An automated,high‐throughput method for targeted quantification of intact insulin and its therapeutic analogs in human serum or plasma coupling mass spectrometric immunoassay with high resolution and accurate mass detection (MSIA‐HR/AM)

The detection and quantification of insulin and its therapeutic analogs is important for medical, sports doping, and forensic applications. Synthetic variants contain slight sequence variations to affect bioavailability. To reduce sample handling bias, a universal extraction method is required for simultaneous extraction of endogenous and variant insulins with subsequent targeted quantification by LC‐MS. A mass spectrometric immunoassay (MSIA), a multiplexed assay for intact insulin and its analogues that couples immunoenrichment with high resolution and accurate mass (HR/AM) spectrometric detection across the clinical range is presented in this report. The assay is sensitive, selective, semi‐automated and can potentially be applied to detect new insulin isoforms allowing their further incorporation into second or third generation assays.     

Design, synthesis, and evaluations of antifungal activity of novel phenyl(2H-tetrazol-5-yl)methanamine derivatives

Fungal infections pose a continuous and serious threat to human health and life. The intrinsic resistance has been observed in many genera of fungi. Many fungal infections are caused by opportunistic pathogens that may be endogenous (Candida infections) or acquired from the environment (Cryptococcus and Aspergillus infections). So, new therapeutic strategies are needed to combat various fungal infections. Fluconazole shows good antifungal activity with relatively low toxicity and is preferred as first line antifungal therapy, but it has suffered from severe drug resistance. So, there is a need to design novel analogues by modification of fluconazole-like structure. A novel series of phenyl(2H-tetrazol-5-yl)methanamine derivatives were synthesized by reaction of α-amino nitrile with sodium azide and ZnCl₂ in presence of isopropyl alcohol. They were evaluated for antifungal activity against Candida albicans and Aspergillus niger and subjected to docking study against 1EA1.     

Plasma membrane proteome of adhesion‐competent endometrial epithelial cells and its modulation by Rab11a

Human endometrial epithelium (EE) is composed of a multitude of proteins, amongst which those localized on the plasma membrane [plasma membrane proteins (PMPs)] are of critical relevance in the early stages of implantation. Evidence supports the key role of few PMPs in implantation. However, many remain unidentified, as efforts have not been made till date to generate the plasma membrane proteome of human EE cells, using a gel‐free approach. This study presents a protein catalog of the PMP enriched fraction of Ishikawa cell line; often used as an in vitro model for embryo‐adhesive EE. Liquid chromatography with tandem mass spectrometry identified 3,598 proteins. Of these, 1,963 proteins were annotated for their membrane localization. Of 1,963 proteins, 1,321 were found to have a transmembrane domain and 43 proteins had glycophosphatidylinositol (GPI) anchor. Extensive data mining revealed endometrial expression of 943 proteins reported in humans and/or rodents. Further, quantitative alterations were observed in the plasma membrane proteome on the perturbation of intracellular trafficking. Silencing of Rab11a (known for its role in plasma membrane organization) expression caused alteration in the abundance of 74 proteins. Caveolin‐1 and EpCAM levels were reduced whereas Rab4a abundance increased in the PMP extracts of Rab11a deficient cells, compared with control cells. Briefly, the study reports the identity of several novel plasma membrane‐localized proteins. A major spin‐off of the study is the identification of novel proteins trafficked by Rab11a to the plasma membrane. Targeted analysis of novel PMPs may reveal their specific roles in endometrial receptivity and implantation.     

Liprin α3: a putative estrogen regulated acrosomal protein

Liprin α3 was reported for the first time using sperm proteomics. Present study reports its localization on sperm and immunochemical characterization. Liprin α3 is identified as a 133 kDa protein in testis and epididymal protein extracts. In testis, immunohistochemical localization was seen in pachytenes, diplotenes, round spermatids whereas it was localized in the epithelial cells and luminal sperm in all the three regions of epididymis. Protein was localized in acrosome of rat sperm, which was further confirmed by sequential treatment of sperm with hypertonic solution. In the spermatogenic cells the protein was found to be located in developing acrosome as evident by its co-localization with Golgi marker. Protein was found to be developmentally regulated. In silico analysis of Liprin α3 revealed presence of the estrogen responsive elements upstream to initiation site and its regulation by estrogen was experimentally validated using a tamoxifen treated rat model. Western blot analysis of epididymosomes showed the presence of Liprin α3, indicating its involvement in trafficking of vesicle. The protein expression was seen in both mouse and human sperm indicating conserved nature and a probable role in acrosome reaction.     

