Bioactive compounds of Jingfang Granules against SARS-CoV-2 virus proteases 3CLpro and PLpro.

OBJECTIVE: Jingfang Granules have been recommended for the prevention and treatment of corona virus disease 2019 (COVID-19). Through chemical analysis and bioactivity evaluation, this study aims to elucidate the potential effective components of Jingfang Granules. METHOD(S): The inhibitory acti-vities of Jingfang Granules extract against 3-chymotrypsin-like protease (3CLpro), papain like protease (PLpro), spike protein receptor-binding domain (S-RBD) and human cyclooxygenase-2 (COX-2) were evaluated using enzyme assay. The antitussive effects were evaluated using the classical ammonia-induced cough model. The chemical constituents of Jingfang Granules were qualitatively and quantitatively analyzed by liquid chromatography-mass spectrometry (LC/MS). The 3CLpro and PLpro inhibitory activities of the major compounds were determined by enzyme assay, molecular docking, and site-directed mutagenesis. RESULT(S): Jingfang Granules exhibited 3CLpro and PLpro inhibitory activities, as well as COX-2 inhibitory and antitussive activities. By investigating the MS/MS behaviors of reference standards, a total of fifty-six compounds were characterized in Jingfang Granules. Sixteen of them were unambiguously identified by comparing with reference standards. The contents of the 16 major compounds were also determined, and their total contents were 2 498.8 mug/g. Naringin, nodakenin and neohesperidin were three dominating compounds in Jingfang Granules, and their contents were 688.8, 596.4 and 578.7 mug/g, respectively. In addition, neohesperidin and naringin exhibited PLpro inhibitory activities, and the inhibition rates at 8 mumol/L were 53.5% and 46.1%, respectively. Prim-O-glucosylcimifugin showed significant inhibitory activities against 3CLpro and PLpro, and the inhibitory rates at 8 mumol/L were 76.8% and 78.2%, respectively. Molecular docking indicated that hydrogen bonds could be formed between prim-O-glucosylcimifugin and amino acid residues H163, E166, Q192, T190 of 3CLpro (binding energy, -7.7 kcal/mol) and K157, D164, R166, E167, T301 of PLpro(-7.3 kcal/mol), respectively. Site-directed mutagenesis indicated amino acid residue K157 was a key active site for the interaction between prim-O-glucosylcimifugin and PLpro. CONCLUSION(S): Prim-O-glucosylcimifugin, neohesperidin, and naringin as the major compounds from Jingfang Granules could inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus proteases 3CLpro and PLpro. The results are valuable for rational clinical use of Jingfang Granules.

Heterologous Production of the D-Cycloserine Intermediate O-acetyl-L-serine in Human Lung Cancer Cells

According to the World Health Organization, Tuberculosis (TB) is the second leading cause of death by a single infectious disease behind COVID-19. Despite a century of effort, the current TB vaccine does not effectively prevent pulmonary TB, promote herd immunity, or prevent transmission. Therefore, we seek to develop a genetic prophylaxis for TB. We have determined D-cycloserine to be the optimal target for this approach due to its relatively short six-enzyme biosynthetic pathway. D-CS is a second-line antibiotic for TB that inhibits bacterial cell wall synthesis. The first committed step towards D-CS synthesis is catalyzed by the L-serine-O-acetyltransferase (DcsE) which converts L-serine and acetyl-CoA to O-acetyl-L-serine (L-OAS). To test if the D-CS pathway could be an effective prophylaxis for TB in human cells, we endeavored to express DcsE in human cells and test its functionality. We overexpressed DcsE tagged with FLAG and GFP in A549 lung cancer cells as determined using fluorescence microscopy. We observed that purified DcsE catalyzed the synthesis of L-OAS as observed by HPLC-MS. Therefore, DcsE synthesized in human cells is a functional enzyme capable of converting L-serine and acetyl-CoA to L-OAS demonstrating the first step towards DCS production in human cells.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

New Sample Prep Products and Accessories for 2023

[...]instrument-based sample preparation technologies are presented. [...]attention is turned to other sample preparation accessories and supporting technologies. First is the Swift HLB-DPX tips. First is the Thermo Scientific AccelerOme sample preparation platform automating sample preparation for liquid chroma-tography-mass spectrometry (LC-MS) proteomics.

