ChatGPT outscored human candidates in a virtual objective structured clinical examination in obstetrics and gynecology.

BACKGROUND: Natural language processing is a form of artificial intelligence that allows human users to interface with a machine without using complex codes. The ability of natural language processing systems, such as ChatGPT, to successfully engage with healthcare systems requiring fluid reasoning, specialist data interpretation, and empathetic communication in an unfamiliar and evolving environment is poorly studied. This study investigated whether the ChatGPT interface could engage with and complete a mock objective structured clinical examination simulating assessment for membership of the Royal College of Obstetricians and Gynaecologists. OBJECTIVE: This study aimed to determine whether ChatGPT, without additional training, would achieve a score at least equivalent to that achieved by human candidates who sat for virtual objective structured clinical examinations in Singapore. STUDY DESIGN: This study was conducted in 2 phases. In the first phase, a total of 7 structured discussion questions were selected from 2 historical cohorts (cohorts A and B) of objective structured clinical examination questions. ChatGPT was examined using these questions and responses recorded in a script. Of note, 2 human candidates (acting as anonymizers) were examined on the same questions using videoconferencing, and their responses were transcribed verbatim into written scripts. The 3 sets of response scripts were mixed, and each set was allocated to 1 of 3 human actors. In the second phase, actors were used to presenting these scripts to examiners in response to the same examination questions. These responses were blind scored by 14 qualified examiners. ChatGPT scores were unblinded and compared with historical human candidate performance scores. RESULTS: The average score given to ChatGPT by 14 examiners was 77.2%. The average historical human score (n=26 candidates) was 73.7 %. ChatGPT demonstrated sizable performance improvements over the average human candidate in several subject domains. The median time taken for ChatGPT to complete each station was 2.54 minutes, well before the 10 minutes allowed. CONCLUSION: ChatGPT generated factually accurate and contextually relevant structured discussion answers to complex and evolving clinical questions based on unfamiliar settings within a very short period. ChatGPT outperformed human candidates in several knowledge areas. Not all examiners were able to discern between human and ChatGPT responses. Our data highlight the emergent ability of natural language processing models to demonstrate fluid reasoning in unfamiliar environments and successfully compete with human candidates that have undergone extensive specialist training.

Characterizing the N- and O-linked glycans of the PGF-CTERM sorting domain-containing S-layer protein of Methanoculleus marisnigri.

The glycosylation of structural proteins is a widespread posttranslational modification in Archaea. Although only a handful of archaeal N-glycan structures have been determined to date, it is evident that the diversity of structures expressed is greater than in the other domains of life. Here, we report on our investigation of the N- and O-glycan modifications expressed by Methanoculleus marisnigri, a mesophilic methanogen from the Order Methanomicrobiales. Unusually, mass spectrometry (MS) analysis of purified archaella revealed no evidence for N- or O-glycosylation of the constituent archaellins, In contrast, the S-layer protein, identified as a PGF-CTERM sorting domain-containing protein encoded by MEMAR_RS02690, is both N- and O-glycosylated. Two N-glycans were identified by NMR and MS analysis: a trisaccharide α-GlcNAc-4-ß-GlcNAc3NGaAN-4-ß-Glc-Asn where the second residue is 2-N-acetyl, 3-N-glyceryl-glucosamide and a disaccharide ß-GlcNAc3NAcAN-4-ß-Glc-Asn, where the terminal residue is 2,3 di-N-acetyl-glucosamide. The same trisaccharide was also found N-linked to a type IV pilin. The S-layer protein is also extensively modified in the threonine-rich region near the C-terminus with O-glycans composed exclusively of hexoses. While the S-layer protein has a predicted PGF-CTERM processing site, no evidence of a truncated and lipidated C-terminus, the expected product of processing by an archaeosortase, was found. Finally, NMR also identified a polysaccharide expressed by M. marisnigri and composed of a repeating tetrasaccharide unit of [-2-ß-Ribf-3-α-Rha2OMe-3-α-Rha - 2-α-Rha-]. This is the first report of N- and O-glycosylation in an archaeon from the Order Methanomicrobiales.

