Severe COVID-19 among Admitted COVID-19 Patients in a Tertiary Care Centre: A Descriptive Cross-sectional Study.

Introduction: Severe COVID-19 patients experience elevated levels of serologic indicators of inflammation which can alter blood cell lineages and cause lymphopenia. The objective of this study was to find out the prevalence of severe COVID-19 among admitted COVID-19 patients in a tertiary care centre. Methods: A descriptive cross-sectional study was conducted in a tertiary care centre from 22 June 2021 to 30 September 2021 after obtaining ethical approval from the Institutional Review Committee (Reference number: IRC-PA-146/2077-78). Confirmed COVID-19 patients by reverse transcriptase polymerase chain reaction and admitted in the COVID block during the study period were included and those who were discharged on request or referred or unavailable blood tests were excluded. A convenience sampling method was used. Point estimate and 95% Confidence Interval were calculated. Results: Among 72 admitted COVID-19 patients, 63 (87.5%) (79.86-95.14, 95% Confidence Interval) patients had severe disease. The mean neutrophil to lymphocyte ratio and mean lymphocyte to C-reactive protein ratio were 11.60±8.15 and 25.55±20.96 respectively. Conclusions: The prevalence of severe COVID-19 was higher than in other studies done in similar settings. We suggest clinical parameter-based early categorisation of COVID-19 cases to utilize limited resources during the pandemic. Keywords: COVID-19; c-reactive protein; lymphocytes; severe acute respiratory syndrome coronavirus.

Complete Stone Clearance after Retrograde Intrarenal Surgery among Patients with Urolithiasis in a Tertiary Care Centre: A Descriptive Cross-sectional Study.

Introduction: Retrograde intra-renal surgery using flexible scopes and laser energy is a newer alternative in stone disease treatment armamentarium. It is claimed to be superior to other modalities for stone clearance, complications and hospital stay. The aim of this study was to find out the prevalence of complete stone clearance after retrograde intra-renal surgery among patients with urolithiasis in a tertiary care centre. Methods: This was a descriptive cross-sectional study conducted in the Department of Urology in a tertiary care centre from 15 June 2021 to 14 May 2022 including adult patients with stone size up to 15 mm. Ethical approval was obtained from the Institutional Review Committee (Reference number: IRC-PA-143/2077-78). Convenience sampling was done. The prevalence of complete stone clearance (no residual fragment >4 mm) was calculated. Point estimation and 95% confidence interval were calculated. Results: Among 42 patients, 36 (85.71%) patients (75.1-96.3, 95% Confidence Interval) achieved complete stone clearance. The mean age was 40.26±14.05 (16-74) years and the stone size was 1.27±0.19 (0.9 -1.5) cm. Similarly, the mean operating time was 51.55±9.34 (40-85) minutes and the hospital stay was 1.33±0.52 (1-3) days. Grade 3 ureteric injury occurred in one case. Residual fragments were seen in 6 cases (14.29%). Sepsis occurred in 4 cases (11.11%). Conclusions: The prevalence of complete stone clearance was similar among patients undergoing retrograde intra-renal surgery in our study when compared to other studies conducted in similar settings. Keywords: laser; miniaturization; postoperative complications.

Comparative evaluation of pharmacokinetics and pharmacodynamics of insulin glargine (Glaritus®) and Lantus® in healthy subjects: a double-blind, randomized clamp study.

AIMS: The objective of the study was to compare the pharmacokinetic (PK) and pharmacodynamic (PD) properties of an insulin glargine formulation, Glaritus® (test) with the innovator's formulation Lantus® (reference) using the euglycemic clamp technique in a single-dose, double-blind, randomized, two sequences, four-period replicate crossover study in healthy volunteers (n = 40). METHODS: Subjects received subcutaneous administration of the insulin glargine (0.4 IU/kg) formulation at two occasions for test and reference and a 20% glucose solution was infused at variable rate to maintain euglycemia for 24 h. RESULTS: Both PK [area under the plasma concentration time curve (AUC0-24 h) and maximum insulin concentration (Cmax)] and PD endpoints [area under glucose infusion rate time curve (AUCGIR0-24) and maximum glucose infusion rate (GIRmax)] demonstrated bioequivalence of Glaritus to Lantus with the 90% confidence interval of geometric mean ratio of test to reference entirely contained within 0.80-1.25. Both formulations showed equivalent geometric least-square mean LSM value (0.08 nmol/L) for Cmax. The geometric LSM AUC0-24 h value for Glaritus® (1.09 h nmol/L) was comparable to Lantus (1.05 h nmol/L). Median Tmax values were also identical (12 h for both), and median t1/2 values were also equal (18 h for both). For GIRTmax, the difference between the means for the two was not statistically significant. No AEs related to study formulations were reported, and both products were well tolerated. CONCLUSIONS: The test product (Glaritus) was found to be bioequivalent to the reference product (Lantus). CLINICAL TRIAL REGISTRATION NUMBER: CTRI/2015/06/005890; http://www.ctri.nic.in/ .

Microproteomic analysis of 10,000 laser captured microdissected breast tumor cells using short-range sodium dodecyl sulfate-polyacrylamide gel electrophoresis and porous layer open tubular liquid chromatography tandem mass spectrometry.

