Cytoplasmic aggregation of mutant FUS causes multistep RNA splicing perturbations in the course of motor neuron pathology.

Dysfunction of the RNA-binding protein (RBP) FUS implicated in RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. Mutations affecting FUS nuclear localization can drive RNA splicing defects and stimulate the formation of non-amyloid inclusions in affected neurons. However, the mechanism by which FUS mutations contribute to the development of ALS remains uncertain. Here we describe a pattern of RNA splicing changes in the dynamics of the continuous proteinopathy induced by mislocalized FUS. We show that the decrease in intron retention of FUS-associated transcripts represents the hallmark of the pathogenesis of ALS and is the earliest molecular event in the course of progression of the disease. As FUS aggregation increases, the pattern of RNA splicing changes, becoming more complex, including a decrease in the inclusion of neuron-specific microexons and induction of cryptic exon splicing due to the sequestration of additional RBPs into FUS aggregates. Crucially, the identified features of the pathological splicing pattern are also observed in ALS patients in both sporadic and familial cases. Our data provide evidence that both a loss of nuclear FUS function due to mislocalization and the subsequent cytoplasmic aggregation of mutant protein lead to the disruption of RNA splicing in a multistep fashion during FUS aggregation.

Antigenic Cartography of SARS-CoV-2.

Antigenic cartography is a tool for interpreting and visualizing antigenic differences between virus variants based on virus neutralization data. This approach has been successfully used in the selection of influenza vaccine seed strains. With the emergence of SARS-CoV-2 variants escaping vaccine-induced antibody response, adjusting COVID-19 vaccines has become essential. This review provides information on the antigenic differences between SARS-CoV-2 variants revealed by antigenic cartography and explores a potential of antigenic cartography-based methods (e.g., building antibody landscapes and neutralization breadth gain plots) for the quantitative assessment of the breadth of the antibody response. Understanding the antigenic differences of SARS-CoV-2 and the possibilities of the formed humoral immunity aids in the prompt modification of preventative vaccines against COVID-19.

Changes in the liver proteome of zebrafish (Danio rerio) exposed to glyphosate and aminomethylphosphonic acid in the presence of a humic substance.

Herbicide exposure can pose a considerable threat to non-target aquatic animals. We aimed to study changes in the liver proteome of a model cyprinid fish species, zebrafish Danio rerio, to provide a molecular basis for the adverse effects of environmentally relevant concentrations of glyphosate (100 µg/L), its breakdown product aminomethylphosphonic acid (AMPA; 100 µg/L), and a mixture of both (50 + 50 µg/L) in the presence of humic acid (20 mg/L), which simulated a component of natural organic matter in the aquatic environment. Proteomic analysis was performed by means of high-performance liquid chromatography-tandem mass spectrometry employing a label-free quantification approach. The results present molecular evidence of the stress responses and disturbance of primary metabolic processes such as immune response, dysregulation in DNA repair, necroptosis and apoptosis signaling pathways, oxidative phosphorylation, cholesterol, lipoprotein, and carbohydrate metabolism. We registered the synergistic effect of the glyphosate and AMPA co-exposure, which was expressed in a substantial increase in the number of dysregulated proteins compared to the solo treatments. Humic acid alleviated the effects of glyphosate and its mixture with AMPA and aggravated the impact of AMPA exposure. RuvB-like 2, a protein taking part in DNA repair, and EIF2S1, involved in the regulation of stress-induced gene expression, were downregulated in the liver of zebrafish from all treatments.

Differential Expression of Stress Adaptation Genes in a Diatom Ulnaria acus under Different Culture Conditions.