Facilitating Overdose Risk Mitigation Among Patients Following a Clinician Office Closure: A Connecticut Case Study of the Opioid Rapid Response Program.

The Opioid Rapid Response Program (ORRP) is a federal program designed to support states in mitigating risks to patients who lose access to a prescriber of opioids or other controlled substances. Displaced patients might face risks of withdrawal, overdose, or other harms. Rapid response efforts to mitigate risks require coordination across multiple parts of the health care system. This case study describes an ORRP-coordinated event, including notification from law enforcement, information sharing with state health officials, state-coordinated response efforts, key observations, and lessons learned. Timely risk mitigation and care continuity required coordination between law enforcement and public health in advance of the disruption and throughout the state-led response. Patients' acute and prolonged health care needs were complex and highlight the importance of investing time and resources in coordinated, multisector state and local preparedness for these types of disruptions.

Identification of a novel N-linked glycan on the archaellins and S-layer protein of the thermophilic methanogen, Methanothermococcus thermolithotrophicus.

Motility in archaea is facilitated by a unique structure termed the archaellum. N-Glycosylation of the major structural proteins (archaellins) is important for their subsequent incorporation into the archaellum filament. The identity of some of these N-glycans has been determined, but archaea exhibit extensive variation in their glycans, meaning that further investigations can shed light not only on the specific details of archaellin structure and function, but also on archaeal glycobiology in general. Here we describe the structural characterization of the N-linked glycan modifications on the archaellins and S-layer protein of Methanothermococcus thermolithotrophicus, a methanogen that grows optimally at 65 °C. SDS-PAGE and MS analysis revealed that the sheared archaella are composed principally of two of the four predicted archaellins, FlaB1 and FlaB3, which are modified with a branched, heptameric glycan at all N-linked sequons except for the site closest to the N termini of both proteins. NMR analysis of the purified glycan determined the structure to be α-d-glycero-d-manno-Hep3OMe6OMe-(1-3)-[α-GalNAcA3OMe-(1-2)-]-ß-Man-(1-4)-[ß-GalA3OMe4OAc6CMe-(1-4)-α-GalA-(1-2)-]-α-GalAN-(1-3)-ß-GalNAc-Asn. A detailed investigation by hydrophilic interaction liquid ion chromatography-MS discovered the presence of several, less abundant glycan variants, related to but distinct from the main heptameric glycan. In addition, we confirmed that the S-layer protein is modified with the same heptameric glycan, suggesting a common N-glycosylation pathway. The M. thermolithotrophicus archaellin N-linked glycan is larger and more complex than those previously identified on the archaellins of related mesophilic methanogens, Methanococcus voltae and Methanococcus maripaludis This could indicate that the nature of the glycan modification may have a role to play in maintaining stability at elevated temperatures.

The Influence of Web-Based Tools on Maternal and Neonatal Outcomes in Pregnant Adolescents or Adolescent Mothers: Mixed Methods Systematic Review.

