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In this study, we investigated the properties of proteolytic enzymes of two species of Aspergillus, Aspergillus flavus 1 (with a high degree of pathogenicity) and Aspergillus ochraceus L-1 (a conditional pathogen), and their effects on various components of the hemostasis system (in vitro) in the case of their penetration into the bloodstream. We showed that micromycete proteases were highly active in cleaving both globular (albuminolysis) and fibrillar (fibrin) proteins, and, to varying degrees, they could coagulate the plasma of humans and animals (due to proteolysis of factors of the blood coagulation cascade) but were not able to coagulate fibrinogen. The proteases of both Aspergillus fully hydrolyzed thrombi in 120–180 min. Micromycetes did not show hemolytic activity but were able to break down hemoglobin.
The relevance of studying the regulation of protein-ligand interactions is due to the emergence of new views on the role of metabolites and their key importance in vital processes. To study the protein-ligand interaction, the AB0 antigen-antibody blood system and the enzyme-substrate system of dehydrogenases were used as a test system, and ethanol was used as an influencing factor. In experiments performed with A and B blood erythrocyte antigens, natural AB0 system antibodies and monoclonal antibodies under the influence of ethanol performed change of the degree of agglutination and the time to onset of erythrocyte agglutination. It was found that ethanol can regulate the enzyme-substrate interactions of dehydrogenases: lactate dehydrogenase (EC 18.104.22.168), glyceraldehyde phosphate dehydrogenase (EC 22.214.171.124), and α-glycerol phosphate dehydrogenase (EC 126.96.36.199). The increase in the activity of studied enzymes under the influence of ethanol in the whole blood hemolysate was 2.5 - 3 times higher than in the isolated medium (with pure enzyme preparations).
Inferences on the development and morphology of extinct brachiopods must be informed by the ontogeny and shell ornamentation of extantbrachiopods. Although the adult shells of extant brachiopods are wellstudied,detailed descriptions of the embryonic and juvenile shells of extant lingulides are lacking. Here, we describe in detail the shells of juveniles of Lingula anatina Lamarck, 1801 from Vietnam and the Republic of the Philippines. The following previously unknown properties of the lingulide shell are described: (i) a distinct border between the protegulum and the brephic shell; (ii) drapes that develop on both the protegulum and brephic shell; and (iii) the notched anterior margin of the brephic shell. The drapes and cogs on the brephic shell may becaused by the formation of setal follicles during the planktonic stage. Specimens of L. anatina from the Philippines have larger brephic shells than those from Vietnam probably because the former have a longer planktonic stage. Based on comparisons of the first-formed shells of extant brachiopods with published data on fossil brachiopods, we suggest that the life cycle of extant lingulides, in which planktotrophic juveniles with a shell hatch from the egg envelopes, is the mostevolutionarily advanced brachiopod life cycle and appeared in the Early Silurian. We suggest criteria for determining the type of life cycle based on the structure of the first-formed shell of brachiopods. Finally, we consider hypothetical scenarios of life cycles of fossil brachiopods including true planktotrophic larvae in the Cambrian linguliforms.
Brachiopoda is a relict phylum of marine benthic animals that have not been adequately studied with modern microscopy methods. Microscopic study may provide useful information on the evolution of the brachiopod body plan and brachiopod phylogeny. Understanding the organisation of the coelomic system is important because of its role in body form and compartmentalisation. Most brachiopods are considered to have a bipartite coelomic system; the only known exception is Lingulida, which have a tripartite coelomic system. In the present study, we provide the first complete 3D reconstruction of the coelomic system in the craniide brachiopod Novocrania anomala (Müller, 1776). Its coelomic system consists of the following five main parts, which are entirely separated from each other: 1) a pair of large brachial canals; 2) a complex system of paired small brachial canals and a perioesophageal coelom; 3) frontal coelomic chambers; 4) a main trunk coelom, which includes several semi-detached muscular chambers and mantle sinuses; and 5) a pair of posterior adductors chambers. These results indicate that the coelomic system of N. anomala (and perhaps of other craniides) is complex and cannot be considered to be bipartite or tripartite. The frontmost part of the coelomic system is represented by a pair of frontal chambers, which are considered to be a part of the lophophore but which are derived from dorsal mantle fold extensions and thus may be a part of the trunk coelomic system. A number of similarities were discovered between craniiformean and rhynchonelliformean coelomic systems, including the prominent dorsal projections of the large brachial canals and the morphological features of the perioesophageal coelom. The complex subdivision of the N. anomala trunk coelom is explained by the location and function of muscles, and by the location of several mesenteries
Detailed characterization of cell type transitions is essential for cell biology in general and particularly for the development of stem cell-based therapies in regenerative medicine. To systematically study such transitions, we introduce a method that simultaneously measures protein expression and thermal stability changes in cells and provide the web-based visualization tool ProteoTracker. We apply our method to study differences between human pluripotent stem cells and several cell types including their parental cell line and differentiated progeny. We detect alterations of protein properties in numerous cellular pathways and components including ribosome biogenesis and demonstrate that modulation of ribosome maturation through SBDS protein can be helpful for manipulating cell stemness in vitro. Using our integrative proteomics approach and the web-based tool, we uncover a molecular basis for the uncoupling of robust transcription from parsimonious translation in stem cells and propose a method for maintaining pluripotency in vitro.
