New anticoagulant protein from medicinal leech.

The saliva of the medicinal leech contains various anticoagulants. Some of them, such as hirudin, are well known. However, it is reasonable to believe that not all anticoagulant proteins from medicinal leech saliva have been identified. We previously performed a comprehensive study of the transcriptome, genome, and proteome of leech salivary gland cells, which led to the discovery of several previously unknown hypothetical proteins that may have anticoagulant properties. Subsequently, we obtained a series of recombinant proteins and investigated their impact on coagulation in in vitro assays. We identified a previously undescribed protein that exhibited a high ability to suppress coagulation. The His-tagged recombinant protein was expressed in Escherichia coli and purified using metal chelate chromatography. To determine its activity, commonly used coagulation methods were used: activated partial thromboplastin time, prothrombin time, and thrombin inhibition clotting assay. Clotting and chromogenic assays for factor Xa inhibition were performed to evaluate anti-Xa activity. We used recombinant hirudin as a control anticoagulant protein in all experiments. The new protein showed significantly greater inhibition of coagulation than hirudin at the same molar concentrations in the activated partial thrombin time assay. However, hirudin demonstrated better results in the direct thrombin inhibition test, although the tested protein also exhibited the ability to inhibit thrombin. The chromogenic analysis of factor Xa inhibition revealed no activity, whereas the clotting test for factor Xa showed the opposite result. Thus, a new powerful anticoagulant protein has been discovered in the medicinal leech. This protein is homologous to antistatin, with 28 % identical amino acid residues. The recombinant protein was expressed in E. coli. This protein is capable of directly inhibiting thrombin, and based on indirect evidence, other proteases of the blood coagulation cascade have been identified.

Upregulation of YciM Expression Reduces Endotoxin Contamination of Recombinant Proteins Produced in Escherichia coli Cells.

Recombinant proteins produced in Escherichia coli are often contaminated with endotoxins, which can be a serious problem for their further application. One of the possible solutions is the use of modified strains with reduced lipopolysaccharide (LPS) levels. We compared two approaches to engineering such strains. The first commonly known approach was modification of LPS biosynthesis pathway by knocking out seven genes in the E. coli genome. The second approach, which has not been previously used, was to increase expression of E. coli protein YciM. According to the published data, elevated expression of YciM leads to the reduction in the amount of the LpxC enzyme involved in LPS biosynthesis. We investigated the impact of YciM coexpression with eGFP on the content of endotoxins in the purified recombinant eGFP samples. Both approaches provided similar outcomes, i.e., decreased the endotoxin levels in the purified protein samples.

RALF peptides modulate immune response in the moss Physcomitrium patens.

Background: RAPID ALKALINIZATION FACTOR (RALFs) are cysteine-rich peptides that regulate multiple physiological processes in plants. This peptide family has considerably expanded during land plant evolution, but the role of ancient RALFs in modulating stress responses is unknown.Results: Here, we used the moss Physcomitrium patens as a model to gain insight into the role of RALF peptides in the coordination of plant growth and stress response in non-vascular plants. The quantitative proteomic analysis revealed concerted downregulation of M6 metalloprotease and some membrane proteins, including those involved in stress response, in PpRALF1, 2 and 3 knockout (KO) lines. The subsequent analysis revealed the role of PpRALF3 in growth regulation under abiotic and biotic stress conditions, implying the importance of RALFs in responding to various adverse conditions in bryophytes. We found that knockout of the PpRALF2 and PpRALF3 genes resulted in increased resistance to bacterial and fungal phytopathogens, Pectobacterium carotovorum and Fusarium solani, suggesting the role of these peptides in negative regulation of the immune response in P. patens. Comparing the transcriptomes of PpRALF3 KO and wild-type plants infected by F. solani showed that the regulation of genes in the phenylpropanoid pathway and those involved in cell wall modification and biogenesis was different in these two genotypes. Conclusion: Thus, our study sheds light on the function of the previously uncharacterized PpRALF3 peptide and gives a clue to the ancestral functions of RALF peptides in plant stress response.

Microarray Profiling of Vaccination-Induced Antibody Responses to SARS-CoV-2 Variants of Interest and Concern.

