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Background & Aim: The new UCOE models we have recently developed, tested on many cell groups (including mouse ES and human iPS cells) and human mAb recombinant production studies as well, shows a powerful resistance to DNA methylation- mediated silencing and provides a higher and stable transfection profile. By the urgent need of vaccine development for COVID-19 during the pandemic, in this study we aimed to produce a potential recombinant vaccine by using the new generation UCOEs models of our own design. Methods, Results & Conclusion(s): Existing new-generation UCOE models and standard plasmid vectors to be used as control group were provided. Then, the sequences related to the PCR method were amplified for sufficient stock generation and cloning experiments. Verification in the plasmid vector was carried out in gel electrophoresis. Transfection of 293T cells was performed with clone plasmids carrying antigen genes and plasmids carrying genetic information of lentivirus units for the production of lentiviral vectors. Afterwards, 293T cells produced lentiviral vectors carrying antigen genes. Harvesting of these vectors was carried out during 48th and 72nd hours. Afterwards, CHO cells were transduced with appropriate quantity of lentiviral vectors. Isolation and purification of targeted proteins from the relevant medium were performed by HPLC and Q-TOF methods. A part of the spike and nucleocapsid gene sequences of COVID-19 were firstly cloned into our UCOE models. These UCOEs plasmids were then transferred into 293T cells along with plasmids carrying the genes that will form the lentivirus vectors (LVs). After harvesting and calculation of LV vector titers, the cloned vectors were then transfected into the CHO cells which the targeted recombinant production of the antigen proteins will be carried out. Antigenic structures were then isolated from the culture medium of CHO cells in following days for confirmation. Using HPLC and qTOF mass spectrometer methods, these structures in the medium were confirmed to be the units of spike and nucleocapsid proteins of the COVID-19 virus. In order to produce large amount of the recombinant antigens, the culture was then carried out with bioreactors in liters. At the final stage, these recombinantly produced antigen proteins were tested on rats to measure their immunogenic responses, and the study recently been completed successfully as a potential recombinant vaccine against COVID-19.Copyright © 2023 International Society for Cell & Gene Therapy
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Aim. To study the effect of inhalation therapy with an active hydrogen (AH) on the protein composition of exhaled breath condensate (EBC) in patients with post-COVID syndrome (PCS). Material and methods. This randomized controlled parallel prospective study included 60 patients after coronavirus disease 2019 (COVID-19) with PCS during the recovery period and clinical manifestations of chronic fatigue syndrome who received standard therapy according to the protocol for managing patients with chronic fatigue syndrome (CFS). The patients were divided into 2 groups: group 1 (main) - 30 people who received standard therapy and AH inhalations (SUISONIA, Japan) for 10 days, and group 2 (control) - 30 medical workers who received only standard therapy. Patients in both groups were comparable in sex and mean age. All participants in the study were sampled with EBC on days 1 and 10. Samples were subjected to tryptic digestion and high-performance liquid chromatography combined with tandem mass spectrometry analysis using a nanoflow chromatograph (Dionex 3000) in tandem with a high-resolution time-of-flight mass spectrometer (timsTOF Pro). Results. A total of 478 proteins and 1350 peptides were identified using high resolution mass spectrometry. The number of proteins in samples after AH therapy, on average, is 12% more than before treatment. An analysis of the distribution of proteins in different groups of patients showed that only half of these proteins (112) are common for all groups of samples and are detected in EBC before, after, and regardless of hydrogen therapy. In addition to the qualitative difference in the EBC protein compositions in different groups, quantitative changes in the concentration of 36 proteins (mainly structural and protective) were also revealed, which together made it possible to reliably distinguish between subgroups before and after treatment. It is worth noting that among these proteins there are participants of blood coagulation (alpha-1-antitrypsin), chemokine- and cytokine-mediated inflammation, and a number of signaling pathways (cytoplasmic actin 2), response to oxidative stress (thioredoxin), glycolysis (glyceraldehyde-3- phosphate dehydrogenase), etc. Conclusion. The use of hydrogen therapy can contribute to the switching of a number of physiological processes, which may affect the success of recovery in PCS patients. In particular, the obtained results indicate the activation of aerobic synthesis of adenosine triphosphate in mitochondria by hydrogen therapy, which correlates well with the decrease in the blood lactate level detected by laboratory studies. At the same time, this therapy can inhibit pro-inflammatory activity, negatively affecting the coagulation and signaling pathways of integrins and apoptosis, and, in addition, activate protective pathways, tricarboxylic acid cycle, FAS signaling, and purine metabolism, which may be essential for effective recovery after COVID-19.Copyright © 2023 Vserossiiskoe Obshchestvo Kardiologov. All rights reserved.