BACKGROUND: Pregnant adolescent women increasingly seek support during pregnancy and the puerperium through digital platforms instead of the traditional support system of family, friends, and the community. However, it is uncertain whether digital, web-based tools are reliable and effective in providing information to the user on a variety of topics such as fetal development, pregnancy outcomes, delivery, and breastfeeding to improve maternal and infant outcomes. OBJECTIVE: We aimed to identify web-based tools designed to promote knowledge, attitudes, and skills of pregnant adolescents or adolescent mothers and determine the efficacy of such web-based tools compared with conventional resources in promoting good pregnancy and infant outcomes. METHODS: A systematic search was conducted using Medline, Scopus, CINAHL, and PsycINFO for articles published from January 2004 to November 2020 to identify randomized trials and observational studies that evaluated digital, web-based platforms to deliver resources to pregnant adolescents. All articles written in the author's languages were included. Two authors independently reviewed abstracts and full-text articles for inclusion and assessed study quality. Risk of bias in each study was assessed using appropriate tools recommended by PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) and the Joanna Briggs Institute. We adopted a qualitative synthesis and presented the results in a narrative format due to the heterogenous nature of the studies. RESULTS: Seven articles met the inclusion criteria and were analyzed. The majority of the studies were graded to be of low to moderate risk for bias. The research methodologies represented were varied, ranging from randomized (n=1) and nonrandomized controlled trials (n=1) and prospective cohort studies (n=1) to mixed methods studies (n=1) and longitudinal surveys (n=3). Four studies included active web-based interventions, and 3 described exposure to web-based tools, including the use of social media and/or other internet content. Web-based tools positively influenced treatment-seeking intentions (intervention 17.1%, control 11.5%, P=.003) and actual treatment-seeking behavior for depression among postpartum adolescents (intervention 14.1%, control 6.5%, P<.001). In contrast, readily available information on the internet may leave adolescents with increased anxiety. The critical difference lies in information curated by health care professionals specifically to address targeted concerns versus self-acquired data sourced from various websites. CONCLUSIONS: Despite almost universal web use, few studies have used this platform for health promotion and disease prevention. Social media interventions or web-based tools have the potential to positively influence both maternal and infant outcomes in adolescent pregnancy, but there is a need for more well-conducted studies to demonstrate the effectiveness of these support programs. The vastness of the information available on the web limits the ability of health care professionals to monitor or control sources of information sought by patients. Thus, it is important to create professionally curated platforms to prevent or limit exposure to potentially misleading or harmful information on the internet while imparting useful knowledge to the user. TRIAL REGISTRATION: PROSPERO International Prospective Register of Systematic Reviews CRD42020195854; https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=195854.

UXT in Sertoli cells is required for blood-testis barrier integrity†.

Spermatogenesis is a complex process that establishes male fertility and involves proper communication between the germline (spermatozoa) and the somatic tissue (Sertoli cells). Many factors that are important for spermatozoa production are also required for Sertoli cell function. Recently, we showed that the transcriptional cofactor ubiquitously expressed transcript (UXT) encodes a protein that is essential in germ cells for spermatogenesis and fertility. However, the role of UXT within Sertoli cells and how it affects Sertoli cell function was still unclear. Here we describe a novel role for UXT in the Sertoli cell's ability to support spermatogenesis. We find that the conditional deletion of Uxt in Sertoli cells results in smaller testis size and weight, which coincided with a loss of germ cells in a subset of seminiferous tubules. In addition, the deletion of Uxt has no impact on Sertoli cell abundance or maturity, as they express markers of mature Sertoli cells. Gene expression analysis reveals that the deletion of Uxt in Sertoli cells reduces the transcription of genes involved in the tight junctions of the blood-testis barrier (BTB). Furthermore, tracer experiments and electron microscopy reveal that the BTB is permeable in UXT KO animals. These findings broaden our understanding of UXT's role in Sertoli cells and its contribution to the structural integrity of the BTB.

Risk factors and prevalence of urinary incontinence in mid-life Singaporean women: the Integrated Women's Health Program.

