Aminic Organoselenium Compounds as Glutathione Peroxidase Mimics and Inhibitors of Ferroptosis.

The synthesis of diarylamine-based organoselenium compounds via the nucleophilic substitution reactions has been described. Symmetrical monoselenides and diselenides were conveniently synthesized by the reduction of their corresponding selenocyanates using sodium borohydride. Selenocyanates were obtained from 2-chloro acetamides by the nucleophilic displacement with potassium selenocyanate. Selenides were synthesized by treating the 2-chloro acetamides with in situ generated sodium butyl selenolate as nucleophile. Further, the newly synthesized organoselenium compounds were evaluated for their glutathione peroxidase (GPx)-like activity in thiophenol assay. This study revealed that the methoxy-substituted organoselenium compounds showed significant effect on the GPx-like activity. The catalytic parameters for the most efficient catalysts were also determined. The anti-ferroptotic activity for all GPx-mimics evaluated in a 4-OH-tamoxifen (TAM) inducible GPx4 knockout cell line using liproxstatin as standard.

A novel mitigation strategy for the prevention of transfusion-transmitted malaria.

BACKGROUND: Malaria is caused by protozoa of the genus Plasmodium and transmitted by Anopheles mosquitos. In the US, blood donors are assessed for malaria risk, including donor travel or previous residence in endemic areas and history of malaria by questionnaire and deferred for three months or three years, respectively. METHODS: The Procleix Plasmodium Assay is a qualitative nucleic acid test based on transcription-mediated amplification (TMA) for the detection of 18S ribosomal RNA of P. falciparum, P. ovale, P. vivax, P. malariae, and P. knowlesi for use on the Procleix Panther system. Analytical sensitivity was evaluated with in vitro transcripts and infected red blood cells. For clinical specificity, 12,800 individual donations and 283 pools of 16 samples from routine US donors were screened. Malaria risk was evaluated by testing 862 donors deferred for 3 years. Reactive results were confirmed with in-house real-time TMA assay and serology. RESULTS: Assay sensitivity was 8.47-11.89 RNA copies/mL and 2.10-6.82 infected red cells/mL. Specificity was 99.99% in 12,800 individual donations and 100% in 283 pools of 16. Of 862 tested deferred donor samples, one donor (0.12%) confirmed positive individually and in pools; he remained confirmed positive for 13 months. The infected donor was a prior resident of a malaria-endemic area in West Africa. CONCLUSIONS: The Procleix Plasmodium Assay showed high sensitivity and specificity and detected Plasmodium RNA in an asymptomatic presenting donor. This assay may prove helpful as a screening test versus the use of risk questions to reduce the number of donors deferred for malaria risk.

Comparative metabolome analysis reveals higher potential of haemoperfusion adsorption in providing favourable outcome in ACLF patients.

BACKGROUND AND AIMS: Acute-on-chronic liver failure (ACLF) is a serious illness associated with altered metabolome, organ failure and high mortality. Need for therapies to improve the metabolic milieu and support liver regeneration are urgently needed. METHODS: We investigated the ability of haemoperfusion adsorption (HA) and therapeutic plasma exchange (TPE) in improving the metabolic profile and survival in ACLF patients. Altogether, 45 ACLF patients were randomized into three groups: standard medical therapy (SMT), HA and TPE groups. Plasma metabolomics was performed at baseline, post-HA and TPE sessions on days 7 and 14 using high-resolution mass spectrometry. RESULTS: The baseline clinical/metabolic profiles of study groups were comparable. We identified 477 metabolites. Of these, 256 metabolites were significantly altered post 7 days of HA therapy (p < .05, FC > 1.5) and significantly reduced metabolites linked to purine (12 metabolites), tryptophan (7 metabolites), primary bile acid (6 metabolites) and arginine-proline metabolism (6 metabolites) and microbial metabolism respectively (p < .05). Metabolites linked to taurine-hypotaurine and histidine metabolism were reduced and temporal increase in metabolites linked to phenylalanine and tryptophan metabolism was observed post-TPE therapy (p < .05). Finally, weighted metabolite correlation network analysis (WMCNA) along with inter/intragroup analysis confirmed significant reduction in inflammatory (tryptophan, arachidonic acid and bile acid metabolism) and secondary energy metabolic pathways post-HA therapy compared to TPE and SMT (p < .05). Higher baseline plasma level of 11-deoxycorticosterone (C03205; AUROC > 0.90, HR > 3.2) correlated with severity (r2 > 0.5, p < .05) and mortality (log-rank-p < .05). Notably, 51 of the 64 metabolite signatures (ACLF non-survivor) were reversed post-HA treatment compared to TPE and SMT(p < .05). CONCLUSION: HA more potentially (~80%) improves plasma milieu compared to TPE and SMT. High baseline plasma 11-deoxycorticosterone level correlates with early mortality in ACLF patients.