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Molnupiravir, a broad-spectrum antiviral is an isopropyl ester prodrug of beta-D-N4-hydroxycytidine. Molnupiravir targets RNA-dependent RNA-polymerase enzyme of the viruses. A new stability-indicating HPLC-method was developed to determine related substances and assay of molnupiravir. Separation was achieved by using Shim-pack GWS C18 column. The method was validated according to current ICH requirements. The calibration plot gave a linear relationship for all known analytes over the concentration range from LOQ to 200%. LOD and LOQ for all known analytes were found in 0.05-0.08 microg mL-1 and 0.12-0.20 microg mL-1, respectively, the mean recovery was found to be 97.79-102.44 %. Study showed that the method, results of robustness, solution stability studies are precise and within the acceptable limits. Molnupiravir was found to degrade in acid, alkali, and oxidative conditions, and was stable in thermal, moisture, and photolytic degradation condition. The method is simple, accurate, precise, and reproducible for routine purity analysis of drug-samples.Copyright © 2022 Indian Drug Manufacturers' Association. All rights reserved.
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Effective drug delivery to the brain is critical for the treatment of glioblastoma (GBM), an aggressive and invasive primary brain tumor that has a dismal prognosis. Radiation therapy, the mainstay of brain tumor treatment, works by inducing DNA damage. Therefore, inhibiting DNA damage response (DDR) pathways can sensitize tumor cells to radiation and enhance cytotoxicity. AZD1390 is an inhibitor of ataxia-telangiectasia mutated kinase, a critical regulator of DDR. Our in vivo studies in the mouse indicate that delivery of AZD1390 to the central nervous system (CNS) is restricted due to active efflux by P-glycoprotein (P-gp). The free fraction of AZD1390 in brain and spinal cord were found to be low, thereby reducing the partitioning of free drug to these organs. Coadministration of an efflux inhibitor significantly increased CNS exposure of AZD1390. No differences were observed in distribution of AZD1390 within different anatomic regions of CNS, and the functional activity of P-gp and breast cancer resistance protein also remained the same across brain regions. In an intracranial GBM patient-derived xenograft model, AZD1390 accumulation was higher in the tumor core and rim compared with surrounding brain. Despite this heterogenous delivery within tumor-bearing brain, AZD1390 concentrations in normal brain, tumor rim, and tumor core were above in vitro effective radiosensitizing concentrations. These results indicate that despite being a substrate of efflux in the mouse brain, sufficient AZD1390 exposure is anticipated even in regions of normal brain. SIGNIFICANCE STATEMENT Given the invasive nature of glioblastoma (GBM), tumor cells are often protected by an intact blood-brain barrier, requiring the development of brain-penetrant molecules for effective treatment. We show that efflux mediated by P-glycoprotein (P-gp) limits central nervous system (CNS) distribution of AZD1390 and that there are no distributional differences within anatomical regions of CNS. Despite efflux by P-gp, concentrations effective for potent radiosensitization are achieved in GBM tumor-bearing mouse brains, indicating that AZD1390 is an attractive molecule for clinical development of brain tumors.Copyright © 2022 American Society for Pharmacology and Experimental Therapy. All rights reserved.
