Cellular Protective Mechanisms Research Articles

In Memoriam: Prof. Olena Petrivna Sotnikova

Professor Olena Petrivna Sotnikova, Head of Pharmacology and Tissue Therapy Laboratory at the Filatov Institute of Eye Diseases and Tissue Therapy, passed away on June 9, 2023. She was born in the town of Makiivka, Donetsk region, on June 26, 1939. After graduating cum laude from the Faculty of General Medicine, Odesa Medical Institute, in 1969, Olena Petrivna began her career as a junior research associate at the Pharmacology and Tissue Therapy Laboratory at the Filatov Institute of Eye Diseases and Tissue Therapy, NAMS of Ukraine. Completing her Cand Sc (Med) thesis, Cytochemical Changes in the Corneal Epithelium and Nasal Mucosa in Impaired Sensory Innervation, under the co-supervision of Drs Sc (Med) V.D. Dragomiretskyi and R.K. Marmur, was her first serious achievement in science. In 1981, O.P. Sotnikova was awarded with a silver medal and the Certificate of Participation in the Exhibition of Economic Achievements of the USSR as a co-developer of a preparation named “aloe tablets covered with a film shell”. She defended her Dr Sc (Med) dissertation and became head of the Pharmacology and Tissue Therapy Laboratory in 1990 and was promoted to professorship in 1999. Professor Sotnikova’s study on molecular and cellular bioregulative and protective mechanisms of tissue preparations in the presence of the impact of adverse factors on the body has become an important contribution to the Acad. Filatov’s fundamental theory of “biogenic stimulators”. Two components had been incorporated into the science and practice activities of the laboratory under her guidance: first, the ocular pharmacological research aimed at the development of single and multicomponent ophthalmic dosage forms from national plant, animal and geo-organic raw materials, and, second, the preclinical evaluation of the curative and preventive efficacy of these forms in ophthalmopathy models. Twelve individuals completed their Cand Sc (Med) degrees under the supervision of Prof. Sotnikova. She produced about 400 publications and authored or co-authored 20 patents. Olena Petrivna was a person with a capital P. She was a remarkably talented scientist and manager, and her job was her passion. She also was a benevolent and empathetic person who had never remained indifferent to the problems of others and was always ready to assist in any way she could. Olena Petrivna’s memory will leave on forever through her numerous published works, scientific ideas and research results, and the apprentices that she mentored.

Cetacean Intracytoplasmic Eosinophilic Globules: A Cytomorphological, Histological, Histochemical, Immunohistochemical, and Proteomic Characterization.

The nature, etiopathogenesis, and clinicopathologic relevance of the prevalent intracytoplasmic eosinophilic globules (IEGs) within hepatocytes of cetaceans are unknown. This study aims to evaluate the presence and characterize the IEGs in the hepatocytes of cetaceans using histochemical and immunohistochemical electron microscopy, Western blot, lectin histochemistry, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry techniques. A total of 95/115 (83%) animals (16 species) exhibited histologically evident intracytoplasmic round to oval, single to multiple, hyaline eosinophilic globules within the hepatocytes. These globules were largely PAS-positive, diastase resistant, and were immunopositive for fibrinogen (FB, 97%), albumin (Alb, 85%), and α1-antitrypsine (A1AT, 53%). The IEG positivity for FB and A1AT were correlated with live-stranding, hepatic congestion and a good nutritional status. The cetaceans lacking IEGs were consistently dead stranded and had poor body conditions. The IEGs in 36 bycaught cetaceans were, all except one, FB-positive and A1AT-negative. The IEGs exhibited morphologic and compositional variations at the ultrastructural level, suggesting various stages of development and/or etiopathogenesis(es). The glycocalyx analysis suggested an FB- and A1AT-glycosylation pattern variability between cetaceans and other animals. The proteomic analyses confirmed an association between the IEGs and acute phase proteins, suggesting a relationship between acute stress (i.e., bycatch), disease, and cellular protective mechanisms, allowing pathologists to correlate this morphological change using the acute hepatocytic cell response under certain stress conditions.

