observed in pts who prematurely stopped VGC. Hospitalizations were longer for CMV infections in D+/R- KTRs . There was a trend toward higher rejection for D+/R- KTRs .Universal VGC Px in D+/R- KTR remains challenging and requires significant resources for monitoring and intervention for neutropenia, including MD involvement and ID referral. Intermittent/premature stop of VGC may have led to VGC-resistant CMV,and stop of MMF may have led to a trend of higher cellular rejection at 1 yr. There is critical need for new CMV agents with a better safety profile.Amit D. Raval, PhD, Merck and Co., Inc. (Employee) Yuexin Tang, PhD, JnJ Merck & Co., Inc.  Cornelius J. Clancy, MD, Merck (Grant/Research Support) Minh-Hong Nguyen, MD, Merck (Grant/Research Support)


Carbapenem (Carb) minimum inhibitory concentration (MIC) breakpoints were lowered by CLSI in 2010 and recognized by FDA in 2012. Adoption of revised breakpoints is often slow, which may lead to under-reporting of Carb non-susceptibility (NS) by facilities. We compare facility-reported rates of Carb-NS ENT to the CLSI MIC breakpoints for a large nationwide collection of isolates in the United States (US) from 2016-2019.All adults with a positive non-contaminant ENT culture  in ambulatory/inpatient settings from up to 300 US hospitals from 2016-2019 were evaluated (BD Insights Research Database). Facility-reported Carb-NS was defined as: susceptible (S), intermediate (I) or R to ertapenem (ETP), imipenem (IPM), meropenem (MEM) and/or doripenem (DOR) per commercial panels. Where available, MICs were interpreted using CLSI 2010 MIC breakpoints (µg/ml): ≤ 0.5 (S), 1 (I), ≥ 2 (R) for ETP and ≤1 (S), 2 (I), and ≥ 4 (R) for IPM/MEM/DOR. For evaluable ENT isolates we compared susceptibility results as reported by the facility to CLSI MIC breakpoints.Overall, 77.4%  and 90.6%  non-duplicate ENT isolates with facility-reported susceptibility results also had interpretable MIC results for ETP and IPM/MEM/DOR, respectively (Tables). ETP S rates were 99.3% and 99.1% as reported by facilities and using CLSI criteria, respectively. S rates of other Carbs were 98.9% and 98.4% by facility reporting and CLSI criteria, respectively. Systematic application of CLSI breakpoints under-reported EPT-I and –R isolates by 24.2% and 16.4%, respectively, and identification of IPM/MEM/DOR-I and –R isolates by 31.3% and 22.7%, respectively.Systematic application of CLSI breakpoints in 2016-19 would have had minimal impact on ENT S rates in the US. However, facility reporting failed to identify 18.8% of ETP I or R and 26.5% of IPM/MEM/DOR I or R isolates. The clinical implications of this observation are unknown. Facilities should know their local epidemiology, decide if under-reporting might be an issue, and assess if there is any impact on their patients.Vikas Gupta, PharmD, BCPS, Becton, Dickinson and Company  Kalvin Yu, MD, BD (Employee) Jason M Pogue, PharmD, BCPS, BCIDP, Merck (Consultant)QPex (Consultant)Shionogi (Consultant)Utility Therapeutics (Consultant)VenatoRX (Consultant) Janet Weeks, PhD, Becton, Dickinson and Company (Employee) Cornelius J. Clancy, MD, Merck (Grant/Research Support)


Scientific Reports 10.1038/s41598-021-98868-y, published online 30 September 2021Correction to: The original version of this Article contained an error in the order of the References 16 and 17, which was incorrectly given as:et al. Vitamin D increases glucocorticoid efficacy via inhibition of mTORC1 in experimental models of multiple sclerosis. Acta Neuropathol. 10.1007/s00401-019-02018-8 (2019).16. Hoepner, R. FASEB J23, 3649–3658 (2009).17. Ayroldi E, Riccardi C. Glucocorticoid‐induced leucine zipper (GILZ): a new important mediator of glucocorticoid action. The correct order of the References is listed below:FASEB J23, 3649–3658 (2009).16. Ayroldi E, Riccardi C. Glucocorticoid‐induced leucine zipper (GILZ): a new important mediator of glucocorticoid action. et al. Vitamin D increases glucocorticoid efficacy via inhibition of mTORC1 in experimental models of multiple sclerosis. Acta Neuropathol. 10.1007/s00401-019-02018-8 (2019).17. Hoepner, R. The original Article has been corrected.


