Neuroprotective Effects of Noncanonical PAR1 Agonists on Cultured Neurons in Excitotoxicity.

Serine proteases regulate cell functions through G protein-coupled protease-activated receptors (PARs). Cleavage of one peptide bond of the receptor amino terminus results in the formation of a new N-terminus ("tethered ligand") that can specifically interact with the second extracellular loop of the PAR receptor and activate it. Activation of PAR1 by thrombin (canonical agonist) and activated protein C (APC, noncanonical agonist) was described as a biased agonism. Here, we have supposed that synthetic peptide analogs to the PAR1 tethered ligand liberated by APC could have neuroprotective effects like APC. To verify this hypothesis, a model of the ischemic brain impairment based on glutamate (Glu) excitotoxicity in primary neuronal cultures of neonatal rats has been used. It was shown that the nanopeptide NPNDKYEPF-NH2 (AP9) effectively reduced the neuronal death induced by Glu. The influence of AP9 on cell survival was comparable to that of APC. Both APC and AP9 reduced the dysregulation of intracellular calcium homeostasis in cultured neurons induced by excitotoxic Glu (100 µM) or NMDA (200 µM) concentrations. PAR1 agonist synthetic peptides might be noncanonical PAR1 agonists and a basis for novel neuroprotective drugs for disorders related to Glu excitotoxicity such as brain ischemia, trauma and some neurodegenerative diseases.

The role of non-commercial cyprinids in maintenance and spread of the opisthorchiasis focus in the middle Ob River basin (Tomsk region, Russia).

The study assessed the role of non-commercial cyprinid species in maintaining the opisthorchiasis focus in the middle Ob River basin, Tomsk region, Russia. The source of O. felineus infection for humans and carnivores is fish of the family Cyprinidae. This is the most numerous family, 14 species live in the middle Ob River basin, which includes 6 commercial species and 8 non-commercial species. This study aimed to investigate the current situation on infestation of non-commercial cyprinids with O. felineus metacercariae and their role in maintaining and spreading the natural focus of opisthorchiasis in the middle Ob River basin. We investigated 4 non-commercial species (tench, sunbleak, common bleak, gudgeon), which are highly abundant in water bodies. Tench, common bleak and gudgeon are objects of amateur fishing. These species are traditionally included in the diet of the local population. Opisthorchis felineus metacercariae were recorded in muscles of all the examined fish species. The identification of metacercariae was confirmed by morphological methods and PCR diagnostics. Tench and sunbleak are the main sources of opisthorchiasis infection in the floodplain lakes of the Ob River basin (the prevalence of tench infection is 89.3% and mean intensity of infection is 11.2 metacercariae per fish, the prevalence of sunbleak infection is 50.9% and the intensity of infection is 4.25 metacercariae per fish). The prevalence of infection in the introduced common bleak from the rivers of the middle Ob River basin is rapidly increasing from 2.4 (2016-2018) to 37.5% (2020-2021), and mean intensity of infection increased from 1 to 4.15. The epizootic state of water bodies in the middle Ob River basin remains unfavorable in relation to opisthorchiasis. Tench, common bleak and sunbleak, along with ide and dace, are the main source of infection for humans and animals, which is evidenced by high infection with Opisthorchis felineus metacercariae in these numerous fish species. They pose the greatest danger of infection of people and animals with opisthorchiasis. These species should be included in the campaign to avoid raw and poorly cooked fish in the diet. In addition, such species as roach, bream and sunbleak also pose the danger of infection with opisthorchiasis, but to a lesser extent.

An Update of Orthopoxvirus Molecular Evolution.

Although variola virus (VARV) has been eradicated through widespread vaccination, other orthopoxviruses pathogenic for humans circulate in nature. Recently, new orthopoxviruses, including some able to infect humans, have been found and their complete genomes have been sequenced. Questions about the orthopoxvirus mutation rate and the emergence of new threats to humankind as a result of the evolution of circulating orthopoxviruses remain open. Based on contemporary data on ancient VARV DNA and DNA of new orthopoxvirus species, an analysis of the molecular evolution of orthopoxviruses was carried out and the timescale of their emergence was estimated. It was calculated that the orthopoxviruses of the Old and New Worlds separated approximately 40,000 years ago; the recently discovered Akhmeta virus and Alaskapox virus separated from other orthopoxviruses approximately 10,000-20,000 years ago; the rest of modern orthopoxvirus species originated from 1700 to 6000 years ago, with the exception of VARV, which emerged in approximately 300 AD. Later, there was a separation of genetic variants of some orthopoxvirus species, so the monkeypox virus West African subtype originated approximately 600 years ago, and the VARV minor alastrim subtype emerged approximately 300 years ago.