Inhibition of Spike Protein of SARS-CoV2 from (Merremia mammosa (Lour) Hall. F.) Bioactive Compounds: Molecular Docking and ADMET Study

Spike protein is a receptor protein that has e role in the entry step of SARS-CoV2. This protein will bind to the ACE2 receptor in the human body and activate TMPRSS2. Inhibition of this protein will prevent the binding of the virus to host cells to spread the infection. This study aims to identify the activity of bioactive compounds of Merremia mammosa (Lour) tuber obtained from LC-MS/MS QTOF analysis of a previous study against the Spike protein of SARS-CoV2 using molecular docking and ADMET analysis. Molecular docking was conducted using SARS-CoV2 spike protein (PDB id. 6M0J) using Maestro Schrodinger software. Results showed that from 206 compounds there are 8 compounds of Merremia mammosa (Lour) that have lower predictive binding energies than standard drugs arbidol, hydroxychloroquine, and chloroquine. Result(s): 206 compounds of Merremia mammosa (Lour) tuber were successfully docked, there were 8 compounds that have docking scores more negative than standard drugs. It indicates that 8 compounds are more active than the positive controls. ADMET study revealed all of those potential ligands had the possibility to be developed as drugs. Conclusion(s): Molecular docking simulations were successfully utilized to identify the potential compounds from Merremia mammosa (Lour) tuber with the activity as an inhibitor for spike protein of SARS-CoV2. Further in vitro assay and purification are needed for future research.Copyright © 2021 Muslim OT et al.

Effects of training and protein/carbohydrate supplementation by food on strength, body composition and bone metabolism in postmenopausal women

Background: Menopause is associated with impairments of health, e.g. cardiovascular disease, changes in body composition, decrease in bone density. Physical activity and nutrition strategies have been demonstrated to counteract some of these disabilities. Aim of the present study was to analyze the impact of 3 months of strength and endurance training combined with protein/carbohydrate supplementation on strength, body composition and bone metabolism in postmenopausal women. Method(s): 62 postmenopausal women were recruited. Measurements: Body composition by BIA. Strength of leg, chest and handgrip. delta44Ca/42Ca in blood and urine as proxies for bone metabolism, samples were analyzed by mass spectrometry. Participants completed 2/week endurance training (walking) for 60 minutes (60-75% km/h of 4mmol threshold) and a whole-body strength training 1/week for 60 minutes (online). In addition, the intervention group (IG) received 100g of sour milk cheese and 76g of white bread (35.3 g carbohydrate, 36.1 g protein, 3.5 g fat, 321 kcal) after each training. Result(s): Training results in an anabolic effect on bone metabolism, here protein/carbohydrate supplementation does not show additive effects. Training resulted in an increase of leg and hand grip strength. For hand grip strength an additive effect could be demonstrated after protein/carbohydrate supplementation. Both groups increased muscle mass and reduced fat mass, although the results were not significant. Discussion(s): Training was effective, showing an increase in strength. Additive effects of the nutritional intervention could be only observed for hand grip strength. This may be due to a weak compliance of the protein/carbohydrate supplementation by a meal while corona pandemic. Also, because of the endurance parts, the training was not specifically designed to increase strength. Nevertheless, even this mild training has a remarkably strong impact on bone metabolism. Conclusion(s): Even if the effects are faint, the data of this study provide evidence that protein/carbohydrate supplementation, also by food, supports the events of training on strength. Training has a strong impact on bone metabolism in postmenopausal women. The subjects respond very individually to training and nutrition interventions. Training consequentially is to be personalized.Copyright © 2023

Synthesis and Analysis of the SARS-CoV-2 ORF7a Accessory Protein Transmembrane Domain and Determination of the Oligomerization State