Effective treatment of glioma requires a nanocarrier that can cross the blood–brain barrier (BBB) to target the tumor lesion. In the current study, elemene (ELE) and cabazitaxel (CTX) liposomes were prepared by conjugating liposomes with transferrin (Tf) and embedding the cell membrane proteins of RG2 glioma cells into liposomes , which exhibited effective BBB infiltration to target glioma.The findings showed that Tf-ELE/CTX@BLIP was highly stable. The liposomes exhibited highly significant homologous targeting and immune evasion in vitro and a 5.83-fold intake rate compared with classical liposome (ELE/CTX@LIP). Bioluminescence imaging showed increased drug accumulation in the brain and increased tumor penetration of Tf-ELE/CTX@BLIP in orthotopic glioma model nude mice. Findings from in vivo studies indicated that the antitumor effect of the Tf-ELE/CTX@BLIP led to increased survival time and decreased tumor volume in mice. The average tumor fluorescence intensity after intravenous administration of Tf-ELE/CTX@BLIP was 65.2, 12.5, 22.1, 6.6, 2.6, 1.5 times less compared with that of the control, CTX solution, ELE solution, ELE/CTX@LIP, ELE/CTX@BLIP, Tf-ELE/CTX@LIP groups, respectively. Histopathological analysis showed that Tf-ELE/CTX@BLIP were less toxic compared with administration of the CTX solution.These findings indicate that the active-targeting biomimetic liposome, Tf-ELE/CTX@BLIP, is a promising nanoplatform for delivery of drugs to gliomas.The online version contains supplementary material available at 10.1186/s12951-021-01048-3. P-gp) occurs on the BBB, which further increases clearance of chemotherapy drugs  = 20 μg/mL) and incubated for 2 h. In addition, RG2 cells were seeded into a 6-well plate (3 × 105 cells/well) and incubated with ELE/CTX@LIP, ELE/CTX@BLIP, Tf-ELE/CTX@LIP and Tf-ELE/CTX@BLIP (Rho B = 20 μg/mL) for 2 h. The medium was removed and cells were fixed with 4% paraformaldehyde, stained with DAPI and washed thrice with PBS. The cellular uptake was determined by confocal laser scanning microscope imaging  and flow cytometry [Tf-ELE/CTX@BLIP was incubated with SPC-A-1 cells, A549 cells, MDA-MB-231 cells, LM-3 cells, U251 cells, C6 cells, and RG2 cells, separately. Cells were inoculated into 6-well plates  for 2 h. Effect on immune evasion was then determined by CLSM and flow cytometry .Cytotoxicity effects of ELE/CTX@LIP, ELE/CTX@BLIP, Tf-ELE/CTX@LIP and Tf-ELE/CTX@BLIP on RG2 cells were determined using a CCK-8 kit. RG2 cells (3000 cells/well) were inoculated in 96-well plates and incubated for 24 h. After incubation, cells were exposed to ELE/CTX@LIP, ELE/CTX@BLIP, Tf-ELE/CTX@LIP and Tf-ELE/CTX@BLIP (CTX concentrations ranging from 0.4 to 200 ng/mL) for 48 h. Non-treated cells were used as negative controls. Medium was evaluated as the blank control. Cytotoxicity was quantitatively determined by measuring the absorbance at 450 nm using a Spark multi-functional microporous plate testing platform .Furthermore, apoptotic cells were determined by FACS analysis . RG2 cells were incubated with ELE/CTX@LIP, ELE/CTX@BLIP, Tf-ELE/CTX@LIP and Tf-ELE/CTX@BLIP and 50 ng/mL CTX for 48 h then treated with the apoptosis detection kit  for 10 min. Percentage of apoptotic cells was determined using a FACS Calibur System. Non-treated cells were used as the negative control .5 cells/well) and incubated for 24 h. Rhodamine 123  was preconditioned with ELE/CTX@LIP, ELE/CTX@BLIP, Tf-ELE/CTX@LIP, Tf-ELE/CTX@BLIP (50 ng/mL) and verapamil (0.625 μg/mL) separately for 30 min then it was exposed to bEnd.3 for 2 h. The medium was removed and bEnd.3 cells were fixed with 4% paraformaldehyde for 30 min and washed thrice with PBS. Cells were then analyzed using fluorescence microscope and flow cytometry. Relative expression level of P-gp in bEnd.3 cells was determined by WB assay. bEnd3 cells in 6-well plates were conditioned with medium and verapamil, ELE/CTX@LIP, ELE/CTX@BLIP, Tf-ELE/CTX@LIP, Tf-ELE/CTX@BLIP were added [bEnd.3 cells were cultured as described in the “re added .Female nude mice  were obtained from Shanghai Slack Laboratory Animal Co. LTD. Mice were allowed to acclimatize at room temperature for 7 days prior to the performing animal studies. Animals were housed under the animal care facility and allowed free access to food/water. All animal experiments were approved by the animal ethics committee of Hangzhou Normal University .7 glioma-luc cells in medium were injected slowly into the brain right striatum of nude mice . The needle was maintained at the injection point for 1 min after injection and then pulled out slowly. Alcohol cotton swabs were used to disinfect the skin, the skin was sewn with a needle and thread. The mice were then placed back to the cage to wake up naturally. Rate of growth of intracranial tumor was monitored by magnetic resonance imaging (MRI) and fluorescence imaging.Glioma-luc cells were injected into the right striatum of nude mice to develop a orthotopic glioma-bearing model. Nude mice were anesthetized through administration of 10% chloral hydrate and the head was immobilized on a stoelting . Approximately 2.5 × 10Cypate = 0.1 mg/mL) were injected into normal mice and glioma-bearing mice through the tail vein. Bioluminescence imaging was performed at fixed times  using a small animals in vivo 3D bioluminescence imaging system .The nude mice were then sacrificed, and brains, liver, heart, spleen, lung, and kidney were collected for quantitative biodistribution analysis and ex vivo bioluminescence imaging [Orthotopic glioma-bearing model was established by injecting RG2 cells into the brain striatum as described above. Cypate, Cypate@LIP, Cypate@BLIP, Tf-Cypate@LIP and Tf-Cypate@BLIP (CP-gp.Orthotopic glioma-bearing model nude mice were randomly assigned into seven groups (6 mice/group) as follows: (1) control ; (2) CTX solution; (3) ELE solution (25 mg/kg