Human leukocyte antigen (HLA) class I molecules play a crucial role in the development of a specific immune response to viral infections by presenting viral peptides at the cell surface where they will be further recognized by T cells. In the present manuscript, we explored whether HLA class I genotypes can be associated with the critical course of Coronavirus Disease-19 by searching possible connections between genotypes of deceased patients and their age at death. HLA-A, HLA-B, and HLA-C genotypes of n = 111 deceased patients with COVID-19 (Moscow, Russia) and n = 428 volunteers were identified with next-generation sequencing. Deceased patients were split into two groups according to age at the time of death: n = 26 adult patients aged below 60 and n = 85 elderly patients over 60. With the use of HLA class I genotypes, we developed a risk score (RS) which was associated with the ability to present severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) peptides by the HLA class I molecule set of an individual. The resulting RS was significantly higher in the group of deceased adults compared to elderly adults [p = 0.00348, area under the receiver operating characteristic curve (AUC ROC = 0.68)]. In particular, presence of HLA-A*01:01 allele was associated with high risk, while HLA-A*02:01 and HLA-A*03:01 mainly contributed to low risk. The analysis of patients with homozygosity strongly highlighted these results: homozygosity by HLA-A*01:01 accompanied early deaths, while only one HLA-A*02:01 homozygote died before 60 years of age. Application of the constructed RS model to an independent Spanish patients cohort (n = 45) revealed that the score was also associated with the severity of the disease. The obtained results suggest the important role of HLA class I peptide presentation in the development of a specific immune response to COVID-19.
Every year, the size of the human population grows; with it, the need for agricultural products increases. This leads to an increment in the volume of waste, including hard-to-degrade keratin-rich ones, such as feathers. Currently, most of the agro-industrial complex protein by-products are utilized by incineration, landfilling, and chemical hydrolysis. Such methods do not meet modern trends in the development of a sustainable economy, negatively affecting the environment and humans, and preventing the reusing of waste. An alternative is biodegradation, which consists of the application of living organisms and their enzymes to recycle by-products. This approach is not only sustainable, but also makes it possible to obtain products of waste hydrolysis that are in demand for the manufacture of fertilizers and feed additives. This brings the development of agriculture closer to a circular economy and makes the recycling process more profitable. This review article emphasizes the significance of keratinolytic microorganisms and keratinases for the improvement of green methods for processing hard-to-degrade protein waste of the agro-industrial complex, which is necessary for sustainable economic development.
The Keap1-Nrf2 signaling axis is a validated and promising target for cellular defense and survival pathways. This minireview discusses the potential off-target effects and their impact on future drug development originating from Keap1-targeting small molecules that function as displacement activators of the redox-sensitive transcription factor Nrf2. We argue that small-molecule displacement activators, similarly to electrophiles, will release both Nrf2 and other Keap1 client proteins from the ubiquitin ligase complex. This non-specificity is likely unavoidable and may result in off-target effects during Nrf2 activation by targeting Keap1. The small molecule displacement activators may also target Kelch domains in proteins other than Keap1, causing additional off-target effects unless designed to ensure specificity for the Kelch domain only in Keap1. A potentially promising and alternative therapeutic approach to overcome this non-specificity emerging from targeting Keap1 is to inhibit the Nrf2 repressor Bach1 for constitutive activation of the Nrf2 pathway and bypass the Keap1-Nrf2 complex.
A preparation of thrombolytic enzymes of micromycete S. strictum 203 was obtained and characterized. The expressed urokinase activity of producer proteinases was determined, and the content of the complex of three alkaline trypsin-like thiol-dependent serine-type proteinases with different isoelectric points (4.5, 7.2 and 11.8) but close molecular weight was detected in the enzyme preparation (about 35 kDa). One of the proteinases (proteinase III) was not glycosylated, and the rest were glycoproteins. The proteinases differed in the spectrum of proteolytic activity in relation to proteins; the thrombus components also turned out to be different. Presumably, the enzymatic urokinase activity causes proteinases to activate plasminogen.