Mutations in surface proteins enable emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to escape a substantial fraction of neutralizing antibodies and may thus weaken vaccine-driven immunity. To compare available vaccines and justify revaccination, rapid evaluation of antibody (Ab) responses to currently circulating SARS-CoV-2 variants of interest (VOI) and concern (VOC) is needed. Here, we developed a multiplex protein microarray-based system for rapid profiling of anti-SARS-CoV-2 Ab levels in human sera. The microarray system was validated using sera samples from SARS-CoV-2-free donors and those diagnosed with COVID-19 based on PCR and enzyme immunoassays. Microarray-based profiling of vaccinated donors revealed a substantial difference in anti-VOC Ab levels elicited by the replication-deficient adenovirus vector-base (Sputnik V) and whole-virion (CoviVac Russia COVID-19) vaccines. Whole-virion vaccine-induced Abs showed minor but statistically significant cross-reactivity with the human blood coagulation factor 1 (fibrinogen) and thrombin. However, their effects on blood clotting were negligible, according to thrombin time tests, providing evidence against the concept of pronounced cross-reactivity-related side effects of the vaccine. Importantly, all samples were collected in the pre-Omicron period but showed noticeable responses to the receptor-binding domain (RBD) of the Omicron spike protein. Thus, using the new express Ab-profiling system, we confirmed the inter-variant cross-reactivity of the anti-SARS-CoV-2 Abs and demonstrated the relative potency of the vaccines against new VOCs.

Autoimmune Effect of Antibodies against the SARS-CoV-2 Nucleoprotein.

COVID-19 caused by SARS-CoV-2 is continuing to spread around the world and drastically affect our daily life. New strains appear, and the severity of the course of the disease itself seems to be decreasing, but even people who have been ill on an outpatient basis suffer post-COVID consequences. Partly, it is associated with the autoimmune reactions, so debates about the development of new vaccines and the need for vaccination/revaccination continue. In this study we performed an analysis of the antibody response of patients with COVID-19 to linear and conformational epitopes of viral proteins using ELISA, chip array and western blot with analysis of correlations between antibody titer, disease severity, and complications. We have shown that the presence of IgG antibodies to the nucleoprotein can deteriorate the course of the disease, induce multiple direct COVID-19 symptoms, and contribute to long-term post-covid symptoms. We analyzed the cross reactivity of antibodies to SARS-CoV-2 with own human proteins and showed that antibodies to the nucleocapsid protein can bind to human proteins. In accordance with the possibility of HLA presentation, the main possible targets of the autoantibodies were identified. People with HLA alleles A01:01; A26:01; B39:01; B15:01 are most susceptible to the development of autoimmune processes after COVID-19.

16S rRNA gene sequencing data of the upper respiratory tract microbiome in the SARS-CoV-2 infected patients.

The SARS-CoV-2 pandemic is a big challenge for humanity. The COVID-19 severity differs significantly from patient to patient, and it is important to study the factors protecting from severe forms of the disease. Respiratory microbiota may influence the patient's susceptibility to infection and disease severity due to its ability to modulate the immune system response of the host organism. This data article describes the microbiome dataset from the upper respiratory tract of SARS-CoV-2 positive patients from Russia. This dataset reports the microbial community profile of 335 human nasopharyngeal swabs collected between 2020-05 and 2021-03 during the first and the second epidemic waves. Samples were collected from both inpatients and outpatients in 4 cities of the Russian Federation (Moscow, Kazan, Irkutsk, Nizhny Novgorod) and sequenced using the 16S rRNA gene amplicon sequencing of V3-V4 region. Data contains information about the patient such as age, sex, hospitalization status, percent of damaged lung tissue, oxygen saturation (SpO2), respiratory rate, need for supplemental oxygen, chest computer tomography severity score, SARS-CoV-2 lineage, and also information about smoking and comorbidities. The amplicon sequencing data were deposited at NCBI SRA as BioProject PRJNA751478.

A vast pool of lineage-specific microproteins encoded by long non-coding RNAs in plants.

