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Malaria remains a major health issue worldwide that affects many people, particularly in developing nations. Since, the malarial parasite has developed resistance against nearly every antimalarial drug now in use, it is imperative to search for novel antimalarial medications. Toxins produced by skin glands of toads have been shown to possess antiparasitic properties against a variety of protozoan parasites because of the bufadienolides they contain. Even though several studies have been conducted to show that toad skin secretions have antimalarial properties, very little information is known about the precise mechanism by which they work against Plasmodium infection. Thus, the goal of this study was to evaluate the antiplasmodial activity of crude skin extracts from Common Asian Toads, Duttaphrynus melanostictus , of different sizes and illustrate how they work against Plasmodium falciparum 3D7 cells. The findings demonstrated a negative correlation between the toad size and percent yield of the extracts. HPTLC and UPLC-MS/MS analysis of the extracts exhibited varied composition of bufadienolides depending on the size of the animal. The extract obtained from small toads containing resibufagin and marinobufagin lactate demonstrated highest antiplasmodial activity and showed lowest cytotoxicity on peripheral blood mononuclear cells. It was discovered that the extract was effective against the trophozoite stage of the parasite. The extract was reported to inhibit Na + /H + ATPase of Plasmodium by binding to sodium-enzyme complex at ATP binding site. The study offers baseline data that can be used to assess the antimalarial potential of individual components in the skin extract derived from small toads., Supplementary Information: The online version contains supplementary material available at 10.1007/s12639-024-01716-9., Competing Interests: Conflict of interestThe authors have no conflict of interest to declare., (© Indian Society for Parasitology 2024. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Background: Skin secretions of toads are widely used in medicine all over the world for their antiviral, anti-infective, and cardiotonic properties. Because these secretions are mostly employed to combat blood parasite infection, it is important to understand their potential toxic effects on human erythrocytes. Therefore, the objective of the current investigation was to elucidate the effects of Duttaphrynus melanostictus (Schneider) skin extracts on the physiology of human erythrocytes., Methods: Toads captured from their natural habitat were separated into three groups according to their body size. Hydroalcoholic extracts of toad skin were prepared by reflux heating. These extracts were then evaluated for their hemolytic and hemoglobin denaturation potential. The effects of the extracts on cytosolic and membrane-bound enzymes of human erythrocytes were assessed., Results: The hemolysis and hemoglobin denaturation caused by these extracts correlated positively with the respective toad sizes. Extracts from medium and large toads led to increased osmotic fragility even at near iso-osmotic concentrations. Biochemical analysis of hemolysate showed that the treatment induced a shift of metabolic flux toward the glutathione pathway. Analysis of membrane-bound enzymes revealed a significant decrease in the activity of Na+/K+ ATPase and acetylcholinesterase. SDS-PAGE analysis of the erythrocyte membrane did not show the band of tropomodulin for the cells treated with 1000 𝜇g/ml extract from large toads., Conclusions: In conclusion, the present study demonstrates that the toxicity of toad skin secretions aggravates with the size of the animal and interferes with the physiology of human erythrocytes, leading to their membrane disruption and rapid lysis., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Abstract: This multi-institutional study assessed the efficacy of Enterococcus faecium NRRL B-2354 as a nonpathogenic Salmonella surrogate for thermal processing of nonfat dry milk powder, peanut butter, almond meal, wheat flour, ground black pepper, and date paste. Each product was analyzed by two laboratories (five independent laboratories total), with the lead laboratory inoculating (E. faecium or a five-strain Salmonella enterica serovar cocktail of Agona, Reading, Tennessee, Mbandaka, and Montevideo) and equilibrating the product to the target water activity before shipping. Both laboratories subjected samples to three isothermal treatments (between 65 and 100°C). A log-linear and Bigelow model was fit to survivor data via one-step regression. On the basis of D80°C values estimated from the combined model, E. faecium was more thermally resistant (P < 0.05) than Salmonella in nonfat dry milk powder (DEf-80°C, 100.2 ± 5.8 min; DSal-80°C, 28.9 ± 1.0 min), peanut butter (DEf-80°C, 133.5 ± 3.1 min; DSal-80°C, 57.6 ± 1.5 min), almond meal (DEf-80°C, 34.2 ± 0.4 min; DSal-80°C, 26.1 ± 0.2 min), ground black pepper (DEf-80°C, 3.2 ± 0.8 min; DSal-80°C, 1.5 ± 0.1 min), and date paste (DEf-80°C, 1.5 ± 0.0 min; DSal-80°C, 0.5 ± 0.0 min). Although the combined laboratory D80°C for E. faecium was lower (P < 0.05) than for Salmonella in wheat flour (DEf-80°C, 9.4 ± 0.1 min; DSal-80°C, 10.1 ± 0.2 min), the difference was ∼7%. The zT values for Salmonella in all products and for E. faecium in milk powder, almond meal, and date paste were not different (P > 0.05) between laboratories. Therefore, this study demonstrated the impact of standardized methodologies on repeatability of microbial inactivation results. Overall, E. faecium NRRL B-2354 was more thermally resistant than Salmonella, which provides support for utilizing E. faecium as a surrogate for validating thermal processing of multiple low-moisture products. However, product composition should always be considered before making that decision., (Published 2022 by the International Association for Food Protection. Not subject to U.S. Copyright.)