Diatoms are a group of unicellular eukaryotes that are essential primary producers in aquatic ecosystems. The dynamic nature of their habitat necessitates a quick and specific response to various stresses. However, the molecular mechanisms of their physiological adaptations are still underexplored. In this work, we study the response of the cosmopolitan freshwater diatom Ulnaria acus (Bacillariophyceae, Fragilariophycidae, Licmophorales, Ulnariaceae, Ulnaria) in relation to a range of stress factors, namely silica deficiency, prolonged cultivation, and interaction with an algicidal bacterium. Fluorescent staining and light microscopy were used to determine the physiological state of cells under these stresses. To explore molecular reactions, we studied the genes involved in the stress response-type III metacaspase (MC), metacaspase-like proteases (MCP), death-specific protein (DSP), delta-1-pyrroline-5-carboxylate dehydrogenase (ALDH12), and glutathione synthetase (GSHS). We have described the structure of these genes, analyzed the predicted amino acid sequences, and measured their expression dynamics in vitro using qRT-PCR. We demonstrated that the expression of UaMC1, UaMC3, and UaDSP increased during the first five days of silicon starvation. On the seventh day, it was replaced with the expression of UaMC2, UaGSHS, and UaALDH. After 45 days of culture, cells stopped growing, and the expression of UaMC1, UaMC2, UaGSHS, and UaDSP increased. Exposure to an algicidal bacterial filtrate induced a higher expression of UaMC1 and UaGSHS. Thus, we can conclude that these proteins are involved in diatoms' adaptions to environmental changes. Further, these data show that the molecular adaptation mechanisms in diatoms depend on the nature and exposure duration of a stress factor.

Identification of cell type-specific correlations between ERK activity and cell viability upon treatment with ERK1/2 inhibitors.

Increased MAPK signaling is a hallmark of various cancers and is a central regulator of cell survival. Direct ERK1/2 inhibition is considered a promising approach to avoid ERK1/2 reactivation caused by upstream kinases BRAF, MEK1/2, and KRAS, as well as by receptor tyrosine kinase inhibitors, but the dynamics and selectivity of ERK1/2 inhibitors are much less studied compared with BRAF or MEK inhibitors. Using ERK1/2 and downstream kinase ELK1 reporter cell lines of lung cancer (H1299; NRASQ61K), colon cancer (HCT-116; KRASG13D), neuroblastoma (SH-SY5Y), and leukemia (U937), we examined the relationship between ERK inhibition and drug-induced toxicity for five ERK inhibitors: SCH772984, ravoxertinib, LY3214996, ulixertinib, and VX-11e, as well as one MEK inhibitor, PD0325901. Comparing cell viability and ERK inhibition revealed different ERK dependencies for these cell lines. We identify several drugs, such as SCH772984 and VX-11e, which induce excessive toxicity not directly related to ERK1/2 inhibition in specific cell lines. We also show that PD0325901, LY3214996, and ulixertinib are prone to ERK1/2 reactivation over time. We distinguished two types of ERK1/2 reactivation: the first could be reversed by adding a fresh dose of inhibitors, while the second persists even after additional treatments. We also showed that cells that became resistant to the MEK1/2 inhibitor PD0325901 due to ERK1/2 reactivation remained sensitive to ERK1/2 inhibitor ulixertinib. Our data indicate that correlation of ERK inhibition with drug-induced toxicity in multiple cell lines may help to find more selective and effective ERK1/2 inhibitors.

The Role of Ubiquitin-Proteasome System in the Biology of Stem Cells.

Selective degradation of cellular proteins by the ubiquitin-proteasome system (UPS) is one of the key regulatory mechanisms in eukaryotic cells. A growing body of evidence indicates that UPS is involved in the regulation of fundamental processes in mammalian stem cells, including proliferation, differentiation, cell migration, aging, and programmed cell death, via proteolytic degradation of key transcription factors and cell signaling proteins and post-translational modification of target proteins with ubiquitin. Studying molecular mechanisms of proteostasis in stem cells is of great importance for the development of new therapeutic approaches aimed at the treatment of autoimmune and neurodegenerative diseases, cancer, and other socially significant pathologies. This review discusses current data on the UPS functions in stem cells.

Analysis of Predicted Amino Acid Sequences of Diatom Microtubule Center Components.

Diatoms synthesize species-specific exoskeletons inside cells under the control of the cytoskeleton and microtubule center. Previous studies have been conducted with the visualization of the microtubule center; however, its composition has not been studied and reliably established. In the present study, several components of MTOC in diatoms, GCP (gamma complex proteins), Aurora A, and centrins have been identified. Analysis of the predicted amino acid sequences of these proteins revealed structural features typical for diatoms. We analyzed the conserved amino acids and the motives necessary for the functioning of proteins. Phylogenetic analysis of GCP showed that all major groups of diatoms are distributed over phylogenetic trees according to their systematic position. This work is a theoretical study; however, it allows drawing some conclusions about the functioning of the studied components and possible ways to regulate them.