Pervasive transcription of eukaryotic genomes results in expression of long non-coding RNAs (lncRNAs) most of which are poorly conserved in evolution and appear to be non-functional. However, some lncRNAs have been shown to perform specific functions, in particular, transcription regulation. Thousands of small open reading frames (smORFs, <100 codons) located on lncRNAs potentially might be translated into peptides or microproteins. We report a comprehensive analysis of the conservation and evolutionary trajectories of lncRNAs-smORFs from the moss Physcomitrium patens across transcriptomes of 479 plant species. Although thousands of smORFs are subject to substantial purifying selection, the majority of the smORFs appear to be evolutionary young and could represent a major pool for functional innovation. Using nanopore RNA sequencing, we show that, on average, the transcriptional level of conserved smORFs is higher than that of non-conserved smORFs. Proteomic analysis confirmed translation of 82 novel species-specific smORFs. Numerous conserved smORFs containing low complexity regions (LCRs) or transmembrane domains were identified, the biological functions of a selected LCR-smORF were demonstrated experimentally. Thus, microproteins encoded by smORFs are a major, functionally diverse component of the plant proteome.

Distinct types of short open reading frames are translated in plant cells.

Genomes contain millions of short (<100 codons) open reading frames (sORFs), which are usually dismissed during gene annotation. Nevertheless, peptides encoded by such sORFs can play important biological roles, and their impact on cellular processes has long been underestimated. Here, we analyzed approximately 70,000 transcribed sORFs in the model plant Physcomitrella patens (moss). Several distinct classes of sORFs that differ in terms of their position on transcripts and the level of evolutionary conservation are present in the moss genome. Over 5000 sORFs were conserved in at least one of 10 plant species examined. Mass spectrometry analysis of proteomic and peptidomic data sets suggested that tens of sORFs located on distinct parts of mRNAs and long noncoding RNAs (lncRNAs) are translated, including conserved sORFs. Translational analysis of the sORFs and main ORFs at a single locus suggested the existence of genes that code for multiple proteins and peptides with tissue-specific expression. Functional analysis of four lncRNA-encoded peptides showed that sORFs-encoded peptides are involved in regulation of growth and differentiation in moss. Knocking out lncRNA-encoded peptides resulted in a decrease of moss growth. In contrast, the overexpression of these peptides resulted in a diverse range of phenotypic effects. Our results thus open new avenues for discovering novel, biologically active peptides in the plant kingdom.

Discovery of novel antimicrobial peptides: A transcriptomic study of the sea anemone Cnidopus japonicus.

As essential conservative component of the innate immune systems of living organisms, antimicrobial peptides (AMPs) could complement pharmaceuticals that increasingly fail to combat various pathogens exhibiting increased resistance to microbial antibiotics. Among the properties of AMPs that suggest their potential as therapeutic agents, diverse peptides in the venoms of various predators demonstrate antimicrobial activity and kill a wide range of microorganisms. To identify potent AMPs, the study reported here involved a transcriptomic profiling of the tentacle secretion of the sea anemone Cnidopus japonicus. An in silico search algorithm designed to discover toxin-like proteins containing AMPs was developed based on the evaluation of the properties and structural peculiarities of amino acid sequences. The algorithm revealed new proteins of the anemone containing antimicrobial candidate sequences, and 10 AMPs verified using high-throughput proteomics were synthesized. The antimicrobial activity of the candidate molecules was experimentally estimated against Gram-positive and -negative bacteria. Ultimately, three peptides exhibited antimicrobial activity against bacterial strains, which suggests that the method can be applied to reveal new AMPs in the venoms of other predators as well.

Corrigendum: Interaction of Bacteroides fragilis Toxin with Outer Membrane Vesicles Reveals New Mechanism of Its Secretion and Delivery.

[This corrects the article on p. 2 in vol. 7, PMID: 28144586.].

Interaction of Bacteroides fragilis Toxin with Outer Membrane Vesicles Reveals New Mechanism of Its Secretion and Delivery.