Choriocarcinoma cells BeWo b30 are used to model human placental trophoblast hypoxia using cobalt (II) chloride and hydroxyquinoline derivative (HD) as chemical inducers of hypoxia-inducible factor (HIF). In this study, it was shown that both substances activate the hypoxic pathway and the epithelial–mesenchymal transition and inhibit the pathways of cell proliferation. However, CoCl2 caused activation of the apoptosis pathway, increased the activity of effector caspases 3 and 7, and increased the expression of the unfolded protein response target DDIT3. The mTORC1 pathway was activated upon exposition to CoCl2, while HD suppressed this pathway, as it happens during real trophoblast hypoxia. Thus, effect of CoCl2 on BeWo cells can be a model of severe hypoxia with activation of apoptosis, while HD mimics moderate hypoxia.
Previous studies have demonstrated the formation of stable complexes between inorganic pyrophosphatase (PPase) and three other Escherichia coli enzymes – cupin-type phosphoglucose isomerase (cPGI), class I fructose-1,6-bisphosphate aldolase (FbaB) and l-glutamate decarboxylase (GadA).
Here, we determined by activity measurements how complex formation between these enzymes affects their activities and oligomeric structure.
cPGI activity was modulated by all partner proteins, but none was reciprocally affected by cPGI. PPase activity was down-regulated upon complex formation, whereas all other enzymes were up-regulated. For cPGI, the activation was partially counteracted by a shift in dimer ⇆ hexamer equilibrium to inactive hexamer. Complex stoichiometry appeared to be 1:1 in most cases, but FbaB formed both 1:1 and 1:2 complexes with both GadA and PPase, FbaB activation was only observed in the 1:2 complexes. FbaB and GadA induced functional asymmetry (negative kinetic cooperativity) in hexameric PPase, presumably by favoring partial dissociation to trimers.
These four enzymes form all six possible binary complexes in vitro, resulting in modulated activity of at least one of the constituent enzymes. In five complexes, the effects on activity were unidirectional, and in one complex (FbaB⋅PPase), the effects were reciprocal. The effects of potential physiological significance include inhibition of PPase by FbaB and GadA and activation of FbaB and cPGI by PPase. Together, they provide a mechanism for feedback regulation of FbaB and GadA biosynthesis.
These findings indicate the complexity of functionally significant interactions between cellular enzymes, which classical enzymology treats as individual entities, and demonstrate their moonlighting activities as regulators.
We review the diversity and development of archegonia, the female reproductive organs of land-plant gametophytes. The archegonium is a uniquely land-plant structure, and studies of its evolution benefit from use of a comparative approach in a phylogenetic context. Archegonia of most land plants share a common developmental motif, here termed a T-shaped pattern. A primary axial cell produces a primary cover cell and a central cell by horizontal division. The upper cell usually divides vertically and the lower one horizontally. In mosses such as Atrichum, the T-shaped stage is shifted towards the end of archegonium development, whereas in vascular plants it appears at the beginning of development, but these stages are still probably homologous. The fully exposed archegonia are traditionally viewed as an ancestral (plesiomorphic) condition in land plants, but there is no direct support for this view. We speculate that the fully exposed condition is derived and synapomorphic for setaphytes (mosses and liverworts). The fully sunken hornwort archegonia may be similar to the ancestral type of land-plant archegonia. Developmental evidence suggests that archegonium necks of setaphytes and tracheophytes are not homologous to each other. The neck wall of pteridophytes is composed of four-celled tiers, and one such tier is present in gymnosperms with motile male gametes. Neck-cell arrangement is much more plastic in archegonia of gymnosperms with sperm cell delivery by pollen tube (siphonogamy), in which the neck plays a role similar to pollen-tube transmitting tissue of angiosperms. Angiosperm synergids are probably homologues of gymnosperm neck cells, and the angiosperm egg cell is probably homologous to the ventral canal cell of gymnosperms. Developmental genetic bases of archegonium diversity in land plants remain to be understood. Even descriptive developmental data are currently missing or controversial for some key lineages of land plants.