Novel Dihydrocoumarins Induced by Radiolysis as Potent Tyrosinase Inhibitors.

A representative naturally occurring coumarin, 4-methylumbelliferone (5), was exposed to 50 kGy of gamma ray, resulting in four newly generated dihydrocoumarin products 1-4 induced by the gamma irradiation. The structures of these new products were elucidated by interpretation of spectroscopic data (NMR, MS, [α]D, and UV). The unusual bisdihydrocoumarin 4 exhibited improved tyrosinase inhibitory capacity toward mushroom tyrosinase with IC50 values of 19.8 ± 0.5 µM as compared to the original 4-methylumbelliferone (5). A kinetic analysis also exhibited that the potent metabolite 4 had non-competitive modes of action. Linkage of the hydroxymethyl group in the C-3 and C-4 positions on the lactone ring probably enhances the tyrosinase inhibitory effect of 4-methylumbelliferone (5). Thus, the novel coumarin analog 4 is an interesting new class of tyrosinase inhibitory candidates that requires further examination.

Biomolecular map of albumin identifies signatures of severity and early mortality in acute liver failure.

BACKGROUND & AIMS: Acute liver failure (ALF) is associated with high mortality. Alterations in albumin structure and function have been shown to correlate with outcomes in cirrhosis. We undertook a biomolecular analysis of albumin to determine its correlation with hepatocellular injury and early mortality in ALF. METHODS: Altogether, 225 participants (200 patients with ALF and 25 healthy controls [HC]) were enrolled. Albumin was purified from the baseline plasma of the training cohort (ALF, n = 40; survivors, n = 8; non-survivors, n = 32; and HC, n = 5); analysed for modifications, functionality, and bound multi-omics signatures; and validated in a test cohort (ALF, n = 160; survivors, n = 53; non-survivors, n = 107; and HC, n = 20). RESULTS: In patients with ALF, albumin is more oxidised and glycosylated with a distinct multi-omics profile than that in HC, more so in non-survivors (p <0.05). In non-survivors, albumin was more often bound (p <0.05, false discovery rate <0.01) to proteins associated with inflammation, advanced glycation end product, metabolites linked to arginine, proline metabolism, bile acid, and mitochondrial breakdown products. Increased bacterial taxa (Listeria, Clostridium, etc.) correlated with lipids (triglycerides [4:0/12:0/12:0] and phosphatidylserine [39:0]) and metabolites (porphobilinogen and nicotinic acid) in non-survivors (r2 >0.7). Multi-omics signature-based probability of detection for non-survival was >90% and showed direct correlation with albumin functionality and clinical parameters (r2 >0.85). Probability-of-detection metabolites built on the top five metabolites, namely, nicotinic acid, l-acetyl carnitine, l-carnitine, pregnenolone sulfate, and N-(3-hydroxybutanoyl)-l-homoserine lactone, showed diagnostic accuracy of 98% (AUC 0.98, 95% CI 0.95-1.0) and distinguish patients with ALF predisposed to early mortality (log-rank <0.05). On validation using high-resolution mass spectrometry and five machine learning algorithms in test cohort 1 (plasma and paired one-drop blood), the metabolome panel showed >92% accuracy/sensitivity and specificity for prediction of mortality. CONCLUSIONS: In ALF, albumin is hyperoxidised and substantially dysfunctional. Our study outlines distinct 'albuminome' signatures capable of distinguishing patients with ALF predisposed to early mortality or requiring emergency liver transplantation. IMPACTS AND IMPLICATIONS: Here, we report that the biomolecular map of albumin is distinct and linked to severity and outcome in patients with acute liver failure (ALF). Detailed structural, functional, and albumin-omics analysis in patients with ALF led to the identification and classification of albumin-bound biomolecules, which could segregate patients with ALF predisposed to early mortality. More importantly, we found albumin-bound metabolites indicative of mitochondrial damage and hyperinflammation as a putative indicator of <30-day mortality in patients with ALF. This preclinical study validates the utility of albuminome analysis for understanding the pathophysiology and development of poor outcome indicators in patients with ALF.

