The division protein FtsZ interacts with the small heat shock protein IbpA in Acholeplasma laidlawii.

Small heat shock proteins (sHSPs) control the proteins stability in the cell preventing their irreversible denaturation. While many mycoplasmas possess the sHSP gene in the genome, Acholeplasma laidlawii is the only mycoplasma capable of surviving in the environment. Here we report that the sHSP IbpA directly interacts with the key division protein FtsZ in A. laidlawii, representing the first example of such interaction in prokaryotes. FtsZ co-immunoprecipitates with IbpA from A. laidlawii crude extract and in vitro binds IbpA with KD ~ 1 µM. Proteins co-localize in the soluble fraction of the cell at 30-37 °C and in the non-soluble fraction after 1 h exposition to cold stress (4 °C). Under heat shock conditions (42 °C) the amount of FtsZ decreases and the protein remains in both soluble and non-soluble fractions. Furthermore, in vitro, FtsZ co-elutes with IbpAHis6 from A. laidlawii crude extract at any temperatures from 4 to 42 °C, with highest yield at 42 °C. Moreover, in vitro FtsZ retains its GTPase activity in presence of IbpA, and the filaments and bundles formation seems to be even improved by sHSP at 30-37 °C. At extreme temperatures, either 4 or 42 °C, IbpA facilitates FtsZ polymerization, although filaments under 4 °C appears shorter and with lower density, while at 42 °C IbpA sticks around the bundles, preventing their destruction by heat. Taken together, these data suggest that sHSP IbpA in A. laidlawii contributes to the FtsZ stability control and may be assisting appropriate cell division under unfavorable conditions.

Inhibitory effects of specific combination of natural compounds against SARS-CoV-2 and its Alpha, Beta, Gamma, Delta, Kappa, and Mu variants.

Despite vaccine availability, the global spread of COVID-19 continues, largely facilitated by emerging SARS-CoV-2 mutations. Our earlier research documented that a specific combination of plant-derived compounds can inhibit SARS-CoV-2 binding to its ACE2 receptor and controlling key cellular mechanisms of viral infectivity. In this study, we evaluated the efficacy of a defined mixture of plant extracts and micronutrients against original SARS-CoV-2 and its Alpha, Beta, Gamma, Delta, Kappa, and Mu variants. The composition containing vitamin C, N-acetylcysteine, resveratrol, theaflavin, curcumin, quercetin, naringenin, baicalin, and broccoli extract demonstrated a highest efficacy by inhibiting the receptor-binding domain (RBD) binding of SARS-CoV-2 to its cellular ACE2 receptor by 90%. In vitro exposure of test pseudo-typed variants to this formula for 1 h before or simultaneously administrated to human pulmonary cells resulted in up to 60% inhibition in their cellular entry. Additionally, this composition significantly inhibited other cellular mechanisms of viral infectivity, including the activity of viral RdRp, furin, and cathepsin L. These findings demonstrate the efficacy of natural compounds against SARS-CoV-2 including its mutated forms through pleiotropic mechanisms. Our results imply that simultaneous inhibition of multiple mechanisms of viral infection of host cells could be an effective strategy to prevent SARS-CoV-2 infection.

Peptidome: Chaos or Inevitability.

Thousands of naturally occurring peptides differing in their origin, abundance and possible functions have been identified in the tissue and biological fluids of vertebrates, insects, fungi, plants and bacteria. These peptide pools are referred to as intracellular or extracellular peptidomes, and besides a small proportion of well-characterized peptide hormones and defense peptides, are poorly characterized. However, a growing body of evidence suggests that unknown bioactive peptides are hidden in the peptidomes of different organisms. In this review, we present a comprehensive overview of the mechanisms of generation and properties of peptidomes across different organisms. Based on their origin, we propose three large peptide groups-functional protein "degradome", small open reading frame (smORF)-encoded peptides (smORFome) and specific precursor-derived peptides. The composition of peptide pools identified by mass-spectrometry analysis in human cells, plants, yeast and bacteria is compared and discussed. The functions of different peptide groups, for example the role of the "degradome" in promoting defense signaling, are also considered.

