Effect of Focal Adhesion Kinase and Vinculin Expression on Migration Parameters of Normal and Tumor Epitheliocytes.

Focal adhesions (FAs) are mechanosensory structures that transform physical stimuli into chemical signals guiding cell migration. Comprehensive studies postulate correlation between the FA parameters and cell motility metrics for individual migrating cells. However, which properties of the FAs are critical for epithelial cell motility in a monolayer remains poorly elucidated. We used high-throughput microscopy to describe relationship between the FA parameters and cell migration in immortalized epithelial keratinocytes (HaCaT) and lung carcinoma cells (A549) with depleted or inhibited vinculin and focal adhesion kinase (FAK) FA proteins. To evaluate relationship between the FA morphology and cell migration, we used substrates with varying stiffness in the model of wound healing. Cells cultivated on fibronectin had the highest FA area values, migration rate, and upregulated expression of FAK and vinculin mRNAs, while the smallest FA area and slower migration rate to the wound were specific to cells cultivated on glass. Suppression of vinculin expression in both normal and tumor cells caused decrease of the FA size and fluorescence intensity but did not affect cell migration into the wound. In contrast, downregulation or inactivation of FAK did not affect the FA size but significantly slowed down the wound closure rate by both HaCaT and A549 cell lines. We also showed that the FAK knockdown results in the FA lifetime decrease for the cells cultivated both on glass and fibronectin. Our data indicate that the FA lifetime is the most important parameter defining migration of epithelial cells in a monolayer. The observed change in the cell migration rate in a monolayer caused by changes in expression/activation of FAK kinase makes FAK a promising target for anticancer therapy of lung carcinoma.

Anti-Inflammatory Effect of Synaptamide in Ischemic Acute Kidney Injury and the Role of G-Protein-Coupled Receptor 110.

The development of drugs for the treatment of acute kidney injury (AKI) that could suppress the excessive inflammatory response in damaged kidneys is an important clinical challenge. Recently, synaptamide (N-docosahexaenoylethanolamine) has been shown to exert anti-inflammatory and neurogenic properties. The aim of this study was to investigate the anti-inflammatory effect of synaptamide in ischemic AKI. For this purpose, we analyzed the expression of inflammatory mediators and the infiltration of different leukocyte populations into the kidney after injury, evaluated the expression of the putative synaptamide receptor G-protein-coupled receptor 110 (GPR110), and isolated a population of CD11b/c+ cells mainly representing neutrophils and macrophages using cell sorting. We also evaluated the severity of AKI during synaptamide therapy and the serum metabolic profile. We demonstrated that synaptamide reduced the level of pro-inflammatory interleukins and the expression of integrin CD11a in kidney tissue after injury. We found that the administration of synaptamide increased the expression of its receptor GPR110 in both total kidney tissue and renal CD11b/c+ cells that was associated with the reduced production of pro-inflammatory interleukins in these cells. Thus, we demonstrated that synaptamide therapy mitigates the inflammatory response in kidney tissue during ischemic AKI, which can be achieved through GPR110 signaling in neutrophils and a reduction in these cells' pro-inflammatory interleukin production.

Lactate protects neurons and astrocytes against ischemic injury by modulating Ca2+ homeostasis and inflammatory response.

Lactate is now considered an additional fuel or signaling molecule in the brain. In this study, using an oxygen-glucose deprivation (OGD) model, we found that treatment with lactate inhibited the global increase in intracellular calcium ion concentration ([Ca2+]) in neurons and astrocytes, decreased the percentage of dying cells, and caused a metabolic shift in astrocytes and neurons toward aerobic oxidation of substrates. OGD resulted in proinflammatory changes and increased expression of cytokines and chemokines, whereas incubation with lactate reduced these changes. Pure astrocyte cultures were less sensitive than neuroglia cultures during OGD. Astrocytes exposed to lipopolysaccharide (LPS) also showed pro-inflammatory changes that were reduced by incubation with lactate. Our study suggests that lactate may have neuroprotective effects under ischemic and inflammatory conditions.