Bile multi-omics analysis classifies lipid species and microbial peptides predictive of carcinoma of gallbladder.

BACKGROUND AND AIMS: Histopathological examination is the gold standard for detection of gallstone (GS) or gallbladder carcinoma (CAGB). Bile concentrated in the gallbladder (GB) is expected to recapitulate metagenomics and molecular changes associated with development of CAGB. APPROACH AND RESULTS: Bile samples were screened for lipidomics and metaproteome (metagenomics) signatures capable of early detection of cancer in GB anomalies. Analysis of the training cohort (n = 87) showed that metastability of bile was reduced in CAGB (p < 0.05). CAGB bile showed significant alteration of lipidome and microbiome as indicated by multivariate partial least squares regression analysis and alpha-diversity and beta-diversity indexes (p < 0.05). Significant reduction of lipid species and increase in bacterial taxa were found to be associated with patients with CAGB, CAGB with GS, and GS (p < 0.05, log fold change >1.5). A multimodular correlation network created using weighted lipid/metaproteomic correlation network analysis showed striking associations between lipid and metaproteomic modules and functionality. CAGB-linked metaproteomic modules/functionality directly correlated with lipid modules, species, clinical parameters, and bile acid profile (p < 0.05). Increased bacterial taxa (Leptospira, Salmonella enterica, Mycoplasma gallisepticum) and their functionality showed a direct correlation with lipid classes such as lysophosphatidylinositol, ceramide 1-phosphate, and lysophosphatidylethanolamine and development of CAGB (r2  > 0.85). Lipid/metaproteomic signature-based probability of detection for CAGB was > 90%, whereas that for GS was > 80% (p < 0.05). Validation of eight lipid species using four machine learning algorithms in two separate cohorts (n = 38; bile [test cohort 1] and paired plasma [test cohort 2]) showed accuracy (99%) and sensitivity/specificity (>98%) for CAGB detection. CONCLUSIONS: Bile samples of patients with CAGB showed significant reduction in lipid species and increase in bacterial taxa. Our study identifies a core set of bile lipidome and metaproteome signatures which may offer universal utility for early diagnosis of CAGB.

Deciphering the role of miRNA in reprogramming plant responses to drought stress.

Drought is the most prevalent environmental stress that affects plants' growth, development, and crop productivity. However, plants have evolved adaptive mechanisms to respond to the harmful effects of drought. They reprogram their: transcriptome, proteome, and metabolome that alter their cellular and physiological processes and establish cellular homeostasis. One of the crucial regulatory processes that govern this reprogramming is post-transcriptional regulation by microRNAs (miRNAs). miRNAs are small non-coding RNAs, involved in the downregulation of the target mRNA via translation inhibition/mRNA degradation/miRNA-mediated mRNA decay/ribosome drop off/DNA methylation. Many drought-inducible miRNAs have been identified and characterized in plants. Their main targets are regulatory genes that influence growth, development, osmotic stress tolerance, antioxidant defense, phytohormone-mediated signaling, and delayed senescence during drought stress. Overexpression of drought-responsive miRNAs (Osa-miR535, miR160, miR408, Osa-miR393, Osa-miR319, and Gma-miR394) in certain plants has led to tolerance against drought stress indicating their vital role in stress mitigation. Similarly, knock down (miR166/miR398c) or deletion (miR169 and miR827) of miRNAs has also resulted in tolerance to drought stress. Likewise, engineered Arabidopsis plants with miR165, miR166 using short tandem target mimic strategy, exhibited drought tolerance. Since miRNAs regulate the expression of an array of drought-responsive genes, they can act as prospective targets for genetic manipulations to enhance drought tolerance in crops and achieve sustainable agriculture. Further investigations toward functional characterization of diverse miRNAs, and understanding stress-responses regulated by these miRNAs and their utilization in biotechnological applications is highly recommended.

Nucleic acid testing for monkeypox in United States blood donor specimens.