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Background: SARS-CoV-2 displays high affinity for ACE2 receptors, expressed on type 2 alveolar cells. These cells produce pulmonary surfactant - a crucial thin layer of surface-active lipid rich fluid - fundamental for proper gas exchange. Aims and objectives: To investigate changes in surfactant lipid composition and the relationship to prolonged symptoms of post covid-19 among patients treated in intensive care unit for covid-19 infection. Method(s): Patients (n=43, 17 female, aged 44-80 years) treated in an intensive care unit with covid-19 infection in average six months prior to enrollment were recruited. Particles in exhaled air were collected with PExA-instrument (PExA AB) and we also conducted body plethysmograph and diffusion capacity of the lungs for carbon monoxide. Twenty-two healthy, non-infected, age- and gender-matched controls were also enrolled. Lipids were analysed using liquid chromatography with a triple quadrupole mass spectrometer. Statistical analyses were performed with Qlucore. Result(s): Preliminary results suggest a significant change in the composition of surfactant lipids. Analysis show significant reductions of all measured phosphatidyl-glycerols (PG, n=14) an increase of all measured phosphatidyl- inositols (PI, n=4), e.g. PG 18:1-18:1 22 % lower (p<0.001, q=0.04) and PI:16:0:18:1 67% higher (p<0.001, q=0.0003) among the post-covid patients compared to controls. Conclusion(s): Our findings suggest that surfactant composition is altered also in the recovery phase after covid-19 infection which could be a key component in the post-covid syndrome with lingering effects on the respiratory system.
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Introduction Argatroban is indicated for treatment of heparin-induced thrombocytopenia, but is also used in critical ill COVID-19 patients presenting with extensive thrombin overload. Direct drug monitoring is not available and argatroban dosing is mainly based on activated partial plasmin time (aPTT), which has limitations in hypercoagulable patients with increased FVIII [1, 2]. The aim of this study was to compare correlation of routine clotting tests (aPTT, ecarin clotting time [ECA-CT] and diluted thrombin time [dTT]) [3] to argatroban plasma levels measured by gold standard mass spectrometry (LC/MS/MS). Method From 06/2021 to 03/2022, 205 samples from 22 COVID-19 ICU patients were analyzed: aPTT and dTT on STA R Max3-Analyzer (Stago Deutschland GmbH, Germany) using the BIOPHEN DTI Kit with Argatroban-calibration (CoaChrom Diagnostica GmbH, Austria);ECA-CT was measured using ClotPro ecarin assay. LC/MS/MS was performed using an RP column, a solvent gradient and an API4000 mass spectrometer with electrospray. Correlation was analyzed using Pearson correlation coefficient r in R version 3.2.4. This study was approved by the Ethics Committee of the Technical University of Dresden, Germany (BO-EK-64022022) and registered with German Clinical Trials Register DRKS00028689. Results From 205 samples with LC/MS/MS analysis, 195 were compared to aPTT, 153 to ECA-CT and 105 to dTT. In 40 samples, dTT was not measureable due high bilirubin values. Compared to LC/MS/MS, correlation of dTT was highest (r = 0.924), followed by ECA-CT (r 0.609) and aPTT (r 0.367;p < 0.001;Fig. 1). When recommended cut-offs for argatroban plasma levels (500-1000 ng/ml according to SmPC) were applied, dTT (when measurable) and ECA-CT better identified critical values of argatroban plasma values > 1000ng/ml than aPTT (Fig. 2). Conclusion Argatroban in critical ill COVID-19 patients should be monitored using dTT. If dTT is not possible or measurements are highly time-sensitive, point-of-care ClotPro ECA-test should be preferably used instead of aPTT. (Table Presented).
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During spring 2020, the COVID-19 pandemic caused massive reductions in emissions from industry and ground and airborne transportation. To explore the resulting atmospheric composition changes, we conducted the BLUESKY campaign with two research aircraft and measured trace gases, aerosols, and cloud properties from the boundary layer to the lower stratosphere. From 16 May to 9 June 2020, we performed 20 flights in the early COVID-19 lockdown phase over Europe and the Atlantic Ocean. We found up to 50% reductions in boundary layer nitrogen dioxide concentrations in urban areas from GOME-2B satellite data, along with carbon monoxide reductions in the pollution hot spots. We measured 20%-70% reductions in total reactive nitrogen, carbon monoxide, and fine mode aerosol concentration in profiles over German cities compared to a 10-yr dataset from passenger aircraft. The total aerosol mass was significantly reduced below 5 km altitude, and the organic aerosol fraction also aloft, indicative of decreased organic precursor gas emissions. The reduced aerosol optical thickness caused a perceptible shift in sky color toward the blue part of the spectrum (hence BLUESKY) and increased shortwave radiation at the surface. We find that the 80% decline in air traffic led to substantial reductions in nitrogen oxides at cruise altitudes, in contrail cover, and in resulting radiative forcing. The light extinction and depolarization by cirrus were also reduced in regions with substantially decreased air traffic. General circulation-chemistry model simulations indicate good agreement with the measurements when applying a reduced emission scenario. The comprehensive BLUESKY dataset documents the major impact of anthropogenic emissions on the atmospheric composition.
