Construction and immunogenicity of a trypsin-independent porcine epidemic diarrhea virus variant.

Porcine epidemic diarrhea virus (PEDV) is a re-emerging enteropathogenic coronavirus that causes high mortality in neonatal piglets. The addition of trypsin plays a crucial role in the propagation of PEDV, but also increases the complexity of vaccine production and increases its cost. Previous studies have suggested that the S2' site and Y976/977 of the PEDV spike (S) protein might be the determinants of PEDV trypsin independence. In this study, to achieve a recombinant trypsin-independent PEDV strain, we used trypsin-dependent genotype 2 (G2) PEDV variant AJ1102 to generate three recombinant PEDVs with mutations in S (S2' site R894G and/or Y976H). The three recombinant PEDVs were still trypsin dependent, suggesting that the S2' site R894 and Y976 of AJ1102 S are not key sites for PEDV trypsin dependence. Therefore, we used AJ1102 and the classical trypsin-independent genotype 1 (G1) PEDV strain JS2008 to generate a recombinant PEDV carrying a chimeric S protein, and successfully obtained trypsin-independent PEDV strain rAJ1102-S2'JS2008, in which the S2 (amino acids 894-1386) domain was replaced with the corresponding JS2008 sequence. Importantly, immunization with rAJ1102-S2'JS2008 induced neutralizing antibodies against both AJ1102 and JS2008. Collectively, these results suggest that rAJ1102-S2'JS2008 is a novel vaccine candidate with significant advantages, including no trypsin requirement for viral propagation to high titers and the potential provision of protection for pigs against G1 and G2 PEDV infections.

Viral enteritis in cattle: To well known viruses and beyond

Livestock products supply about 13 percent of energy and 28 percent of protein in diets consumed worldwide. Diarrhea is a leading cause of sickness and death of beef and dairy calves in their first month of life and also affecting adult cattle, resulting in large economic losses and a negative impact on animal welfare. Despite the usual multifactorial origin, viruses are generally involved, being among the most important causes of diarrhea. There are several viruses that have been confirmed as etiological agents (i.e., rotavirus and coronavirus), and some viruses that are not yet confirmed as etiological agents. This review summarizes the viruses that have been detected in the enteric tract of cattle and tries to deepen and gather knowledge about them.Copyright © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Screening for Potential Traditional Herbal Inhibitors Against 3-Chymo-trypsin-like Main Protease (3CLpro) from Four Different Coronaviruses: An in silico Approach

Background: The recent outbreak of the COVID-19 pandemic has raised a global health concern due to the unavailability of any vaccines or drugs. The repurposing of traditional herbs with broad-spectrum anti-viral activity can be explored to control or prevent a pandemic. Objective(s): The 3-chymotrypsin-like main protease (3CLpro), also referred to as the "Achilles' heel" of the coronaviruses (CoVs), is highly conserved among CoVs and is a potential drug target. 3CLpro is essential for the virus' life cycle. The objective of the study was to screen and identify broad--spectrum natural phytoconstituents against the conserved active site and substrate-binding site of 3CLpro of HCoVs. Method(s): Herein, we applied the computational strategy based on molecular docking to identify potential phytoconstituents for the non-covalent inhibition of the main protease 3CLpro from four different CoVs, namely, SARS-CoV-2, SARS-CoV, HCoV-HKU1, and HCoV-229E. Result(s): Our study shows that natural phytoconstituents in Triphala (a blend of Emblica officinalis fruit, Terminalia bellerica fruit, and Terminalia chebula fruit), namely chebulagic acid, chebulinic acid, and elagic acid, exhibited the highest binding affinity and lowest dissociation constants (Ki), against the conserved 3CLpro main protease of SARSCoV-2, SARS-CoV, HCoV-HKU1, and HCoV-229E. Besides, phytoconstituents of other herbs like Withania somnifera, Glycyrrhiza glabra, Hyssopus officinalis, Camellia sinensis, Prunella vulgaris, and Ocimum sanctum also showed good binding affinity and lower Ki against the active site of 3CLpro. The top-ranking phyto-constituents' binding interactions clearly showed strong and stable interactions with amino acid residues in the catalytic dyad (CYS-HIS) and substrate-binding pocket of the 3CLpro main proteases. Conclusion(s): This study provides a valuable scaffold for repurposing traditional herbs with anti--CoV activity to combat SARS-CoV-2 and other HCoVs until the discovery of new therapies.Copyright © 2021 Bentham Science Publishers.