Thymodepressin-Unforeseen Immunosuppressor.

The paper summarizes the available information concerning the biological properties and biomedical applications of Thymodepressin. This synthetic peptide drug displays pronounced immunoinhibitory activity across a wide range of conditions in vitro and in vivo. The history of its unforeseen discovery is briefly reviewed, and the current as well as potential expansion areas of medicinal practice are outlined. Additional experimental evidence is obtained, demonstrating several potential advantages of Thymodepressin over another actively used immunosuppressor drug, cyclosporin A.

Delta Sleep-Inducing Peptide Recovers Motor Function in SD Rats after Focal Stroke.

Background and Objectives: Mutual effect of the preliminary and therapeutic intranasal treatment of SD rats with DSIP (8 days) on the outcome of focal stroke, induced with intraluminal middle cerebral occlusion (MCAO), was investigated. Materials and Methods: The groups were the following: MCAO + vehicle, MCAO + DSIP, and SHAM-operated. DSIP or vehicle was applied nasally 60 (±15) minutes prior to the occlusion and for 7 days after reperfusion at dose 120 µg/kg. The battery of behavioral tests was performed on 1, 3, 7, 14, and 21 days after MCAO. Motor coordination and balance and bilateral asymmetry were tested. At the end of the study, animals were euthanized, and their brains were perfused, serial cryoslices were made, and infarction volume in them was calculated. Results: Although brain infarction in DSIP-treated animals was smaller than in vehicle-treated animals, the difference was not significant. However, motor performance in the rotarod test significantly recovered in DSIP-treated animals. Conclusions: Intranasal administration of DSIP in the course of 8 days leads to accelerated recovery of motor functions.

Intranasal and intrapulmonary vaccination with an M protein-deficient respiratory syncytial virus (RSV) vaccine improves clinical signs and reduces viral replication in infant baboons after an RSV challenge infection.

Respiratory syncytial virus (RSV) is the major viral respiratory pathogen for human infants and children. Despite a severe global burden incurred by annual RSV epidemics, there is no licensed RSV vaccine. We have developed an RSV vaccine from a human RSV strain from which the gene for the viral M protein has been deleted ("Mnull RSV"). RSV infects airway cells and produces each of its proteins. The M protein is responsible for reassembling the various other synthesized viral proteins into new, intact virus. In the absence of the M protein, therefore, reassembly does not occur, and the Mnull RSV does not replicate. We vaccinated 2-week old infant baboons with Mnull RSV either intranasally (IN) or directly into the lung (intratracheal, or IT), then infected these animals by inoculating human RSV directly into the lung. IN vaccination induced inconsistent serum RSV neutralizing antibody (NA) responses, but provided moderate reductions in respiratory rates, overall signs of illness and viral replication in bronchoalveolar lavage (BAL) fluid following infection. Intratracheal vaccination induced much stronger RSV NA responses, which persisted for at least 4-6 months. Following RSV infection, animals vaccinated by the IT route had much greater reductions in tachypnea and work of breathing than animals vaccinated IN, and had undetectable amounts of virus in BAL fluids. These results support the further development of IT Mnull RSV vaccination to reduce the impact of RSV infection in humans.

DSIP-Like KND Peptide Reduces Brain Infarction in C57Bl/6 and Reduces Myocardial Infarction in SD Rats When Administered during Reperfusion.