The pulsation of the stolon coenosarc of the colonial hydroid Dynamena pumila (L., 1758) was recorded using time-lapse microvideo recording. The study included several experiments: control (without air exposure) and a series of consecutive air exposure periods: 5, 10, 20, 30 min and 1 h. In general, comparison of the experiments with same colony in control and in a series of air exposure periods up to 30 min demonstrated no significant changes in the coenosarc pulsations and the hydroplasmic flow. Air exposure for 20 and 30 min caused a decrease in the amplitude of growth pulsations, and in growth increment by more than a third. The limit of permissible air exposure for D. pumila colonies is between 10 and 20 min. Such a short air exposure period does not lead to disruption of the distribution system and the growth of the growing tips, and therefore can be used in laboratory studies of colonies. A longer air exposure period, namely one hour, is critical for the colony, since it leads to disruption of its structure integrity. Tissue degradation occurs, and the inner contents begin to pour out through the holes in the burst perisarc of the growing tip.
The best known polymers (alginate, carrageenan, and acrylamide) were examined for their potential use as carriers of mycelium cells of Aspergillus ochraceus VKM-F4104D, a producer of fibrinolytic protease activator of protein C in human blood plasma during its deep cultivation. The morpho-cytological features of the development of A. ochraceus VKM-F4104D mycelium in granules of carrageenan and calcium alginate are shown. The conditions for the cultivation of micromycete immobilized in granules of 3.5% calcium alginate were optimized. The immobilized cells of the producer mycelium demonstrated increased stability and were able to synthesize protein-C protease-activator in blood plasma at the maximum level for 10 or more days. In addition, immobilized mycelial cells during cultivation can be used repeatedly (up to five cycles).
Extracellular proteinase of the opportunistic Aspergillus fumigatus D-1 strain (molecular weight ~33 kDa, pI 4.6) was isolated. It was shown that proteinase hydrolyzes casein, fibrin, fibrinogen, albumin, and hemoglobin to varying degrees. However, proteolytic activity with respect to globular proteins of blood plasma was comparable to fibrinolytic activity. Proteinase did not coagulate human fibrinogen and bovine fibrinogen; it also did not coagulate human and rabbit blood plasma without dilution and when diluted twice. The plasminogen-activating activity of A. fumigatus D-1 extracellular proteinase was found, which may indicate its ability to indirect fibrinolysis.
Breast cancer (BC) is the leading cause of death from malignant neoplasms among women worldwide, and metastatic BC presents the biggest problems for treatment. Previously, it was shown that lower expression of ELOVL5 and IGFBP6 genes is associated with a higher risk of the formation of distant metastases in BC. In this work, we studied the change in phenotypical traits, as well as in the transcriptomic and proteomic profiles of BC cells as a result of the stable knockdown of ELOVL5and IGFBP6 genes. The knockdown of ELOVL5 and IGFBP6 genes was found to lead to a strong increase in the expression of the matrix metalloproteinase (MMP) MMP1. These results were in good agreement with the correlation analysis of gene expression in tumor samples from patients and were additionally confirmed by zymography. The knockdown of ELOVL5 and IGFBP6 genes was also discovered to change the expression of a group of genes involved in the formation of intercellular contacts. In particular, the expression of the CDH11 gene was markedly reduced, which also complies with the correlation analysis. The spheroid formation assay showed that intercellular adhesion decreased as a result of the knockdown of the ELOVL5 and IGFBP6 genes. Thus, the obtained data indicate that malignant breast tumors with reduced expression of the ELOVL5 and IGFBP6 genes can metastasize with a higher probability due to a more efficient invasion of tumor cells.
The review addresses and highlights the main results of research on the physicohemical properties of single-phase and composite materials based on transition metal oxides in relation to their practical application as electrode materials for symmetrical solid oxide fuel cells. The electronic structures and thermodynamic stability of transition metal oxides with the perovskite structure are discussed. A detailed consideration is given to the thermal behaviour, chemical stability, electrical conductivity and electrochemical properties of a broad range of electrode materials based on iron-, chromium- and manganese-containing perovskite-like oxides and oxides that crystallize in other structure types. The analysis revealed the most promising compositions of electrode materials for symmetrical solid oxide fuel cells and effective approaches to the improvement of their functional characteristics.
Since 2003, the world has been confronted with three new betacoronaviruses that cause human respiratory infections: SARS-CoV, which causes severe acute respiratory syndrome (SARS), MERS-CoV, which causes Middle East respiratory syndrome (MERS), and SARS-CoV-2, which causes Coronavirus Disease 2019 (COVID-19). The mechanisms of coronavirus transmission and dissemination in the human body determine the diagnostic and therapeutic strategies. An important problem is the possibility that viral particles overcome tissue barriers such as the intestine, respiratory tract, blood-brain barrier, and placenta. In this work, we will 1) consider the issue of endocytosis and the possibility of transcytosis and paracellular trafficking of coronaviruses across tissue barriers with an emphasis on the intestinal epithelium; 2) discuss the possibility of antibody-mediated transcytosis of opsonized viruses due to complexes of immunoglobulins with their receptors; 3) assess the possibility of the virus transfer into extracellular vesicles during intracellular transport; and 4) describe the clinical significance of these processes. Models of the intestinal epithelium and other barrier tissues for in vitro transcytosis studies will also be briefly characterized.