The ongoing SARS-CoV-2 pandemic continues to sicken millions worldwide and fundamentally change the way people interact with each other. In order to better characterize the SARS-CoV-2 virus and potentially develop methods of inhibition for further spread of the disease, this research project focused on synthesizing and characterizing the trans-membrane region of the accessory protein ORF7a. ORF7a has been implicated in proper viral assembly, leading to the idea that inhibition of this protein could prevent viral copies from being produced and halt the spread of the virus. The goal of this project was to determine the oligomerization state of the protein through a fluorescence assay in order to better understand the quaternary structure of the ORF7a complex and how it folds. The fluorescence assay is performed using three different samples of the synthesized peptide: one labeled with a TAMRA fluorophore, one labeled with a NBD fluorophore, and the last is unlabeled. After determining the oligomerization state of the protein, potential inhibitors could be synthesized and tested for their efficacy at inhibiting the function of the protein. Further applications of these inhibitors on other viruses can be explored due to the highly conserved nature of transmembrane domains across multiple viral families. Synthesis of the protein was done using a Solid Phase Peptide Synthesis (SPPS) technique and multiple batches of all three samples of peptide have been generated. Characterization and purification were done using High Performance Liquid Chromatography (HPLC) as well as Liquid Chromatography Mass Spectrometry (LCMS). Current research focuses on the purification and quantification of purified ORF7a oligopeptide for implementation of the fluorescence assay. -Hampden-Sydney College Office of Undergraduate Research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Cracking the Code of Complex Drug Modalities via Multidimensional Liquid Chromatography Coupled to Mass Spectrometry

Reducing the molecule complexity is achieved by reducing the molecule size after enzymatic digestion to produce smaller fragments more amenable to LC separation and tandem mass spectrometry (MS/MS) sequencing. Non-denaturing CEX chromatography, size-exclusion chromatogra- phy (SEC), hydrophobic interaction chromatography (HIC), and protein A modes can be easily coupled to reversed-phase LC (RPLC) because of the high aqueous conditions, enabling the versatile 4D-LC-MS systems with the use of alternative modes to 1D CEX, such as SEC or Protein A (6,7). [...]the nanopar-ticle size and free drug concentration are determined at the particle Level, whereas the encapsulated drug and lipids forming the layer are commonly characterized at the molecuar level after denaturing the lipid nanoparticle (LNP) via a surfactant. [...]MDLC-MS setups present a formidable opportunity to unify the characterization of drug delivery systems at the molecular and particle evels, which would enable their high throughput analysis.

Dried blood spot (DBS) sample analysis for drug and metabolomic profiling in oncology clinical trials: Cost-effective decentralized sampling modality for precision Oncology

The COVID19 pandemic accelerated opportunities for innovation within the decentralization process of clinical trials with opportunities for implementation of patient-centric workflows for efficiency and cost-reduction. Decentralized sample collection, particularly whole blood using dried blood spots (DBS) provides the ideal mechanism for patient driven sample collection with ease of access to sample generation, drug level assessments and metabolomic prMegofiling, providing longitudinal real-time measure of drug specific pharmacodynamic readout for safety and efficacy. In this study, we report the development of a protocol for the capture and comprehensive profiling of metabolomics using dried blood spots from a cohort of 49 healthy volunteer donors. Using liquid chromatography combined with mass spectrometric (UPLC-MS/MS) methods an untargeted metabolomic approach resulted in the identification of >800 biochemicals of which a significant subset was found to be presented in corresponding matched plasma (from whole blood) samples. The biochemicals identified from the DBS samples included metabolites that were part of the lipid, amino acid, nucleotide, peptide, cofactors, carbohydrate and energy super pathways. A significant number of metabolites identified in the DBS samples were xenobiotics including those representing the biotransformation products of drugs. The overall metabolite profiles were analyzed for precision and accuracy of measure, variability in performance and dynamic range to establish benchmarks for evaluation. An additional cohort with a longitudinal sampling as part of the protocol provided the reproducibility of the analytic method for inter-day variability of metabolite performance over time. Although metabolomic profiles varied between individuals from a population perspective, there was minimal variation observed within individuals when samples were profiled longitudinally over several weeks. Thus, the protocols for DBS collection and the corresponding capture of a large set of metabolites with reproducible performance provides an opportunity for its implementation in oncological clinical trials as part of a de-centralized clinical trial solution.