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Background: Bersama abyssinica is a common tree in Africa, with diverse medical uses in different areas. The plant is well-known in Tanzania for treating respiratory disorders such as TB, tonsillitis, bronchitis and asthma, The plant has lately been utilized for treatment of COVID-19 symptoms. Water extract of leaf and stem bark has been registered as a herbal medication known as 'Coviba Dawa' for the treatment of viral and bacterial respiratory infections due to its broad medical benefits. The aim of this work was to test for the cytotoxicity and antiviral effects of bioactive ingredients from B. abyssinica extracts against the Delta variant of the SARS-CoV-2 coronavirus. Method(s): Bersama abyssinica leaves and stem barks were dried under shade in room temperature and then pulverized to obtain small pieces before soaking into different solvents. One hundred grams of each, leaves and stem barks were extracted in petroleum ether, dichloromethane, ethyl acetate and ethanol. Water extract was obtained by decoction of 100g of stem bark into water. Phenols, flavonoids, tannins with antioxidant activity were confirmed as components of the extracts. Analysis of aqueous extracts of bark stem bark and leaves was done by LC-MS/MS (Q-orbitrap-Ultra High Performance Thermofisher Company). Antiviral screening and cytotoxicity experiments were conducted in a Biosafety Level 3 (BSL-3) Laboratory facility according to previously established Standard operating procedures (SOPs). Result(s): The existence of four phenolic compounds in B. abyssinica water extract;2,4-di-tert-butylphenol, 4-formyl-2-methoxyphenyl propionate, 7,8-Dihydroxy-4-methylcoumarin, and 2,3, 6-trimethoxyflavone with antioxidant activity was confirmed by LC-MS/MS analysis. In-vitro testing of the extracts revealed antiviral activity against coronavirus the delta B1 variant of SARS-CoV2. Conclusion(s): We recommend further studies to characterize the medicinal value of B. abyssinica metabolites as potential antiviral and antibacterial agents.
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Aim To elucidate the effective components of Ganoderma applanatum and its mechanism of preventing the coronavirus disease 2019(COVID-19).Methods To begin with, UHPLC-Q-Exactive-Orbitrap-MS was established to identify the main chemical constituents of G.applanatum.Then, the predicted targets of G.applanatum were selected by Swiss Target Prediction.GO analysis and KEGG analysis of core target genes were performed using the DAVID database.Finally, to explore the potential mechanism of G.applanatum against COVID-19, core functional components-core target-metabolism path network diagram was constructed using Cytoscape 3.8.0, and molecular docking was used to analyze the binding force of the core effective compounds with angiotensin-converting enzyme II(ACE2)and three SARS CoV-2 proteins, nonstructural protein-15 Endoribonuclease(NSP15), the receptor-binding domain of spike protein(RBD of S protein), and main protease(Mpro/3CLpro).Results Sixty-two components were identified from G.applanatum by UHPLC-Q-Exactive-Orbitrap-MS study;30 active components were closely associated with 32 core targets including IL6, PTGS2, and MAPK1;KEGG analysis showed that it might treat COVID-19 through signaling pathways, such as PI3K-Akt signaling pathway, TNF signaling pathway, tuberculosis, and so on;molecular docking analysis showed that 1,4-Dihydroxy-2-naphthoic acid, parthenolide, 7,8-Dihydroxycoumarin, and other vital compounds had a certain degree of affinity with ACE2 and three SARS CoV-2 proteins.Conclusion This study clarifies the chemical composition and the potential mechanism of G.applanatum, providing a scientific basis for screening the effective ingredients of G.applanatum. Copyright © 2022 Publication Centre of Anhui Medical University. All rights reserved.