BACKGROUND: The 2022 multi-country outbreak of monkeypox (mpox) resulted in blood collection and public health agencies closely monitoring for changes in transmission dynamics that could pose a threat to the blood supply. While mpox virus (MPXV) is not known to be transfusion transmissible, there have been several studies demonstrating the detection of MPXV in blood. We evaluated the performance characteristics of a research use only (RUO) nucleic acid amplification test for MPXV. The assay was developed to detect MPXV DNA in plasma and serum specimens from human blood donors. METHODS AND MATERIALS: The sensitivity of the RUO MPXV Assay was determined using a synthetic DNA sequence, purified full-length genomic DNA, and a chemically inactivated virus. Specificity was determined using fresh plasma samples collected from blood donors during the outbreak. Plasma samples collected from donors considered at increased risk for exposure to mpox were also tested. RESULTS: For sensitivity, the 95% limit of detection (LOD) ranged from 0.26 copies/mL (inactivated virus) to 31.65 copies/mL (synthetic DNA) to 166.61 copies/mL (for full-length DNA). All donor samples tested with the RUO MPXV Assay were nonreactive, resulting in a specificity of 100% (95% CI, 99.93%-100.00%). DISCUSSION: The RUO MPXV Assay was developed as a potential blood donation screening assay in response to the outbreak. While not directly comparable, the 95% LOD fiducial limits obtained from partial- and full-length DNA analysis were similar to other manufacturers' MPXV assays. Additionally, this assay demonstrated high specificity for screening blood donors.

Inflammatory responses during trichomoniasis: The role of Toll-like receptors and inflammasomes.

Toll-like receptors (TLRs) and inflammasomes belong to the pattern recognition receptors (PRRs) of innate immunity identifying conserved compounds produced by pathogens or discharged by injured cells. Different cell subsets in the human urogenital system, such as epithelial cells and infiltrating leukocytes, express different kinds of TLRs (such as TLR2, TLR3, TLR4, TLR5 and TLR9) as well as inflammasomes (such as NLRP3, NLRC4 and AIM2). Various types of the Trichomonas vaginalis-derived components such as glycosyl-phosphatidylinositol (GPI), T. vaginalis virus (TVV), Lipophosphoglycan (LPG) and flagellin can be recognized by TLR2, TLR3, TLR4 and TLR5, respectively, leading to the production of proinflammatory cytokines and chemokines in the cervicovaginal mucosa. The T. vaginalis-induced inflammasomes can lead to pyroptosis as well as the release of IL-1ß and IL-18 promoting innate and adaptive immune responses. The PRR-mediated responses to T. vaginalis may contribute to the induction of protective immune responses, local inflammation, promotion of co-infections, or even the development of malignancies, for example, prostate cancer. The protective or pathogenic roles of the TLRs and inflammasomes during trichomoniasis are highlighted in this review. A better understanding of PRR-mediated responses provides invaluable insights to develop effective immunotherapeutic strategies against T. vaginalis infection.

Glutathione Peroxidase-like Antioxidant Activity of 1,3-Benzoselenazoles: Synthesis and In Silico Molecular Docking Studies as Pancreatic Lipase Inhibitors.

The synthesis of 1,3-benzoselenazoles was achieved by the reaction of corresponding bis[3-amino-N-(p-tolyl)benzamide-2-yl] diselenide, bis[3-amino-N-(4-methoxyphenyl)benzamide-2-yl] diselenide, and bis[3-amino-N-(4-(dimethylamino)phenyl) benzamide-2-yl] diselenide with aryl aldehydes. The 1,3-benzoselenazoles continued to exist as planar molecules due to the presence of secondary Se···O interactions as revealed by the single-crystal X-ray analysis. The presence of secondary Se···O interactions in 1,3-benzoselenazoles was confirmed using natural bond orbital (NBO) and atoms in molecules (AIM) calculations. Nucleus-independent chemical shift (NICS) values suggested the presence of aromatic character in a five-membered benzoselenazole heterocyclic ring. The glutathione peroxidase (GPx)-like antioxidant activity of all 1,3-benzoselenazoles was assessed using a thiophenol assay, exhibiting greater antioxidant activity than Ph2Se2 used as a reference. The most active catalyst carrying a strong electron-donating group (-NMe2) at the ortho-position to the benzoselenazole ring was further investigated at different concentrations of thiophenol, H2O2, and 1,3-benzoselenazoles as catalyst for determining their catalytic parameters. Moreover, the potential applications of all 1,3-benzoselenazoles against pancreatic lipase (PL) have been identified using in silico interactions between the active sites of the 1LPB protein as evaluated using a molecular docking study.

