Fluorometric Liposome Screen for Inhibitors of a Physiologically Important Bacterial Ion Channel

The bacterial K+ homeostasis machinery is widely conserved across bacterial species, and different from that in animals. Dysfunction in components of the machinery has an impact on intracellular turgor, membrane potential, adaptation to changes in both extracellular pH and osmolarity, and in virulence. Using a fluorescence-based liposome flux assay, we have performed a high-throughput screen to identify novel inhibitors of the KtrAB ion channel complex from Bacillus subtilis, a component of the K+ homeostasis machinery that is also present in many bacterial pathogens. The screen identified 41 compounds that inhibited K+ flux and that clustered into eight chemical groups. Many of the identified inhibitors were found to target KtrAB with an in vitro potency in the low µM range. We investigated the mechanisms of inhibition and found that most molecules affected either the membrane component of the channel, KtrB alone or the full KtrAB complex without a preference for the functional conformation of the channel, thus broadening their inhibitory action. A urea derivative molecule that inhibited the membrane component of KtrAB affected cell viability in conditions in which KtrAB activity is essential. With this proof-of-concept study, we demonstrate that targeting components of the K+ homeostasis machinery has the potential as a new antibacterial strategy and that the fluorescence-based flux assay is a robust tool for screening chemical libraries.This work was supported by FEDER funds through COMPETE 2020-POCI, Portugal 2020, and FCT – Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior: POCI-01-0145-FEDER-029863 (PTDC/BIA-BQM/29863/2017), and by “Fundação Luso-Americana para o Desenvolvimento” FLAD Life Science 2020 awarded to JM-C. We acknowledge FCT fellowship SFRH/BPD/105672/2015 and contract DL 57/2016/CP1355/CT0026 awarded to AF, fellowship SFRH/BPD/107785/2015 to AP, and fellowship SFRH/BD/123761/2016 to CT-D

Dysregulation of T cell receptor N-glycosylation: A molecular mechanism involved in ulcerative colitis

The incidence of inflammatory bowel disease is increasing worldwide and the underlying molecular mechanisms are far from being fully elucidated. Herein, we evaluated the role of N-glycosylation dysregulation in T cells as a key mechanism in the ulcerative colitis (UC) pathogenesis. The evaluation of the branched N-glycosylation levelsandprofile of intestinalTcell receptor (TCR)wereassessedin colonic biopsies fromUCpatientsand healthy controls. Expression alterations of the glycosyltransferase gene MGAT5 were also evaluated. We demonstrated thatUCpatients exhibit a dysregulation ofTCRbranchedN-glycosylationonlamina propriaTlymphocytes. Patients with severe UC showed the most pronounced defect on N-glycan branching in T cells. Moreover, UC patients showed a significant reduction of MGAT5 gene transcription in T lymphocytes. In this study, we disclose for the first time that a deficiency in branched N-glycosylation on TCR due to a reduced MGAT5 gene expression is a new molecular mechanism underlying UC pathogenesis, being a potential novel biomarker with promising clinical and therapeutic applications.This work was supported by grants from the Portuguese Foundation for Science and Technology (FCT), project grants (PTDC/ CVT/111358/2009; PTDC/BBB-EBI/0786/2012; EXPL/ BIM-MEC/0149/2012), ‘financiados no âmbito do Programa Operacional Temático Factores de Competitividade (COMPETE) e comparticipado pelo fundo Comunitário Europeu FEDER’, e do Quadro de Referência Estratégia Nacio-nal QREN. This work was further supported by a portuguese grant from ‘Grupo de Estudo da Doenc¸a Inflamatória Intestinal’ (GEDII). S.S.P. (SFRH/BPD/63094/2009); S.C. (SFRH/BD/ 77386/2011) also acknowledge FCT. IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education, and is partially supported by FCT

Persistence of Mycoplasma genitalium Following Azithromycin Therapy

BACKGROUND: To determine clinical outcomes and cure rates for M.genitalium genital infection in men and women following azithromycin 1 g. METHODOLOGY: Patients attending Melbourne Sexual Health Centre between March 2005 and November 2007 with urethritis/epididymitis, cervicitis/pelvic inflammatory disease and sexual contacts of M.genitalium were tested for M.genitalium by polymerase chain reaction (PCR). M.genitalium-infection was treated with 1 g of azithromycin and a test-of-cure (toc) was performed one month post-azithromycin. Response to azithromycin, and response to moxifloxacin (400 mg daily for 10 days) in individuals with persistent infection post-azithromycin, was determined. PRINCIPAL FINDINGS: Of 1538 males and 313 females tested, 161 males (11%) and 30 females (10%) were infected with M.genitalium. A toc was available on 131 (69%) infected individuals (median = 36 days [range 12-373]). Of 120 individuals prescribed azithromycin only pre-toc, M.genitalium was eradicated in 101 (84%, 95% confidence intervals [CI]: 77-90%) and persisted in 19 (16%, 95% CI: 10-23%). Eleven individuals with persistent infection (9%, 95% CI: 5-15%) had no risk of reinfection from untreated-partners, while eight (7%, 95% CI: 3-12%) may have been at risk of reinfection from doxycycline-treated or untreated-partners. Moxifloxacin was effective in eradicating persistent infection in all cases not responding to azithromycin. Patients with persistent-M.genitalium were more likely to experience persistent symptoms (91%), compared to patients in whom M.genitalium was eradicated (17%), p<0.0001. CONCLUSION: Use of azithromycin 1 g in M.genitalium-infected patients was associated with unacceptable rates of persistent infection, which was eradicated with moxifloxacin. These findings highlight the importance of follow-up in M.genitalium-infected patients prescribed azithromycin, and the need to monitor for the development of resistance. Research to determine optimal first and second-line therapeutic agents for M.genitalium is needed