ACE2-Independent Bat Sarbecovirus Entry and Replication in Human and Bat Cells.

Hundreds of sarbecoviruses have been found in bats, but only a fraction of them have the ability to infect cells using angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV and -2. To date, only ACE2-dependent sarbecoviruses have been isolated from field samples or grown in the laboratory. ACE2-independent sarbecoviruses, comprising the majority of the subgenus, have not been propagated in any type of cell culture, as the factors and conditions needed for their replication are completely unknown. Given the significant zoonotic threat posed by sarbecoviruses, cell culture models and in vitro tools are urgently needed to study the rest of this subgenus. We previously showed that the exogenous protease trypsin could facilitate cell entry of viral-like particles pseudotyped with spike protein from some of the ACE2-independent sarbecoviruses. Here, we tested if these conditions were sufficient to support bona fide viral replication using recombinant bat sarbecoviruses. In the presence of trypsin, some of the spike proteins from clade 2 viruses were capable of supporting bat sarbecovirus infection and replication in human and bat cells. Protease experiments showed a specific viral dependence on high levels of trypsin, as TMPRSS2 and furin had no effect on clade 2 virus entry. These results shed light on how sarbecoviruses transmit and coexist in their natural hosts, provide key insights for future efforts to isolate and grow these viruses from field samples, and further underscore the need for broadly protective, universal coronavirus vaccines. IMPORTANCE Our studies demonstrate that some unexplored sarbecoviruses are capable of replicating in human and bat cells in an ACE2-independent way but need a high trypsin environment. We found that trypsin is not compensated by other known proteases involved in some coronavirus entry. This work provides important information that the trypsin-dependent entry may be a widely employed mechanism for coronaviruses and will help for further understanding the biological features of the less-studied viruses.

Effect of Adropin on Pancreas Exocrine Function in a Rat Model: A Preliminary Study.

The aim was to investigate the potential effect of adropin (ADR) on pancreatic-biliary juice (PBJ) secretion (volume, protein content, trypsin activity) in a rat model. The animals were divided into control and five experimental groups: adropin, CCK-8 (CCK-8 stimulation), capsaicin (capsaicin deactivation of afferents), vagotomy (vagotomy procedure), and vagal stimulation (vagal nerve stimulation). The experiment consisted of four phases, during which vehicle (0.9% NaCl) and three ADR boluses (5, 10, and 20 µg/kg BW) were administered i.v. every 30 min. PBJ samples were collected from each rat at 15 min intervals after boluses. Exogenous ADR failed to affect the pancreatic responses after vagotomy and the capsaicin pretreatment and reduced the PBJ volume, protein outputs, and trypsin activity in the adropin, CCK-8, and vagal stimulation groups in a dose-dependent manner. In all these groups, volume of PBJ was reduced only by the highest dose of ADR (p < 0.001 for adropin group and p < 0.01 for CCK-8 and vagal stimulation groups), and the protein outputs were reduced by the administration of ADR 10 µg/kg BW (adropin and CCK-8 groups, p < 0.01 in both cases) and 20 µg/kg BW (p < 0.001 for adropin and CCK-8 groups, p < 0.01 for vagal stimulation group). The 10 µg/kg BW dose of ADR reduced the trypsin output in the CCK-8 group (p < 0.01), and the highest ADR dose reduced the trypsin output in the CCK-8 (p < 0.001) and vagal stimulation (p < 0.01) groups. In conclusion, adropin in the analyzed doses exhibits the negative feedback pathway. This mechanism seems to participate in the regulation of pancreatic juice secretion via an indirect vagal mechanism.

Clinical Trial Subgroup Analyses to Investigate Clinical and Immunological Outcomes of Convalescent Plasma Therapy in Severe COVID-19.