Phytohormones Affect Differentiation Status of Human Skin Fibroblasts via UPR Activation.

Normalization of secretory activity and differentiation status of mesenchymal cells, including fibroblasts, is an important biomedical problem. One of the possible solutions is modulation of unfolded protein response (UPR) activated during fibroblast differentiation. Here, we investigated the effect of phytohormones on the secretory activity and differentiation of cultured human skin fibroblasts. Based on the analysis of expression of genes encoding UPR markers, abscisic acid (ABA) upregulated expression of the GRP78 and ATF4 genes, while gibberellic acid (GA) upregulated expression of CHOP. Evaluation of the biosynthetic activity of fibroblasts showed that ABA promoted secretion and synthesis of procollagen I and synthesis of fibronectin, as well as the total production of collagen and non-collagen proteins of the extracellular matrix (ECM). ABA also stimulated the synthesis of smooth muscle actin α (α-SMA), which is the marker of myofibroblasts, and increased the number of myofibroblasts in the cell population. On the contrary, GA increased the level of fibronectin secretion, but reduced procollagen I synthesis and the total production of the ECM collagen proteins. GA downregulated the synthesis of α-SMA and decreased the number of myofibroblasts in the cell population. Our results suggest that phytohormones modulate the biosynthetic activity of fibroblasts and affect their differentiation status.

Lung inflammation is associated with lipid deposition.

Lung inflammation, pneumonia, is an acute respiratory disease of varying etiology that has recently drawn much attention during the COVID-19 pandemic as lungs are among the main targets for SARS-CoV-2. Multiple other etiological agents are associated with pneumonias. Here, we describe a newly-recognized pathology, namely abnormal lipid depositions in the lungs of patients who died from COVID-19 as well as from non-COVID-19 pneumonias. Our analysis of both semi-thin and Sudan III-stained lung specimens revealed extracellular and intracellular lipid depositions irrespective of the pneumonia etiology. Most notably, lipid depositions were located within vessels adjacent to inflamed regions, where they apparently interfere with the blood flow. Structurally, the lipid droplets in the inflamed lung tissue were homogeneous and lacked outer membranes as assessed by electron microscopy. Morphometric analysis of lipid droplet deposition area allowed us to distinguish the non-pneumonia control lung specimens from the macroscopically intact area of the pneumonia lung and from the inflamed area of the pneumonia lung. Our measurements revealed a gradient of lipid deposition towards the inflamed region. The pattern of lipid distribution proved universal for all pneumonias. Finally, lipid metabolism in the lung tissue was assessed by the fatty acid analysis and by expression of genes involved in lipid turnover. Chromato-mass spectrometry revealed that unsaturated fatty acid content was elevated at inflammation sites compared to that in control non-inflamed lung tissue from the same individual. The expression of genes involved in lipid metabolism was altered in pneumonia, as shown by qPCR and in silico RNA-seq analysis. Thus, pneumonias of various etiologies are associated with specific lipid abnormalities; therefore, lipid metabolism can be considered to be a target for new therapeutic strategies.

Ectopic expression of HIV-1 Tat modifies gene expression in cultured B cells: implications for the development of B-cell lymphomas in HIV-1-infected patients.

An increased frequency of B-cell lymphomas is observed in human immunodeficiency virus-1 (HIV-1)-infected patients, although HIV-1 does not infect B cells. Development of B-cell lymphomas may be potentially due to the action of the HIV-1 Tat protein, which is actively released from HIV-1-infected cells, on uninfected B cells. The exact mechanism of Tat-induced B-cell lymphomagenesis has not yet been precisely identified. Here, we ectopically expressed either Tat or its TatC22G mutant devoid of transactivation activity in the RPMI 8866 lymphoblastoid B cell line and performed a genome-wide analysis of host gene expression. Stable expression of both Tat and TatC22G led to substantial modifications of the host transcriptome, including pronounced changes in antiviral response and cell cycle pathways. We did not find any strong action of Tat on cell proliferation, but during prolonged culturing, Tat-expressing cells were displaced by non-expressing cells, indicating that Tat expression slightly inhibited cell growth. We also found an increased frequency of chromosome aberrations in cells expressing Tat. Thus, Tat can modify gene expression in cultured B cells, leading to subtle modifications in cellular growth and chromosome instability, which could promote lymphomagenesis over time.