Procollagen Triple Helix Assembly: An Unconventional Chaperone-Assisted Folding Paradigm

Fibers composed of type I collagen triple helices form the organic scaffold of bone and many other tissues, yet the energetically preferred conformation of type I collagen at body temperature is a random coil. In fibers, the triple helix is stabilized by neighbors, but how does it fold? The observations reported here reveal surprising features that may represent a new paradigm for folding of marginally stable proteins. We find that human procollagen triple helix spontaneously folds into its native conformation at 30–34°C but not at higher temperatures, even in an environment emulating Endoplasmic Reticulum (ER). ER-like molecular crowding by nonspecific proteins does not affect triple helix folding or aggregation of unfolded chains. Common ER chaperones may prevent aggregation and misfolding of procollagen C-propeptide in their traditional role of binding unfolded polypeptide chains. However, such binding only further destabilizes the triple helix. We argue that folding of the triple helix requires stabilization by preferential binding of chaperones to its folded, native conformation. Based on the triple helix folding temperature measured here and published binding constants, we deduce that HSP47 is likely to do just that. It takes over 20 HSP47 molecules to stabilize a single triple helix at body temperature. The required 50–200 µM concentration of free HSP47 is not unusual for heat-shock chaperones in ER, but it is 100 times higher than used in reported in vitro experiments, which did not reveal such stabilization

Predictors of linkage to care following community-based HIV counseling and testing in rural Kenya

Despite innovations in HIV counseling and testing (HCT), important gaps remain in understanding linkage to care. We followed a cohort diagnosed with HIV through a community-based HCT campaign that trained persons living with HIV/AIDS (PLHA) as navigators. Individual, interpersonal, and institutional predictors of linkage were assessed using survival analysis of self-reported time to enrollment. Of 483 persons consenting to follow-up, 305 (63.2%) enrolled in HIV care within 3 months. Proportions linking to care were similar across sexes, barring a sub-sample of men aged 18–25 years who were highly unlikely to enroll. Men were more likely to enroll if they had disclosed to their spouse, and women if they had disclosed to family. Women who anticipated violence or relationship breakup were less likely to link to care. Enrolment rates were significantly higher among participants receiving a PLHA visit, suggesting that a navigator approach may improve linkage from community-based HCT campaigns.Vestergaard Frandse

The study of vancomycin use and its adverse reactions associated to patients of a brazilian university hospital

<p>Abstract</p> <p>Background</p> <p>Vancomycin is an antibiotic of growing importance in the treatment of hospital infections, with particular emphasis on its value in the fight against methicillin-resistant <it>Staphylococcus aureus</it>. However its usage profile must be evaluated to assure maximum benefit and minimum risk.</p> <p>Findings</p> <p>A cross-sectional retrospective study was carried out among inpatients that received vancomycin in a Brazilian quaternary hospital. The occurrence of adverse reactions reported was evaluated in medical records relating to patients taking vancomycin during a one year period. Males comprised 52% (95% CI: 41.7-60.2%) of the sample population, with a mean age of 50.6 (95% CI: 47.2-54.0) years and mean treatment period of 9.7 (95% CI: 8.0-11.5) Days. It was verified that nephrotoxicity occurred in 18.4% (95% CI: 11.3-27.5) of patients, Red man syndrome occurred in 2% (95% CI 0.2-7.2), while the occurrence of thrombocytopenia was 7.1% (95% CI: 2.9-14.2).</p> <p>Conclusions</p> <p>It may be noted that even after 50 years of use, adverse reactions associated with vancomycin continue with high frequency, presenting a public health problem, especially considering its current use in cases of multidrug resistant infections. In this context, we emphasize the importance of intensive pharmacovigilance in hospital as a surveillance tool after drug approval by the sanitary authority.</p

Dynamic Activation and Repression of the Plasmodium falciparum rif Gene Family and Their Relation to Chromatin Modification

The regulation of variant gene expression in Plasmodium falciparum is still only partially understood. Regulation of var genes, the most studied gene family involved in antigenic variation, is orchestrated by a dynamic pattern of inherited chromatin states. Although recent evidence pointed to epigenetic regulation of transcribed and repressed rif loci, little is known about specific on/off associated histone modifications of individual rif genes. To investigate the chromatin marks for transcribed and repressed rif loci, we cultivated parasites and evaluated the transcriptional status of chosen rif targets by qRT-PCR and performed ChIP assays using H3K9ac and H3K9me3 antibodies. We then monitored changes in the epigenetic patterns in parasites after several reinvasions and also evaluated the “poised” mark in trophozoites and schizonts of the same erythrocytic cycle by ChIP using H3K4me2 specific antibodies. Our results show that H3K9 is acetylated in transcribed rif loci and trimethylated or even unmodified in repressed rif loci. These transcriptional and epigenetic states are inherited after several reinvasions. The poised modification H3K4me2 showed a tendency to be more present in loci in trophozoites that upon progression to schizonts strongly transcribe the respective locus. However, this effect was not consistently observed for all monitored loci. While our data show important similarities to var transcription-associated chromatin modifications, the observed swiftly occurring modifications at rif loci and the absence of H3K9 modification point to a different dynamic of recruitment of chromatin modifying enzymes