A structural analogue of the DSIP, peptide KND, previously showed higher detoxification efficacy upon administration of the cytotoxic drug cisplatin, compared to DSIP. DSIP and KND were investigated using the model of acute myocardial infarction in male SD rats and the model of acute focal stroke in C57Bl/6 mice. A significant decrease in the myocardial infarction area was registered in KND-treated animals relative to saline-treated control animals (19.1 ± 7.3% versus 42.1 ± 9.2%). The brain infarction volume was significantly lower in animals intranasally treated with KND compared to the control saline-treated animals (7.4 ± 3.5% versus 12.2 ± 5.6%). Injection of KND in the first minute of reperfusion in the models of myocardial infarction and cerebral stroke reduced infarction of these organs, indicating a pronounced cardioprotective and neuroprotective effect of KND and potentiality for the treatment of ischemia-reperfusion injuries after transient ischemic attacks on the heart and brain, when administered during the reperfusion period. A preliminary pilot study using the model of myocardial infarction with the administration of DSIP during occlusion, and the model of cerebral stroke with the administration of KND during occlusion, resulted in 100% mortality in animals. Thus, in the case of ischemia-reperfusion injuries of the myocardium and the brain, use of these peptides is only possible during reperfusion.

Inhibition of ACE2 Expression by Ascorbic Acid Alone and its Combinations with Other Natural Compounds.

BACKGROUND: Angiotensin-converting enzyme II or ACE2 is an integral membrane protein present on many types of cells, including vascular endothelial cells and lung alveolar epithelial cells. This receptor serves as the entry point for SARS-coronaviruses (SARS-CoVs), including a novel coronavirus 2019-nCoV. Limited availability of these receptors can thwart cellular entry of this virus. METHODS: We tested the effects of ascorbic acid (vitamin C) on cellular expression of ACE2 at the protein and RNA levels in human small alveolar epithelial cells and microvascular endothelial cells. In addition, we investigated whether combinations of ascorbic acid with other natural compounds can affect ACE2 expression. RESULTS: The results show that ascorbic acid itself has moderate but consistent lowering effects on ACE2 expression at the cellular, protein, and RNA levels. Some natural compounds were effective in lowering ACE2 cellular expression, with the highest inhibitory effects observed for baicalin (75%) and theaflavin (50%). Significantly, combinations of these and other test compounds with ascorbic acid further decreased ACE2 expression. The highest impact of ascorbate on ACE2 expression was noted when combined with theaflavin (decrease from 50% to 87%), zinc (decrease from 22% to 62%), and with 10-undecenoic acid (from 18% to 53%). Ascorbic acid showed moderate additional benefits in decreasing ACE2 expression when combined with N-acetylcysteine and baicalin. CONCLUSION: Our study provides valuable experimental confirmation of the efficacy of micronutrients in controlling ACE2 expression-the coronavirus cellular "entry" point. It further validates the importance of nutrient interactions in various aspects of cellular metabolism and in considering potential therapeutic applications of nutrient-based approaches. The study shows that ascorbic acid and its combination with some natural compounds could be included in developing preventive and therapeutic approaches toward the current pandemic.

Possible role of small secreted peptides (SSPs) in immune signaling in bryophytes.

Plants utilize a plethora of peptide signals to regulate their immune response. Peptide ligands and their cognate receptors involved in immune signaling share common motifs among many species of vascular plants. However, the origin and evolution of immune peptides is still poorly understood. Here, we searched for genes encoding small secreted peptides in the genomes of three bryophyte lineages-mosses, liverworts and hornworts-that occupy a critical position in the study of land plant evolution. We found that bryophytes shared common predicted small secreted peptides (SSPs) with vascular plants. The number of SSPs is higher in the genomes of mosses than in both the liverwort Marchantia polymorpha and the hornwort Anthoceros sp. The synthetic peptide elicitors-AtPEP and StPEP-specific for vascular plants, triggered ROS production in the protonema of the moss Physcomitrella patens, suggesting the possibility of recognizing peptide ligands from angiosperms by moss receptors. Mass spectrometry analysis of the moss Physcomitrella patens, both the wild type and the Δcerk mutant secretomes, revealed peptides that specifically responded to chitosan treatment, suggesting their role in immune signaling.

Vitamin C inhibits the calcification process in human vascular smooth muscle cells.