TRIM28 Is a Novel Regulator of CD133 Expression Associated with Cancer Stem Cell Phenotype.

CD133 is an extensively studied marker of the most malignant tumor cell population, designated as cancer stem cells (CSCs). However, the function of this glycoprotein and its involvement in cell regulatory cascades are still poorly understood. Here we show a positive correlation between the level of CD133 plasma membrane expression and the proliferative activity of cells of the Caco-2, HT-29, and HUH7 cancer cell lines. Despite a substantial difference in the proliferative activities of cell populations with different levels of CD133 expression, transcriptomic and proteomic profiling revealed only minor distinctions between them. Nonetheless, a further in silico assessment of the differentially expressed transcripts and proteins revealed 16 proteins that could be involved in the regulation of CD133 expression; these were assigned ranks reflecting the apparent extent of their involvement. Among them, the TRIM28 transcription factor had the highest rank. The prominent role of TRIM28 in CD133 expression modulation was confirmed experimentally in the Caco2 cell line clones: the knockout, though not the knockdown, of the TRIM28 gene downregulated CD133. These results for the first time highlight an important role of the TRIM28 transcription factor in the regulation of CD133-associated cancer cell heterogeneity.

CTLs From Patients With Atherosclerosis Show Elevated Adhesiveness and Distinct Integrin Expression Patterns on 2D Substrates.

Atherosclerosis is the major cause of cardiovascular disease that is characterized by plaque formation in the blood vessel wall. Atherosclerotic plaques represent sites of chronic inflammation with diverse cell content that is shifted toward the prevalence of cytotoxic T-lymphocytes (CTLs) upon plaque progression. The studies of CTL recruitment to atherosclerotic plaques require adequate in vitro models accounting for CTL interactions with chemokine-ligands and extracellular matrix fibers via surface chemokine receptors and integrins. Here we applied such a model by investigating CTL adhesion and migration on six types of coated surfaces. We assessed adhesion and motility metrics, the expression of chemokine receptors, and integrins in CTLs of patients with atherosclerosis and healthy donors. Using fibronectin, platelet-poor plasma from patients with atherosclerosis, and conditioned medium from atherosclerotic plaques we revealed the role of substrate in CTL adhesiveness: fibronectin alone and fibronectin combined with platelet-poor plasma and conditioned medium elevated the CTL adhesiveness - in patients the elevation was significantly higher than in healthy donors (p = 0.02, mixed 2-way ANOVA model). This was in line with our finding that the expression levels of integrin-coding mRNAs were elevated in the presence of fibronectin (p < 0.05) and ITGB1, ITGA1, and ITGA4 were specifically upregulated in patients compared to healthy donors (p < 0.01). Our experimental model did not affect the expression levels of mRNAs CCR4, CCR5, and CX3CR1 coding the chemokine receptors that drive T-lymphocyte migration to plaques. Thus, we demonstrated the substrate-dependence of integrin expression and discriminated CTLs from patients and healthy donors by adhesion parameters and integrin expression levels.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)-Specific T Cells and Antibodies in Coronavirus Disease 2019 (COVID-19) Protection: A Prospective Study.