ELE); (4) ELE/CTX@LIP; (5) ELE/CTX@BLIP; (6) Tf-ELE/CTX@LIP; (7) Tf-ELE/CTX@BLIP, to explore the anti-tumor effect in vivo. Treatments were administered by intravenous injection through the tail at day 1, 3, 5, 7, 9 and 11. Group 2, 4, 5, 6, 7 was administered with 2.5 mg/kg (CTX) on the first time and 0.625 mg/kg for the subsequent 5 injections. Bioluminescence imaging was performed at day 1, 5, 10, and 15 to explore tumor growth. Body weight and survival time were recorded every 3 days. Nude mice were euthanized 2 h post-treatment and their brains were collected for H&E staining, TUNEL immunofluorescence staining and detection of glioma Glioma-bearing nude mice were assigned into seven groups and were administered with (1) control ; (2) CTX solution; (3) ELE solution; (4) ELE/CTX@LIP; (5) ELE/CTX@BLIP; (6) Tf-ELE/CTX@LIP; (7) Tf-ELE/CTX@BLIP, 6 times and every other day. Whole blood samples were collected from retro-orbital sinus of glioma-bearing nude mice 2 h post-treatment. A portion of the complete blood samples was used for complete blood count analysis. The supernatant (serum) of the portion of blood obtained after centrifugation  was used for determination of biochemical indexes of liver and kidney, including total bilirubin, blood urea nitrogen, uric acid, alanine transaminase, creatinine and aspartate transaminase level. Liver, heart, spleen, lung and kidney sections were stained with H&E to evaluate effects of free ELE, CTX solution and liposomes toxicity .t test for comparison between two groups and ANOVA for comparison among multiple groups. Statistical difference was defined as significant for *p < 0.05 and highly significant for **p < 0.01.GraphPad Prism 8.0.2.263 software  was used for statistical analysis. Data were expressed as mean ± SD. Experimental data were analyzed using two-tailed Student Additional file 1: Table S1. Encapsulation efficiency stability of the drug in 4 liposomes at 3 months (n = 3). Fig. S1. Diameter and ζ-potential of Tf-ELE/CTX@BLIP, Tf-ELE/CTX@LIP, ELE/CTX@BLIP and ELE/CTX@LIP. Fig. S2. 7-days stability of TF-ELE/CTX@BLIP diameter in different medium. Fig. S3. Flow cytometry analysis of RG2 glioma cells after incubation with Tf-ELE/CTX@BLIP, Tf-ELE/CTX@LIP, ELE/CTX@BLIP and ELE/CTX@LIP for 2 h. Rho B = 20 μg/mL. Fig. S4. CLSM images of RG2, U251 and C6 glioma cells treated with Tf-ELE/CTX@BLIP for 2 h. Scale bar = 50 μm. Fig. S5. Flow cytometry analysis of RAW264.7 cells treated with Tf-ELE/CTX@BLIP, Tf-ELE/CTX@LIP, ELE/CTX@BLIP and ELE/CTX@LIP for 2 h. Rho B = 20 μg/mL. Fig. S6. WB analysis of P-gp in bEnd.3 cells. Relative protein expression was calculated. Cells preconditioned with (1) control; (2) verapamil; (3) ELE/CTX@LIP; (4) ELE/CTX@BLIP; (5) Tf-ELE/CTX@LIP; (6) Tf-ELE/CTX@BLIP. . Fig. S7. Corresponding quantitative fluorescent analysis of brain, liver, heart, spleen, lung and kidney at 48 h post-injection in glioma-beard mice. *p < 0.05. Fig. S8. In vivo fluorescence imaging of Tf-Cypate@BLIP, Tf-Cypate@LIP, Cypate@BLIP and Cypate@LIP in normal mice. Cypate = 0.5 mg/kg. Fig. S9. Averaged fluorescent intensity of saline, free CTX, free ELE, ELE/CTX@LIP, ELE/CTX@BLIP, Tf-ELE/CTX@LIP and Tf-ELE/CTX@BLIP in nude mice bearing orthotopic glioma brain within 15 days of treatments. Fig. S10. H&E staining of brain sections of orthotopic glioma-bearing mice in different formulation groups. Fig. S11. Biochemical parameter analysis after treated with saline, free CTX, free ELE, ELE/CTX@LIP, ELE/CTX@BLIP, Tf-ELE/CTX@LIP and Tf-ELE/CTX@BLIP. (A) BLI-T: total bilirubin, (B) BUN: blood urea nitrogen, (C) URIC: uric acid, (D) ALT: alanine transaminase, (E) CRE: creatinine, (F) AST: aspartate transaminase.


Compared with WT mice, S−/−tat1 had reduced neutrophils, Th1, and Th17 cell infiltration. To evaluate corticosteroid sensitivity, mice were treated with either vehicle, 1 or 3 mg/kg fluticasone propionate (FP). Corticosteroids significantly reduced eosinophil infiltration and cytokine levels in both c-di-GMP + MA-challenged WT and −/−Stat1 mice. However, histological and functional analyses show that corticosteroids did not reduce airway inflammation, epithelial mucous cell abundance, airway smooth muscle mass, and AHR in c-di-GMP + MA-challenged WT or −/−Stat1 mice. Collectively, our data suggest that increased Th1 inflammation is associated with a decrease in corticosteroid sensitivity. However, increased airway pathology and AHR persist in the absence of STAT1 indicate corticosteroid insensitivity in structural airway cells is a STAT1 independent process.Corticosteroid insensitivity in asthma limits the ability to effectively manage severe asthma, which is characterized by persistent airway inflammation, airway hyperresponsiveness (AHR), and airflow obstruction despite corticosteroid treatment. Recent reports indicate that corticosteroid insensitivity is associated with increased interferon-γ (IFN-γ) levels and T-helper (Th) 1 lymphocyte infiltration in severe asthma. Signal transducer and activator of transcription 1 (STAT1) activation by IFN-γ is a key signaling pathway in Th1 inflammation; however, its role in the context of severe allergic airway inflammation and corticosteroid sensitivity remains unclear. In this study, we challenged wild-type (WT) and  Corticosteroids have broad anti-inflammatory effects that contribute to the effective management of asthma symptoms and exacerbations. In contrast to the corticosteroid-sensitive mild asthma, people with severe asthma exhibit greater airway inflammation, remodeling, airway hyperresponsiveness (AHR), and more frequent exacerbations despite treatment with higher doses of corticosteroids , 2. Alth+ T lymphocytes in bronchoalveolar lavage samples  levels . Studies samples , 9. Alth samples . These o samples , 11–13.  