Objective: This retrospective observational study analyzes the bacteriological profile of pathogens causing burn wound infections in a tertiary care center., Materials and Methods: This study was conducted at Father Muller Medical College Hospital, Karnataka, India, from January 2014 through December 2016. The specimens (ie, pus or a wound swab) were collected from patients with suspected of infection and processed as per standard microbiological techniques. The antibiotic sensitivity testing was performed by the Kirby Bauer's disk diffusion test on Mueller-Hinton agar as per Clinical and Laboratory Standards Institute guidelines., Results: During the study period, a total of 124 eligible patient samples were collected; 22 samples were excluded as there was no significant growth/colonization. Among the 102 patients included in the study, 56 (54.9%) were females and the majority (33, 32.35%) of the patients were between 18 to 30 years. Acinetobacter species and Pseudomonas aeruginosa (26.56% each) were the most common pathogen among gram-negative bacteria and Staphylococcus aureus (36, 11.25%) was the most common gram-positive bacteria. Methicillin resistance was 30.5% among the Staphylococcus aureus isolates. Most of Acinetobacter species isolates were resistant to piperacillin tazobactum (84.71%), meropenem (80%), and amikacin (87.06%). Other gram-negative bacteria were also emerging with multidrug resistance., Conclusions: The current study revealed the non-fermenting Gram-negative bacteria as the leading cause of burn wound infection and are highly resistant to available high-level antibacterial agents.

SUMOylation is a reversible post-translational modification (PTM) regulating all nuclear processes. Identification of SUMOylation sites by mass spectrometry (MS) has been hampered by bulky tryptic fragments, which thus far necessitated the use of mutated SUMO. Here we present a SUMO-specific protease-based methodology which circumvents this problem, dubbed Protease-Reliant Identification of SUMO Modification (PRISM). PRISM allows for detection of SUMOylated proteins as well as identification of specific sites of SUMOylation while using wild-type SUMO. The method is generic and could be widely applied to study lysine PTMs. We employ PRISM in combination with high-resolution MS to identify SUMOylation sites from HeLa cells under standard growth conditions and in response to heat shock. We identified 751 wild-type SUMOylation sites on endogenous proteins, including 200 dynamic SUMO sites in response to heat shock. Thus, we have developed a method capable of quantitatively studying wild-type mammalian SUMO at the site-specific and system-wide level.