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Curcumae Longae Rhizoma (CLR) is the rhizome of Curcuma longa L. Pharmacological studies show that CLR can be used to treat cervical cancer, lung cancer, lupus nephritis, and other conditions. In this paper, we review botany, traditional application, phytochemistry, pharmacological activity, and pharmacokinetics of CLR. The literature from 1981 to date was entirely collected from online databases, such as Web of Science, Google Scholar, China Academic Journals full-text database (CNKI), Wiley, Springer, PubMed, and ScienceDirect. The data were also obtained from ancient books, theses and dissertations, and Flora Reipublicae Popularis Sinicae. There are a total of 275 compounds that have been isolated from CLR, including phenolic compounds, volatile oils, and others. The therapeutic effect of turmeric has been expanded from breaking blood and activating qi in the traditional sense to antitumor, anti-inflammatory, antioxidation, neuroprotection, antibacterial, hypolipidemic effects, and other benefits. However, the active ingredients and mechanisms of action related to relieving disease remain ill defined, which requires more in-depth research and verification at a clinical level.
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Multidrug-resistant K. pneumoniae bacterial strains producing extended range of beta-lactamases or carbapenemases are of serious clinical concern. The aim of the study was to determine the resistance factors of K. pneumoniae strains isolated from the lower respiratory tract of patients diagnosed with community-acquired pneumonia during the COVID-19 pandemic. Materials and methods. The study of resistance to antimicrobial drugs included 138 strains of K. pneumoniae isolated from the sputum of patients treated in infectious diseases monohospitals in the city of Tyumen and the Tyumen region within the period from May 2020 to June 2021. Among the strains examined, 51.4% of them were isolated from SARS-CoV-2 positive patients. The presence of resistance genes was determined by PCR in 71 strains of K. pneumoniae (34 strains from COVID-19-positive and 37 strains from COVID-19-negative patients). Identification of isolated bacterial strains was carried out according to the protein spectra by using a desktop time-of-flight mass spectrometer with matrix laser desorption MALDI-TOF MS (Bruker, Germany). The belonging of the strains to the hypermucoid phenotype was determined using the string test. Sensitivity to antimicrobial drugs was assessed in the disk diffusion method on Muller-Hinton medium. The sensitivity of culture strains to bacteriophage preparations was determined by the drop method (spot-test). In the study, we used “Polyvalent Sextaphage Pyobacteriophage” and “Purified Polyvalent Klebsiella Bacteriophage”, JSC NPO Microgen, Russia. Detection of resistance genes to beta-lactam antibiotics by real-time PCR was carried out using the BakRezista kit (OOO DNA-technology, Russia). The results of the study evidence that K. pneumoniae bacteria isolated from COVID-19-positive and COVID-19-negative patients diagnosed with community-acquired pneumonia displayed a high resistance to antimicrobial drugs and commercial phage-containing drugs. Resistance of K. pneumoniae strains was recorded from 50% (to aminoglycosides and carbapenems) to 90% (to inhibitor-protected penicillins). Sensitivity to bacteriophages was noted on average in no more than 20% of strains. It is important to emphasize that strains isolated from COVID-19-positive patients more often showed a hypermucoid phenotype, suggesting a high bacterial virulence, and also showed greater resistance to all groups of antibacterial drugs examined in the study, which is confirmed by the presence of resistance genes of the ESBL group and carbapenemase. The results of the study suggest that the high level of resistance of K. pneumoniae strains isolated from COVID-19-positive patients is associated with immunosuppression provoked by the SARS-CoV-2 virus, which contributes to their colonization by more virulent strains.
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Background: SARS-CoV-2 displays high affinity for ACE2 receptors as a vector of pathogenesis. ACE2 receptors are highly expressed on surfactant producing type 2 alveolar cells. These cells produce pulmonary surfactant - a crucial thin layer of surface-active fluid mainly composed of lipids, lining the alveolar epithelial surface. The main function, to reduce the surface tension, is fundamental for proper gas exchange. Aims and Objectives: To investigate changes in surfactant lipid composition and the relationship to longstanding symptoms of post Covid-19 among patients treated in intensive care for Covid-19 infection. Methods: We recruited 43 patients (17 women, aged 44-80 years) who had previously been treated in ICU in a major Swedish hospital, in average 6 months before inclusion. The participants answered a questionnaire regarding symptoms, we collected particles in exhaled air with PExA-instrument (PExA AB) and conducted pulmonary function tests, body plethysmography, and diffusion capacity of the lungs for carbon monoxide. Twenty-two healthy, non-infected, ageand gender-matched controls were enrolled. Lipids were analysed using liquid chromatography with a triple quadrupole mass spectrometer. Statistical analyses were performed with Qlucore. Results: Early results suggest a significant change in the composition of surfactant lipids among post-Covid -19 patients treated in intensive care compared to controls. Early analysis show significant reductions of all measured phosphatidyl-glycerols (PG, n = 14) an increase of all measured phosphatidyl-inositols (PI, n = 4), for example were PG 18:1-18:1 22% lower (p < 0.001, q = 0.04) and PI:16:0:18:1 67% higher (p < 0.001, q = 0.0003) in subjects post-Covid compared to controls. Conclusions: Our findings suggest that surfactant composition is altered also in the recovery after Covid-19 infection, which could be a key component in the post-Covid syndrome and the lingering effects on the respiratory system.