The impact of the cytoplasmic ubiquitin ligase TNFAIP3 gene variation on transcription factor NF-κB activation in acute kidney injury

Nuclear factor κB (NF-κB) activation is a deleterious molecular mechanism that drives acute kidney injury (AKI) and manifests in transplanted kidneys as delayed graft function. The TNFAIP3 gene encodes A20, a cytoplasmic ubiquitin ligase and a master negative regulator of the NF- κB signaling pathway. Common population-specific TNFAIP3 coding variants that reduce A20's enzyme function and increase NF- κB activation have been linked to heightened protective immunity and autoimmune disease, but have not been investigated in AKI. Here, we functionally identified a series of unique human TNFAIP3 coding variants linked to the autoimmune genome-wide association studies single nucleotide polymorphisms of F127C; namely F127C;R22Q, F127C;G281E, F127C;W448C and F127C;N449K that reduce A20's anti-inflammatory function in an NF- κB reporter assay. To investigate the impact of TNFAIP3 hypomorphic coding variants in AKI we tested a mouse Tnfaip3 hypomorph in a model of ischemia reperfusion injury (IRI). The mouse Tnfaip3 coding variant I325N increases NF- κB activation without overt inflammatory disease, providing an immune boost as I325N mice exhibit enhanced innate immunity to a bacterial challenge. Surprisingly, despite exhibiting increased intra-kidney NF- κB activation with inflammation in IRI, the kidney of I325N mice was protected. The I325N variant influenced the outcome of IRI by changing the dynamic expression of multiple cytoprotective mechanisms, particularly by increasing NF- κB-dependent anti-apoptotic factors BCL-2, BCL-XL, c-FLIP and A20, altering the active redox state of the kidney with a reduction of superoxide levels and the enzyme super oxide dismutase-1, and enhancing cellular protective mechanisms including increased Foxp3+ T cells. Thus, TNFAIP3 gene variants represent a kidney and population-specific molecular factor that can dictate the course of IRI.

Molecular Investigation of DKK3 in Cerebral Ischemic/Reperfusion Injury

Dickkopf-3 (Dkk3) is an atypical member of the Dkk family of Wnt inhibitors, which has been implicated in the pathophysiology of neurodegenerative disorders. Its role in the mechanisms of cellular degeneration and protection is still unknown. The aim of our work is to investigate the endogenous activation of the DKK3 pathway in a model of transient occlusion of the middle cerebral artery in rats. In particular, the animals were subjected to 1 h of ischemia followed by different reperfusion times (1 h, 6 h, 12 h and 24 h) to evaluate the downstream pathway and the time course of its activation. Western blot analysis showed increased Dkk3 expression in animals with the highest time of reperfusion. The increased levels of Dkk3 were accompanied by reduced Wnt3a, Frz1 and PIWI1a expression in the cytosol while FOXM1 and β-catenin decreased in the nucleus. These molecular changes led to an increase in the apoptotic pathway, as showed by the increased expression of Caspase 3 and Bax and the reduced levels of Bcl-2, and to a decrease in neurogenesis, as shown by the decreased expression of Tbr2, Ngn2 and Pax6. In the second part of the study, we decided to employ curcumin, an activator of the Wnt/β-catenin signaling, to investigate its effect on Dkk3. In particular, curcumin was administered 1 and 6 h after ischemia, and animals were sacrificed 24 h later when the expression of Dkk3 was higher. Our data displayed that curcumin administration decreased Dkk3 expression, and increased Wnt3a, Frz1 and PIWI1a levels. Well in line with these data, curcumin administration increased nuclear β-catenin and FOXM1 expression. The down-regulation of Dkk3 by curcumin led to reduced apoptosis and increased neurogenesis. Summarizing, our results showed that Dkk3 acts as an inhibitor of Wnt/β-catenin signaling during cerebral ischemia. Additionally, its inhibition and the contextual activation of the Wnt/β-catenin pathway are protective against ischemic stroke.

Response to endoplasmic reticulum stress in arginine vasopressin neurons.