Objective: To assess the clinical and immunological benefits of passive immunization using convalescent plasma therapy (CPT). Materials and Methods: A series of subclass analyses were performed on the previously published outcome data and accompanying clinical metadata from a completed randomized controlled trial (RCT) (Clinical Trial Registry of India, number CTRI/2020/05/025209). The subclass analyses were performed on the outcome data and accompanying clinical metadata from a completed RCT (patient recruitment between May 15, 2020 and October 31, 2020). Data on the plasma abundance of a large panel of cytokines from the same cohort of patients were also used to characterize the heterogeneity of the putative anti-inflammatory function of convalescent plasma (CP) in addition to passively providing neutralizing antibodies. Results: Although the primary clinical outcomes were not significantly different in the RCT across all age groups, significant immediate mitigation of hypoxia, reduction in hospital stay, and significant survival benefit were registered in younger (<67 years in our cohort) patients with severe coronavirus disease 2019 and acute respiratory distress syndrome on receiving CPT. In addition to neutralizing the antibody content of CP, its anti-inflammatory proteome, by attenuation of the systemic cytokine deluge, significantly contributed to the clinical benefits of CPT. Conclusion: Subgroup analyses revealed that clinical benefits of CPT in severe coronavirus disease 2019 are linked to the anti-inflammatory protein content of CP apart from the anti-severe acute respiratory syndrome coronavirus 2 neutralizing antibody content.

Human interaction targets of SARS-COV-2 spike protein: A systematic review

Objectives: The development of effective targeted therapy and drug-design approaches against the SARS-CoV-2 is a universal health priority. Therefore, it is important to assess possible therapeutic strategies against SARS-CoV-2 via its most interaction targets. The present study aimed to perform a systematic review on clinical and experimental investigations regarding SARS-COV-2 interaction targets for human cell entry. Methods: A systematic search using relevant MeSH terms and keywords was performed in PubMed, Scopus, Embase, and Web of Science (ISI) databases up to July 2021. Two reviewers independently assessed the eligibility of the studies, extracted the data, and evaluated the methodological quality of the included studies. Additionally, a narrative synthesis was done as a qualitative method for data gathering and synthesis of each outcome measure. Results: A total of 5610 studies were identified, and 128 articles were included in the systematic review. Based on the results, spike antigen was the only interaction protein from SARS-CoV-2. However, the interaction proteins from humans varied including different spike receptors and several cleavage enzymes. The most common interactions of the spike protein of SARS-CoV-2 for cell entry were ACE2 (entry receptor) and TMPRSS2 (for spike priming). A lot of published studies have mainly focused on the ACE2 receptor followed by the TMPRSS family and furin. Based on the results, ACE2 polymorphisms as well as spike RBD mutations affected the SARS-CoV-2 binding affinity. Conclusion: The included studies shed more light on SARS-CoV-2 cellular entry mechanisms and detailed interactions, which could enhance the understanding of SARS-CoV-2 pathogenesis and the development of new and comprehensive therapeutic approaches.

Effect of Urinary Trypsin Inhibitor (Ulinastatin) Therapy in COVID-19.

Purpose: End-organ damage in coronavirus disease-2019 (COVID-19) is linked to "cytokine storm" and excessive release of inflammatory mediators. Various novel therapies have been used in COVID-19 including urinary trypsin inhibitor therapy. This study explores the efficacy of ulinastatin in COVID-19. Materials and methods: We retrieved the medical records of patients admitted during one month and did a propensity score analysis to create matched treatment and control groups. We analyzed these groups and the outcomes were presented with appropriate statistics. Survival curve was prepared to compare the survival effect of ulinastatin therapy at the end of hospitalization, among both the groups. Results: A total of 736 patients were admitted, and after adjusting the data with propensity score matching, 55 cases were selected by the system. On the final outcome analysis, we found that intensive care unit (ICU) length of stay [median (interquartile range) days 3 (3.5-7.8) vs 2 (0-4); p-value 0.28] in control vs intervention groups, and in hospital mortality (odds ratio: 0.491, CI 95%: 0.099-2.44, p-value 0.435) were not statistically different among the groups. In survival plot analysis also, there was no statistical difference (p-value 0.414) among both the groups.Conclusion: In this retrospective study, we conclude that the final outcome of the ICU length of stay, and overall, in hospital mortality were not different among both the groups. Hence, adequately powered randomized control trials are urgently required to confirm any benefit of ulinastatin therapy in COVID-19 treatment. How to cite this article: Jain A, Kasliwal R, Jain SS, Jain R, Gupta D, Gupta P, et al. Effect of Urinary Trypsin Inhibitor (Ulinastatin) Therapy in COVID-19. Indian J Crit Care Med 2022;26(6):696-703.