INTRODUCTION AND HYPOTHESIS: The objective was to identify the prevalence and risk factors for urinary incontinence (UI) in healthy midlife Singaporean women. METHODS: Healthy women, aged 45-69 years, were assessed for UI and sociodemographic characteristics, including ethnicity, menopausal status, parity, and body mass index (BMI). UI subtypes corresponding to stress (SUI) alone, urge (UUI) alone, mixed (MUI), and leakage (drops only) incontinence were classified using the Urinary Distress Inventory 6 (UDI-6). Risk factors were examined using Chi-squared tests, followed by sequential multivariate logistic regression to estimate adjusted odds ratios (aOR and 95% confidence intervals). RESULTS: A total of 1,119 women (mean age 56.2 ± 5.2) completed the UDI-6. 52.3% reported any UI; MUI and SUI were the most common, each affecting 20% of women. Post-menopausal women had a lower risk (aOR 0.5 [0.3-0.9]) of SUI, but a higher risk (aOR 4.4 [1.0-19.9]) of UUI compared with premenopausal women. Higher education was negatively associated (aOR 0.3 [0.2-0.7]) with UUI, but positively associated with MUI (aOR 2.3 [1.3-4.0]). Parity (1-2 children) increased the risk of SUI (aOR 1.8 [1.0-3.1]), but reduced the risk of UUI (aOR 0.4 [0.2-0.9]). Obesity was associated with increased risk for MUI (aOR 2.2 [1.4-3.4]) and leakage (aOR 2.0 [1.0-4.1]). Malays and Indians had a higher risk of MUI, having (aOR 2.1 (1.2-3.7) and 1.7 (1.1-2.7) respectively compared with Chinese, a difference mediated by higher BMI. CONCLUSION: Urinary incontinence is a major morbidity prevalent in healthy midlife Asian women. Post-menopausal status, education level, parity, BMI (and its link with ethnicity) are independent risk factors in this population, and should be incorporated into counseling and targeted interventions.

Well-being, obstetrics and gynaecology and COVID-19: Leaving no trainee behind.

The COVID-19 pandemic has significantly disrupted training in obstetrics and gynaecology. Past pandemics have been shown to result in significant psychological morbidity. As specialty trainees continue frontline work, they will face unprecedented work environments and may face delays in progression due to postponed examinations, case log shortfalls and inadequate clinical rotations. This contributes to burnout, anxiety and depression. We share technology-based suggestions as well as institutional, departmental and self-care tips on how to maintain trainees' mental well-being during the fight against COVID-19.

Patterns of patient coping following hospital discharge from medical and surgical units: A pilot study.

A pilot study was conducted to determine the feasibility of a longitudinal investigation of patients' coping during the early postdischarge period. Recruitment was conducted on a general medical unit and a surgical orthopedic unit. Forty-four participants were recruited with 95% retention. Demographic characteristics plus measures of discharge risk and perceived readiness (expected coping) were collected before discharge. Measures of coping (experienced) and the use of supports and services were collected on the first day postdischarge, the end of the first week, and during weeks 3 and 5. Considerable variability was evident in coping scores, and not all participants exhibited improvement over time. Four patterns of coping were identified: ongoing recovery, initial shock, bumpy road, and progressive decline. Further investigation is required to validate the observed coping patterns. A better understanding of conditions affecting patient coping during the transition from hospital to home will support efforts to reduce unplanned use of acute care services.

The S-layer protein of a Clostridium difficile SLCT-11 strain displays a complex glycan required for normal cell growth and morphology.

Clostridium difficile is a bacterial pathogen that causes major health challenges worldwide. It has a well-characterized surface (S)-layer, a para-crystalline proteinaceous layer surrounding the cell wall. In many bacterial and archaeal species, the S-layer is glycosylated, but no such modifications have been demonstrated in C. difficile. Here, we show that a C. difficile strain of S-layer cassette type 11, Ox247, has a complex glycan attached via an O-linkage to Thr-38 of the S-layer low-molecular-weight subunit. Using MS and NMR, we fully characterized this glycan. We present evidence that it is composed of three domains: (i) a core peptide-linked tetrasaccharide with the sequence -4-α-Rha-3-α-Rha-3-α-Rha-3-ß-Gal-peptide; (ii) a repeating pentasaccharide with the sequence -4-ß-Rha-4-α-Glc-3-ß-Rha-4-(α-Rib-3-)ß-Rha-; and (iii) a nonreducing end-terminal 2,3 cyclophosphoryl-rhamnose attached to a ribose-branched sub-terminal rhamnose residue. The Ox247 genome contains a 24-kb locus containing genes for synthesis and protein attachment of this glycan. Mutations in genes within this locus altered or completely abrogated formation of this glycan, and their phenotypes suggested that this S-layer modification may affect sporulation, cell length, and biofilm formation of C. difficile In summary, our findings indicate that the S-layer protein of SLCT-11 strains displays a complex glycan and suggest that this glycan is required for C. difficile sporulation and control of cell shape, a discovery with implications for the development of antimicrobials targeting the S-layer.