Background: The Qualitative aspect of health care delivery is one of the major factors in reducing morbidity and mortality in a health care setup. The expanding suburban secondary health care delivery facilities of the Municipal Corporation of Greater Mumbai are an important part of the healthcare backbone of Mumbai and therefore the quality of care delivered here needed standardization., Material and Methods: The project was completed over a period of one year from Jan to Dec, 2013 and implemented in three phases. The framework with components and sub-components were developed and formats for data collection were standardized. The benchmarks were based on past performance in the same hospital and probability was used for development of normal range. An Excel spreadsheet was developed to facilitate data analysis., Results: The indicators comprise of 3 components--Statutory Requirements, Patient care & Cure and Administrative efficiency. The measurements made, pointed to the broad areas needing attention., Conclusion: The Indicators for patient care and monitoring standards can be used as a self assessment tool for health care setups for standardization and improvement of delivery of health care services.

SUMOylation is a reversible post-translational modification essential for genome stability. Using high-resolution MS, we have studied global SUMOylation in human cells in a site-specific manner, identifying a total of >4,300 SUMOylation sites in >1,600 proteins. To our knowledge, this is the first time that >1,000 SUMOylation sites have been identified under standard growth conditions. We quantitatively studied SUMOylation dynamics in response to SUMO protease inhibition, proteasome inhibition and heat shock. Many SUMOylated lysines have previously been reported to be ubiquitinated, acetylated or methylated, thus indicating cross-talk between SUMO and other post-translational modifications. We identified 70 phosphorylation and four acetylation events in proximity to SUMOylation sites, and we provide evidence for acetylation-dependent SUMOylation of endogenous histone H3. SUMOylation regulates target proteins involved in all nuclear processes including transcription, DNA repair, chromatin remodeling, precursor-mRNA splicing and ribosome assembly.

Regulatory protein phosphorylation controls normal and pathophysiological signaling in eukaryotic cells. Despite great advances in mass-spectrometry-based proteomics, the extent, localization, and site-specific stoichiometry of this posttranslational modification (PTM) are unknown. Here, we develop a stringent experimental and computational workflow, capable of mapping more than 50,000 distinct phosphorylated peptides in a single human cancer cell line. We detected more than three-quarters of cellular proteins as phosphoproteins and determined very high stoichiometries in mitosis or growth factor signaling by label-free quantitation. The proportion of phospho-Tyr drastically decreases as coverage of the phosphoproteome increases, whereas Ser/Thr sites saturate only for technical reasons. Tyrosine phosphorylation is maintained at especially low stoichiometric levels in the absence of specific signaling events. Unexpectedly, it is enriched on higher-abundance proteins, and this correlates with the substrate KM values of tyrosine kinases. Our data suggest that P-Tyr should be considered a functionally separate PTM of eukaryotic proteomes., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Transforming growth factor-β (TGF-β) signaling promotes cell motility by inducing epithelial-to-mesenchymal transitions (EMTs) in normal physiology and development, as well as in pathological conditions, such as cancer. We performed a time-resolved analysis of the proteomic and phosphoproteomic changes of cultured human keratinocytes undergoing EMT and cell cycle arrest in response to stimulation with TGF-β. We quantified significant changes in 2079 proteins and 2892 phosphorylation sites regulated by TGF-β. We identified several proteins known to be involved in TGF-β-induced cellular processes, such as the cytostatic response, extracellular matrix remodeling, and epithelial dedifferentiation. In addition, we identified proteins involved in other cellular functions, such as vesicle trafficking, that were not previously associated with TGF-β signaling. Although many TGF-β responses are mediated by phosphorylation of the transcriptional regulators of the SMAD family by the TGF-β receptor complex, we observed rapid kinetics of changes in protein phosphorylation, indicating that many responses were mediated through SMAD-independent TGF-β signaling. Combined analysis of changes in protein abundance and phosphorylation and knowledge of protein interactions and transcriptional regulation provided a comprehensive representation of the dynamic signaling events underlying TGF-β-induced changes in cell behavior. Our data suggest that in epithelial cells stimulated with TGF-β, early signaling is a mixture of both pro- and antiproliferative signals, whereas later signaling primarily inhibits proliferation., (Copyright © 2014, American Association for the Advancement of Science.)