Chronic Administration of Non-Constitutive Proteasome Inhibitor Modulates Long-Term Potentiation and Glutamate Signaling-Related Gene Expression in Murine Hippocampus.

Proteasomes degrade most intracellular proteins. Several different forms of proteasomes are known. Little is known about the role of specific proteasome forms in the central nervous system (CNS). Inhibitors targeting different proteasome forms are used in clinical practice and were shown to modulate long-term potentiation (LTP) in hippocampal slices of untreated animals. Here, to address the role of non-constitutive proteasomes in hippocampal synaptic plasticity and reveal the consequences of their continuous inhibition, we studied the effect of chronic administration of the non-constitutive proteasome inhibitor ONX-0914 on the LTP induced by two different protocols: tetanic stimulation and theta-burst stimulation (TBS). Both the tetanus- and TBS-evoked potentiation contribute to the different forms of hippocampal-dependent memory and learning. Field-excitatory postsynaptic potentials (fEPSPs) in hippocampal slices from control animals and animals treated with DMSO or ONX-0914 were compared. LTP induced by the TBS was not affected by ONX-0914 administration; however, chronic injections of ONX-0914 led to a decrease in fEPSP slopes after tetanic stimulation. The observed effects correlated with differential expression of genes involved in synaptic plasticity, glutaminergic synapse, and synaptic signaling. Obtained results indicate that non-constitutive proteasomes are likely involved in the tetanus-evoked LTP, but not the LTP occurring after TBS, supporting the relevance and complexity of the role of specific proteasomes in synaptic plasticity, memory, and learning.

Antigenic Cartography Indicates That the Omicron BA.1 and BA.4/BA.5 Variants Remain Antigenically Distant to Ancestral SARS-CoV-2 after Sputnik V Vaccination Followed by Homologous (Sputnik V) or Heterologous (Comirnaty) Revaccination.

The rapid emergence of evasive SARS-CoV-2 variants is an ongoing challenge for COVID-19 vaccinology. Traditional virus neutralization tests provide detailed datasets of neutralization titers against the viral variants. Such datasets are difficult to interpret and do not immediately inform of the sufficiency of the breadth of the antibody response. Some of these issues could be tackled using the antigenic cartography approach. In this study, we created antigenic maps using neutralization titers of sera from donors who received the Sputnik V booster vaccine after primary Sputnik V vaccination and compared them with the antigenic maps based on serum neutralization titers of Comirnaty-boosted donors. A traditional analysis of neutralization titers against the WT (wild-type), Alpha, Beta, Delta, Omicron BA.1, and BA.4/BA.5 variants showed a significant booster humoral response after both homologous (Sputnik V) and heterologous (Comirnaty) revaccinations against all of the studied viral variants. However, despite this, a more in-depth analysis using antigenic cartography revealed that Omicron variants remain antigenically distant from the WT, which is indicative of the formation of insufficient levels of cross-neutralizing antibodies. The implications of these findings may be significant when developing a new vaccine regimen.

The TZM-bl Reporter Cell Line Expresses Kynureninase That Can Neutralize 2F5-like Antibodies in the HIV-1 Neutralization Assay.

Induction of broadly neutralizing antibodies targeting ectodomain of the transmembrane (TM) glycoprotein gp41 HIV-1 provides a basis for the development of a universal anti-viral vaccine. The HeLa cell-derived TZM-bl reporter cell line is widely used for the estimation of lentiviruses neutralization by immune sera. The cell line is highly permissive to infection by most strains of HIV, SIV, and SHIV. Here we demonstrated that TZM-bl cells express a 48 kDa non-glycosylated protein (p48) recognized by broadly neutralizing monoclonal antibody (mAb) 2F5 targeting the ELDKWA (aa 669-674) epitope of gp41TM of HIV-1. A significant amount of p48 was found in the cell supernatant. The protein was identified as human kynureninase (KYNU), which has the ELDKWA epitope. The protein is further called "p48 KYNU". The HIV-1 neutralization by mAb 2F5 and 4E10 in the presence of p48KYNU was tested on Jurkat and TZM-bl cells. It was demonstrated that p48KYNU reduces neutralization by 2F5-like antibodies, but it has almost no effect on mAb 4E10. Therefore, p48KYNU can attenuate HIV-1 neutralization by 2F5-like antibodies and hence create false-negative results. Thus, previously tested immune sera that recognized the ELDKWA-epitope and demonstrated a "weak neutralization" of HIV-1 in TZM-bl assay should be reevaluated.