Arginine vasopressin (AVP) is an antidiuretic hormone synthesized principally in the hypothalamic supraoptic and paraventricular nuclei. The immunoglobulin heavy chain binding protein (BiP), one of the most abundant endoplasmic reticulum (ER) chaperones, is highly expressed in AVP neurons, even under basal conditions. Moreover, its expression is upregulated in proportion to the increase in AVP expression under dehydration. These data suggest that AVP neurons are constantly exposed to ER stress. BiP knockdown in AVP neurons induces ER stress and autophagy, resulting in AVP neuronal loss, indicating that BiP is pivotal in maintaining the AVP neuron system. Furthermore, inhibition of autophagy after BiP knockdown exacerbates AVP neuronal loss, suggesting that autophagy induced under ER stress is a protective cellular mechanism by which AVP neurons cope with ER stress. Familial neurohypophysial diabetes insipidus (FNDI) is an autosomal dominant disorder caused by mutations in the AVP gene. It is characterized by delayed-onset progressive polyuria and eventual AVP neuronal loss. In AVP neurons of FNDI model mice, mutant protein aggregates are confined to a specific compartment of the ER, called the ER-associated compartment (ERAC). The formation of ERACs contributes to maintaining the function of the remaining intact ER, and mutant protein aggregates in ERACs undergo autophagic-lysosomal degradation without isolation or translocation from the ER, representing a novel protein degradation system in the ER.

Genetic control of tolerance to drought stress in soybean

BackgroundDrought stress limits the production of soybean [Glycine max (L.) Merr.], which is the most grown high-value legume crop worldwide. Breeding for drought tolerance is a difficult endeavor and understanding the genetic basis of drought tolerance in soybean is therefore crucial for harnessing the genomic regions involved in the tolerance mechanisms. A genome-wide association study (GWAS) analysis was applied in a soybean germplasm collection (the EUCLEG collection) of 359 accessions relevant for breeding in Europe, to identify genomic regions and candidate genes involved in the response to short duration and long duration drought stress (SDS and LDS respectively) in soybean.ResultsThe phenotypic response to drought was stronger in the long duration drought (LDS) than in the short duration drought (SDS) experiment. Over the four traits considered (canopy wilting, leaf senescence, maximum absolute growth rate and maximum plant height) the variation was in the range of 8.4−25.2% in the SDS, and 14.7−29.7% in the LDS experiments. The GWAS analysis identified a total of 17 and 22 significant marker-trait associations for four traits in the SDS and LDS experiments, respectively. In the genomic regions delimited by these markers we identified a total of 12 and 16 genes with putative functions that are of particular relevance for drought stress responses including stomatal movement, root formation, photosynthesis, ABA signaling, cellular protection and cellular repair mechanisms. Some of these genomic regions co-localized with previously known QTLs for drought tolerance traits including water use efficiency, chlorophyll content and photosynthesis.ConclusionOur results indicate that the mechanism of slow wilting in the SDS might be associated with the characteristics of the root system, whereas in the LDS, slow wilting could be due to low stomatal conductance and transpiration rates enabling a high WUE. Drought-induced leaf senescence was found to be associated to ABA and ROS responses. The QTLs related to WUE contributed to growth rate and canopy height maintenance under drought stress. Co-localization of several previously known QTLs for multiple agronomic traits with the SNPs identified in this study, highlights the importance of the identified genomic regions for the improvement of agronomic performance in addition to drought tolerance in the EUCLEG collection.

Factors of non-specific resistance of bee hemolymph when feeding probiotic drug ‘Bilakt’