Synthesis, Reactions, and Antioxidant Properties of Bis(3-amino-1-hydroxybenzyl)diselenide.

Bis(3-amino-1-hydroxybenzyl)diselenide containing two ortho groups was synthesized from 7-nitro-3H-2,1-benzoxaselenole and in situ generated sodium benzene tellurolate (PhTeNa). One-pot synthesis of 1,3-benzoselenazoles was achieved from bis(3-amino-1-hydroxybenzyl)diselenide and aryl aldehydes using acetic acid as a catalyst. The X-ray crystal structure of chloro-substituted benzoselenazole revealed a planar structure with T-shaped geometry around the Se atom. Both natural bond orbital and atoms in molecules calculations confirmed the presence of secondary Se···H interactions in bis(3-amino-1-hydroxybenzyl)diselenide and Se···O interactions in benzoselenazoles, respectively. The glutathione peroxidase (GPx)-like antioxidant activities of all compounds were evaluated using a thiophenol assay. Bis(3-amino-1-hydroxybenzyl)diselenide and benzoselenazoles showed better GPx-like activity compared to that of the diphenyl diselenide and ebselen, used as references, respectively. Based on 77Se{1H} NMR spectroscopy, a catalytic cycle for bis(3-amino-1-hydroxybenzyl)diselenide using thiophenol and hydrogen peroxide was proposed involving selenol, selenosulfide, and selenenic acid as intermediates. The potency of all GPx mimics was confirmed by their in vitro antibacterial properties against the biofilm formation of Bacillus subtilis and Pseudomonas aeruginosa. Additionally, molecular docking studies were used to evaluate the in silico interactions between the active sites of the TsaA and LasR-based proteins found in Bacillus subtilis and Pseudomonas aeruginosa.

Structure-function analysis of DEAD-box helicase DDX43.

DDX43 (DEAD-box helicase 43), also known as HAGE (helicase antigen gene), is a member of the DEAD-box protein family. It contains a K homology (KH) domain in its N terminus, a helicase core domain in its C terminus, and a flexible linker domain in between. DDX43 expression is low or undetectable in normal tissue, but is overexpressed in many tumors; therefore, it is considered a potential target molecule for cancer therapy. We, along with other groups, have shown that DDX43 is an ATP-dependent RNA and DNA helicase, and the KH domain is required for its ATPase and unwinding activity. Electrophoretic mobility shift assay (EMSA), SELEX (systematic evolution of ligands by exponential enrichment), chromatin immunoprecipitation (ChIP)-seq, crosslinking immunoprecipitation (CLIP)-seq, and nuclear magnetic resonance (NMR) showed that the KH domain prefers to bind pyrimidine-rich ssDNA and ssRNA, such as TTGT in the promoter regions of genes. Moreover, the KH domain facilitates the substrate specificity and processivity of the DDX43 helicase. No animal model has been generated for DDX43; cellular studies have revealed that DDX43 has roles in piRNA amplification, tumorigenesis, RAS signaling, and innate immunity. Structural and functional studies of DDX43 will not only advance our understanding of DEAD-box helicases and KH domains, but also shed light on the application of DDX43 as therapeutics, where its key binding sites can be targeted by small molecules and natural products as an alternative approach in treating DDX43 overexpressed cancers.

Factors associated with late gestational age of diagnosis and/or delayed referral of fetuses with major structural malformations: A study in a tertiary care hospital in South India.

OBJECTIVES: To evaluate the factors related to delayed diagnosis or referral of women with major congenital malformations in a tertiary care hospital in South India, with a specific emphasis on the socioeconomic and psychosocial aspects. METHODS: A retrospective analysis was conducted on 107 pregnant women with confirmed fetal anomalies at Jawaharlal Institute of Postgraduate Medical Education and Research. Both qualitative and quantitative data were collected on factors related to the patient, physician, and healthcare system that could have contributed to the delay. RESULTS: In our study of 107 patients, 98.1% had at least one antenatal check-up before 20 weeks. Among them, 75.2% (79/107) were advised to undergo a mid-trimester anomaly scan, and 70.8% (56/79) actually underwent the scan. However, 62.5% (35/56) of those who had the scan performed at the appropriate time had undetected abnormalities. Retrospective analysis showed that 28.5% (10/35) of these anomalies could have been detected as early as 12-14 weeks of gestation. Additionally, 24.3% (26/107) of women were advised to undergo scans at or after 20 weeks and 4.7% (5/107) had anomalies detected during the scan but were unaware about the findings until subsequent visits. Inadequate healthcare professional training, busy schedules, lack of awareness, and communication gaps were identified as potential reasons for delayed diagnosis. Psychosocial factors such as denial, fear, emotional distress, and anxiety also influenced decision-making and contributed to delayed medical care. CONCLUSION: Addressing healthcare provider training, communication between the healthcare professionals and the patients, and psychosocial factors are essential to prevent delays and improve pregnancy management. Future interventions should focus on awareness, providing comprehensive information, and support for parents facing fetal anomalies.