MicroRNAs (miRNAs) are short RNA molecules regulating multiple cellular processes through posttranscriptional gene silencing. Over the last decade, miRNA have been found in extracellular space and have been consistently shown to mediate functional communication between cells. While it remains widely accepted that miRNA transfer between cells occurs via extracellular vesicles (EVs), multiple other carriers of cell-free miRNA have been described. In addition, some studies have demonstrated that both miRNA and their binding partners, Argonaute proteins, remain hardly detectable in common isolates of EVs. In this work, we summarize the state-of-the-art mechanisms of miRNA sorting and secretion, discuss methodological challenges associated with extracellular miRNA research, and suggest experimental steps to resolve current inconsistencies in the field of miRNA-mediated cell-cell communication.
Fig mosaic virus (FMV) (genus Emaravirus in the family Fimoviridae) is considered the etiological agent of fig mosaic disease (FMD), which is recorded in most of the fig-growing areas with an average global infection rate of 33%. The multipartite FMV genome is composed of six negative monocistronic ssRNAs, each of which is separately encapsidated (Preising et al. 2021). Although FMD-like symptoms, which include mosaic, chlorotic ringspots, and oak leaf patterns, were observed in approximately a third of 400 fig accessions in the Nikita Botanical Gardens, Yalta, Russia (Mitrofanova et al. 2016), FMV has not been identified as the causal agent of the disease. In June of 2020, total RNA was isolated from symptomatic leaves of 59 trees (32 years old) representing 31 local and 27 introduced Ficus carica L. cultivars and a single F. pseudocarica Miq. tree using an RNeasy Plant Mini kit (Qiagen, U.S.A.). FMV was tested by reverse transcription PCR using primer sets E5 (Elbeaino et al. 2009) and EMARAVGP (Walia et al. 2009), which amplify a 302-bp fragment of RNA1 and a 468-bp fragment of RNA2, respectively. PCR products of the expected sizes were generated in all samples, indicating a high FMV incidence in the plantings. The genome sequences of FMV isolates from F. carica cultivars Bleuet, Kraps di Hersh, Smena, and Temri and F. pseudocarica were determined by high-throughput sequencing on a MiSeq Illumina platform. Double-stranded RNA was isolated from FMV-positive leaves using a Viral Gene-spin Viral DNA/RNA Extraction Kit (iNtRON, Korea), followed by cDNA library preparation with the NEBNext Ultra II RNA Library Prep Kit (New England Biolabs, U.S.A.). On average, 695,000 quality-filtered 150-bp pair-ended reads per a library were produced and used in a de novo assembly using the metaSpades program version 3.14 (Nurk et al. 2017). In each of five samples, BLASTn analysis found six FMV-related contigs. The contigs spanned 99 to 100% of corresponding genomic segments of the most closely related isolates. In addition to FMV, fig cryptic virus-related contigs were also detected in some samples. The FMV contigs covering RNA1 to RNA6 had the highest identity to corresponding genomic segments of isolates AM941711 (96.5 to 96.6%), FM864225 (94.4 to 94.6%), FM991954 (97.9 to 98.2%), AB697863 (96.4 to 96.6%), AB697879 (93.3 to 93.4%), and AB697895 (95.4 to 97.0%), respectively. Five Russian isolates shared 99.2 to 100% nucleotide sequence identity, depending on the genomic segment. Their sequences were deposited in GenBank under accession numbers MW201216 to MW201230 and MW208662 to MW208676. Phylogenetic analysis of six open reading frames (ORFs) showed that ORF1 to ORF3 and ORF6 of the Russian isolates clustered with FMV isolates from Italy, whereas ORF4 grouped with the isolate JTT-Pa (AB697863) from Japan. ORF5 of the Russian isolates formed a separate cluster with the isolates SB1 and SB2 from Serbia and JTT-Vi from Japan (AB697879 to AB697884). Incongruency of phylogenetic relationship among the genomic segments suggests reassortment among ancestors of the Russian FMV isolates. In addition, similar to SB1, SB2, and JTT-Vi, ORF5 of the Russian isolates encodes a protein of 486 amino acid (aa) residues, in contrast to the corresponding protein of Italian isolates consisting of 502 aa. To the best of our knowledge, this is the first report of FMV in Russia. This finding not only expands the information on the geographical distribution of FMV but also extends knowledge on F. pseudocarica as a natural host of the virus.