Precise proteomic profiling of limited levels of disease tissue represents an extremely challenging task. Here, we present an effective and reproducible microproteomic workflow for sample sizes of only 10,000 cells that integrates selective sample procurement via laser capture microdissection (LCM), sample clean-up and protein level fractionation using short-range SDS-PAGE, followed by ultrasensitive LC-MS/MS analysis using a 10 µm i.d. porous layer open tubular (PLOT) column. With 10,000 LCM captured mouse hepatocytes for method development and performance assessment, only 10% of the in-gel digest, equivalent to ∼1000 cells, was needed per LC-MS/MS analysis. The optimized workflow was applied to the differential proteomic analysis of 10,000 LCM collected primary and metastatic breast cancer cells from the same patient. More than 1100 proteins were identified from each injection with >1700 proteins identified from three LCM samples of 10,000 cells from the same patient (1123 with at least two unique peptides). Label free quantitation (spectral counting) was performed to identify differential protein expression between the primary and metastatic cell populations. Informatics analysis of the resulting data indicated that vesicular transport and extracellular remodeling processes were significantly altered between the two cell types. The ability to extract meaningful biological information from limited, but highly informative cell populations demonstrates the significant benefits of the described microproteomic workflow.

In situ proteomic analysis of human breast cancer epithelial cells using laser capture microdissection: annotation by protein set enrichment analysis and gene ontology.

Identification of molecular signatures that allow detection of the transition from normal breast epithelial cells to malignant invasive cells is a critical component in the development of diagnostic, therapeutic, and preventative strategies for human breast cancer. Substantial efforts have been devoted to deciphering breast cancer etiology at the genome level, but only a limited number of studies have appeared at the proteome level. In this work, we compared individual in situ proteome profiles of nonpatient matched nine noncancerous, normal breast epithelial (NBE) samples with nine estrogen receptor (ER)-positive (luminal subtype), invasive malignant breast epithelial (MBE) samples by combining laser capture microdissection (LCM) and quantitative shotgun proteomics. A total of 12,970 unique peptides were identified from the 18 samples, and 1623 proteins were selected for quantitative analysis using spectral index (SpI) as a measure of protein abundance. A total of 298 proteins were differentially expressed between NBE and MBE at 95% confidence level, and this differential expression correlated well with immunohistochemistry (IHC) results reported in the Human Protein Atlas (HPA) database. To assess pathway level patterns in the observed expression changes, we developed protein set enrichment analysis (PSEA), a modification of a well-known approach in gene expression analysis, Gene Set Enrichment Analysis (GSEA). Unlike single gene-based functional term enrichment analyses that only examines pathway overrepresentation of proteins above a given significance threshold, PSEA applies a weighted running sum statistic to the entire expression data to discover significantly enriched protein groups. Application of PSEA to the expression data in this study revealed not only well-known ER-dependent and cellular morphology-dependent protein abundance changes, but also significant alterations of downstream targets for multiple transcription factors (TFs), suggesting a role for specific gene regulatory pathways in breast tumorigenesis. A parallel GOMiner analysis revealed both confirmatory and complementary data to PSEA. The combination of the two annotation approaches yielded extensive biological feature mapping for in depth analysis of the quantitative proteomic data.

Profiling the glycoforms of the intact alpha subunit of recombinant human chorionic gonadotropin by high-resolution capillary electrophoresis-mass spectrometry.

With the rapid growth of complex heterogeneous biological molecules, effective techniques that are capable of rapid characterization of biologics are essential to ensure the desired product characteristics. To address this need, we have developed a method for analysis of intact glycoproteins based on high-resolution capillary electrophoretic separation coupled to an LTQ-FT mass spectrometer. We evaluated the performance of this method on the alpha subunit of mouse cell line-derived recombinant human chorionic gonadotrophin (r-alpha hCG), a protein that is glycosylated at two sites and is part of the clinically relevant gonadotrophin family. Analysis of r-alpha hCG, using capillary electrophoresis (CE) with a separation time under 20 min, resulted in the identification of over 60 different glycoforms with up to nine sialic acids. High-resolution CE-Fourier transform mass spectrometry (FT-MS) allowed separation and analysis of not only intact glycoforms with different numbers of sialic acids but also intact glycoforms that differed by the number and extent of neutral monosaccharides. The high mass resolution of the FT-MS enabled a limited mass range to be targeted for the examination of the protein glycoforms, simplifying the analysis without sacrificing accuracy. In addition, the limited mass range resulted in a fast scan speed that enhanced the reproducibility of the relative quantitation of individual glycoforms. The intact glycoprotein analysis was complemented with the analysis of the tryptic glycopeptides and glycans of r-alpha hCG to enable the assignment of glycan structures to individual sites, resulting in a detailed characterization of the protein. Samples of r-alpha hCG obtained from a CHO cell line were also analyzed and briefly shown to be significantly different from the murine cell line product. Taken together, the results suggest that the CE coupled to high-resolution FT-MS can be one of the effective tools for in-process monitoring as well as for final product characterization.