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Background-aim: With recent emergences in new infectious diseases and their variants, there is a need to develop a faster and more specific analytical tool to detect different respiratory infectious diseases such as SARS-CoV-2 or influenza viruses. Not only their symptoms are similar at early stages, but also, they are both enveloped viruses with several common biological properties, often leading to challenges in disease identification. Among different viral components, nucleocapsid protein or nucleoprotein (NP) is highly conserved, less post-translational modifications possessed, and mostly specific for each infectious disease virus types. Therefore, targeting NP could be more advantageous to the method development, achieving much simpler and robust method with minimal subsequent modifications. This study describes a targeted approach for simultaneous detection of NPs from different respiratory infectious diseases using immunoprecipitation (IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Multiple viruses, SARS-CoV-2, influenza virus A and B types, respiratory syncytial virus, and human coronavirus (HCoV-229E), were selected to show that this method can distinguish different disease viruses. Methods: Sample collected via nasopharyngeal swabs in viral transport media was directly subjected to IP using Thermo Scientific™ Pierce™ MS-Compatible IP Kit (Streptavidin). The IP purified samples were then digested using SMART Digest™ Trypsin Kits and analyzed by Thermo Scientific™ Vanquish™ MD HPLC system hyphenated to Thermo Scientific™ TSQ Altis MD mass spectrometer. Data processing was performed using TraceFinder™ LDT software 1.0. Results: Combining IP and LC-MS/MS resulted in a highly targeted approach with the high sensitivity and specificity. The method detected sub tens to hundreds amol of peptides on LC column. Also, it simplified the overall sample preparation process eliminating prior protein precipitation and post sample clean-up. Since the NPs mostly remain unchanged or less modified regardless of variants, the method doesn’t need tremendous alterations once established. Conclusions: This targeted approach can be applied to other enveloped viruses’ detection. Automated IP method is available with KingFisher system so it could lead to a faster turn-around time and higher throughput of the method.
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Background: Although vaccination efforts have been deployed worldwide over the past 10 months, there are still gaps in our understanding surrounding the immune response to SARS-CoV-2 infection and vaccination, including changes to the antibody repertoire. One way of tracking the immune response over time is through measuring IgG Fc glycosylation, which provides insight into the inflammatory state of an infected individual, antibody effector function, antibody half-life, and more. Therefore we set out to interrogate bulk IgG changes in glycosylation in both natural infection and vaccinated cohorts in order to determine potential insight into protection from severe disease and responsiveness to vaccination. Methods: We evaluated 98 plasma samples from COVID-19 patients with either mild or severe COVID-19. Symptomatic patients were characterized as mild or severe based on hospital admission. We also evaluated plasma from 228 vaccinated individuals (Pfizer-BioNTech). Bulk IgG glycosylation analysis was measured through a quadrupole orbitrap mass spectrometry. Neutralization potential was assessed through a spike pseudotyped neutralization assay. Spike antibody levels were measured using a Luminex assay and ELISA. Results: We found that inflammatory glycans (fucosylated agalactosylated, G0F) on bulk IgG were elevated in hospitalized COVID-19 patients and increased over time in this population when compared to mild infection. Mild patients had an anti-inflammatory glycosylation pattern (afucosylated galactosylated, G2) which increased over time. Siaylation levels were elevated in mild individuals, increased over time, and correlated with increased RBD antibody levels. Interestingly, when we assessed COVID-19 vaccinated individuals with low Spike antibody levels and low neutralization, they had the same glycosylation pattern (G0F) as that of hospitalized COVID-19 patients. Additionally, a small longitudinal vaccinated cohort (out to 8 months) revealed a decrease in G0F associated with peak IgG concentrations and neutralization (Fig 1). Conclusion: Inflammatory glycan signatures, such as an elevation in G0F glycans, can be used as prognostic tools, not only to predict the severity of COVID-19 disease, but also to predict patient responsiveness to COVID-19 vaccines. This is the first report identifying a shift in glycan signature to be associated with COVID-19 disease severity and vaccine responsiveness, which can guide future studies into SARS-CoV-2 protective immunity and vaccine development.