Unique and ancestral features in trunk kidney microanatomy and ultrastructure of omul Coregonus migratorius.

This study presents novel data on the microanatomy and ultrastructure of the omul Coregonus migratorius trunk kidney. Adult individuals of C. migratorius were sampled in the Barguzin Bay of Lake Baikal. Active leuko- and erythropoiesis were found in the interstitium of the mesonephros. For the first time, cells with radially arranged vesicles have been described in the renal interstitium of C. migratorius. The quantitative characteristics of blood cells and ultrastructural parameters of leukocytes reflected the functioning of the non-specific defence system in the organism. The share of the renal interstitium, morphological diversity of the epithelial cells of the nephron tubules, the ultrastructural features of the renal corpuscles and nephron tubules and the number of mitochondria in leukocytes and ion-transporting cells were typical for representatives of the whitefish Coregonus lavaretus complex and thus considered ancestral features of the present-day C. migratorius population reflecting its adaptive potential to living in an ultra-deep Lake Baikal.

Responses of hepatic biotransformation and antioxidant enzymes in Japanese medaka (Oryzias latipes) exposed to humic acid.

Humic substances, a major component of natural organic matter in surface waters, can induce biotransformation enzyme activities and influence antioxidant defense in fish. The study aimed to provide a molecular basis for the stress responses, the synthesis of biotransformation, and antioxidant enzymes in particular. Adult medaka fish (Hd-rR strain) were exposed to environmentally relevant concentrations of humic acid for 96 h. The actual humic acid concentrations in water were determined photometrically and expressed as organic carbon concentrations. Liquid chromatography with tandem mass spectrometry was used for protein profile analysis of medaka liver samples. The relative amount of isozymes was determined using the label-free quantification approach. Hepatic biotransformation enzyme activities were measured as well. Thus, ethoxyresorufin-O-deethylase activity showed a pronounced induction at the highest tested concentration (9.4 mg C/L). Various biotransformation and antioxidant isozymes responded to humic acid differently, reflecting a balanced interplay of proteins that ensures the metabolism of humic acid in fish liver. Some isozymes were not affected by humic acid. The study provides new insight into the molecular mechanisms of the fish stress response to the humic acid-related challenge.

Planar Gas Expansion under Intensive Nanosecond Laser Evaporation into Vacuum as Applied to Time-of-Flight Analysis.

A computational investigation of the dynamics of gas expansion due to intense nanosecond laser evaporation into vacuum has been carried out. The problem is solved in a one-dimensional approximation, which simplifies calculations and at the same time allows one to analyze the main features of the expansion dynamics. For analysis we use three different approaches. Two of them are based on kinetic analysis via the direct simulation Monte Carlo (DSMC) method and numerical solution of the model Bhatnagar-Gross-Krook (BGK) equation. The third one focuses on derivation of an analytical continuum solution. Emphasis is placed on the analysis of the velocity distribution function and the average energy of particles passing through the time-of-flight detector on the normal to the evaporation surface, which is important for interpreting experimental measurements. The formulated problem is quite difficult as the considered flow is time-dependent, contains discontinuities in boundary conditions and involves large variations of local Knudsen numbers as well as steep gradients of the velocity distribution function. Data were obtained on the particle energy in the time-of-flight distribution for the range of regimes from the free molecular flow to continuum one. The maximum attainable average energy of particles in the time-of-flight distribution is determined. The non-monotonicity of the energy increase was found, which is explained based on analysis of the velocity distribution of particles.

A Comprehensive Analysis of Human Endogenous Retroviruses HERV-K (HML.2) from Teratocarcinoma Cell Lines and Detection of Viral Cargo in Microvesicles.