Are hypopressive and other exercise programs effective for the treatment of pelvic organ prolapse?

INTRODUCTION AND HYPOTHESIS: Pelvic floor muscle training (PFMT) is effective for the treatment of pelvic organ prolapse (POP), but other exercise programs have also been promoted and used. The aim of this review was to evaluate the effect of hypopressive and other exercise programs besides PFMT for POP. METHODS: A literature search was conducted on Ovid Medline, EMBASE, CINAHL, Cochrane, PEDro, and Scopus databases from January 1996 to 30 December 2021. Only randomized controlled trials (RCTs) were included. The keywords were combinations of "pelvic organ prolapse" or "urogenital prolapse," and "exercise therapy," "hypopressive exercise," "Kegel," "pelvic floor muscle training," "pelvic floor muscle exercises," "Pilates," "treatment," "yoga," "Tai Chi." Methodological quality was assessed using the PEDro rating scale (0-10). RESULTS: Seven RCTs containing hypopressive exercise, yoga or breathing and hip muscle exercises in an inverted position were retrieved and analyzed. PEDro score ranged from 4 to 7. There was no additional effect of adding hypopressive exercise to PFMT, and PFMT was more effective than hypopressive exercise alone. The studies that included the term "yoga" included regular PFMT and thus can be classified as PFMT. Hip exercises in an inverted position added to PFMT vs PFMT alone showed better improvement in some secondary outcomes but not in the primary outcome, POP stage. CONCLUSIONS: There are few RCTs assessing the effects of other exercise programs besides PFMT in the treatment of POP. To date, there is no evidence that other exercise programs are more effective than PFMT for POP.

Strenuous physical activity, exercise, and pelvic organ prolapse: a narrative scoping review.

INTRODUCTION AND HYPOTHESIS: High-intensity physical activity and exercise have been listed as possible risk factors for pelvic organ prolapse (POP). The aim of the present study is to conduct a literature review on the prevalence and incidence of POP in women who engage in regular physical activity. In addition, we review the effects of a single exercise or a single session of exercise on pelvic floor support. Finally, the effect of exercises on POP in the early postpartum period is reviewed. METHODS: This is a narrative scoping review. We searched PubMed and Ovid Medline, the Physiotherapy Evidence Database (PEDro), and the Cochrane Database of Systematic Reviews up to May 2022 with the following MeSH terms: "physical activity" AND "exercise" AND "pelvic floor" AND "pelvic organ prolapse". RESULTS: Eight prevalence studies were retrieved. Prevalence rates of symptomatic POP varied between 0 (small study within different sports) and 23% (Olympic weightlifters and power lifters). Parity was the only factor associated with POP in most studies. Three studies evaluated the pelvic floor after a single exercise or one session of exercise and found increased vaginal descent or increased POP symptoms. One prospective cohort study reported the development of POP after 6 weeks of military parashot training, and one randomized trial reported increased POP symptoms after transverse abdominal training. There is scant knowledge on exercise and POP in the postpartum period. CONCLUSIONS: Prevalence of POP in sports varies widely. Experimental and prospective studies indicate that strenuous exercise increased POP symptoms and reduced pelvic floor support.

The KH domain facilitates the substrate specificity and unwinding processivity of DDX43 helicase.