COV2MS: A tool for simultaneous longitudinal epidemiological monitoring of a variety of pathogens

Background-aim: A lesson learned already in the early phase of the COVID-19 pandemic is the need for diagnostic tools that target different biomolecules, using orthogonal experimental setups and fit-for-purpose specification of detection, in addition to the well accepted reverse transcription polymerase chain reaction (RT-PCR). Methods: The Cov-MS effort developed an isotope dilution (based on QconCAT technology) - liquid chromatography mass spectrometry (LC-MS) method that allows accurate, high throughput measurement of SARS-CoV-2 nucleocapsid (NCAP) protein. It uses Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) technology to enrich and quantify proteotypic peptides of the NCAP protein from trypsin-digested samples from COVID-19 patients. The method is for a bigger part automatable (in terms of the sample preparation, digestion, peptide enrichment and LC-MS measurements). Results: The Cov2MS assay is compatible with most matrices including nasopharyngeal swabs, saliva and blood plasma, with a sensitivity into the attomole range thanks to the peptide enrichment. The latter also reduces dependency upon LC and allows shortening of LC run time, resulting in the analysis of up to 500 samples per day per MS instrument. There is a strong positive correlation between the SISCAPA antigen assay and qPCR detection up to a Cycle threshold (Ct) of 30. Importantly, peptide enrichment allowed detection of NCAP protein in a pooled sample containing a single PCR positive patient mixed with 31 PCR negative samples, without loss in sensitivity. Finally, we also demonstrated that it is feasible to rapidly adapt the method for the incorporation of ever emerging variants of concern (VoC), and even other types of respiratory viruses (e.g. Influenza A and B). Conclusions: In conclusion, since the Cov2MS assay is insensitive to pooling and easily multiplexed, it can provide longitudinal epidemiological monitoring of large numbers of pathogens within a population and can be applied as an early warning system.

Simultaneous detection of respiratory infectious diseases using immunoprecipitation and liquid chromatography-tandem mass spectrometry

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.

Search and investigation of potential peptide agents of interaction between human organism and its microbiome

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.

An improved heamagglutination inhibition test for rapid diagnosis and stereotyping of avian infectious bronchitis virus

Infectious bronchitis virus (IBV) causes an acute, highly contagious viral respiratory disease in poultry with huge economic impact and extremely difficult to control due to its multiple serotypes. The disease could be prevented by rapid diagnosis either molecular or serological test. However, the later test is inexpensive such as heamagglutination inhibition test (HI), but IBV fail to give Heamagglutination (HA) reaction without pretreatment. Therefore, we designed this study for preparation of IBV antigen by treating with different enzymes for HA reaction. IBV local isolates were characterized by SDS-PAGE and RT-PCR. The indigenous isolate HA antigens were treated with different proteolytic enzymes trypsin, neuraminidase and phospholipase C. The prepared antigen were stored at -86oC and used for HA test. All antigen prepared by different enzyme were found to give significant HA titer up to 7 log2 . During stability test antigen prepared by phospholipase C were found most stable up to six month by giving constant 7 log2 HA titer, while neuraminidase induced antigen were stable up to five months (7 log2). Trypsin treated antigen were readily lost its activity from 7 log2 to 2 log2 after two months of incubation. During specificity test all antigens showed specific effect on IBV by eliciting agglutination of RBCs while other avian viruses avian influenza (AI), new castle disease virus (NDV) and infectious bursal disease virus (IBDV) were not affected by enzymatic inductions. Therefore, the antigen prepared by phospholipase C has been found to be more effective for HI test for rapid diagnosis of IBV during infection.

Identification and analysis of exogenous peptides in human blood serum and plasma: Search for potential agents of interaction between the intestinal microbiota and the human body

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.