About 8% of our genome is composed of sequences from Human Endogenous Retroviruses (HERVs). The HERV-K (HML.2) family, here abbreviated HML.2, is able to produce virus particles that were detected in cell lines, malignant tumors and in autoimmune diseases. Parameters and properties of HML.2 released from teratocarcinoma cell lines GH and Tera-1 were investigated in detail. In most experiments, analyzed viruses were purified by density gradient centrifugation. HML.2 structural proteins, reverse transcriptase (RT) activity, viral RNA (vRNA) and particle morphology were analyzed. The HML.2 markers were predominantly detected in fractions with a buoyant density of 1.16 g/cm3. Deglycosylation of TM revealed truncated forms of transmembrane (TM) protein. Free virions and extracellular vesicles (presumably microvesicles-MVs) with HML.2 elements, including budding intermediates, were detected by electron microscopy. Viral elements and assembled virions captured and exported by MVs can boost specific immune responses and trigger immunomodulation in recipient cells. Sequencing of cDNA clones demonstrated exclusive presence of HERV-K108 env in HML.2 from Tera-1 cells. Not counting two recombinant variants, four known env sequences were found in HML.2 from GH cells. Obtained results shed light on parameters and morphology of HML.2. A possible mechanism of HML.2-induced diseases is discussed.

Bacterial Communities in Areas of Oil and Methane Seeps in Pelagic of Lake Baikal.

We have assessed the diversity of bacteria near oil-methane (area I) and methane (area II) seeps in the pelagic zone of Lake Baikal using massive parallel sequencing of 16S rRNA, pmoA, and mxaF gene fragments amplified from total DNA. At depths from the surface to 100 m, sequences belonging to Cyanobacteria dominated. In the communities to a depth of 200 m of the studied areas, Proteobacteria dominated the deeper layers of the water column. Alphaproteobacteria sequences were predominant in the community near the oil-methane seep, while the community near the methane seep was characterized by the prevalence of Alpha- and Gammaproteobacteria. Among representatives of these classes, type I methanotrophs prevailed in the 16S rRNA gene libraries from the near-bottom area, and type II methanotrophs were detected in minor quantities at different depths. In the analysis of the libraries of the pmoA and mxaF functional genes, we observed the different taxonomic composition of methanotrophic bacteria in the surface and deep layers of the water column. All pmoA sequences from area I were type II methanotrophs and were detected at a depth of 300 m, while sequences of type I methanotrophs were the most abundant in deep layers of the water column of area II. All mxaF gene sequences belonged to Methylobacterium representatives. Based on comparative analyses of 16S rRNA, pmoA, and mxaF gene fragment libraries, we suggest that there must be a wider spectrum of functional genes facilitating methane oxidation that were not detected with the primers used.

FUS(1-359) transgenic mice as a model of ALS: pathophysiological and molecular aspects of the proteinopathy.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that leads to the eventual death of motor neurons. Described cases of familial ALS have emphasized the significance of protein misfolding and aggregation of two functionally related proteins, FUS (fused in sarcoma) and TDP-43, implicated in RNA metabolism. Herein, we performed a comprehensive analysis of the in vivo model of FUS-mediated proteinopathy (ΔFUS(1-359) mice). First, we used the Noldus CatWalk system and confocal microscopy to determine the time of onset of the first clinical symptoms and the appearance of FUS-positive inclusions in the cytoplasm of neuronal cells. Second, we applied RNA-seq to evaluate changes in the gene expression profile encompassing the pre-symptomatic and the symptomatic stages of disease progression in motor neurons and the surrounding microglia of the spinal cord. The resulting data show that FUS-mediated proteinopathy is virtually asymptomatic in terms of both the clinical symptoms and the molecular aspects of neurodegeneration until it reaches the terminal stage of disease progression (120 days from birth). After this time, the pathological process develops very rapidly, resulting in the formation of massive FUS-positive inclusions accompanied by a transcriptional "burst" in the spinal cord cells. Specifically, it manifests in activation of a pro-inflammatory phenotype of microglial cells and malfunction of acetylcholine synapse transmission in motor neurons. Overall, we assume that the highly reproducible course of the pathological process, as well as the described accompanying features, makes ΔFUS(1-359) mice a convenient model for testing potential therapeutics against proteinopathy-induced decay of motor neurons.

Evolutionary history of the chitin synthases of eukaryotes.