Vascular calcification is a pathophysiological process that is associated with coronary atherosclerosis, and is a prognostic marker of cardiovascular morbidity and mortality. The process of arterial wall calcification is triggered and accompanied by pro-osteogenic phenotypical modifications of resident smooth muscle cells (SMC). Vitamin C (ascorbic acid) is an essential nutrient required to support the production of extracellular matrix components and maintain healthy connective tissue. In this study we investigated the effects of ascorbic acid on cultured human aortic SMC calcification process in vitro. Our results demonstrate that supplementation of SMC cultures with ascorbic acid significantly decreases calcium accumulation in SMC-produced and -deposited extracellular matrix. These effects were accompanied by a reduction in cell-associated alkaline phosphatase activity. Significantly, treatment of cultured SMC with HMG-CoA reductase inhibitors, simvastatin and mevastatin, resulted in increased calcium accumulation in cultured SMC. These effects were blocked by ascorbic acid. The effects of ascorbic acid supplementation on pro-osteogenic modification were compared in different cell types. Analysis of the expression of osteogenic markers in cultured human aortic SMC, human dermal fibroblasts and immortalized human osteoblasts (hFOB) revealed cell type-specific responses to ascorbate supplementation. We conclude that ascorbic acid supplementation can actively and beneficially interfere with the process of arterial wall calcification, with potential implications for human health.

Novel platform for the preparation of synthetic orally active peptidomimetics with hemoregulating activity. II. Hemosuppressor activity of 2,5-diketopiperazine-based cyclopeptides.

The novel chemical platform formed by the branched piperazine-2,5-dione peptide derivatives (2,5-diketopiperazines) for creating non-invasive biologically active peptidomimetics has been developed. A successful application of this approach to orally available hemostimulatory peptidomimetics was demonstrated for all-L cyclopeptide from the Glu-Trp-peptide family. In the 1980s, we have separated and characterized a number of dipeptides from the thymus homogenate. The most active peptide Glu-Trp has been studied and developed into the immunostimulating drug Thymogen. The inversion of the amino acid optical form endows the dipeptides with suppressor properties: D-Glu-D-Trp-OH and D-Glu-(D-Trp)-OH, inhibit proliferation of hemopoietic progenitors in the intact bone marrow. Based on the peptide D-Glu-(D-Trp)-OH, the new immunosuppressive drug Thymodepressin has been prepared. In this work, we applied the platform mentioned above to the design and synthesis of orally active hemosuppressive Thymodepressin® analogs. The novel data for the hemosuppressor activity of the dipeptide D-Glu(D-Trp-OH)-OH and its cyclopeptide analogs are discussed. A new example is presented of a rare phenomenon when enantiomeric molecules demonstrate reciprocal (i.e., opposite) biological activities.

Identification of Antimicrobial Peptides from Novel Lactobacillus fermentum Strain.

Antimicrobial peptides (AMPs) are natural antagonistic tools of many bacteria and are considered as attractive antimicrobial agents for the treatment of bacteria with multidrug resistance. Lactic acid bacteria from the gastrointestinal tract of animals and human produce various AMPs inhibiting the growth of pathogens. Here we report the isolation and identification of novel Lactobacillus fermentum strain HF-D1 from the human gut producing AMPs which prevents the growth of P. aeruginosa and S. marcescens. The active fraction of peptides was obtained from the culture liquid by precipitation at 80% saturation of ammonium sulphate. For peptides identification, the precipitate was treated with guanidine hydrochloride to desorb from proteins, separated with ultrafiltration on spin columns with 10,000 MWCO, desalted with a reversed-phase chromatography and subjected to LC-MS/MS analysis. The in silico analysis of the identified 1111 peptides by using ADAM, CAMPR3 and AMPA prediction servers led to identification of the linear peptide with highly probable antimicrobial activity and further investigation of its antibacterial activity mechanism is promising. By using the dereplication algorithm, the peptide highly similar to non-ribosomal cyclic AMPs originally isolated from Staphylococcus epidermidis has been identified. This indicates that L. fermentum HF-D1 represents a novel strain producing antimicrobial peptides targeting P. aeruginosa and S. marcescens.

Maternal immunization with RSV fusion glycoprotein vaccine and substantial protection of neonatal baboons against respiratory syncytial virus pulmonary challenge.