In covid-19 infection, plasma extra cellular vesicle tissue factor activity does not correlate with d-dimer levels

Objective: Identify a plasma-based activity, or biomarker, that defines the mechanism(s) by which Covid-19 disease triggers excessive coagulation. Introduction: While acute respiratory syndrome is the fundamental feature of severe Covid-19 disease, having a high level of the coagulation biomarker D-dimer upon admission is associated with increased thrombosis and mortality. As such, hospitalized patients are often placed on anticoagulant heparins. How Covid-19 triggers excessive coagulation is unresolved. Sars-CoV-2 infection could expose existing tissue factor (TF) to blood or, via cytokines, induce TF expression on cells that are in direct contact with blood. Extracellular vesicles (EV) are lipid bound microparticles released by all types of healthy and damaged cell and Covid-19 patient plasma EV TF activity has been recently reported. Cellular activation and damage due to SARS-CoV-2 could also release polyanionic nucleic acids and polyphosphates and generate neutrophil extracellular traps as contact surfaces for clot formation. Methods: Study 1. We attempted to identify excessive coagulation pathway activities in Covid-19 plasma-based, Ca++-induced thrombin generation assays. Assays were performed in the absence and presence of selective extrinsic (TF) and intrinsic (contact activation) pathway inhibitors (n=296 plasma samples). D-dimer levels were also determined. In a smaller study, Covid-19 patient samples were collected directly into citrate or citrate plus corn trypsin inhibitor, then processed for analysis. Study 2. We conducted studies to evaluate the extent to which EV TF activity contributes to the Covid-19-associated coagulopathies. Plasma EVs were isolated and EV TF activity determined by the difference in FXa activity in the absence vs presence of anti-TF antibody. D-dimer and tissue factor pathway inhibitor a (TFPIa) antigen levels were measured. Data from 232 samples collected from 96 Covid-19 positive patients and 18 samples from 14 healthy controls were analyzed. For each study analysis, patient samples were organized into groups based on the disease severity outcomes as follows: hospitalization (Hospitalization;n=37);intensive care (ICU;n=16);mechanical ventilation (Ventilation;n=22);or fatality (Deceased;n=22). Result: Study 1. Covid-19 samples showed considerable thrombin generation variability with some samples failing to generate thrombin;pathway selective inhibitors reduced thrombin generation while heparinase treatment increased thrombin generation. Upon analysis, thrombin generation parameters showed no significant correlations to either D-dimer levels or disease severity. Instead, plasma prepared from blood collected directly into corn trypsin inhibitor revealed that contact activation that occurred post-sample collection dominates procoagulant activity. Study 2. Figure 1, shows EV TF activities, D-dimer and TFPIα levels obtained for Covid-19 samples, with data segregated based on disease severity outcomes. Statistically significant difference versus the Hospitalized group are shown. TFPIa levels were highest in heparin IV patients (24.4+1.5 nM) vs Heparin-SQ (12.8+0.9 nM) vs enoxaparin (10.8 +0.7 nM) (p value:<0.0001). It is known that heparin treatment increases circulating TFPIα, however an increase in TFPIα might also further increase circulating TF/FVIIa/XaTFPI inhibitory complex, which would dissociate in citrated plasma, and might account for the increase in EV TF in other studies. Conclusions: Contact activation that occurs post-sample collection is sufficient to obscure endogenous triggers of coagulation, if present, in Covid-19 patients' plasma. D-dimer and TFPIα strongly correlate with disease severity although the latter is likely affected by heparin treatment. The most severe Covid-19 patients with high D-dimer did not show detectible plasma EV TF activity. Plasma EV TF activity does not appear to adequately represent the mechanism responsible for elevated D-dimer levels in Covid-19 cases.

Optimization of Soybean Protein Extraction Using By-Products from NaCl Electrolysis as an Application of the Industrial Symbiosis Concept