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Symbiotic interaction between the human body and its microbiota is an important issue of modern biomedicine and personalized medicine. However, little is known on molecular mechanisms of that relationship. Bearing in mind the ubiquitous participation of peptides in biomolecular interactions and regulatory processes we attempted direct search of blood peptides originated from microbial proteins. LC-MS/MS analysis was carried out of blood serum and plasma samples taken from 20 healthy donors on Q Exactive HF-X Hybrid Quadrupole-Orbitrap mass-spectrometer. Sample preparation was carried out based on our previously developed method of peptide desorption from the surface of major blood plasma proteins followed by standard chromatographic steps. Human microbiota protein sequences were taken from NIH Human Microbiome Project. As a result, out of 13,625 identified peptides 912 were unique fragments of microbial precursors, which is about 6.69% of the total amount of detected bloodstream peptides. In 30 cases peptide identification was confirmed by mass-spectral study of individual synthetic samples. Absolute quantification by the mass-spectrometric method of multiple reaction monitoring (MRM) confirmed the presence of bacterial peptides in plasma and serum in the range of approximately 0.1 nM to 1 uM, which is comparable to physiologically significant hormone concentrations in human blood in normal conditions. The abundance of microbiota peptides reaches its maximum 5h after a meal. Most of the peptides correlate with the bacterial composition of the small intestine and are likely obtained by hydrolysis of membrane proteins with trypsin, chymotrypsin and pepsin - the main proteases of the gastrointestinal tract. The isolated fraction of peripheral blood mononuclear cells showed increase secretion of proinflammatory cytokines, colony stimulating factors and chemoattractants as the response to the addition of some of the identified microbiotic peptides. Such peptides - identified both in the plasma and in the serum - have properties of resistance to fibrinolysis, since the pool of peptides is preserved after passing through the digestive tract. The physicochemical properties of the identified bacterial peptides are consistent with those required for the selective permeability of mucosal barriers. Our approach to the identification of microbiota peptides in the blood serum and plasma may be useful for determining the microbiota composition of hard-to-reach intestinal areas, such as the small intestine, and for monitoring the permeability of the intestinal mucosal barrier.
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Symbiotic interaction between the human body and its microbiota is an important issue of modern biomedicine and personalized medicine. However, little is known on molecular mechanisms of that relationship. Bearing in mind the ubiquitous participation of peptides in biomolecular interactions and regulatory processes we attempted direct search of blood peptides originated from microbial proteins. LC-MS/MS analysis was carried out of blood serum and plasma samples taken from 20 healthy donors on Q Exactive HF-X Hybrid Quadrupole-Orbitrap mass-spectrometer. Sample preparation was carried out based on our previously developed method of peptide desorption from the surface of major blood plasma proteins followed by standard chromatographic steps. Mascot and X! Tandem search engines were used for peptide identification. Human protein sequences were taken from UniProt Knowledgebase and sequences of human microbiota proteins-from NIH Human Microbiome Project (HMP). As a result, out of 13625 identified peptides 912 were unique fragments of microbial precursors, which is 6.69% of the total amount of detected bloodstream peptides. In 30 cases peptide identification was confirmed by mass-spectral study of individual synthetic samples. Absolute quantification by the mass-spectrometric method of multiple reaction monitoring (MRM) confirmed the presence of bacterial peptides in plasma and serum in the range of approximately 0.1 nMol/L to 1 mkMol/L, which is comparable to physiologically significant hormone concentrations in human blood in normal conditions. Analysis of the in silico obtained hydrolyzates of microbiotic proteins showed that significant number of the identified peptides are derived from the precursor proteins as a result of hydrolysis with trypsin, chymotrypsin and pepsin, the main proteases of the gastrointestinal system. 60% of the identified “microbial” peptides are derived from the intestine flora, about 20% - from oral microbiota and 20% fall on the remaining microbiotic communities. Most of the precursor proteins refer to intracellular, cytoplasmic proteins. The isolated fraction of peripheral blood mononuclear cells showed increase secretion of proinflammatory cytokines, colony stimulating factors and chemoattractants as the response to the addition of some of the identified microbiotic peptides. The data obtained serve as a basis for the ongoing study of the functional properties of microbiome derived peptides.