The paper presents the results of the feeding the ‘Bilakt’ probiotic on factors of non-specific resistance in bees. Hemolymph samples were taken on the 7th, 14th, and 21st days, and the activity of lysozyme and phagocytosis, bactericidal activity were determined. According to the research results, the lysozyme activity in the hemolymph of 3–5-day-old larvae and nurse bees of the experimental groups after 21 days was reliably 1.5 times higher than the control group and before feeding. The increase in the hemolymph bactericidal factor in 3–5-day-old larvae, and in nurse bees — threefold on the 21st day after the end of feeding with ‘Bilakt’ drug. Phagocytic activity before the beginning of the experiment in the hemolymph of 3–5-day-old larvae was 39.5%, on the 1st day after the end of feeding with ‘Bilakt’ it increased and exceeded this indicator by 29%. The phagocytic index before feeding was 2.04 ± 0.11. On the 1st day after the end of feeding, it increased by 18.4%, respectively. The phagocytic activity of hemolymph of nurse bees was 45.8%. Accordingly, the phagocytic index was 2.2 ± 0.12. On the 1st day after the end of ‘Bilakt’ feeding, phagocytic activity increased by 40.9%, the phagocytic index was 3.24 ± 0.1, which was 32.1% higher than the initial level. Research results indicate that the use of ‘Bilakt’ helps to improve the general physiological condition of sick bees by stimulating the cellular and humoral mechanisms of protection of insects from pathogens, i.e. increasing the non-specific protective properties of both the body of 3–5-day-old larvae and bee adults

Rat Hepatocytes Mitigate Cadmium Toxicity by Forming Annular Gap Junctions and Degrading Them via Endosome-Lysosome Pathway.

Gap junction protein connexin 43 (Cx43) plays a critical role in gap junction communication in rat hepatocytes. However, those located between hepatocytes are easily internalized following exposure to poisons. Herein, we investigated the potential of buffalo rat liver 3A (BRL 3A) cells to generate annular gap junctions (AGJs) proficient at alleviating cadmium (Cd) cytotoxic injury through degradation via an endosome-lysosome pathway. Our results showed that Cd-induced damage of liver microtubules promoted Cx43 internalization and increased Cx43 phosphorylation at Ser373 site. Furthermore, we established that Cd induced AGJs generation in BRL 3A cells, and AGJs were subsequently degraded through the endosome-lysosome pathway. Overall, our results suggested that Cx43 internalization and the generation of AGJs were cellular protective mechanisms to alleviate Cd toxicity in rat hepatocytes.

Effects of Molecular Crowding and Betaine on HSPB5 Interactions, with Target Proteins Differing in the Quaternary Structure and Aggregation Mechanism

The aggregation of intracellular proteins may be enhanced under stress. The expression of heat-shock proteins (HSPs) and the accumulation of osmolytes are among the cellular protective mechanisms in these conditions. In addition, one should remember that the cell environment is highly crowded. The antiaggregation activity of HSPB5 and the effect on it of either a crowding agent (polyethylene glycol (PEG)) or an osmolyte (betaine), or their mixture, were tested on the aggregation of two target proteins that differ in the order of aggregation with respect to the protein: thermal aggregation of glutamate dehydrogenase and DTT-induced aggregation of lysozyme. The kinetic analysis of the dynamic light-scattering data indicates that crowding can decrease the chaperone-like activity of HSPB5. Nonetheless, the analytical ultracentrifugation shows the protective effect of HSPB5, which retains protein aggregates in a soluble state. Overall, various additives may either improve or impair the antiaggregation activity of HSPB5 against different protein targets. The mixed crowding arising from the presence of PEG and 1 M betaine demonstrates an extraordinary effect on the oligomeric state of protein aggregates. The shift in the equilibrium of HSPB5 dynamic ensembles allows for the regulation of its antiaggregation activity. Crowding can modulate HSPB5 activity by affecting protein-protein interactions.

The enhanced degradation behavior of oxytetracycline by black soldier fly larvae with tetracycline resistance genes in the larval gut: Kinetic process and mechanism