Correct classification of cancer patients into subtypes is a prerequisite for acute diagnosis and effective treatment. Currently this classification relies mainly on histological assessment, but gene expression analysis by microarrays has shown great promise. Here we show that high accuracy, quantitative proteomics can robustly segregate cancer subtypes directly at the level of expressed proteins. We investigated two histologically indistinguishable subtypes of diffuse large B-cell lymphoma (DLBCL): activated B-cell-like (ABC) and germinal-center B-cell-like (GCB) subtypes, by first developing a general lymphoma stable isotope labeling with amino acids in cell culture (SILAC) mix from heavy stable isotope-labeled cell lines. This super-SILAC mix was combined with cell lysates from five ABC-DLBCL and five GCB-DLBCL cell lines. Shotgun proteomic analysis on a linear ion trap Orbitrap mass spectrometer with high mass accuracy at the MS and MS/MS levels yielded a proteome of more than 7,500 identified proteins. High accuracy of quantification allowed robust separation of subtypes by principal component analysis. The main contributors to the classification included proteins known to be differentially expressed between the subtypes such as the transcription factors IRF4 and SPI1/PU.1, cell surface markers CD44 and CD27, as well as novel candidates. We extracted a signature of 55 proteins that segregated subtypes and contained proteins connected to functional differences between the ABC and GCB-DLBCL subtypes, including many NF-κB-regulated genes. Shortening the analysis time to single-shot analysis combined with use of the new linear quadrupole Orbitrap analyzer (Q Exactive) also clearly differentiated between the subtypes. These results show that high resolution shotgun proteomics combined with super-SILAC-based quantification is a promising new technology for tumor characterization and classification.

Mass spectrometry (MS)-based proteomics now enables the analysis of thousands of phosphorylation sites in single projects. Among a wide range of analytical approaches, the combination of high resolution MS scans in an Orbitrap analyzer with low resolution MS/MS scans in a linear ion trap has proven to be particularly successful ("high-low" strategy). Here we investigate if the improved sensitivity of higher energy collisional dissociation (HCD) on an LTQ-Orbitrap Velos instrument allows a "high-high" strategy. A high resolution MS scan was followed by up to 10 HCD MS/MS scans, and we achieved cycle times of about 3 s making the method compatible with chromatographic time scales. Fragment mass accuracy increased about 50-fold compared to the "high-low" strategy. Unexpectedly, the HCD approach mapped up to 16,000 total phosphorylation sites in one day's measuring time--the same or better than the standard high-low strategy. Reducing the target values from a standard of 30,000 to 5000 ions did not severely affect identification rates but did decrease identification and localization scores for phosphorylation sites. We conclude that HCD in the new configuration is now a viable method for large-scale phosphoproteome analysis alongside collisional induced dissociation, (CID) and electron capture/transfer dissociation (ECD/ETD).

Serine/threonine protein kinases (STPKs) represent a burgeoning concept in prokaryotic signaling and have been implicated in a range of control mechanisms. This paper describes the enzymatic and molecular characterization of PknH, a mycobacterial STPK. After cloning and expression as a Glutathione-S-transferase fusion protein in E. coli, PknH was found to phosphorylate itself and exogenous substrates like myelin basic protein and histone. The kinase activity of PknH was inhibited by the kinase inhibitors staurosporine and H-7. The results confirmed that PknH is a transmembrane protein and is restricted to members of the Mycobacterium tuberculosis complex. In addition, transcriptional analysis of pknH in M. tuberculosis under various stress conditions revealed that exposure to low pH and heat shock decreased the level of pknH transcription significantly. This is the first report describing differential expression of a mycobacterial kinase in response to stress conditions which can indicate its ability to regulate cellular events promoting bacterial adaptation to environmental change.

The measurement of renal parenchymal volume using a calibrated computed tomography image processing method has been evaluated clinically on a cohort of patients with chronic pyelonephritis. Comparison of renal volume with function as assessed by 99Tcm DTPA renography demonstrated a simple linear relationship in patients who were normotensive and aproteinuric. The implications of this result on the interpretation of prognostic factors determining declining renal function in chronic pyelonephritis are discussed.