An Optimized and High-Throughput Method for Histone Propionylation and Data-Independent Acquisition Analysis for the Identification and Quantification of Histone Post-translational Modifications.

Histones are DNA binding proteins that allow for packaging of the DNA into the nucleus. They are abundantly present across the genome and thus serve as a major site of epigenetic regulation through the use of post-translational modifications (PTMs). Aberrations in histone expression and modifications have been implicated in a variety of human diseases and thus are a major focus of disease etiology studies. A well-established method for studying histones and PTMs is through the chemical derivatization of isolated histones followed by liquid chromatography and mass spectrometry analysis. Using such an approach has allowed for a swath of discoveries to be found, leading to novel therapeutics such as histone deacetylase (HDAC) inhibitors that have already been applied in the clinic. However, with the rapid improvement in instrumentation and data analysis pipelines, it remains important to temporally re-evaluate the established protocols to improve throughput and ensure data quality. Here, we optimized the histone derivatization procedure to increase sample throughput without compromising peptide quantification. An implemented spike-in standard peptide further serves as a quality control to evaluate the propionylation and digestion efficiencies as well as reproducibility in chromatographic retention and separation. Last, the application of various data-independent acquisition (DIA) strategies was explored to ensure low variation between runs. The output of this study is a newly optimized derivatization protocol and mass spectrometry method that maintains high identification and quantification of histone PTMs while increasing sample throughput.

Medical laboratory staff satisfaction and their perspective on the role of health institutions to combat COVID-19 pandemic.

OBJECTIVE: To assess the facilities and challenges encountered in the clinical laboratories, satisfaction of the medical laboratory staff (MLS) toward their profession and their views on the role of related health institutions during the first wave of the Coronavirus Disease 2019 (COVID-19) pandemic in Nepal. METHODS: A web-based cross-sectional study was conducted among registered MLS in Nepal. Data were collected using a structured self-reported questionnaire on the Google Docs platform. RESULTS: A total of 301 respondents were enrolled in the study; of which 180 were male and 121 were female. Of the 301 respondents, a lack of infrastructure was reported by 241 (80.1%), a lack of skill development training by 204 (67.8%), limited availability of diagnostics kits by 151 (50.2%), overburdened by the workload by 142 (47.2%) and difficulty in sample management by 129 (42.9%). A total of 244 of 301 respondents (81.1%) believed that stakeholder institutions should collaborate with the government during the pandemic. The level of satisfaction during the pandemic (130 of 301; 43.19%) was found to have decreased compared with before the pandemic (203 of 301; 67.4%). CONCLUSION: MLS were not fully satisfied with the available resources during the pandemic.

Predicted N-terminal N-linked glycosylation sites may underlie membrane protein expression patterns in Saccharomyces cerevisiae.

N-linked glycosylation is one type of posttranslational modification that proteins undergo during expression. The following describes the effects of N-linked glycosylation on high-level membrane protein expression in yeast with an emphasis on Saccharomyces cerevisiae. N-linked glycosylation is highlighted here as an important consideration when preparing membrane protein gene constructs for expression in S. cerevisiae, which continues to be used as a workhorse in both research and industrial applications. Non-native N-linked glycosylation commonly occurs during the heterologous expression of mammalian proteins in many yeast species which can have important immunological consequences when used in the production of biotherapeutic proteins or peptides. Further, non-native N-linked glycosylation can lead to improper protein folding and premature degradation, which can impede high-level expression yields and hinder downstream analysis. Multiple strategies are presented in this article, which suggest different methods that can be implemented to circumvent the unwanted consequences of N-linked glycosylation during the expression process. These considerations may have long-term benefits for high-level protein production in S. cerevisiae across a broad spectrum of expression targets with special emphasis placed on G-protein coupled receptors, one of the largest families of membrane proteins.

In vitro Antimicrobial Synergy Testing of Extensively Drug-Resistant Clinical Isolates at an Organ Transplant Center in Nepal.

PURPOSE: Inappropriate use of broad-spectrum antibiotics contributes to the emergence of multidrug-resistant (MDR) bacteria. Finding novel antimicrobial agents and strategies based on synergistic combinations are essential to combat MDR infections. This study was designed to determine in vitro synergy of different antimicrobials against extensively drug-resistant (XDR) Gram-negative clinical isolates. METHODS: A descriptive, cross-sectional study was conducted at Human Organ Transplant Center, Nepal, for five months. Clinical isolates were checked for their drug-resistance properties including extended-spectrum beta-lactamase- (ESBL-) and metallo-beta-lactamase- (MBL-) production. The XDR isolates were further tested for antimicrobial synergy, and the results were interpreted as synergistic, additive, indifferent or antagonistic determining fractional inhibitory concentration of the antibiotics. RESULTS: Out of total 1155 clinical samples, 308 showed significant growth. Escherichia coli was the most common isolate (n=142) followed by Klebsiella pneumoniae, Acinetobacter calcoaceticus baumannii (Acb) complex, Pseudomonas aeruginosa and miscellaneous bacteria. Out of the culture positive isolates, 21.4% were MDR and 10.06% were XDR. The XDR population comprised K. pneumoniae (18.42%), E. coli (9.86%), Acb complex (7.41%) and P. aeruginosa (4.17%). Among the culture positive isolates, 4.5% and 5.8% were ESBL- and MBL-producers, respectively. Colistin, polymyxin B, and tigecycline were the antibiotics effective in majority of MDR isolates as compared to carbapenems. The combination of antibiotics - meropenem and colistin showed the highest proportion of "synergy" among all XDR E. coli whereas the combination of amikacin and colistin showed synergistic effect in XDR K. pneumoniae. CONCLUSION: A significant proportion of isolates were MDR among which a large fraction was XDR. The combination of meropenem, amikacin and colistin with one another in pair showed beneficial activity in vitro. Such combinations can be utilized as effective therapy for XDR infections. Further studies are required to confirm these findings, and accordingly treatment protocols should be developed in the management of such infections.

COVID-19: benefits and risks of passive immunotherapeutics.

Passive immunotherapeutics (PITs), including convalescent plasma, serum, or hyperimmune immunoglobulin, have been of clinical importance during sudden outbreaks since the early twentieth century for the treatment of viral diseases such as severe acute respiratory syndrome (SARS), middle east respiratory syndrome (MERS) and swine flu (H1N1). With the recent SARS-CoV-2 pandemic, wherein effective antivirals and vaccines are still lacking, an interest in convalescent plasma therapy as a lifesaving option has resurfaced due to its capacity for antigenic neutralization and reducing viremia. This review summarizes convalescent blood products (CBPs) in terms of current technologies and the shortcomings related to the collection, manufacture, pathogen inactivation, and banking of CBPs, with a specific focus on their plausible applications, benefits, and risks in the COVID-19 pandemic.