Black soldier fly larvae (larvae) can digest organic wastes and degrade contaminants such as oxytetracycline (OTC). However, compared to the kinetic processes and enhanced mechanisms used in the traditional microbial degradation of OTC, those employed by larvae are largely uncharacterized. To obtain further details, a combined analysis of larval development, larval nutritional values (crude protein, crude fat and the composition of fatty acids) and the expression of tetracycline resistance genes (TRGs) in the larval gut was performed for the degradation of OTC added to substrates and for oxytetracycline bacterial residue (OBR). When the larvae were exposed to the substrates, the degradation processes were enhanced significantly (P < 0.01), with a 4.74–7.86-fold decrease in the degradation half-life (day−1) and a 3.34–5.74-fold increase in the final degradation efficiencies. This result was attributed to the abundant TRGs (with a detection rate of 35.90%∼52.14%) in the larval gut. The TRGs presented the resistance mechanisms of cellular protection and efflux pumps, which ensured that the larvae could tolerate elevated OTC concentrations. Investigation of the TRGs indicated that enzymatic inactivation enhanced OTC degradation by larvae. These findings demonstrate that the larval degradation of antibiotic contaminants is an efficient method based on abundant TRGs in the larval gut, even though OTC degradation results in OBR. In addition, a more optimized system for higher reductions in antibiotic levels and the expansion of larval bioremediation to other fields is necessary.

Assessing the effects of nickel on, e.g., Medicago sativa L. nodules using multidisciplinary approach.

Industrial wastes and fertilizers can introduce excessive levels of nickel (Ni) into the environment, potentially causing threats to plants, animals, as well as human beings. However, the number of studies on the effects of Ni toxicity on nodules is fairly limited. To address this issue, the effects of increasing Ni concentration on alfalfa nodules were assessed at chemical, biochemical, and transcriptomic levels. For this purpose, plants were grown in soils supplied with Ni (control, 0 mg/kg; C1, 50 mg/kg; C2, 150 mg/kg; C3, 250 mg/kg; and C4, 500 mg/kg) for 90 days. Ni loads in leaves, roots, and nodules were monitored after the exposure period. A set of biochemical biomarkers of oxidative stress was determined in nodules including antioxidants and metal homeostasis as well as lipid peroxidation. Gene expression levels of the main targets involved in oxidative stress and metal homeostasis were assessed. Our data indicated a high concentration of Ni in leaves, roots, and nodules where values reached 25.64 ± 3.04 mg/kg, 83.23 ± 5.16 mg/kg, and 125.71 ± 4.53 mg/kg in dry weight, respectively. Moreover, a significant increase in nodule biomass was observed in plants exposed to C4 in comparison to control treatment and percentage increased by 63%. Then, lipid peroxidation increased with a rate of 95% in nodules exposed to C4. Enzymatic activities were enhanced remarkably, suggesting the occurrence of oxidative stress, with increased superoxide dismutase (SOD), glutathione reductase (GR), and ascorbate peroxidase (APX). Our results showed also a significant upregulation of SOD, GR and APX genes in nodules. Nodule homoglutathione (HGSH) levels increased with the different Ni concentrations, with a remarkable decrease of glutathione S-transferase (GST) activity and glutathione (GSH) content for the highest Ni concentration with 43% and 52% reduction, respectively. The phytochelatin (PC) and metallothionein (MT) concentrations increased in nodules, which implied the triggering of a cellular protection mechanism for coping with Ni toxicity. The results suggested that Ni promotes a drastic oxidative stress in alfalfa nodules, yet the expression of MT and PC to reduce Ni toxicity could be used as Ni stress bioindicators. Our findings provide new insights into the central role of alfalfa nodules in limiting the harmful effects of soil pollution. Therefore, nodules co-expressing antioxidant enzymes may have high phytoremediation potential.

Quantification of Reactive Oxygen Species Using 2',7'-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in Müller Glial Cells.

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the modification of proteins, lipids, and DNA, which finally may lead to alteration in cellular function and cell death. Therefore, it is beneficial for cells to increase their antioxidant defense in response to detrimental insults, either by activating an antioxidant pathway like Keap1/Nrf2 or by improving redox scavengers (vitamins A, C, and E, β-carotene, and polyphenols, among others). Inflammation and oxidative stress are involved in the pathogenesis and progression of retinopathies, such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Since Müller glial cells (MGCs) play a key role in the homeostasis of neural retinal tissue, they are considered an ideal model to study these cellular protective mechanisms. In this sense, quantifying ROS levels with a reproducible and simple method is essential to assess the contribution of pathways or molecules that participate in the antioxidant cell defense mechanism. In this article, we provide a complete description of the procedures required for the measurement of ROS with DCFH-DA probe and flow cytometry in MGCs. Key steps for flow cytometry data processing with the software are provided here, so the readers will be able to measure ROS levels (geometric means of FITC) and analyze fluorescence histograms. These tools are highly helpful to evaluate not only the increase in ROS after a cellular insult but also to study the antioxidant effect of certain molecules that can provide a protective effect on the cells.