Featured ApplicationThis study shows the use of a by-product from the manufacture of a novel antiseptic/disinfectant (HOCl) to obtain a protein isolate from defatted soybean flour (a co-product from the soybean oil industry);an optimization process was carried out to create an industrial symbiosis.Defatted soybean flour is generated during the oil extraction process of soybean, and it has a protein content of ~50%. On the other hand, an alkaline solution of NaOH is produced during the electrolysis process of NaCl in a novel method used to make a potent disinfectant/antiseptic (HOCl). In the present work, we suggest using these two products to produce soy protein isolate (SPI), aiming to create an industrial symbiosis. A Box–Behnken experimental design was executed, and a surface response analysis was performed to optimize temperature, alkaline solution, and time used for SPI extraction. The SPI produced at optimal conditions was then characterized. The experimental results fit well with a second-order polynomial equation that could predict 93.15% of the variability under a combination of 70 °C, alkaline solution 3 (pH 12.68), and 44.7 min of the process. The model predicts a 49.79% extraction yield, and when tested, we obtained 48.30% within the confidence interval (46.66–52.93%). The obtained SPI was comparable in content and structure with a commercial SPI by molecular weight and molecular spectroscopy characterization. Finally, the urease activity (UA) test was negative, indicating no activity for trypsin inhibitor. Based on the functional properties, the SPI is suitable for food applications.

Interfering with Host Proteases in SARS-CoV-2 Entry as a Promising Therapeutic Strategy.

Due to its fast international spread and substantial mortality, the coronavirus disease COVID-19 evolved to a global threat. Since there is currently no causative drug against this viral infection available, science is striving for new drugs and other approaches to treat the new disease. Studies have shown that the cell entry of coronaviruses into host cells takes place through the binding of the viral spike (S) protein to cell receptors. Priming of the S protein occurs via hydrolysis by different host proteases. The inhibition of these proteases could impair the processing of the S protein, thereby affecting the interaction with the host-cell receptors and preventing virus cell entry. Hence, inhibition of these proteases could be a promising strategy for treatment against SARSCoV- 2. In this review, we discuss the current state of the art of developing inhibitors against the entry proteases furin, the transmembrane serine protease type-II (TMPRSS2), trypsin, and cathepsin L.

Treatment of patients with Covid-19 with a high dose of ulinastatin

Currently, there are no specific therapeutic agents available for the treatment of coronavirus disease 2019 (Covid-19). The present study aimed to assess the efficacy of high-dose ulinastatin for the treatment of patients with Covid-19. A total of 12 patients hospitalized with confirmed severe acute respiratory syndrome coronavirus 2 infection were treated with a high dose of ulinastatin alongside standard care. Changes in clinical manifestations, laboratory examinations and chest images were retrospectively analyzed. A total of 10 patients with severe Covid-19 and two patients with moderate Covid-19 received ulinastatin treatment. The average age of the patients was 68.0±11.9 years (age range, 48-87 years). In total, nine of the 12 patients (75.0%) had one or more comorbidities. The most common symptoms on admission were fever (8/12, 66.7%), cough (5/12, 41.7%) and dyspnea (5/12, 41.7%). The percentage of lymphocytes was decreased in 41.7% of patients (5/12) and 58.3% of patients (7/12) had elevated hypersensitive C-reactive protein (CRP) levels (mean, 49.70±77.70 mg/l). The white blood cell levels and the percentage of lymphocytes returned to normal in all of the patients, and CRP was significantly decreased and returned to normal in 83.3% of patients (10/12;mean, 6.87±6.63 mg/l) on day 7 after ulinastatin treatment. Clinical symptoms were relieved synchronously. The peripheral oxygen saturation improved and 66.7% of the patients (8/12) did not require further oxygen therapy 7 days after ulinastatin treatment. No patients required intensive care unit admission or mechanical ventilation. All patients revealed different degrees of absorption of pulmonary lesions after treatment. Compared with the standard care group, ulinastatin treatment significantly prevented illness deterioration. In conclusion, these preliminary data revealed that high-dose ulinastatin treatment was safe and exhibited a potential beneficial effect for patients with Covid-19. [ FROM AUTHOR] Copyright of Experimental & Therapeutic Medicine is the property of Spandidos Publications UK Ltd and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

In vitro interaction of potential antiviral TMPRSS2 inhibitors with human serum albumin and cytochrome P 450 isoenzymes.