The K-homology (KH) domain is a nucleic acid-binding domain present in many proteins. Recently, we found that the DEAD-box helicase DDX43 contains a KH domain in its N-terminus; however, its function remains unknown. Here, we purified recombinant DDX43 KH domain protein and found that it prefers binding ssDNA and ssRNA. Electrophoretic mobility shift assay and NMR revealed that the KH domain favors pyrimidines over purines. Mutational analysis showed that the GXXG loop in the KH domain is involved in pyrimidine binding. Moreover, we found that an alanine residue adjacent to the GXXG loop is critical for binding. Systematic evolution of ligands by exponential enrichment, chromatin immunoprecipitation-seq, and cross-linking immunoprecipitation-seq showed that the KH domain binds C-/T-rich DNA and U-rich RNA. Bioinformatics analysis suggested that the KH domain prefers to bind promoters. Using 15N-heteronuclear single quantum coherence NMR, the optimal binding sequence was identified as TTGT. Finally, we found that the full-length DDX43 helicase prefers DNA or RNA substrates with TTGT or UUGU single-stranded tails and that the KH domain is critically important for sequence specificity and unwinding processivity. Collectively, our results demonstrated that the KH domain facilitates the substrate specificity and processivity of the DDX43 helicase.

Virulence gene mutations as a differentiator of clinical phenotypes: insights from community-acquired uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) remains an important cause of urinary tract infection during pregnancy. Multiple molecular virulence determinants and antibiotic resistant genes facilitate its pathogenesis and virulence phenotype. Hence it is hypothesized that there will be considerable variation in genes among the isolates from symptomatic as well as asymptomatic bacteriuria (ABU) during pregnancy. The aim of this study was to decipher the genetic variation among the two phenotypes. Six different UPEC isolates collected from urine specimens of consecutive pregnant females (five, symptomatic bacteriuria and one, ABU) were tested for their growth kinetics, and biofilm formation. A total of 87 virulence determinants and 56 antibiotic resistance genes were investigated using whole-genome sequencing, to identify putative drives of virulence phenotype. In this analysis, we identified eight different types of fully functional toxin antitoxin (TA) systems [HipAB, YefM-YoeB, YeeU-YeeV (CbtA), YhaV-PrlF, ChpBS, HigAB, YgiUT and HicAB] in the isolates from symptomatic bacteriuria; whereas partially functional TA system with mutations were observed in the asymptomatic one. Isolates of both the groups showed equivalent growth characteristics and biofilm-formation ability. Genes for an iron transport system (Efe UOB system, Fhu system except FhuA) were observed functional among all symptomatic and asymptomatic isolates, however functional mutations were observed in the latter group. Gene YidE was observed predominantly associated with the biofilm formation along with few other genes (BssR, BssS, YjgK, etc.). This study outlines putative critical relevance of specific variations in the genes for the TA system, biofilm formation, cell adhesion and colonization among UPEC isolates from symptomatic and asymptomatic bacteriuria among pregnant women. Further functional genomic study in the same cohort is warranted to establish the pathogenic role of these genes.

Runx proteins mediate protective immunity against Leishmania donovani infection by promoting CD40 expression on dendritic cells.

The level of CD40 expression on dendritic cells (DCs) plays a decisive role in disease protection during Leishmania donovani (LD) infection. However, current understanding of the molecular regulation of CD40 expression remains elusive. Using molecular, cellular and functional approaches, we identified a role for Runx1 and Runx3 transcription factors in the regulation of CD40 expression in DCs. In response to lipopolysaccharide (LPS), tumor necrosis factor alpha (TNFα) or antileishmanial drug sodium antimony gluconate (SAG), both Runx1 and Runx3 translocated to the nucleus, bound to the CD40 promoter and upregulated CD40 expression on DCs. These activities of Runx proteins were mediated by the upstream phosphatidylinositol 3-kinase (PI3K)-Akt pathway. Notably, LD infection attenuated LPS- or TNFα-induced CD40 expression in DCs by inhibiting PI3K-Akt-Runx axis via protein tyrosine phosphatase SHP-1. In contrast, CD40 expression induced by SAG was unaffected by LD infection, as SAG by blocking LD-induced SHP-1 activation potentiated PI3K-Akt signaling to drive Runx-mediated CD40 upregulation. Adoptive transfer experiments further showed that Runx1 and Runx3 play a pivotal role in eliciting antileishmanial immune response of SAG-treated DCs in vivo by promoting CD40-mediated type-1 T cell responses. Importantly, antimony-resistant LD suppressed SAG-induced CD40 upregulation on DCs by blocking the PI3K-Akt-Runx pathway through sustained SHP-1 activation. These findings unveil an immunoregulatory role for Runx proteins during LD infection.