Toxic effects and mechanisms of common pesticides (Roundup and chlorpyrifos) and their mixtures in a zebrafish model (Danio rerio)

Agrochemicals can adversely affect biodiversity, environment and human health, and commonly occur in mixtures with poorly characterized toxic mechanisms and health hazards. Here, we evaluated the individual and mixture toxicities of Roundup and chlorpyrifos in environmentally relevant concentrations to zebrafish using molecular and biochemical indices. Studied pesticides alone and in combination caused depletion of total antioxidant capacity and cellular thiols, overproduction of ROS, accumulation of oxidative lesions and elevated DNA damage in zebrafish liver. Notably, low concentration of Roundup induced a hormesis-like effect by stimulating the protective cellular mechanisms. Chlorpyrifos showed stronger prooxidant effects than Roundup and additionally caused nitrosative and carbonyl stress in zebrafish. At the organismal level, studied pesticides and their mixtures induced hepato- and neurotoxicity. The effects of the studied pesticides on biomarkers of apoptosis, endocrine disruption and immune disorders were generally weak and inconsistent. The multibiomarker assessment showed that chlorpyrifos is considerably more toxic than Roundup to zebrafish. The toxic effects of the pesticide mixtures were mostly driven by chlorpyrifos, with minimal or mitigating effects of Roundup addition. These findings elucidate the toxic mechanisms of common pesticides in a model vertebrate and demonstrate that health hazards of pesticide mixtures cannot be predicted from the effects of single pesticides.

Exogenous Glutathione Protects IPEC-J2 Cells against Oxidative Stress through a Mitochondrial Mechanism.

The accumulation of reactive oxygen species (ROS) triggers oxidative stress in cells by oxidizing and modifying various cellular components, preventing them from performing their inherent functions, ultimately leading to apoptosis and autophagy. Glutathione (GSH) is a ubiquitous intracellular peptide with multiple functions. In this study, a hydrogen peroxide (HO)-induced oxidative damage model in IPEC-J2 cells was used to investigate the cellular protection mechanism of exogenous GSH against oxidative stress. The results showed that GSH supplement improved the cell viability reduced by HO-induced oxidative damage model in IPEC-J2 cells in a dose-dependent manner. Moreover, supplement with GSH also attenuated the HO-induced MMP loss, and effectively decreased the HO-induced mitochondrial dysfunction by increasing the content of mtDNA and upregulating the expression TFAM. Exogenous GSH treatment significantly decreased the ROS and MDA levels, improved SOD activity in HO-treated cells and reduced HO-induced early apoptosis in IPEC-J2 cells. This study showed that exogenous GSH can protect IPEC-J2 cells against apoptosis induced by oxidative stress through mitochondrial mechanisms.

P2X2 receptor subunit interfaces are missense variant hotspots, where mutations tend to increase apparent ATP affinity.

Background and PurposeP2X receptors are trimeric ligand‐gated ion channels that open a cation‐selective pore in response to ATP binding to their large extracellular domain. The seven known P2X subtypes can assemble as homotrimeric or heterotrimeric complexes and contribute to numerous physiological functions, including nociception, inflammation and hearing. The overall structure of P2X receptors is well established, but little is known about the range and prevalence of human genetic variations and the functional implications of specific domains.Experimental ApproachHere, we examine the impact of P2X2 receptor inter‐subunit interface missense variants identified in the human population or by structural predictions. We test both single and double mutants through electrophysiological and biochemical approaches.Key ResultsWe demonstrate that predicted extracellular domain inter‐subunit interfaces display a higher‐than‐expected density of missense variations and that the majority of mutations that disrupt putative inter‐subunit interactions result in channels with higher apparent ATP affinity. Lastly, we show that double mutants at the subunit interface show significant energetic coupling, especially if located in close proximity.Conclusion and ImplicationsWe provide the first structural mapping of the mutational distribution across the human population in a ligand‐gated ion channel and show that the density of missense mutations is constrained between protein domains, indicating evolutionary selection at the domain level. Our data may indicate that, unlike other ligand‐gated ion channels, P2X2 receptors have evolved an intrinsically high threshold for activation, possibly to allow for additional modulation or as a cellular protection mechanism against overstimulation.