Species-Specific Proteins in the Oviducts of Snail Sibling Species: Proteotranscriptomic Study of Littorina fabalis and L. obtusata.

Genus Littorina subgenus Neritrema (Mollusca, Caenogastropoda) includes the "obtusata" group of closely related species (Littorina obtusata and L. fabalis). The anatomy of the adult reproductive system (pallial oviduct) is the only reliable feature used for species identification in females of these species. Reproductive system anatomy and reproduction-associated proteins often diverge between sibling species. Despite being of high evolutionary interest, the molecular basis of this divergence remains poorly understood. We performed proteotranscriptomic comparison of oviducts of L. obtusata and L. fabalis by RNA-seq on Illumina HiSeq 2500 and two-dimensional protein electrophoresis (2D DIGE) with MS/MS identification of the species-specific proteins. The interspecies differences in the oviduct were associated with (1) metabolic proteins reflecting overall physiological differences between L. obtusata and L. fabalis, (2) receptor proteins, and (3) transcripts related to transposable elements (TEs). Various receptors identified may recognize a wide variety of ligands from pathogen-associated molecular patterns to specific carbohydrates on the sperm surface. Therefore, these may participate in immune defense as well as in sperm storage and regulation. Species-specificity of multiple TE sequences (coding for reverse transcriptase and ribonuclease H) may indicate the important role of these genomic elements in the Littorina species divergence, which has not been reported previously.

Comparative Analysis of Proteins of Functionally Different Body Parts of the Fish Parasites Triaenophorus nodulosus and Triaenophorus crassus.

PURPOSE: Studies of proteins expressed in the morphological structures of the parasite are necessary for elucidating the biological functions of unknown proteins and understanding the molecular basis of parasitism. The research aim was to investigate the spatial distribution of major proteins in scolex, immature and gravid proglottids of Triaenophorus nodulosus and Triaenophorus crassus. METHODS: Protein extracts of worm body parts were analyzed using two-dimensional difference gel electrophoresis (DIGE) and mass spectrometry. RESULTS: Comparison of the protein repertoire of the adult worm and the encysted plerocercoid revealed differences between the worm body parts, life stages and parasite species. The content of proteins associated with the cytoskeleton and musculature (actin, myosin regulatory light chain, and tropomyosin 2) decreased with distance from the scolex. Mature proglottids were rich in transforming growth factor-beta-induced protein, propionyl-CoA carboxylase, glutamate dehydrogenase and beta-tubulin. Interspecific variation in T. nodulosus and T. crassus was found in the content of the myosin, paramyosin, the major vault protein and an uncharacterized secreted protein TRINITY_DN24645. Differential expression of TRINITY_DN24645, paramyosin and tropomyosin 2 was found between plerocercoids and adult worms. CONCLUSION: The present study provides the first characteristics of the spatial distribution of the major proteins of T. crassus and T. nodulosus. Comparison of the protein composition of plerocercoids and adult parasites indicates a significant similarity in the proteomic organization of Triaenophorus sp. in the second intermediate and final hosts. The gradual change in the morphological organization of tapeworms in the longitudinal direction coincided with the expression of some structural and metabolic proteins.

Premating barriers in young sympatric snail species.

Sympatric coexistence of recently diverged species raises the question of barriers restricting the gene flow between them. Reproductive isolation may be implemented at several levels, and the weakening of some, e.g. premating, barriers may require the strengthening of the others, e.g. postcopulatory ones. We analysed mating patterns and shell size of mates in recently diverged closely related species of the subgenus Littorina Neritrema (Littorinidae, Caenogastropoda) in order to assess the role of premating reproductive barriers between them. We compared mating frequencies observed in the wild with those expected based on relative densities using partial canonical correspondence analysis. We introduced the fidelity index (FI) to estimate the relative accuracy of mating with conspecific females and precopulatory isolation index (IPC) to characterize the strength of premating barriers. The species under study, with the exception of L. arcana, clearly demonstrated preferential mating with conspecifics. According to FI and IPC, L. fabalis and L. compressa appeared reliably isolated from their closest relatives within Neritrema. Individuals of these two species tend to be smaller than those of the others, highlighting the importance of shell size changes in gastropod species divergence. L. arcana males were often found in pairs with L. saxatilis females, and no interspecific size differences were revealed in this sibling species pair. We discuss the lack of discriminative mate choice in the sympatric populations of L. arcana and L. saxatilis, and possible additional mechanisms restricting gene flow between them.