samples , 11, imp−Stat1/− mice exhibit reduced Th1 responses and IFN-γ production  is a transcription factor that is activated by IFN-γ and mediates proinflammatory responses in immune and airway structural cells . Given toduction , 16. Preoduction . Given t−Stat1/− mice using a chronic mixed allergen (MA) model augmented with c-di-GMP. We hypothesized that ablation of STAT1 would disrupt Th1 inflammation and increase corticosteroid sensitivity in chronic severe allergic airway inflammation. However, our data demonstrate reduced corticosteroid sensitivity in both WT and −Stat1/− mice. Examination of immunological, functional, and structural aspects of chronic allergic airway inflammation shows that persistent airway remodeling remains increased in −Stat1/− mice despite treatment with corticosteroids. Our studies demonstrate that corticosteroid insensitivity remains in the absence of STAT1 signaling during severe allergic airway inflammation.In this study, we examined corticosteroid sensitivity in wild-type (WT) and −Stat1/− (Stock No. 012606) mice were procured from Jackson Laboratory . Mice were maintained in Research Building III at the Abigail Wexner Research Institute and provided food and water ad libitum. Adult male and female mice were mated to generate pup litters that were randomly assigned a treatment group. Newborn male and female mice were intranasally (in) sensitized and challenged with PBS or mixed allergens (MA), which consists of 10 µg Alternaria alternata , 10 µg Aspergillus fumigatus , 10 µg Dermatophagoides pteronyssinus , and 10 µg ovalbumin (OVA) plus 0.5 µg c-di-GMP , three times per week for 7 wk. During the last 2 wk, mice were intraperitoneally (ip) injected with vehicle (PBS containing 0.015% DMSO) or 1 mg/kg or 3 mg/kg FP  on the same day as MA challenge. Necropsy was performed ∼24 h after last allergen challenge  and hallenge A. Mice wg for 5 min. Supernatant was collected and stored at −80°C for further analyses. Cellular pellets were resuspended in PBS for total and differential cell count analysis. Total counts were performed using a hemocytometer. Cytospins were generated on microscope slides and differentially stained using a modified Wright-Giemsa Stain . Cells were imaged on a bright-field microscope and counted by a blinded investigator until a total of 200 cells had been counted.Bronchoalveolar lavage fluid (BAL) was harvested as previously described . BrieflyOnce anesthetized, the trachea was exposed and cannulated with a 19-gauge blunt tip cannula. The mouse was placed on a 37°C heating pad and attached to Y-tubing on the Flexivent . A series of measurements including Snapshot  maneuvers were performed following nebulization with PBS, 6.25, 12.5, 25, and 50 mg/mL methacholine . Airway hyperresponsiveness is reported as total resistance in response to methacholine.Lung tissue was homogenized in RIPA buffer, processed, and protein concentration measured using Bradford assay. Each well in custom Meso scale U-Plex Multiplex ELISA plates  was coated with antibodies against IL-4, IL-17A, and IFN-γ. Analytes were measured in whole lung homogenates following manufacturer instructions. IL-13 levels in BAL were measured using ELISA  according to the manufacturer’s instructions.Samples were quantified by BCA assay (Pierce) and 25 µg of protein was analyzed per sample. Samples were separated on a 14% SDS-PAGE gel and transferred to nitrocellulose membrane (Bio-Rad). Membranes were blocked for 1 h at room temperature in 1% milk/TBS-T and then incubated in primary antibody overnight at 4°C. Primary antibodies were purchased from Cell Signaling : monoclonal rabbit anti-Stat1 antibody at 1:1,000 dilution, Cat. No. 14994S; polyclonal rabbit antiphospho-Stat1 (Ser727) antibody at 1:500 dilution, Cat. No. 9177S; monoclonal rabbit anti-Stat6 at 1:500, Cat. No. 5397S; and monoclonal rabbit anti-phospho-Stat6 (Tyr641) at 1:500, Cat. No. 56554S. The following day, samples were washed in Tris-buffered saline-Tween 20 (TBST), incubated with secondary antibodies  for 1 h at room temperature, washed with TBST, and imaged on an Odyssey Clx (LI-COR). Protein quantification was performed using ImageStudio Software (LI-COR).2O. Lungs were processed, paraffin embedded, cut into 6 µm sections, and stained with hematoxylin and eosin . H&E slides were analyzed and scored for inflammation  and bronchial-associated lymphoid tissues (BALTs) were quantified by a blinded investigator. Inflammation scores are based on the degree of immune cell infiltration/aggregation around peribronchiolar and perivascular spaces. To quantify BALTs, compact lymphoid nodules were counted in proximal and distal regions. To quantify abundance of mucous cells in the airway epithelium, left lung lobe sections were stained with Alcian Blue-Periodic Acid Schiff (AB-PAS) and photomicrographs of four different airways were taken at ×100 using an Olympus BX-40 light microscope and digital camera . PAS-positive and -negative cells were quantified by a blinded investigator using ImageJ (NIH) and reported as a percentage of total airway epithelial cells counted.Left lung lobes were inflated with 10% neutral-buffered formalin at 25 cm HFormalin-fixed, paraffin embedded left lung lobe sections (6 µm) were used for immunohistochemical staining for α-smooth muscle actin (α-SMA). Sections were deparaffinized with xylene (2 × 5 min each) and rehydrated with graded ethanol . Antigen retrieval was performed with 10 mM sodium citrate at 100°C for 1 h. The slides were blocked for 1 h in TBS containing 4% goat serum and 0.04% Triton X-100. After washing, slides were incubated overnight in monoclonal mouse anti-α-SMA at 1:100 dilution . After incubation, slides were washed and incubated with anti-mouse-FITC secondary antibody  at 1:1,000 dilution. Negative control slides followed the same protocol but without addition of a primary antibody. Slides were counterstained with DAPI, and images taken at ×100 using Lionheart . Smooth muscle actin area was