Antibiotic-Resistant Acinetobacter baumannii Is Susceptible to the Novel Iron-Sequestering Anti-infective DIBI In Vitro and in Experimental Pneumonia in Mice.

Acinetobacter baumannii is a major cause of nosocomial infections especially hospital-acquired pneumonia. This bacterium readily acquires antibiotic resistance traits and therefore, new treatment alternatives are urgently needed. The virulence of A. baumannii linked to iron acquisition suggests a potential for new anti-infectives that target its iron acquisition. DIBI, a 3-hydroxypyridin-4-one chelator, is a purpose-designed, iron-sequestering antimicrobial that has shown promise for treating microbial infection. DIBI was investigated for its in vitro and in vivo activities against clinical A. baumannii isolates. DIBI was inhibitory for all isolates tested with very low MICs (2 µg/ml, equivalent to 0.2 µM), i.e., at or below the typical antibiotic MICs reported for antibiotic-sensitive strains. DIBI inhibition is Fe specific, and it caused an iron-restricted bacterial physiology that led to enhanced antibiotic killing by several discrete antibiotics. DIBI also strongly suppressed recovery growth of the surviving population following antibiotic exposure. A low intranasal dose (11 µmol/kg) of DIBI after intranasal challenge with hypervirulent ciprofloxacin (CIP)-resistant A. baumannii LAC-4 significantly reduced bacterial burdens in mice, and DIBI also suppressed the spread of the infection to the spleen. Treatment of infected mice with CIP alone (20 mg/kg, equivalent to 60 µmol/kg) was ineffective given LAC-4's CIP resistance, but if combined with DIBI, the treatment efficacy improved significantly. Our evidence suggests that DIBI restricts host iron availability to A. baumannii growing in the respiratory tract, bolstering the host innate iron restriction mechanisms. DIBI has potential as a sole anti-infective or in combination with conventional antibiotics for the treatment of A. baumannii pneumonia.

Pharmacokinetics of Prenylflavonoids following Oral Ingestion of Standardized Epimedium Extract in Humans.

Leaves of the Epimedium plant are traditionally consumed for bone health and other indications. The aim of this study was to establish the safety and pharmacokinetics of the metabolites of prenylflavonoids (icariin, icariside I, icariside II, icaritin, and desmethylicaritin) following single doses of a defined Epimedium prenylflavonoid extract in humans. A single oral dose of 370, 740, or 1110 mg of a standardized Epimedium prenylflavonoid extract was administered to 30 healthy male subjects in a randomized, placebo-controlled trial. Serum samples were collected over a 48-h period and analyzed by liquid chromatography-tandem mass spectrometry and non-compartmental pharmacokinetic modelling. Epimedium prenylflavonoid extracts were well tolerated and no adverse effects were observed. The principle metabolites detected in the serum were icariside II and desmethylicaritin. Icariside II had a T max of between 4.1 - 4.3 h, reaching a maximum AUC0→∞ of 23.0 (17.5, 29.9) h×ng/mL (median [IQR: interquartile range]) with the highest dose of the Epimedium prenylflavonoid. On the other hand, desmethylicaritin had a delayed T max of 24.1 - 24.4 h and reached a maximum AUC0→∞ of 126.1 (62.4, 202.9) h×ng/mL. The median maximum plasma concentration and AUC0→∞ of desmethyliciaritin showed an increase with higher doses of the Epimedium prenylflavonoid (p < 0.05). Icariin, icariside I, and icaritin levels were below detection limits. Levels of Epimedium prenylflavonoid metabolites observed in this study were consistent with levels demonstrated to have anti-osteoporotic effects in cellular and animal studies. Coupled with the favorable safety profile of the extract observed, further studies are required to explore the utility of Epimedium prenylflavonoid extracts to prevent osteoporosis in postmenopausal women.