Abundance of Opisthorchis felineus Metacercariae in cyprinid fish in the middle Ob River basin (Tomsk region, Russia).

Infection with the liver fluke, Opisthorchis felineus, caused by the consumption of infected raw or undercooked cyprinid fish is common in humans and carnivores in the middle Ob River basin (Tomsk region, Russia) and can lead to diseases in humans. The goal of this study was the status of fish infection with O. felineus metacercariae in order to assess the role of fish in the infection of the human population in the middle Ob River basin. Of the 14 Cyprinidae species recorded in the middle Ob River basin, we examined 6 cyprinid species for prevalence and intensity of infection with O. felineus metacercariae. Five of these species (Leuciscus idus, Leuciscus leuciscus, Rutilus rutilus, Abramis brama, and Carassius gibelio) are of commercial value, while the common bleak is an object of amateur fishing. In addition, we conducted a survey of the fish consumption habits as part of a community-based cross-sectional study in the rural Shegarsky district, Tomsk region, Russian Federation. O. felineus metacercariae were observed in muscles of all examined species except for the Prussian carp. The ide is the main infection source in the Ob River (prevalence of infection, 100%, and intensity, 50.5 metacercariae per fish) and the common dace in the Tom River (91.1% and 12.7 metacercariae). Although the two alien species (bream and common bleak) are susceptible to infection with O. felineus metacercariae, the prevalence of infection in these fish and in the roach did not exceed 3%. The prevalence and intensity of infection in fish increased with age and size. The infection characteristics of fish in different water flows were different. The cyprinid species account on average for 69.8% of the commercial fish harvested in the Tomsk region. According to epidemiological survey, among 600 participants, 87.5% (n = 525) of respondents consumed river fish, with the most popular fish being cyprinids including Prussian carp, dace, ide and bream, followed by pike and perch. Thus, the epizootological state of the water flows in the middle Ob River basin is adverse with respect to opisthorchiasis, as indicated by high infection rates of liver fluke metacercariae in ide and common dace, which are abundant species of high commercial value. An extremely high prevalence of infection suggests a strong transmission cycle with a high rate of infection from infected humans and/or animal reservoir hosts to snails and fish. In addition to treating humans, there should be a strong focus on identifying these potential reservoir hosts to reduce subsequent infection in humans. Furthermore, since the intensity of infection in humans is determined by the presence of fish species such as ide and dace in the diet, they should be included in a dietary change campaign by eliminating the consumption of raw fish.

LOSP: A putative marker of parasperm lineage in male reproductive system of the prosobranch mollusk Littorina obtusata.

Reproductive isolation is the key attribute of biological species and establishment of the reproductive barriers is an essential event for speciation. Among the mechanisms of reproductive isolation, gamete incompatibility due to the variability of gamete interaction proteins may drive fast divergence even in sympatry. However, the number of available models to study this phenomenon is limited. In case of internally fertilized invertebrates, models to study gamete incompatibility and sperm competition mechanisms are restricted to a single taxon: insects. Here, we propose a group of closely related Littorina species as a new model for such studies. Particularly since periwinkles are already thoroughly studied in terms of morphology, physiology, ecology, phylogeny, and ecological speciation. Earlier, we have identified the first species-specific Littorina sperm protein (LOSP) with no known conservative domains or homologies. LOSP is relatively abundant component of sperm extracts and might be involved in gamete incompatibility. Here, we characterize its definitive localization and mRNA expression pattern in the male reproductive system by immunocytochemistry and RNA in situ hybridization. We demonstrate that LOSP distribution is limited to the parasperm cells. Losp gene expression occurs only at the early stages of parasperm development. The protein is stored within granules of mature parasperm and, most likely, is released after ejaculation inside female reproductive system. Thus, LOSP is the only described molluscan paraspermal protein to date, and there is a possibility for LOSP to be involved in gamete incompatibility since heterospermy is a common phenomenon among Littorina.