analyzed using ImageJ (NIH). Data were normalized to length of airway basement membrane to report as airway smooth muscle (ASM) mass per µm basement membrane.12–18 primers and Superscript IV (Thermo Fisher Scientific Applied Biosystems). Quantitative real-time PCR with TaqMan using Muc5ac (Mm0127618_m1), Muc5b (Mm00466391_m1), and GAPDH (Mm99999915_g1) primer sets was performed (Life Technologies) according to the manufacturer’s instructions. Samples were run on a QuantStudio 3 96-well Real-Time PCR system (Thermo Fisher Scientific Applied Biosystems). Results were analyzed using the comparative Ct method for TaqMan assays.RNA (300–1000 ng) from profiled samples was cDNA transcribed using Oligo dTg for 10 min, and resuspended in 10 mL of DMEM medium. Cells were counted using a hemocytometer. For Th stimulation, two million cells were incubated in DMEM medium containing leukocyte activation cocktail with Golgi plug , protein transported inhibitor containing Monensin , and 10% FBS for 37°C for 4 h. Cells were collected and permeabilized/fixed using fixation/permeabilization buffer and 1× permeabilization/wash buffer  and resuspended in 200 μL of PBS. Single cell suspensions were used in flow cytometry analysis.Lungs were perfused with 5 mL of PBS through right ventricle of the heart, and then placed in C-tubes  and digested for 30 min in 432 U/mL Collagenase Type IV  and 64 U/mL DNAase I  in DMEM medium. Lung tissue was homogenized using Gentlemacs octo dissociator (Miltenyi Biotec), strained through 70 μm nylon cell strainer, centrifuged at 300 1 isotypes were used. All antibodies were purchased from BioLegend. Data were acquired using a BD LSRII flow cytometer  and analyzed using FlowJo software (v. 10.7.1).Single cell suspensions were stained with various fluorochrome conjugated anti-mouse monoclonal antibodies for cellular phenotyping as follows: FITC-conjugated anti-CD45 (30-F11), BV421-conjugated anti-CD3 (17A2), PE-conjugated anti-CD4 (A161A1), Alexa 700-conjugated anti-IFNγ (XMG1.2), APC-conjugated anti-IL-4 (11B11), and APC-conjugated anti-IL-17A (TC11-18H10.1). To control for intracellular cytokine staining, APC (RTK2071) and Alexa fluor 700 (RTK2071) IgGP < 0.05.Data were analyzed by performing two-way ANOVA with Bonferroni post hoc analysis for multiple comparisons. Data were analyzed and graphed using GraphPad Prism 9 Software . Values are presented a means ± standard error and significant differences indicated by −Stat1/− mice challenged with c-di-GMP + MA exhibited increased total BAL immune cells compared with PBS-challenged WT mice  and  WT mice B. BAL to WT mice C. Differ WT mice D. In con−/− mice E. Treatm WT mice B, wherea−/− mice D and E. −/− mice C.+CD4+ T cell numbers were significantly increased in c-di-GMP + MA-challenged WT and −Stat1/−  and IL-4 and IL-13 expression. Th2 lymphocytes were increased in c-di-GMP + MA-challenged WT and −Stat1/− mice and were not reduced with 1 or 3 mg/kg FP treatment  cells and IL-17A levels were significantly higher in c-di-GMP + MA-challenged WT and −Stat1/− mice  nodules quantified. Compared with PBS challenge, c-di-GMP + MA challenge significantly increased airway inflammation in WT and −/− mice A and B. −/− mice B. To anagenotype C. In regcontrols D. A signmg/kg FP C.−Stat1/−mice exhibited significant increases in mucous cells compared with PBS-challenged mice  mass in lung sections stained for α-smooth muscle actin. C-di-GMP + MA-challenged WT and mg/kg FP .−Stat1/− mice showed significantly increased AHR compared with PBS-challenged mice. Treatment with 1 mg or 3 mg/kg fluticasone propionate did not significantly reduce AHR in WT and −Stat1/− mice  was measured using forced oscillation maneuvers following administration of methacholine (0–50 mg/mL). C-di-GMP + MA-challenged WT and −/− mice . To analcontrols .−Stat1/− mice that exhibit dysfunctional Th1 signaling  and c-di-GMP exhibited reduced AHR even in the absence of corticosteroids, implicating Th1 inflammation in increased AHR . Although we observed that corticosteroids significantly reduced IL-4 and IL-13 expression levels in −Stat1/− mice, their levels did not return to baseline. IL-4 and IL-13 at relatively low levels have been shown to induce mucus production, AHR, and remodeling  expression and mucus production are associated with increased Th2-associated inflammation  model, Ifnγ−/− and −Irf5/− mice exhibited evidence of increased Th2 inflammation with greater eosinophil infiltration than allergen-challenged WT mice , and persistent airway remodeling could be contributing factors in the lack of reduced airway inflammation and corticosteroid sensitivity in −Stat1/− mice.Our findings in  WT mice . However−Stat1/− mice, persistent airway inflammation and corticosteroid insensitivity may involve enhanced Th2 inflammation. Negative regulation of Th2 inflammatory responses by Th1 inflammation is an established and important immune regulatory mechanism , R01 HL155095 (to R. D. Britt), R01 AI121405 (to M. Guerau-de-Arellano), R03 AI151769 (to M. Guerau-de-Arellano), and startup funds from the Abigail Wexner Research Institute at Nationwide Children’s Hospital.No conflicts of interest, financial or otherwise, are declared by the authors.B.W.L., M.H.G., and R.D.B.J. conceived and designed research; B.W.L., D.J., S.A.A., J.W., M.G., S.G., E.C., A.V.B., and R.D.B.J. performed experiments; B.W.L., D.J., S.A.A., J.W., M.G., S.G., E.C., A.V.B., M.G.-d.-A., and R.D.B.J. analyzed data; B.W.L., M.G.-d.-A., M.H.G., and R.D.B.J. interpreted results of experiments; B.W.L. and R.D.B.J. prepared figures; B.W.L., M.G.-d.-A., M.H.G., and R.D.B.J. drafted manuscript; B.W.L., D.J., S.A.A., J.W., M.G., S.G., E.C., A.V.B., M.G.-d.-A., M.H.G., and R.D.B.J. edited and revised manuscript; B.W.L., D.J., S. A.A., J.W., M.G., S.G., E.C., A.V.B., M.G.-d.-A., M.H.G., and R.D.B.J. approved final version of manuscript.