A novel glycan modifies the flagellar filament proteins of the oral bacterium Treponema denticola.

While protein glycosylation has been reported in several spirochetes including the syphilis bacterium Treponema pallidum and Lyme disease pathogen Borrelia burgdorferi, the pertinent glycan structures and their roles remain uncharacterized. Herein, a novel glycan with an unusual chemical composition and structure in the oral spirochete Treponema denticola, a keystone pathogen of periodontitis was reported. The identified glycan of mass 450.2 Da is composed of a monoacetylated nonulosonic acid (Non) with a novel extended N7 acyl modification, a 2-methoxy-4,5,6-trihydroxy-hexanoyl residue in which the Non has a pseudaminic acid configuration (L-glycero-L-manno) and is ß-linked to serine or threonine residues. This novel glycan modifies the flagellin proteins (FlaBs) of T. denticola by O-linkage at multiple sites near the D1 domain, a highly conserved region of bacterial flagellins that interact with Toll-like receptor 5. Furthermore, mutagenesis studies demonstrate that the glycosylation plays an essential role in the flagellar assembly and motility of T. denticola. To our knowledge, this novel glycan and its unique modification sites have not been reported previously in any bacteria.

Role of the Unconventional Prefoldin Proteins URI and UXT in Transcription Regulation.

The Unconventional prefoldin RPB5 interacting protein (URI), also known as RPB5-Mediating Protein (RMP) has been shown to play several regulatory roles in different cellular compartments including the mitochondria, as a phosphatase binding protein; in the cytoplasm, as a chaperone-like protein; and in the nucleus, as a transcriptional regulator through binding to RPB5 and RNA polymerase II (polII). This chapter focuses on the role URI plays in transcriptional regulation in the prostate cell. In prostate cells, URI is tightly bound to another prefoldin-like protein called UXT, a known androgen receptor (AR) cofactor. Part of a multiprotein complex, URI and UXT act as transcriptional repressors, and URI regulates KAP1 through PP2A phosphatase activity. The discovery of the interaction of URI and UXT with KAP1, AR, and PP2A, as well as the numerous interactions between URI and components of the R2TP/prefoldin-like complex, RPB5, and nuclear proteins involved in DNA damage response, chromatin remodeling and gene transcription, reveal a pleiotropic effect of the URI/UXT complex on nuclear processes. The mechanisms by which URI/UXT affect transcription, chromatin structure and regulation, and genome stability, remain to be elucidated but will be of fundamental importance considering the many processes affected by alterations of URI/UXT and other prefoldins and prefoldin-like proteins.

Clinical utility of scores on the Blaylock Risk Assessment Screen (BRASS): An analysis of administrative data.

PURPOSE: Project was undertaken to examine the utility of the Blaylock Risk Assessment Screen (BRASS) in identifying patients who may experience discharge complications as indicated by longer hospital stays or readmission within 30-days of a discharge to home. BACKGROUND: Before measures can be put in place to facilitate discharge planning and to prevent unplanned readmission by recently discharged patients, those at risk of such events must be identified. METHODS: Project involved an analysis of 13-months of administrative data from one tertiary care hospital. Utility of the BRASS was examined in terms of its sensitivity and specificity as well as its positive and negative predictive values. RESULTS: Majority (83%) of hospital discharges were to home. Approximately 7% of patients experienced at least one readmission within 30-days of being discharged to home. Using scores of 10 or higher as an indicator of risk, BRASS exhibited a high degree of specificity suggesting it is useful for 'ruling in' those who have the outcomes-of-interest. However low sensitivity indicates many who experienced the outcomes were incorrectly classified by the BRASS as low risk. The low positive predictive value for 30-day readmission also suggests many who were classified by the BRASS as being 'at risk' were not readmitted. CONCLUSION: The observed rate of 30-day readmission is likely conservative as the analysis involved data from only one acute care facility. One explanation for the low positive predictive value for 30-day readmission is that completion of the BRASS on admission enabled the implementation of preventive measures.