Charge Detection Mass Spectrometry: What's the "Big" Deal?

In 2002, the first fully humanized mAb was approved by the U.S. Food and Drug Administration (FDA) (3). [...]the biopharmaceutical industry is still in its infancy and new, more complex products are in development and will ikely dominant the market in the future. [...]the most common forms of LC-MS have limitations when characterizing large macromolecules (4). [...]in this column, we discuss the potential for charge detection MS (CDMS) as an analytical tool for characterizing large, complex, and heterogenous biopharmaceuticals. [...]in October 2021 at the American Association for Mass Spectrometry (ASMS) annua meeting in Philadelphia, TrueMass presented the first commercial CDMS (6,7). The cylinder is often inside an electrostatic linear ion trap (ELIT) instrument, where ions oscillate back and forth. [...]the oscillation frequency gives the m/z and the charge is determined by the magnitude.

Infection metallomics for critical care in the post-COVID era.

Infection metallomics is a mass spectrometry (MS) platform we established based on the central concept that microbial metallophores are specific, sensitive, noninvasive, and promising biomarkers of invasive infectious diseases. Here we review the in vitro, in vivo, and clinical applications of metallophores from historical and functional perspectives, and identify under-studied and emerging application areas with high diagnostic potential for the post-COVID era. MS with isotope data filtering is fundamental to infection metallomics; it has been used to study the interplay between "frenemies" in hosts and to monitor the dynamic response of the microbiome to antibiotic and antimycotic therapies. During infection in critically ill patients, the hostile environment of the host's body activates secondary bacterial, mycobacterial, and fungal metabolism, leading to the production of metallophores that increase the pathogen's chance of survival in the host. MS can reveal the structures, stability, and threshold concentrations of these metal-containing microbial biomarkers of infection in humans and model organisms, and can discriminate invasive disease from benign colonization based on well-defined thresholds distinguishing proliferation from the colonization steady state.

Omics and systems view of innate immune pathways.

Multiomics approaches to studying systems biology are very powerful techniques that can elucidate changes in the genomic, transcriptomic, proteomic, and metabolomic levels within a cell type in response to an infection. These approaches are valuable for understanding the mechanisms behind disease pathogenesis and how the immune system responds to being challenged. With the emergence of the COVID-19 pandemic, the importance and utility of these tools have become evident in garnering a better understanding of the systems biology within the innate and adaptive immune response and for developing treatments and preventative measures for new and emerging pathogens that pose a threat to human health. In this review, we focus on state-of-the-art omics technologies within the scope of innate immunity.

An N-glycopeptide MS/MS data analysis workflow leveraging two complementary glycoproteomic software tools for more confident identification and assignments.

Complete coverage of all N-glycosylation sites on the SARS-CoV2 spike protein would require the use of multiple proteases in addition to trypsin. Subsequent identification of the resulting glycopeptides by searching against database often introduces assignment errors due to similar mass differences between different permutations of amino acids and glycosyl residues. By manually interpreting the individual MS2 spectra, we report here the common sources of errors in assignment, especially those introduced by the use of chymotrypsin. We show that by applying a stringent threshold of acceptance, erroneous assignment by the commonly used Byonic software can be controlled within 15%, which can be reduced further if only those also confidently identified by a different search engine, pGlyco3, were considered. A representative site-specific N-glycosylation pattern could be constructed based on quantifying only the overlapping subset of N-glycopeptides identified at higher confidence. Applying the two complimentary glycoproteomic software in a concerted data analysis workflow, we found and confirmed that glycosylation at several sites of an unstable Omicron spike protein differed significantly from those of the stable trimeric product of the parental D614G variant.