Chalara sp. is able to biotransform the epigenetic modifier vorinostat to form unique, aniline-containing polyketides named chalanilines. Here, we sought to expand the chemical diversity of chalaniline A-type molecules by changing the aniline moiety in the precursor vorinostat. In total, twenty-three different vorinostat analogs were prepared via two-step synthesis, and nineteen were incorporated by the fungus into polyketides. The highest yielding substrates were selected for large-scale precursor-directed biosynthesis and five novel compounds, including two fluorinated chalanilines, were isolated, purified, and structurally characterized. Structure elucidation relied on 1D and 2D NMR techniques and was supported by low- and high-resolution mass spectrometry. All compounds were tested for their bioactivity but were not active in antimicrobial or cell viability assays. Aminofulvene-containing natural products are rare, and this high-yielding, precursor-directed process allows for the diversification of this class of compounds.The plant endophyte  Tolypocladium sp. which results from highly reactive intermediates that can detoxify various synthetic and naturally derived antifungals via nucleophilic substitutions +  and an m/z value of 416.0904 [M + Na]+ , resulting in a molecular formula of C22H16FNO5. The UV spectrum showed maxima at 380, 308, and 244 nm, representing the chalaniline A-type backbone. The 1H NMR spectrum exhibited a broad hydroxyl peak (δH 13.75), an N-H resonance (δH 11.84) with a large coupling constant (14.5 Hz) to one methine (δH 8.74), seven aromatic/olefinic hydrogens, one methoxy, and one methyl group , 2J (26.2 and 21.4 Hz), and 3J (9.4 Hz) C-F coupling constants in the carbon spectrum, and the proton spectrum revealed two 3J (10.7 and 8.0 Hz) H-F coupling constants , as well as the correlation between H-11 and C-2′ supported the structural assignment. The placement of the fluorine at C-3′ was confirmed by C-F coupling constant analysis  was isolated as a yellow amorphous solid and the HRESIMS provided an m/z value of 394.1088 [M + H] +  and m/z value of 416.0909 [M + Na]+ for the sodium adduct . The 1H NMR spectrum of 2 was very similar to the spectrum of 1, the only differences can be found in the number of aromatic signals and the coupling and integration pattern for the fluoro-benzene moiety. Two sets of aromatic signals, with each an integration of ~2H, supported the symmetric, para-substituted fluoro-aniline incorporation , 2J C-F coupling of 23.0 Hz for C-3′/5′, 3J C-F coupling of 8.3 Hz for C 2′/6′, and 4J C-F coupling (2.6 Hz) for C-1′  and the 4-fluoro anilino ring C-2′/6′, and also from the H-11 to C-2′  was isolated as an amorphous yellow solid. The HRESIMS gave an m/z value of 406.1284 [M + H] +  and m/z value of 428.1102 [M + Na]+ for the sodium adduct . The 1H NMR spectrum of 3 showed a similar pattern of signals as previously reported for chalaniline A  + , indicative of a regioisomer of 3. The para-substitution in the 4-methoxy aniline moiety became evident in the proton NMR with two doublets each with an integration of ~2H , as well as the correlation between bridging methine (H-11) to the fulvene  assisted in the structure assembly  was isolated as a yellow amorphous solid and the HRESIMS gave an m/z value of 426.1341 [M + H]+ , and an m/z value of 448.1160 [M + Na]+ for the sodium adduct  supporting a molecular formula of C26H19NO5. The 1H and 13C NMR spectra were similar to chalaniline A with additional signals for the naphthyl moiety instead of the phenyl in the aromatic region  and melanoma (SK-MEL-5) cancer cell models by measuring the reduction of the tetrazolium salt MTT -2,5-diphenyltetrazolium bromide) by metabolically active cells + , m/z 416.0904 [M + Na]+ 3-Fluoro chalaniline A (2): yellow amorphous solid; IR (ATR): νmax = 3430, 2925, 2853, 1712, 1651, 1589, 1508, 1466, 1365, 1209, 1098 cm−1; UV (MeCN) λmax: 380, 308, 244 nm; 13C NMR  and 1H NMR  see m/z 394.1088 [M + H]+ , m/z 416.0909 [M + Na]+, 4-Fluoro chalaniline A (3): yellow amorphous solid; IR (ATR): νmax = 3290, 2924, 2850, 1703, 1647, 1601, 1510, 1470, 1252, 1190, 1050, 840, 735 cm−1; UV (MeCN) λmax = 383, 310, 246 nm; 13C NMR  and 1H NMR  see m/z 406.1284 [M + H]+ , m/z 428.1102 [M + Na]+  3-Methoxy chalaniline A (4): yellow amorphous solid; IR (ATR): νmax = 3290, 2924, 1703, 1647, 1510, 1490, 1252, 1050, 820, 745 cm−1; UV (MeCN) λmax: 382, 308, 244 nm; 13C NMR  and 1H NMR  see m/z 406.1286 [M + H]+, 4-Methoxy chalaniline A (5): yellow amorphous solid; IR (ATR): νmax = 3410, 2926, 2852, 1737, 1647, 1614, 1465,1372, 1307, 1207, 1098, 830, 767 cm−1; UV (MeCN) λmax: 374, 305, 242, 214 nm; 13C NMR  and 1H NMR  see m/z 426.1341 [M + H]+ , m/z 448.1160 [M + Na]+ Naphthyl chalaniline A (Staphylococcus aureus (ATCC 25923), methicillin-resistant Staphylococcus aureus (ATCC BAA-41), multidrug-resistant Staphylococcus aureus (ATCC BAA-44), Pseudomonas aeruginosa (ATCC 15442), Candida albicans (ATCC 90027), Candida krusei (ATCC 34135), and Mycobacterium smegmatis (ATCC 14468) in microbroth assays performed following an established protocol [Antimicrobial Assays. Extracts and fractions were tested for inhibitory activity against protocol ,31. FracCell proliferation assay. Cytotoxic activities of extracts and pure compounds were evaluated against colon (HCT-116) and melanoma (SK-MEL-5) cancer models by measuring the reduction of the tetrazolium salt MTT -2, 5-diphenyltetrazolium bromide) by metabolically active cells following standard procedures ,33.