Globally, human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory infection in infants and young children. There are no licensed vaccines despite the high worldwide disease burden. RSV fusion (F) glycoprotein vaccine is the most advanced candidate for maternal immunization. In this report, a baboon maternal immunization model was used to assess the immunogenicity and protection of infants against pulmonary challenge with human RSV/A. Vaccination in the third trimester produced high anti-RSV F IgG titers and virus-neutralizing antibodies. Infants born to immunized females had high levels of serum RSV antibodies that were comparable to maternal levels at birth and persisted for over 50 days with a half-life of 14-24 days. Furthermore, infants from immunized females and challenged with RSV/A were healthy, developed less severe disease, and had only mild pulmonary inflammatory changes whereas infants born to non-vaccinated females developed more severe disease with marked to moderate interstitial pneumonia, pulmonary edema, and bronchiolar obstruction. These results support the further development of the RSV F vaccine for maternal immunization.

Challenges of the Human Proteome Project: 10-Year Experience of the Russian Consortium.

This manuscript collects all the efforts of the Russian Consortium, bottlenecks revealed in the course of the C-HPP realization, and ways of their overcoming. One of the main bottlenecks in the C-HPP is the insufficient sensitivity of proteomic technologies, hampering the detection of low- and ultralow-copy number proteins forming the "dark part" of the human proteome. In the frame of MP-Challenge, to increase proteome coverage we suggest an experimental workflow based on a combination of shotgun technology and selected reaction monitoring with two-dimensional alkaline fractionation. Further, to detect proteins that cannot be identified by such technologies, nanotechnologies such as combined atomic force microscopy with molecular fishing and/or nanowire detection may be useful. These technologies provide a powerful tool for single molecule analysis, by analogy with nanopore sequencing during genome analysis. To systematically analyze the functional features of some proteins (CP50 Challenge), we created a mathematical model that predicts the number of proteins differing in amino acid sequence: proteoforms. According to our data, we should expect about 100 000 different proteoforms in the liver tissue and a little more in the HepG2 cell line. The variety of proteins forming the whole human proteome significantly exceeds these results due to post-translational modifications (PTMs). As PTMs determine the functional specificity of the protein, we propose using a combination of gene-centric transcriptome-proteomic analysis with preliminary fractionation by two-dimensional electrophoresis to identify chemically modified proteoforms. Despite the complexity of the proposed solutions, such integrative approaches could be fruitful for MP50 and CP50 Challenges in the framework of the C-HPP.

Idiotypic and anti-idiotypic antibodies against polycyclic aromatic hydrocarbon in human blood serum are new biomarkers of lung cancer.

Evaluation of epidemiologic risk factor in relation to lung cancer invoked by polycyclic aromatic hydrocarbons has been inconsistent. To address this issue, we conducted a prospective evaluation of new biomarkers for lung cancer classified according levels of idiotypic and anti-idiotypic antibodies against polycyclic aromatic hydrocarbons in human blood serum. The blood serums of 557 lung cancer patients and 227 healthy donors were analysis of these antibodies by ELISA. Collected data were regrouped and analyzed by gender, smoking, and age as predictors of risk lung cancer factors. Also, the data of lung cancer patients were additionally analyzed by stages and types of lung cancer, surgery, and chemotherapy. It was suggested to use ratio of idiotypic and anti-idiotypic antibodies rather than distinguish level each of them separately. The ratio of levels in healthy people was 3.32 times higher than in lung cancer patients. This approach gave more precisely results and great prognostic value. The logistic regression model (AUC = 0.9) and neural networks (AUC = 0.95) were built to compare lung cancer patients and healthy donors by predictors. The ELISA data of 49 people random sampled from the originally ELISA data and ELISA data of 52 coal miners as a group of lung cancer risk were confirmed logistic regression model. So, suggested idiotypic and anti-idiotypic antibodies against polycyclic aromatic hydrocarbons were not only shown difference between healthy donors and lung cancer patients also elicited group of lung cancer risk among healthy people.

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.