Characteristics and outcomes of hospital admissions for COVID-19 and influenza in the Toronto area

BACKGROUND:Patient characteristics, clinical care, resource use and outcomes associated with admission to hospital for coronavirus disease 2019 (COVID-19) in Canada are not well described.METHODS:We described all adults with COVID-19 or influenza discharged from inpatient medical services and medical–surgical intensive care units (ICUs) between Nov. 1, 2019, and June 30, 2020, at 7 hospitals in Toronto and Mississauga, Ontario. We compared patient outcomes using multivariable regression models, controlling for patient sociodemographic factors and comorbidity level. We validated the accuracy of 7 externally developed risk scores to predict mortality among patients with COVID-19.RESULTS:There were 1027 hospital admissions with COVID-19 (median age 65 yr, 59.1% male) and 783 with influenza (median age 68 yr, 50.8% male). Patients younger than 50 years accounted for 21.2% of all admissions for COVID-19 and 24.0% of ICU admissions. Compared with influenza, patients with COVID-19 had significantly greater in-hospital mortality (unadjusted 19.9% v. 6.1%, adjusted relative risk [RR] 3.46, 95% confidence interval [CI] 2.56–4.68), ICU use (unadjusted 26.4% v. 18.0%, adjusted RR 1.50, 95% CI 1.25–1.80) and hospital length of stay (unadjusted median 8.7 d v. 4.8 d, adjusted rate ratio 1.45, 95% CI 1.25–1.69). Thirty-day readmission was not significantly different (unadjusted 9.3% v. 9.6%, adjusted RR 0.98, 95% CI 0.70–1.39). Three points-based risk scores for predicting in-hospital mortality showed good discrimination (area under the receiver operating characteristic curve [AUC] ranging from 0.72 to 0.81) and calibration.INTERPRETATION:During the first wave of the pandemic, admission to hospital for COVID-19 was associated with significantly greater mortality, ICU use and hospital length of stay than influenza. Simple risk scores can predict in-hospital mortality in patients with COVID-19 with good accuracy.

International studies report that patients admitted to hospital with coronavirus disease 2019 (COVID-19) have high rates of critical illness and mortality.^1–5 Two small Canadian case series have described care for critically ill patients with COVID-19 and found mortality rates of up to 25%.^6,7 However, outcomes of patients admitted to hospital for COVID-19 in Canada are not well described, particularly outside of intensive care units (ICUs). Case fatality rates for COVID-19 vary dramatically worldwide,⁸ and outcomes of patients admitted to hospital for COVID-19 in Canada may differ from other countries because of differences in populations, public health and health care systems.Seasonal influenza is a useful comparator for COVID-19^9–11 as it is another respiratory virus, familiar to the general public, with high rates of morbidity and mortality. The purpose of this study was to describe patient characteristics, resource use, clinical care and outcomes for patients admitted to hospital with COVID-19 in Ontario, Canada, using influenza as a comparator. We also validated the performance of various prognostic risk scores for in-hospital mortality among patients with COVID-19.     

Functional characterization, homology modeling and docking studies of β-glucosidase responsible for bioactivation of cyanogenic hydroxynitrile glucosides from Leucaena leucocephala (subabul)

Glycosyl hydrolase family 1 β-glucosidases are important enzymes that serve many diverse functions in plants including defense, whereby hydrolyzing the defensive compounds such as hydroxynitrile glucosides. A hydroxynitrile glucoside cleaving β-glucosidase gene (Llbglu1) was isolated from Leucaena leucocephala, cloned into pET-28a (+) and expressed in E. coli BL21 (DE3) cells. The recombinant enzyme was purified by Ni–NTA affinity chromatography. The optimal temperature and pH for this β-glucosidase were found to be 45 °C and 4.8, respectively. The purified Llbglu1 enzyme hydrolyzed the synthetic glycosides, pNPGlucoside (pNPGlc) and pNPGalactoside (pNPGal). Also, the enzyme hydrolyzed amygdalin, a hydroxynitrile glycoside and a few of the tested flavonoid and isoflavonoid glucosides. The kinetic parameters K _m and V _max were found to be 38.59 μM and 0.8237 μM/mg/min for pNPGlc, whereas for pNPGal the values were observed as 1845 μM and 0.1037 μM/mg/min. In the present study, a three dimensional (3D) model of the Llbglu1 was built by MODELLER software to find out the substrate binding sites and the quality of the model was examined using the program PROCHEK. Docking studies indicated that conserved active site residues are Glu 199, Glu 413, His 153, Asn 198, Val 270, Asn 340, and Trp 462. Docking of rhodiocyanoside A with the modeled Llbglu1 resulted in a binding with free energy change (ΔG) of ?5.52 kcal/mol on which basis rhodiocyanoside A could be considered as a potential substrate.