Latent Upregulation of Nlrp3, Nlrc4 and Aim2 Differentiates between Asymptomatic and Symptomatic Trichomonas vaginalis Infection.

Trichomonas vaginalis is a parasitic protozoan that causes trichomoniasis. The involvement of NLRP3 inflammasome in trichomoniasis has been discussed in recent studies. The present study aimed to find out the involvement of Nlrp3, Nlrc4, and Aim2 in the BALB/c mouse model infected with symptomatic and asymptomatic isolates of T. vaginalis by quantitative real-time PCR and immunohistochemistry. Our results showed a significantly increased expression of Nlrp3 in the vaginal tissue of the symptomatic group on the 2nd dpi and 14th dpi in the asymptomatic group, respectively. The cervical tissue of asymptomatic groups expressed higher Nlrp3 on 14th dpi than the symptomatic group. The Nlrc4 was expressed on 14th dpi in the vaginal and cervical tissues of mice infected with asymptomatic group as compared to the symptomatic group. Aim2 expression in vaginal tissue was highest at early time points in both the infected groups as compared to controls. However, in cervical tissues, a significant increase of Aim2 expression was observed on 14th dpi in asymptomatic as compared to the symptomatic group. The significantly higher expression of caspase-1 and caspase-4 was observed in cervical tissues of the asymptomatic group on 14th dpi as compared to the symptomatic group, respectively. All NLRs together resulted in higher IL-1ß expression in the vaginal tissues of the symptomatic and asymptomatic groups. We conclude from this study that early expression of Nlrp3, Nlrc4, and Aim2 was seen in the symptomatic group as compared to the late-onset asymptomatic in the vaginal and cervical tissues.

International urogynecology consultation chapter 3 committee 2; conservative treatment of patient with pelvic organ prolapse: Pelvic floor muscle training.

INTRODUCTION AND HYPOTHESIS: This manuscript from Chapter 3 of the International Urogynecology Consultation (IUC) on Pelvic Organ Prolapse (POP) describes the current evidence and suggests future directions for research on the effect of pelvic floor muscle training (PFMT) in prevention and treatment of POP. METHODS: An international group of four physical therapists, four urogynecologists and one midwife/basic science researcher performed a search of the literature using pre-specified search terms on randomized controlled trials (RCTs) in Ovid Medline, EMBASE, CINAHL, Cochrane, PEDro and Scopus databases for publications between 1996 and 2021. Full publications or expanded abstracts in English or in other languages with abstracts in English were included. The PEDro rating scale (0-10) was used to evaluate study quality. Included RCTs were reviewed to summarize the evidence in six key sections: (1) evidence for PFMT in prevention of POP in the general female population; (2) evidence for early intervention of PFMT in the peripartum period for prevention and treatment of POP; (3) evidence for PFMT in treatment of POP in the general female population; (4) evidence for perioperative PFMT; (5) evidence for PFMT on associated conditions in women with POP; (6) evidence for the long-term effect of PFMT on POP. Full publications in English or in other languages with abstracts in English and expanded abstracts presented at international condition specific societies were included. Internal validity was examined by the PEDro rating scale (0-10). RESULTS: After exclusion of duplicates and irrelevant trials, we classified and included 2 preventive trials, 4 trials in the post-partum period, 11 treatment trials of PFMT for POP in the general female population in comparison with no treatment or lifestyle interventions, 10 on PFMT as an adjunct treatment to POP surgery and 9 long-term treatment trials. Only three treatment studies compared PFMT with the use of a pessary. The RCTs scored between 4 and 8 on the PEDro scale. No primary prevention studies were found, and there is sparse and inconsistent evidence for early intervention in the postpartum period. There is good evidence/recommendations from 11 RCTs that PFMT is effective in reducing POP symptoms and/or improving POP stage (by one stage) in women with POP-Q stage I, II and III in the general female population, but no evidence from 9/10 RCTs that adding PFMT pre- and post -surgery for POP is effective. There are few long-term follow-up studies, and results are inconsistent. There are no serious adverse effects or complications reported related to PFMT. CONCLUSIONS: There are few studies on prevention and in the postpartum period, and the effect is inconclusive. There is high-level evidence from 11 RCTs to recommend PFMT as first-line treatment for POP in the general female population. PFMT pre- and post-POP surgery does not seem to have any additional effect on POP. PFMT is effective and safe but needs thorough instruction and supervision to be effective.