URI Regulates KAP1 Phosphorylation and Transcriptional Repression via PP2A Phosphatase in Prostate Cancer Cells.

URI (unconventional prefoldin RPB5 interactor protein) is an unconventional prefoldin, RNA polymerase II interactor that functions as a transcriptional repressor and is part of a larger nuclear protein complex. The components of this complex and the mechanism of transcriptional repression have not been characterized. Here we show that KAP1 (KRAB-associated protein 1) and the protein phosphatase PP2A interact with URI. Mechanistically, we show that KAP1 phosphorylation is decreased following recruitment of PP2A by URI. We functionally characterize the novel URI-KAP1-PP2A complex, demonstrating a role of URI in retrotransposon repression, a key function previously demonstrated for the KAP1-SETDB1 complex. Microarray analysis of annotated transposons revealed a selective increase in the transcription of LINE-1 and L1PA2 retroelements upon knockdown of URI. These data unveil a new nuclear function of URI and identify a novel post-transcriptional regulation of KAP1 protein that may have important implications in reactivation of transposable elements in prostate cancer cells.

The Type B Flagellin of Hypervirulent Clostridium difficile Is Modified with Novel Sulfonated Peptidylamido-glycans.

Glycosylation of flagellins is a well recognized property of many bacterial species. In this study, we describe the structural characterization of novel flagellar glycans from a number of hypervirulent strains of C. difficile We used mass spectrometry (nano-LC-MS and MS/MS analysis) to identify a number of putative glycopeptides that carried a variety of glycoform substitutions, each of which was linked through an initial N-acetylhexosamine residue to Ser or Thr. Detailed analysis of a LLDGSSTEIR glycopeptide released by tryptic digestion, which carried two variant structures, revealed that the glycopeptide contained, in addition to carbohydrate moieties, a novel structural entity. A variety of electrospray-MS strategies using Q-TOF technology were used to define this entity, including positive and negative ion collisionally activated decomposition MS/MS, which produced unique fragmentation patterns, and high resolution accurate mass measurement to allow derivation of atomic compositions, leading to the suggestion of a taurine-containing peptidylamido-glycan structure. Finally, NMR analysis of flagellin glycopeptides provided complementary information. The glycan portion of the modification was assigned as α-Fuc3N-(1→3)-α-Rha-(1→2)-α-Rha3OMe-(1→3)-ß-GlcNAc-(1→)Ser, and the novel capping moiety was shown to be comprised of taurine, alanine, and glycine. This is the first report of a novel O-linked sulfonated peptidylamido-glycan moiety decorating a flagellin protein.

Role of Glycosyltransferases Modifying Type B Flagellin of Emerging Hypervirulent Clostridium difficile Lineages and Their Impact on Motility and Biofilm Formation.