Chitin synthases are widespread among eukaryotes and known to have a complex evolutionary history in some of the groups. We have reconstructed the chitin synthase phylogeny using the most taxonomically comprehensive dataset currently available and have shown the presence of independently formed paralogous groups in oomycetes, ciliates, fungi, and all diatoms except raphid pennates. There were also two cases of horizontal gene transfer (HGT): transfer from fungus to early diatoms gave rise to diatom paralogous group, while transfer from raphid pennate diatom to Acantamoeba ancestor is, to our knowledge, restricted to a single gene in amoeba. Early evolution of chitin synthases is heavily obscured by paralogy, and further sequencing effort is necessary.

New PCR diagnostic systems for the detection and quantification of porcine cytomegalovirus (PCMV).

Pigs are frequently infected with porcine cytomegalovirus (PCMV). Infected adult animals may not present with symptoms of disease, and the virus remains latent. However, the virus may be transmitted to human recipients receiving pig transplants. Recently, it was shown that pig-to-non-human-primate xenotransplantations showed 2 to 3 times lower transplant survival when the donor pig was infected with PCMV. Therefore, highly sensitive methods are required to select virus-free pigs and to examine xenotransplants. Seven previously established PCR detection systems targeting the DNA polymerase gene of PCMV were examined by comparison of thermodynamic parameters of oligonucleotides, and new diagnostic nested PCR and real-time PCR systems with improved parameters and high sensitivity were established. The detection limit of conventional PCR was estimated to be 15 copies, and that of the nested PCR was 5 copies. The sensitivity of the real-time PCR with a TaqMan probe was two copies. An equal efficiency of the newly established detection systems was shown by parallel testing of DNA from sera and blood of six pigs, identifying the same animals as PCMV infected. These new diagnostic PCR systems will improve the detection of PCMV and therefore increase the safety of porcine xenotransplants.

Glyphosate and aminomethylphosphonic acid impact on redox status and biotransformation in fish and the mitigating effects of diet supplementation.

Fish reared under seminatural conditions can be challenged by exposure to herbicides. Farming facilities relying on the surrounding area's water quality can be affected by glyphosate and aminomethylphosphonic acid (AMPA) contamination. This review summarizes findings on how glyphosate and AMPA in the amounts registered in surface waterbodies affect redox status and biotransformation in fish and covers the aspect of diet supplementation for oxidative stress relief. Environmentally relevant concentrations of glyphosate and AMPA can alter the transcription and catalytic activities of antioxidant enzymes, decrease the content of reduced glutathione, and increase the accumulation of lipid peroxidation products, all of which are signs of a redox imbalance. Glyphosate has been shown to affect complex I in the mitochondrial respiratory chain and dysregulate iron transport-related genes, causing redox disturbance. Relatively high but environmentally realistic glyphosate concentrations can initiate the induction of cytochrome P450 biotransformation enzymes, alter the regulation of ABC exporters, and cause the inhibition of the redox-sensitive Nrf2 signaling pathway. Studies on reducing herbicide toxicity through dietary supplementation are a promising area of research. Natural functional supplements have been proven to have great potential for mitigating glyphosate-induced oxidative stress and thereby improving fish health, which in turn means maintaining productivity in fish farms that use natural water. However, data on the effects of AMPA on fish are scarce, and studies on the alleviation of its toxicity in fish are lacking. Considering the variety of AMPA contamination routes, one cannot underestimate the need for further research.

Ultrastructure of the mesonephros of whitefishes from the Lake Baikal basin.

The article presents a study of the mesonephros ultrastructure of Baikal omul Coregonus migratorius, Baikal whitefish Coregonus baicalensis, and a cross between Baikal whitefish and humpback whitefish (C. baicalensis × Coregonus pidschian). The mesonephros ultrastructure was studied using electron microscopy methods. The results of the study show that the number of mature granulocytes is a systematic feature and does not depend on the ecology of fish. The quantitative characteristics of blood cells and the ultrastructural features of leukocytes in the mesonephros are associated with the functioning of the nonspecific defence system in fish. Morphological diversity of epithelial cells in nephron tubules is the ancestral characteristic of the modern omul population, associated with geological and climatic events in the history of Lake Baikal. The development of haematopoietic tissue in the mesonephros, the ultrafine structure of ion-transporting interstitial cells, as well as some ultrastructural features found in the nephron, reflect the adaptive capabilities of the species to live in the ultra-deep Lake Baikal.