Predicted 3D model of the M protein of Porcine Epidemic Diarrhea Virus and analysis of its immunogenic potential.

The membrane protein M of the Porcine Epidemic Diarrhea Virus (PEDV) is the most abundant component of the viral envelope. The M protein plays a central role in the morphogenesis and assembly of the virus through protein interactions of the M-M, M-Spike (S) and M-nucleocapsid (N) type. The M protein is known to induce protective antibodies in pigs and to participate in the antagonistic response of the cellular antiviral system coordinated by the type I and type III interferon pathways. The 3D structure of the PEDV M protein is still unknown. The present work exposes a predicted 3D model of the M protein generated using the Robetta protocol. The M protein model is organized into a transmembrane and a globular region. The obtained 3D model of the PEDV M protein was compared with 3D models of the SARS-CoV-2 M protein created using neural networks and with initial machine learning-based models created using trRosetta. The 3D model of the present study predicted four linear B-cell epitopes (RSVNASSGTG and KHGDYSAVSNPSALT peptides are noteworthy), six discontinuous B-cell epitopes, forty weak binding and fourteen strong binding T-cell epitopes in the CV777 M protein. A high degree of conservation of the epitopes predicted in the PEDV M protein was observed among different PEDV strains isolated in different countries. The data suggest that the M protein could be a potential candidate for the development of new treatments or strategies that activate protective cellular mechanisms against viral diseases.

Epigenetic regulation of thermomorphogenesis and heat stress tolerance.

Many environmental conditions fluctuate and organisms need to respond effectively. This is especially true for temperature cues that can change in minutes to seasons and often follow a diurnal rhythm. Plants cannot migrate and most cannot regulate their temperature. Therefore, a broad array of responses have evolved to deal with temperature cues from freezing to heat stress. A particular response to mildly elevated temperatures is called thermomorphogenesis, a suite of morphological adaptations that includes thermonasty, formation of thin leaves and elongation growth of petioles and hypocotyl. Thermomorphogenesis allows for optimal performance in suboptimal temperature conditions by enhancing the cooling capacity. When temperatures rise further, heat stress tolerance mechanisms can be induced that enable the plant to survive the stressful temperature, which typically comprises cellular protection mechanisms and memory thereof. Induction of thermomorphogenesis, heat stress tolerance and stress memory depend on gene expression regulation, governed by diverse epigenetic processes. In this Tansley review we update on the current knowledge of epigenetic regulation of heat stress tolerance and elevated temperature signalling and response, with a focus on thermomorphogenesis regulation and heat stress memory. In particular we highlight the emerging role of H3K4 methylation marks in diverse temperature signalling pathways.

The Role of Triacylglycerol in the Protection of Cells against Lipotoxicity under Drought in Lolium multiflorum/Festucaarundinacea Introgression Forms.