P. aeruginosa from bloodstream infections (BSI) to those from other infection types.Ceftolozane/tazobactam (C/T), an antipseudomonal cephalosporin combined with a β-lactamase inhibitor, was approved for treatment of complicated urinary tract (cUTI) and intraabdominal infections (cIAI), and hospital-acquired/ventilator-associated bacterial pneumonia (HAP/VAP). Imipenem/relebactam (IMI/REL) is a combination of imipenem/cilastatin with relebactam, an inhibitor of class A and C β-lactamases. IMI/REL was approved for HAP/VAP and for infections due to aerobic gram-negative organisms in adults with limited treatment options . We compared the activity of C/T and IMI/REL against Pa isolates were collected. MICs were determined using CLSI broth microdilution and breakpoints.As part of the SMART program, 24 hospitals in the US and 8 in Canada each collected up to 250 consecutive gram-negative isolates per year in 2018-2019 from patients with BSI, lower respiratory tract infections (LRTI), IAI, and UTI. A total of 2351 Pa isolates from BSI tended to show higher susceptibility than IAI, UTI, and especially LRTI isolates (Table). Susceptibility to the tested comparator β-lactams was 11-12 percentage points lower among LRTI than BSI isolates, while C/T and IMI/REL susceptibility was only 2-5% lower. Even among BSI isolates, the comparator β-lactams were active against only 75-88% of isolates, while C/T and IMI/REL were active against >95%. Only amikacin showed higher activity. Analyzing coverage by either C/T or IMI/REL, 98.7% of Pa isolates from BSI were susceptible to one or both agents. C/T and IMI/REL maintained activity against 89% and 69% of meropenem-nonsusceptible (MEM-NS) Pa isolates from BSI (n=36), respectively, and 87% and 76% of piperacillin/tazobactam (P/T)-NS Pa (n=38).Results TablePa susceptibility to commonly used β-lactams like MEM and P/T was < 90%, 7-23% lower than C/T and IMI/REL. Given the desirability of β-lactams among clinicians and the >98% coverage by either C/T or IMI/REL of Pa isolates from BSI, both agents represent important options in the treatment of patients with BSI.Even among BSI isolates, which were generally more susceptible than those from other infection types, Sibylle Lob, PhD, IHMA (Employee)Pfizer, Inc. (Independent Contractor) Meredith Hackel, PhD MPH, IHMA (Employee)Pfizer, Inc. (Independent Contractor) C. Andrew DeRyke, PharmD, Merck & Co., Inc.  Kelly Harris, PharmD, BCPS, Merck & Co. Inc (Employee) Katherine Young, MS, Merck (Employee) Mary Motyl, PhD, Merck & Co., Inc.  Daniel F. Sahm, PhD, IHMA (Employee)Pfizer, Inc. (Independent Contractor)


Lactococcus lactis may represent a potential approach for IBD therapy.Inflammatory bowel diseases (IBDs) are generally characterized by persistent abdominal pain and diarrhea caused by chronic inflammation in the intestine. Cathelicidins are antimicrobial peptides with pleiotropic roles in anti-infection, wound healing, and immune modulation. However, the sensitivity to the acidic environment and short half-life of cathelicidins limit their application in IBD treatment. Recombinant cathelicidin-related antimicrobial peptide (CRAMP)-producing L. lactis NZ9000 and explore the role and mechanism of recombinant L. lactis NZ9000 expressing CRAMP in colitis.The aim of this study was to develop recombinant CRAMP-producing L. lactis NZ9000 with different plasmids pMG36e (L.L-pMU45CR) or pNZ8148 (L.L-pNU45CR), which use a Usp45 secretion signal to drive the secretion of CRAMP. Bacterial suspensions were orally supplemented to mice with a syringe for 4 days after dextran sodium sulfate (DSS) treatment. Body weight change, disease active score, colon length, and colonic histology were determined. The expression of tight junction  and cytokines  in colon was performed by qPCR. The expression of p-ERK, p-p38, and p-p65 was determined by Western blot analysis.We constructed two strains of CRAMP-producing L. lactis NZ9000 strains protected against colitis, as shown by reduced weight loss and disease activity score, improved colon shortening, and histopathological injury. In addition, CRAMP-producing L. lactis NZ9000 restored gut barrier by upregulating ZO-1, ZO-2, and occludin. Moreover, CRAMP-producing L. lactis NZ9000 regulated the colonic cytokines profile with reduced IL-6, IL-1β, and TNF-α production, and increased IL-10 production. By further analysis, we found that CRAMP-producing L. lactis NZ9000 reduced the expression of p-p38 and p-p65.Both CRAMP-producing L. lactis NZ9000 attenuated dextran sulfate sodium-induced colitis by colonic colonization and inhibiting p38/NF-κB signaling. Orally administered recombinant CRAMP-secreting L. lactis NZ9000 represents a potential strategy for colitis therapy.Together, our data suggested that CRAMP-secreting  Lactococcus lactis NZ9000 were constructed with different plasmids pMG36e (L.L-pMU45CR) or pNZ8148 (L.L-pNU45CR), which use a Usp45 secretion signal to drive the secretion of CRAMP.Two strains of recombinant CRAMP-producing L. lactis NZ9000 strains protected mice from colitis via suppressed activation of p-p38/NF-κB signaling, thus resulting in a restored cytokines profile and an improved gut barrier integrity.CRAMP-producing L. lactis NZ9000 represents a novel intervention strategy for colitis treatment.CRAMP-producing Inflammatory bowel diseases (IBDs) are inflammatory conditions in the intestine, which generally include ulcerative colitis (UC) and Crohn’s disease (CD) . Both UCCathelicidins are antimicrobial peptides found in humans and mice, which includes LL-37 and cathelicidin-related antimicrobial peptide (CRAMP), that are homologous in nature, . These hLactococcus lactis (L. Lactis) is widely used in food fermentation and oral delivery of therapeutic proteins (Lactococcus lactis NZ9000 (L. Lactis NZ9000) plays a key role in industrial fermentation  or pNZ8148 (L.L pNU45CR) plasmids, respectively. We also evaluated the role and mechanism of CRAMP-secreting L. lactis NZ9000 in experimental colitis.In this study, we constructed two recombinant Lactococcus lactis NZ9000  was grown in M17 medium  containing 0.5% (w/v) glucose  at 30°C. CRAMP gene (Lactococcus lactis NZ9000 was transformed with pMG36e-Usp45-CRAMP (L.L-pMU45CR) and pNZ8148-Usp45-CRAMP (L.L-pNU45CR) by electroporation (Lactococcus lactis NZ9000 was transformed with empty pMG36e (L.L-pMVectro) and empty pNZ8148 (L.L-pNVector) as controls. Nisin  