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The Astrobotic M1 mission, as the first mission of NASA s Commercial Lunar Payload Services (CLPS) program, is scheduled to land in the Lacus Mortis region of the Moon in early 2022. Among its payloads it will carry the Peregrine Ion Trap Mass Spectrometer (PITMS), an instrument supplied by NASA GSFC that is dedicated to the investigation of the lunar exosphere, and which includes as its core component the ESA-provided Exospheric Mass Spectrometer (EMS). EMS was developed under an ESA contract by an academic/industrial consortium led by Open University (UK) using a fast track development approach that aimed at delivery of a Proto-Flight Model (PFM) instrument with drastically reduced development time. The chosen development approach and the demanding project schedule (18 months from contract start to flight hardware delivery) posed a number of specific project management challenges for successful development of the instrument within the ESA flight hardware development framework and under constraints imposed by the Covid-19 pandemic. With an as-built envelope of HWD of 168 149 127mm, a mass of 1.48kg and a typical power consumption during measurement not exceeding 9.5 Watts the instrument has modest resource requirements. EMS can be re-used in different future application scenarios, including investigations of the lunar exosphere, analysis of gases evolved from acquired samples, and monitoring aspects of the environmental impact of landers and robotic and human activities on the lunar environment. © 2021 International Astronautical Federation, IAF. All rights reserved.
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Purpose of Study Postrenal causes of acute kidney injury (AKI) can generally be determined by imaging but differentiation of prerenal (PR) from intrinsic renal (IR) can be more difficult. A point of care test to quickly differentiate these two entities would be useful. Mass spectrometry (MS) can visualize small molecules in urine. We utilized a portable, single quadrupole mass spectrometer with a simple atmospheric pressure ionization interface to measure small molecules in urine. The interface allows for direct analysis of samples without the need for chromatography or other time-consuming sample preparation. The goal was to distinguish PR and IR AKI by urine analyte profiles. Methods Used Inpatients that developed AKI were eligible for enrolment. Patients with COVID-19 were excluded. Informed consent was obtained under a protocol approved by the UAMS IRB. Patients were categorized as either PR (n=9) or IR (n=7) AKI etiology using the diagnosis by the on-service nephrology attending. Etiologies for IR AKI included tumor lysis syndrome, contrast-induced nephropathy, myeloma light chain disease, pyelonephritis, and cisplatin-induced kidney injury. Two microliters of urine was dispensed onto a stainless steel probe without prior processing and analyzed by MS with an Advion Expression CMS Mass Spectrometer. Peaks within the 20 to 500 m/z range were recorded. MS spectra were processed and binned in 1 m/z increments for peak clustering using MATLAB. The frequency of binned peaks in the PR AKI group were compared to the IR group and marked as peaks of interest if the difference in number of peaks across the two categories was four or more. Missing peak values were replaced with the minimum value for that peak across all samples. Summary of Results Eleven peaks met our initial criteria for difference in frequency between intrinsic and prerenal cases. From the eleven, the mean intensity was different between the groups for only the sample at m/z 242 (p=0.03). In the PR group, mean and SD of the peak intensities were 1.7E+07 ±1.2E+07 compared to 7.0E+06 ±7.7E+06 Using a cut-off value of 1.55E+07, overall accuracy was 75%. The predictor correctly classified 6/9 in the PR group (67%) and 6/7 in the IR group (86%). Conclusions Point of care MS has the potential to rapidly differentiate PR from IR and potentially to further phenotype causes of AKI. Based on previous studies, the peaks we identified are likely small molecule metabolites. Larger sample size will be needed to better phenotype patients and will enable the use of machine learning algorithms to classify the kidney disease that is present. Further correlation of the different analytes in prerenal and intrarenal AKIs is needed, however this study shows the potential utility of mass spectrometry to rapidly phenotype AKI in the clinical setting.