The only recognized virulence factor of enterotoxigenic Bacteroides fragilis (ETBF) that accompanies bloodstream infections is the zinc-dependent non-lethal metalloprotease B. fragilis toxin (BFT). The isolated toxin stimulates intestinal secretion, resulting in epithelial damage and necrosis. Numerous publications have focused on the interrelation of BFT with intestinal inflammation and colorectal neoplasia, but nothing is known about the mechanism of its secretion and delivery to host cells. However, recent studies of gram-negative bacteria have shown that outer membrane vesicles (OMVs) could be an essential mechanism for the spread of a large number of virulence factors. Here, we show for the first time that BFT is not a freely secreted protease but is associated with OMVs. Our findings indicate that only outer surface-exposed BFT causes epithelial cell contact disruption. According to our in silico models confirmed by Trp quenching assay and NMR, BFT has special interactions with outer membrane components such as phospholipids and is secreted during vesicle formation. Moreover, the strong cooperation of BFT with polysaccharides is similar to the behavior of lectins. Understanding the molecular mechanisms of BFT secretion provides new perspectives for investigating intestinal inflammation pathogenesis and its prevention.

Characterization of secretomes provides evidence for adipose-derived mesenchymal stromal cells subtypes.

INTRODUCTION: This study was aimed at deciphering the secretome of adipose-derived mesenchymal stromal cells (ADSCs) cultured in standard and hypoxic conditions to reveal proteins, which may be responsible for regenerative action of these cells. METHODS: Human ADSCs were isolated from 10 healthy donors and cultured for 3-4 passages. Cells were serum deprived and cell purity was assessed using multiple cell surface markers. Conditioned media was collected and analyzed using LC-MS with a focus on characterizing secreted proteins. RESULTS: Purity of the ADSC assessed as CD90+/CD73+/CD105+/CD45-/CD31- cells was greater than 99 % and viability was greater than 97 %. More than 600 secreted proteins were detected in conditioned media of ADSCs. Of these 100 proteins were common to all cultures and included key molecules involved in tissue regeneration such as collagens and collagen maturation enzymes, matrix metalloproteases, matricellular proteins, macrophage-colony stimulating factor and pigment epithelium derived factor. Common set of proteins also included molecules, which contribute to regenerative processes but were not previously associated with ADSCs. These included olfactomedin-like 3, follistatin-like 1 and prosaposin. In addition, ADSCs from the different subjects secreted proteins, which were variable between different cultures. These included proteins with neurotrophic activities, which were not previously associated with ADSCs, such as mesencephalic astrocyte-derived neurotrophic factor, meteorin and neuron derived neurotrophic factor. Hypoxia resulted in secretion of 6 proteins, the most prominent included EGF-like repeats and discoidin I-like domains 3, adrenomedullin and ribonuclease 4 of RNase A family. It also caused the disappearance of 8 proteins, including regulator of osteogenic differentiation cartilage-associated protein. CONCLUSIONS: Human ADSCs with CD90+/CD73+/CD105+/CD45-/CD31-/PDGFRß+/NG2+/CD146+(-) immunophenotype secrete a large array of proteins, the most represented group is comprised of extracellular matrix components. Number of secreted proteins is largely unaffected by prolonged hypoxia. Variability in the secretion of several proteins from cultured ADSCs of individual subjects suggests that these cells exist as a heterogeneous population containing functionally distinct subtypes, which differ in numbers between donors.

Recombinant fragilysin isoforms cause E-cadherin cleavage of intact cells and do not cleave isolated E-cadherin.

The fragilysin (BFT) is a protein secreted by enterotoxigenic Bacteroides fragilis strains. BFT contains zinc-binding motif which was found in the metzincins family of metalloproteinases. In this study, we generated three known recombinant isoforms of BFT using Escherichia coli, tested their activity and examined whether E-cadherin is a substrate for BFTs. BFT treatment of HT-29 cells induced endogenous E-cadherin cleavage, and this BFT activity requires the native structure of zinc-binding motif. At the same time recombinant BFTs did not cleave recombinant E-cadherin or E-cadherin in isolated cell fractions. It indicates that E-cadherin may be not direct substrate for BFT. We also detected and identified proteins released into the cultural medium after HT-29 cells treatment with BFT. The role of these proteins in pathogenesis and cell response to BFT remains to be determined.

Complete Genome Sequence of an Enterotoxigenic Bacteroides fragilis Clinical Isolate.

Here we present the complete genome sequence of Bacteroides fragilis isolate BOB25. It is an enterotoxigenic isolate that was obtained from a stool sample of a patient with dysbiosis.