Recent progress in online detection methods of bioaerosols

The aerosol transmission of coronavirus disease in 2019, along with the spread of other respiratory diseases, caused significant loss of life and property;it impressed upon us the importance of real-time bioaerosol detection. The complexity, diversity, and large spatiotemporal variability of bioaerosols and their external/internal mixing with abiotic components pose challenges for effective online bioaerosol monitoring. Traditional methods focus on directly capturing bioaerosols before subsequent time-consuming laboratory analysis such as culture-based methods, preventing the high-resolution time-based characteristics necessary for an online approach. Through a comprehensive literature assessment, this review highlights and discusses the most commonly used real-time bioaerosol monitoring techniques and the associated commercially available monitors. Methods applied in online bioaerosol monitoring, including adenosine triphosphate bioluminescence, laser/light-induced fluorescence spectroscopy, Raman spectroscopy, and bioaerosol mass spectrometry are summarized. The working principles, characteristics, sensitivities, and efficiencies of these real-time detection methods are compared to understand their responses to known particle types and to contrast their differences. Approaches developed to analyze the substantial data sets obtained by these instruments and to overcome the limitations of current real-time bioaerosol monitoring technologies are also introduced. Finally, an outlook is proposed for future instrumentation indicating a need for highly revolutionized bioaerosol detection technologies.

High Time-Resolution Monitoring of Volatile Organic Compounds During Multi-Surface Disinfection Activities in Buildings with PTR-TOF-MS

Increased usage of chemical disinfectants during the COVID-19 pandemic may impact the chemical composition of indoor air in residential and commercial buildings. This study characterized gas-phase concentrations of volatile organic compounds (VOCs) during multi-surface disinfection activities in a tiny house research facility. This unique facility provided a controlled, yet realistic environment for simulating whole-building disinfection events. VOCs were measured in real-time (1 Hz) in the bulk air of the tiny house with a proton transfer reaction time-of-flight mass spectrometer (PTR-TOF-MS). In addition, particle number (PN) size distributions were measured with a high-resolution electrical low-pressure impactor (HR-ELPI+). PTR-TOF-MS measurements demonstrate that chemical disinfectant spray products applied to multiple surfaces can substantially increase indoor VOC concentrations. © 2022 17th International Conference on Indoor Air Quality and Climate, INDOOR AIR 2022. All rights reserved.

A New Cellular Interactome of SARS-CoV-2 Nucleocapsid Protein and Its Biological Implications.

There is still much to uncover regarding the molecular details of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. As the most abundant protein, coronavirus nucleocapsid (N) protein encapsidates viral RNAs, serving as the structural component of ribonucleoprotein and virion, and participates in transcription, replication, and host regulations. Virus-host interaction might give clues to better understand how the virus affects or is affected by its host during infection and identify promising therapeutic candidates. Considering the critical roles of N, we here established a new cellular interactome of SARS-CoV-2 N by using a high-specific affinity purification (S-pulldown) assay coupled with quantitative mass spectrometry and immunoblotting validations, uncovering many N-interacting host proteins unreported previously. Bioinformatics analysis revealed that these host factors are mainly involved in translation regulations, viral transcription, RNA processes, stress responses, protein folding and modification, and inflammatory/immune signaling pathways, in line with the supposed actions of N in viral infection. Existing pharmacological cellular targets and the directing drugs were then mined, generating a drug-host protein network. Accordingly, we experimentally identified several small-molecule compounds as novel inhibitors against SARS-CoV-2 replication. Furthermore, a newly identified host factor, DDX1, was verified to interact and colocalize with N mainly by binding to the N-terminal domain of the viral protein. Importantly, loss/gain/reconstitution-of-function experiments showed that DDX1 acts as a potent anti-SARS-CoV-2 host factor, inhibiting the viral replication and protein expression. The N-targeting and anti-SARS-CoV-2 abilities of DDX1 are consistently independent of its ATPase/helicase activity. Further mechanism studies revealed that DDX1 impedes multiple activities of N, including the N-N interaction, N oligomerization, and N-viral RNA binding, thus likely inhibiting viral propagation. These data provide new clues to better depiction of the N-cell interactions and SARS-CoV-2 infection and may help inform the development of new therapeutic candidates.