was used for 4 h to induce gene expression in L.L-pNU45CR and L.L-pNVector as controls (AMP gene  containiAMP gene  was syntAMP gene  (pMG36e-AMP gene  (pNZ8148poration . Lactococontrols . Bacterin = 5) as follows: [1] control (without DSS), [2] 3% DSS + sterilized water, [3] 3% DSS + L.L-pMVector, [4] 3% DSS + L.L-pNVector, [5] 3% DSS + L.L-pMU45CR, and [6] 3% DSS + L.L- pNU45CR. After 7 days of DSS treatment, bacterial suspensions (1010 CFU) were orally administrated with syringe once daily for 4 days. Bacterial suspensions were prepared as described below. The bacterial cells were centrifuged at 3,000 g at 4°C for 20 min and washed twice with PBS. These bacterial cells were resuspended at concentrations of 5 × 1010 CFU/mL based on optical density (OD600). Mice were gavaged with 200 µL of bacterial suspension (1010 CFU) with syringe. To check the number of CFUs, plate counts were performed on M17 agar supplemented with 0.5% glucose at 30°C without agitation. After 24 h, the plates were observed.Male C57BL/6 mice (7–8 weeks old) were purchased from Su Pu Si Biotechnology Co. Ltd  and were randomly divided into six groups  according to the manufacturer’s instructions.Colon tissues were fixed and embedded in paraffin via standard methods , and sec-∆∆ct method was used for calculation and normalized to β-actin. All primers were synthesized by Thermo Fisher Scientific . Primers were designed with Primer 5 software . To check the specificity of primers, blast program  was used for colonic RNA isolation. SuperRT cDNA synthesis kit  was used for reverse transcription. SYBR Green  was used for quantitation. The 2 program  was usedL. Lactis cultures at a given optical density of 600 nm (OD600) were harvested by centrifugation at 3,000´g for 20 min at 4°C. The equivalent of 1 mL of 1 OD600 unit of culture (cell or supernatant) was concentrated in a 100 μL final volume as described below, and 10 μL was loaded for SDS-PAGE. Supernatants were precipitated by the addition of 10% trichloroacetic acid, harvesting by centrifugation at 10,000 × g at 4°C. The resulting pellet was dissolved in a 1:20 volume of 50 mM NaOH. Cell pellets were resuspended in 70 μL of TES containing lysozyme (1 mg/mL). After 30 min of incubation at 37°C, cells were lysed with 30 μL of 20% SDS. Equal volumes of 2× loading buffer were added to all samples. RIPA buffer with phosphatase and protease inhibitors was purchased from Songon Biotech  and used in the preparation of colon samples. Electrophoresis and transfer were performed as described before  and statistically analyzed by GraphPad Prism software  using one-way analysis of variance followed by Tukey’s post-hoc test. L.L-pMU45CR or L.L-pNU45CR. As shown in L.L-pMU45CR and L.L-pNU45CR expressed CRAMP, and the levels of CRAMP were higher in supernatants than in bacterial cells  to confirm whether CRAMP with secretion signal peptides (Usp45 + CRAMP) were expressed in al cells . CollectL. lactis NZ9000 on colitis, we treated mice with recombinant L. lactis NZ9000 for 4 days after dextran sodium sulfate (DSS)-induced colitis. We found that both L.L-pMU45CR and L.L-pNU45CR attenuated colitis in DSS-treated mice, as shown by reduced weight change  L. lactis, which transit with diet, could survival through acidic conditions in gastric juice (L. lactis NZ9000 is able to colonize the colon and has been used to express bioactive molecules in the treatment of colitis, such as theme oxygenase-1 and insulin-like growth factor I (L. lactis are varied due to the characteristics of heterologous proteins. Usp45 is a signal peptide widely applied in driving protein secretion (L. lactis NZ9000 with Usp45 signal peptides.ic juice . Recombifactor I , 19. Theecretion . We achiL. Lactis-treated mice. However, the recombinant CRAMP producing L. lactis may not have a direct effect on serum CRAMP, because bioactive peptides were not able to cross the gut wall intact, except dipeptides and tripeptides (Cathelicidins have multifunctional roles, such as anti-microbial, anti-inflammation, anti-apoptosis activities, and wound healing. Serum LL-37 levels are positively correlated with recovery in IBD patients . We obsepeptides , 39. Conpeptides , 41.L. lactis increased the expression of IL-10. Similarly, cathelicidins increased the expression of IL-10 in human mononuclear cells (L.L-pMU45CR and L.L-pNU45CR reduced IL-6, TNF-α, and IL-1β expression by suppressing the expression of p-p38 and p-p65. These results revealed that recombinant CRAMP-producing L. lactis regulates proinflammatory cytokine expression by p-p38/NF-κB p-p65 signaling.Cytokines exert major impacts on intestinal inflammation and related clinical symptoms in IBD. The unbalanced cytokines profile between proinflammatory and regulatory cytokines promotes mucosal inflammation. IL-10 regulates intestinal homeostasis, and its deficiency leads to spontaneous colitis in mice . We founar cells , 44. Bloar cells , 45, 46.Escherichia coli is increased in IBD patients and can disrupt tight junctions (L. lactis restored the expression of tight junctions by regulating the cytokines profile in colitis, although it is unclear whether recombinant CRAMP-producing L. lactis regulates adherent-invasive E. coli.Epithelial tight junctions are crucial in regulating intestinal barrier and permeability. Disruption of intestinal barrier results in the transfer of intestinal bacteria and antigens into submucosa, thus subsequently leading to inflammatory response, such as transcription factor activation and immune cell infiltration. Tight junctions are regulated by intestinal microbes and cytokines in IBD. Adherent-invasive unctions . Severalunctions . Previouunctions  and epidunctions . TogetheL. lactis NZ9000 strains protected mice from colitis via suppressed activation of p-p38/NF-κB p-p65 signaling, thus resulting in a restored cytokines profile and improved gut barrier integrity (L. lactis NZ9000 may represent a potential strategy for IBD therapy.Our data revealed that two CRAMP-secreting ntegrity . Our dat


Correction to: BMC Public Health 22, 354 (2022)https://doi.org/10.1186/s12889-022-12776-yThe original publication of this article contained an incorrect Fig. 1. The incorrect and correct Fig. 1 are included in this correction article .The original article has been updated.Fig. 1 Incorrect version of figure 1 as originally publishedFig. 2 Correct version of figure 1 as corrected