Triacylglycerol is a key lipid compound involved in maintaining homeostasis of both membrane lipids and free fatty acids (FFA) in plant cells under adverse environmental conditions. However, its role in the process of lipid remodeling has not been fully recognized, especially in monocots, including grass species. For our study, two closely related introgression forms of Lolium multiflorum (Italian ryegrass) and Festuca arundinacea (tall fescue), distinct in their level of drought tolerance, were selected as plant models to study rearrangements in plant lipidome under water deficit and further re-watering. The low drought tolerant (LDT) form revealed an elevated level of cellular membrane damage accompanied by an increased content of polyunsaturated FFA and triacylglycerol under water deficit, compared with the high drought tolerant (HDT) form. However, the LDT introgression form demonstrated also the ability to regenerate its membranes after stress cessation. The obtained results clearly indicated that accumulation of triacylglycerol under advanced drought in the LDT form could serve as a cellular protective mechanism against overaccumulation of toxic polyunsaturated FFA and other lipid intermediates. Furthermore, accumulation of triacylglycerol under drought conditions could serve also as storage of substrates required for further regeneration of membranes after stress cessation. The rearrangements in triacylglycerol metabolism were supported by the upregulation of several genes, involved in a biosynthesis of triacylglycerol. With respect to this process, diacylglycerol O-acyltransferase DGAT2 seems to play the most important role in the analyzed grasses.

Harnessing shear stress preconditioning to improve cell viability in 3D post-printed biostructures using extrusion bioprinting

Extrusion-based three dimensional bioprinting has emerged as a promising fabrication technique in the field of tissue engineering. However, it still suffers from reduced cell survival post-printing due to process-induced shear stress. Great research efforts have been made to reduce such an effect, yet none have investigated the ability of preconditioned cells to tolerate stress induced by printing. In this study, we investigated the effect of shear stress preconditioning of C2C12 murine myoblasts on post-printing cell viability using a custom-built device to induce shear stress. Here, we present the first study investigating the link between shear stress preconditioning, cellular protective mechanisms and cell survival post-printing. We hypothesized that moderate shear stress preconditioning increases the ability of cells to tolerate bioprinting induced stress, thus improving their viability after printing. We used HSP70 expression as an indicator of cellular stress and of the activation of cell adaptation/repair mechanisms. Our results showed an increase in level and a translocation of HSP70 in shear stress preconditioned cells compared to non-conditioned cells. In addition, our results demonstrated that exposure to shear stress prior to printing enhances viability of cells after printing. In particular, viability of preconditioned cells printed via a nozzle was 7.8% higher than that of non-conditioned cells while viability of preconditioned cells printed via a needle was 6.6%, higher than that of non-conditioned cells. Taken together, these findings indicate that shear stress cell preconditioning is a promising method to enhance cell viability post-printing, which is of vital importance in tissue engineering applications.

Efficiency of human plague vaccination in tuvinian natural plague focus. Message 2: dynamics of immune status indicators after revaccination

Relevance. In Russia, the live plague vaccine (LPV) is used for specific prophylaxis of plague. Immunological monitoring of humans vaccinated by LPV in order to search for informative diagnostic markers, as well as to improve the tactics of epidemiological surveillance of plague enzootic territories is an urgent area of research.The aim is to assess the parameters of cellular and humoral immunity in humans revaccinated by LPV who permanently reside on territory of the Tuvinian natural plague focus.Materials and methods. The study involved 76 volunteers from the Republic of Tuva, revaccinated by LPV. Blood sampling was performed before vaccination and 1, 3, and 6 months after revaccination. The study included the determination of cytokine production (IFN-γ, IL-4, TNF-α), specific antibodies, immunoglobulins (IgM, IgG, IgA and IgE) and lymphocyte subpopulation composition (CD3, CD4, CD8, CD16, CD19, immunoregulatory index).Results. A decrease in IgG and IgM and an increase in IgA were found after vaccination with LPV and their increase after revaccination. Correlation relationships were revealed between immunoglobulins, B cells and IL-4. Revaccination leads to an increase seroconversion. The activation of humoral immunity in humans vaccinated against plague is also evidenced by dynamics of changes in the subpopulation composition: an increase in B-lymphocytes and natural killer cells, a decrease in T-helpers and immunoregulatory index, and cellular immunity stimulation is an increase in spontaneous and induced production of pro- and anti-inflammatory cytokines.Conclusion. It has been shown that LPV is capable of causing the body’s immune restructuring and activating the cellular and humoral mechanisms of immunological protection. For a complete understanding of the development and preservation of antiplague immunity, it is necessary to continue the annual immunological monitoring of the population living on the territory of the Tuvinian natural plague focus, using additional modern research methods.