Synthesis and structure-activity relationship of novel 1,4-diazabicyclo[2.2.2]octane derivatives as potent antimicrobial agents.

A series of new quaternary 1,4-diazabicyclo[2.2.2]octane derivatives was synthesized and evaluated for activity against several strains of both Gram positive and Gram negative bacteria and one strain of fungus under different inoculum size. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against six species of microorganisms were tested. Results show a clear structure-activity relationship between alkyl chain length of substitutions of 1,4-diazabicyclo[2.2.2]octane tertiary amine sites and antimicrobial activity. In the case of compounds 4a-4k, MIC was found to decrease with the increase of the alkyl chain length from ethyl to dodecyl and then to increase at higher chain length (n > 14). The MIC values were found to be low for the compounds 4f and 4g with alkyl chains ranging from 10 to 12 carbons in length (1.6 µg/ml) and were comparable to the reference drug Ciprofloxacin. Also, time-kill assay was performed to examine the bactericidal kinetics. Results indicated that 4f and 4g had rapid killing effects against Staphylococcus aureus, and eliminated 100% of the initial inoculum of bacteria in 2.5 h at the concentration of 10 µg/ml. In addition, compound 4g eliminate more than 99.9% of the initial inoculum of Ps. aeruginosa after 2.5 h of interaction but the activity of compound 4f against this species seems to be weak. Thus, 4g had strong bactericidal activity and could rapidly kill Gram positive S. aureus, as well as Gram negative Ps. aeruginosa at low and high inoculum size.

The origin of the variola virus.

The question of the origin of smallpox, one of the major menaces to humankind, is a constant concern for the scientific community. Smallpox is caused by the agent referred to as the variola virus (VARV), which belongs to the genus Orthopoxvirus. In the last century, smallpox was declared eradicated from the human community; however, the mechanisms responsible for the emergence of new dangerous pathogens have yet to be unraveled. Evolutionary analyses of the molecular biological genomic data of various orthopoxviruses, involving a wide range of epidemiological and historical information about smallpox, have made it possible to date the emergence of VARV. Comparisons of the VARV genome to the genomes of the most closely related orthopoxviruses and the examination of the distribution their natural hosts' ranges suggest that VARV emerged 3000 to 4000 years ago in the east of the African continent. The VARV evolution rate has been estimated to be approximately 2 × 10-6 substitutions/site/year for the central conserved genomic region and 4 × 10-6 substitutions/site/year for the synonymous substitutions in the genome. Presumably, the introduction of camels to Africa and the concurrent changes to the climate were the particular factors that triggered the divergent evolution of a cowpox-like ancestral virus and thereby led to the emergence of VARV.

A retrospective study of the orthopoxvirus molecular evolution.

The data on the structure of conserved genes of the Old and New World orthopoxviruses and unclassified Yoka poxvirus were used for a Bayesian dating of their independent evolution. This reconstruction estimates the time when an orthopoxvirus ancestor was transferred to the North American continent as approximately 50 thousand years ago (TYA) and allows for relation of this time interval with the global climate changes (with one of the short-term warmings during the Last Ice Age). The onset of the Yoka poxvirus evolution was assessed as approximately 90TYA. Availability of a large number of genome sequences of various cowpox virus strains provided for a comprehensive analysis of the orthopoxvirus evolutionary history. Such a study is especially topical in view of the postulated role of this virus in the evolution of various orthopoxviruses, namely, as an progenitor virus. The computations have demonstrated that the orthopoxviruses diverged from the ancestor virus to form the extant species about 10TYA, while the forbear of horsepox virus separated about 3TYA. An independent evolution of taterapox, camelpox, and variola viruses commenced approximately 3.5TYA. Study of the geographic distribution areas of the hosts of these three orthopoxviruses suggests the hypothesis on the region of their origin. It is likely that these viruses first emerged in Africa, in the region of the Horn of Africa, and that the introduction of camels to East Africa induced their divergent evolution.

Molecular dating in the evolution of vertebrate poxviruses.