Clostridium difficile is the principal cause of nosocomial infectious diarrhea worldwide. The pathogen modifies its flagellin with either a type A or type B O-linked glycosylation system, which has a contributory role in pathogenesis. We study the functional role of glycosyltransferases modifying type B flagellin in the 023 and 027 hypervirulent C. difficile lineages by mutagenesis of five putative glycosyltransferases and biosynthetic genes. We reveal their roles in the biosynthesis of the flagellin glycan chain and demonstrate that flagellar post-translational modification affects motility and adhesion-related bacterial properties of these strains. We show that the glycosyltransferases 1 and 2 (GT1 and GT2) are responsible for the sequential addition of a GlcNAc and two rhamnoses, respectively, and that GT3 is associated with the incorporation of a novel sulfonated peptidyl-amido sugar moiety whose structure is reported in our accompanying paper (Bouché, L., Panico, M., Hitchen, P., Binet, D., Sastre, F., Faulds-Pain, A., Valiente, E., Vinogradov, E., Aubry, A., Fulton, K., Twine, S., Logan, S. M., Wren, B. W., Dell, A., and Morris, H. R. (2016) J. Biol. Chem. 291, 25439-25449). GT2 is also responsible for methylation of the rhamnoses. Whereas type B modification is not required for flagellar assembly, some mutations that result in truncation or abolition of the glycan reduce bacterial motility and promote autoaggregation and biofilm formation. The complete lack of flagellin modification also significantly reduces adhesion of C. difficile to Caco-2 intestinal epithelial cells but does not affect activation of human TLR5. Our study advances our understanding of the genes involved in flagellar glycosylation and their biological roles in emerging hypervirulent C. difficile strains.

Effects of growth conditions on archaellation and N-glycosylation in Methanococcus maripaludis.

In this study, the effects of growth conditions on archaellation in Methanococcus maripaludis were examined. Cells were grown in a variety of media, including complex, minimal and with formate as the electron donor, with different nitrogen sources, varied salinities and at a variety of growth temperatures. Of the conditions tested, Western blot results showed that major archaellin FlaB2 levels only varied detectably as a result of growth temperature. Whilst the amount of FlaB2 was similar for cells grown at < 35 °C, protein levels decreased at 38 °C and were barely detectable at 42 °C. Quantitative reverse transcription PCR experiments demonstrated that the flaB2 transcript levels were almost undetectable at 42 °C. Electron microscopy confirmed that the FlaB2 levels detected by Western blots corresponded to the state of archaellation, with cells grown at 42 °C being mostly non-archaellated. Unexpectedly, a lower apparent molecular mass for FlaB2 was observed in Western blots of cells grown at temperatures >38 °C, suggestive of a truncation in the attached N-linked tetrasaccharide at higher growth temperatures. MS analysis of archaella isolated from cells grown at 40 °C confirmed that FlaB2 was now decorated with a trisaccharide in which the third sugar was also lacking the attached threonine and acetamidino modifications found in the WT glycan.

A genome-wide RNA interference screen identifies new regulators of androgen receptor function in prostate cancer cells.

The androgen receptor (AR) is a mediator of both androgen-dependent and castration-resistant prostate cancers. Identification of cellular factors affecting AR transcriptional activity could in principle yield new targets that reduce AR activity and combat prostate cancer, yet a comprehensive analysis of the genes required for AR-dependent transcriptional activity has not been determined. Using an unbiased genetic approach that takes advantage of the evolutionary conservation of AR signaling, we have conducted a genome-wide RNAi screen in Drosophila cells for genes required for AR transcriptional activity and applied the results to human prostate cancer cells. We identified 45 AR-regulators, which include known pathway components and genes with functions not previously linked to AR regulation, such as HIPK2 (a protein kinase) and MED19 (a subunit of the Mediator complex). Depletion of HIPK2 and MED19 in human prostate cancer cells decreased AR target gene expression and, importantly, reduced the proliferation of androgen-dependent and castration-resistant prostate cancer cells. We also systematically analyzed additional Mediator subunits and uncovered a small subset of Mediator subunits that interpret AR signaling and affect AR-dependent transcription and prostate cancer cell proliferation. Importantly, targeting of HIPK2 by an FDA-approved kinase inhibitor phenocopied the effect of depletion by RNAi and reduced the growth of AR-positive, but not AR-negative, treatment-resistant prostate cancer cells. Thus, our screen has yielded new AR regulators including drugable targets that reduce the proliferation of castration-resistant prostate cancer cells.