Two years of volatile organic compound online in situ measurements at the Site Instrumental de Recherche par Télédétection Atmosphérique (Paris region, France) using proton-transfer-reaction mass spectrometry

Volatile organic compounds (VOCs) have direct influences on air quality and climate. They indeed play a key role in atmospheric chemistry as precursors of secondary pollutants, such as ozone (O3) and secondary organic aerosols (SOA). In this respect, long-term datasets of in situ atmospheric measurements are crucial for characterizing the variability of atmospheric chemical composition, its sources, and trends. The ongoing establishment of the Aerosols, Cloud, and Trace gases Research InfraStructure (ACTRIS) allows implementation of the collection and provision of such high-quality datasets. In this context, online and continuous measurements of O3, nitrogen oxides (NOx), and aerosols have been carried out since 2012 at the SIRTA (Site Instrumental de Recherche par Télédétection Atmosphérique) observatory, located in the Paris region, France. Within the last decade, VOC measurements were conducted offline at SIRTA, until the implementation of real-time monitoring which started in January 2020 using a proton-transfer-reaction quadrupole mass spectrometer (PTR-Q-MS).The dataset acquired during the first 2 years of online VOC measurements provides insights into their seasonal and diurnal variabilities. The additional long-term datasets obtained from co-located measurements (NOx, aerosol physical and chemical properties, meteorological parameters) are used to better characterize the atmospheric conditions and to further interpret the obtained results. Results also include insights into VOC main sources and the influence of meteorological conditions and air mass origin on their levels in the Paris region. Due to the COVID-19 pandemic, the year 2020 notably saw a quasi-total lockdown in France in spring and a lighter one in autumn. Therefore, the focus is placed on the impact of these lockdowns on the VOC variability and sources. A change in the behaviour of VOC markers for anthropogenic sources was observed during the first lockdown, reflecting a change in human activities. A comparison with gas chromatography data from the Paris city centre consolidates the regional representativity of the SIRTA station for benzene, while differences are observed for shorter-lived compounds with a notable impact of their local sources. This dataset could be further used as input for atmospheric models and can be found at 10.14768/f8c46735-e6c3-45e2-8f6f-26c6d67c4723 (Simon et al., 2022a).

Analytical and biomedical applications of microfluidics in traditional Chinese medicine research

Traditional Chinese medicine (TCM) has significant benefits in the prevention and treatment of diseases due to its unique theoretical system and research techniques. However, there are still key issues to be resolved in the full interpretation and use of TCM, such as vague active compounds and mechanism of action. Therefore, it is promising to promote the research on TCM through innovative strategies and advanced cutting-edge technologies. Microfluidic chips have provided controllable unique platforms for biomedical applications in TCM research with flexible composition and large-scale integration. In this review, the analysis and biomedical applications of microfluidics in the field of TCM are highlighted, including quality control of Chinese herbal medicines (CHMs), delivery of CHMs, evaluation of pharmacological activity as well as disease diagnosis. Finally, potential challenges and prospects of existing microfluidic technologies in the inheritance and innovation of TCM are discussed.Copyright © 2022 Elsevier B.V.

Authentication of Tinospora cordifolia derived herbal supplements using high resolution mass spectrometry-based metabolomics approach – A pilot study

Tinospora cordifolia herbal supplements have recently gained prominence due to their promising immunomodulatory and anti-viral effects against SARS-CoV-2. Mislabelling or diluting Tinospora supplements for profit may harm public health. Thus, validating the label claim of these supplements in markets is critical. This study investigated how high resolution mass spectrometry-based metabolomics and chemometrics can be used to distinguish Tinospora cordifolia from two other closely related species (T. crispa and T. sinensis). The Orthogonal Partial Least Square Discriminant Analysis (OPLS-DA) and PLS-DA based chemometric models predicted the species identity of Tinospora with 94.44% accuracy. These classification models were trained using 54 T. cordifolia, 21 T. crispa, and 21 T. sinensis samples. We identified 7 biomarkers, including corydine, malabarolide, ecdysterone, and reticuline, which discriminated Tinospora cordifolia from the two other species. The label claim of 25 commercial Tinospora samples collected from different parts of India was verified based on the relative abundance of the biomarker compounds, of which 20 were found authentic. The relative abundance of biomarkers significantly varied in the 5 suspicious market samples. This pilot study demonstrates a robust metabolomic approach for authenticating Tinospora species, which can further be used in other herbal matrices for product authentication and securing quality. © 2023 Elsevier B.V.