OBJECTIVES: The goal of this work was to study the evolutionary history of the vertebrate poxviruses using the Bayesian relaxed clock and a large set of highly conserved vitally important viral genes. METHODS: Phylogenetic analysis was performed by the maximum likelihood method using the Paup program. The dating method of Bayes, realized in the Multidivtime, was made. RESULTS: The rate of poxviral evolution is estimated as 0.5-7 × 10(-6) nucleotide substitutions per site per year. We inferred that the modern viruses of the genus Avipoxvirus diverged from the ancestor nearly 249 ± 69 thousand years ago (Tya). The progenitor of the genus Orthopoxvirus separated approximately 166 ± 43 Tya. The separation of the forebear of the genus Leporipoxvirus took place about 137 ± 35 Tya. The next to diverge was the ancestor of the genus Yatapoxvirus. The progenitor of Capripoxvirus and Suipoxvirus diverged 111 ± 29 Tya. CONCLUSION: The evolutionary analysis based on the historical data and utilizing the Bayesian relaxed clock allowed us to determine the molecular evolution rates of the AT-rich genomes of the vertebrate poxviruses and assess the times of their emergences. Involvement of a large set of the conserved genes controlled by stabilizing selection allowed us to perform molecular dating of the vertebrate poxvirus history.

Recombinant analogs of a novel milk pro-apoptotic peptide, lactaptin, and their effect on cultured human cells.

We recently isolated and characterized a human milk peptide, lactaptin, which induced apoptosis of cultured human MCF-7 cells. Lactaptin was identified as a proteolytic fragment of human kappa-casein. Here, we generated two recombinant analogs of the peptide, RL1 and RL2, containing truncated and complete amino acid sequences of lactaptin, respectively. Analogs were produced in E.coli, purified and assayed for biological activity on cultured human MCF-7 cells. RL1 was shown to induce only a small decrease in cell viability, whereas RL2 lowered the viability of MCF-7 cells by 60%. This reduction in MCF-7 cell viability was associated with apoptosis, which was indicated by phosphatidilserine externalization and caspase-7 activation. The viability of A549 and Hep-2 cells was also reduced by RL2, albeit to a lesser degree than seen with MCF-7 cells; this reduced viability was not accompanied by apoptosis. Non-malignant human mesenchymal stem cells (MSC) were completely resistant to RL2 action.

Designing and engineering of DNA-vaccine construction encoding multiple CTL-epitopes of major HIV-1 antigens.

A synthetic T cell immunogen (TCI) has been designed as a candidate DNA-based vaccine against Human immunodeficiency virus (HIV)-1 using cytotoxic T lymphocytes (CD8(+) CTL) and T-helper lymphocytes (CD4(+) Th) epitopes retrieved from the Los Alamos HIV Molecular Immunology Database. The protein 392 amino acids in length contains about eighty CTL-epitopes, many of which are overlapping and are totally restricted by ten different HLA class I molecules. To be able to detect CTL responses induced by a DNA vaccine in experimental animals, additional epitopes, restricted by mouse and Macaque rhesus major histocompatibility complex (MHC) class I molecules, were included in the target immunogen. The gene encoding the TCI protein was assembled, cloned into vector plasmids and expressed in a prokaryotic and a eukaryotic system. The presence of HIV-1 protein fragments in the immunogen structure was ascertained by ELISA and immunoblotting using panels of HIV-1-positive sera and monoclonal antibodies to p24. It has been demonstrated that DNA vaccine can induce both specific T cell responses (CTL and blast transformation) and specific antibodies in mice immunized with pcDNA-TCI.

Comparative analysis using a mouse model of the immunogenicity of artificial VLP and attenuated Salmonella strain carrying a DNA-vaccine encoding HIV-1 polyepitope CTL-immunogen.

Two systems have been examined for delivery of DNA-vaccine encoding a HIV-1 polyepitope CTL-immunogen (TCI). One is intended for i.m. injection and is in the form of an artificial virus like particle containing eukaryotic expression plasmid pcDNA-TCI encapsulated within a spermidine-polyglucin conjugate. The other is intended for mucosal immunization and is based on attenuated Salmonella typhimurium strain 7207, which can deliver pcDNA-TCI directly into professional antigen-presenting cells (APC). After immunization, the artificial VLP and recombinant Salmonella induced an enhanced HIV specific serum antibody, proliferative and CTL responses compared to those induced by naked pcDNA-TCI. The most significant responses were produced when pcDNA-TCI was delivered by Salmonella.