The interactions of four sulfonylated Phe(3-Am)-derived inhibitors (MI-432, MI-463, MI-482 and MI-1900) of type II transmembrane serine proteases (TTSP) such as transmembrane protease serine 2 (TMPRSS2) were examined with serum albumin and cytochrome P450 (CYP) isoenzymes. Complex formation with albumin was investigated using fluorescence spectroscopy. Furthermore, microsomal hepatic CYP1A2, 2C9, 2C19 and 3A4 activities in presence of these inhibitors were determined using fluorometric assays. The inhibitory effects of these compounds on human recombinant CYP3A4 enzyme were also examined. In addition, microsomal stability assays (60-min long) were performed using an UPLC-MS/MS method to determine depletion percentage values of each compound. The inhibitors showed no or only weak interactions with albumin, and did not inhibit CYP1A2, 2C9 and 2C19. However, the compounds tested proved to be potent inhibitors of CYP3A4 in both assays performed. Within one hour, 20%, 12%, 14% and 25% of inhibitors MI-432, MI-463, MI-482 and MI-1900, respectively, were degraded. As essential host cell factor for the replication of the pandemic SARS-CoV-2, the TTSP TMPRSS2 emerged as an important target in drug design. Our study provides further preclinical data on the characterization of this type of inhibitors for numerous trypsin-like serine proteases.

Porcine Deltacoronavirus Utilizes Sialic Acid as an Attachment Receptor and Trypsin Can Influence the Binding Activity.

Porcine deltacoronavirus (PDCoV) is a novel coronavirus that causes diarrhea in nursing piglets. Studies showed that PDCoV uses porcine aminopeptidase N (pAPN) as an entry receptor, but the infection of pAPN-knockout cells or pigs with PDCoV revealed that pAPN might be not a critical functional receptor, implying there exists an unidentified receptor involved in PDCoV infection. Herein, we report that sialic acid (SA) can act as an attachment receptor for PDCoV invasion and facilitate its infection. We first demonstrated that the carbohydrates destroyed on the cell membrane using NaIO4 can alleviate the susceptibility of cells to PDCoV. Further study showed that the removal of SA, a typical cell-surface carbohydrate, could influence the PDCoV infectivity to the cells significantly, suggesting that SA was involved in the infection. The results of plaque assay and Western blotting revealed that SA promoted PDCoV infection by increasing the number of viruses binding to SA on the cell surface during the adsorption phase, which was also confirmed by atomic force microscopy at the microscopic level. In in vivo experiments, we found that the distribution levels of PDCoV and SA were closely relevant in the swine intestine, which contains huge amount of trypsin. We further confirmed that SA-binding capacity to PDCoV is related to the pre-treatment of PDCoV with trypsin. In conclusion, SA is a novel attachment receptor for PDCoV infection to enhance its attachment to cells, which is dependent on the pre-treatment of trypsin on PDCoV. This study paves the way for dissecting the mechanisms of PDCoV-host interactions and provides new strategies to control PDCoV infection.

Structural and functional significance of the amino acid differences Val35Thr, Ser46Ala, Asn65Ser, and Ala94Ser in 3C-like proteinases from SARS-CoV-2 and SARS-CoV.

Three dimensional structures of (chymo)trypsin-like proteinase (3CLpro) from SARS-CoV-2 and SARS-CoV differ at 8 positions. We previously found that the Val86Leu, Lys88Arg, Phe134His, and Asn180Lys mutations in these enzymes can change the orientation of the N- and C-terminal domains of 3CLpro relative to each other, which leads to a change in catalytic activity. This conclusion was derived from the comparison of the structural catalytic core in 169 (chymo)trypsin-like proteinases with the serine/cysteine fold. Val35Thr, Ser46Ala, Asn65Ser, Ala94Ser mutations were not included in that analysis, since they are located far from the catalytic tetrad. In the present work, the structural and functional roles of these variable amino acids at positions 35, 46, 65, and 94 in the 3CLpro sequences of SARS-CoV-2 and SARS-CoV have been established using a comparison of the same set of proteinases leading to the identification of new conservative elements. Comparative analysis showed that, in addition to interdomain mobility, which could modulate catalytic activity, the 3CLpro(s) can use for functional regulation an autolytic loop and the unique Asp33-Asn95 region (the Asp33-Asn95 Zone) in the N-terminal domain. Therefore, all 4 analyzed mutation sites are associated with the unique structure-functional features of the 3CLpro from SARS-CoV-2 and SARS-CoV. Strictly speaking, the presented structural results are hypothetical, since at present there is not a single experimental work on the identification and characterization of autolysis sites in these proteases.