BACKGROUND: During the ongoing coronavirus disease 2019 (COVID-19) pandemic, many individuals were infected with and have cleared the virus, developing virus-specific antibodies and effector/memory T cells. An important unanswered question is what levels of T-cell and antibody responses are sufficient to protect from the infection. METHODS: In 5340 Moscow residents, we evaluated anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin M (IgM)/immunoglobulin G (IgG) titers and frequencies of the T cells specific to the membrane, nucleocapsid, and spike proteins of SARS-CoV-2, using interferon gamma (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assay. Additionally, we evaluated the fractions of virus-specific CD4+ and CD8+ T cells using intracellular staining of IFN-γ and interleukin 2 followed by flow cytometry. We analyzed the COVID-19 rates as a function of the assessed antibody and T-cell responses, using the Kaplan-Meier estimator method, for up to 300 days postinclusion. RESULTS: We showed that T-cell and antibody responses are closely interconnected and are commonly induced concurrently. Magnitudes of both responses inversely correlated with infection probability. Individuals positive for both responses demonstrated the highest levels of protectivity against the SARS-CoV-2 infection. A comparable level of protection was found in individuals with antibody response only, whereas the T-cell response by itself granted only intermediate protection. CONCLUSIONS: We found that the contribution of the virus-specific antibodies to protection against SARS-CoV-2 infection is more pronounced than that of the T cells. The data on the virus-specific IgG titers may be instructive for making decisions in personalized healthcare and public anti-COVID-19 policies. Clinical Trials Registration. NCT04898140.

From apolar gastrula to polarized larva: Embryonic development of a marine hydroid, Dynamena pumila.

BACKGROUND: In almost all metazoans examined to this respect, the axial patterning system based on canonical Wnt (cWnt) signaling operates throughout the course of development. In most metazoans, gastrulation is polar, and embryos develop morphological landmarks of axial polarity, such as blastopore under control/regulation from cWnt signaling. However, in many cnidarian species, gastrulation is morphologically apolar. The question remains whether сWnt signaling providing the establishment of a body axis controls morphogenetic processes involved in apolar gastrulation. RESULTS: In this study, we focused on the embryonic development of Dynamena pumila, a cnidarian species with apolar gastrulation. We thoroughly described cell behavior, proliferation, and ultrastructure and examined axial patterning in the embryos of this species. We revealed that the first signs of morphological polarity appear only after the end of gastrulation, while molecular prepatterning of the embryo does exist during gastrulation. We have shown experimentally that in D. pumila, the direction of the oral-aboral axis is highly robust against perturbations in cWnt activity. CONCLUSIONS: Our results suggest that morphogenetic processes are uncoupled from molecular axial patterning during gastrulation in D. pumila. Investigation of D. pumila might significantly expand our understanding of the ways in which morphological polarization and axial molecular patterning are linked in Metazoa.

Myosin 1C isoform A is a novel candidate diagnostic marker for prostate cancer.

Early diagnosis of prostate cancer is a challenging issue due to the lack of specific markers. Therefore, a sensitive diagnostic marker that is expressed or upregulated exclusively in prostate cancer cells would facilitate diagnostic procedures and ensure a better outcome. We evaluated the expression of myosin 1C isoform A in 5 prostate cell lines, 41 prostate cancer cases, and 11 benign hyperplasias. We analyzed the expression of 12 surface molecules on prostate cancer cells by flow cytometry and analyzed whether high or low myosin 1C isoform A expression could be attributed to a distinct phenotype of prostate cancer cells. Median myosin 1C isoform A expression in prostate cancer samples and cancer cell lines was 2 orders of magnitude higher than in benign prostate hyperplasia. Based on isoform A expression, we could also distinguish clinical stage 2 from clinical stage 3. Among cell lines, PC-3 cells with the highest myosin 1C isoform A level had diminished numbers of CD10/CD13-positive cells and increased numbers of CD29 (integrin ß1), CD38, CD54 (ICAM1) positive cells. The surface phenotype of clinical samples was similar to prostate cancer cell lines with high isoform A expression and could be described as CD10-/CD13- with heterogeneous expression of other markers. Both for cell lines and cancer specimens we observed the strong correlation of high myosin 1C isoform A mRNA expression and elevated levels of CD29 and CD54, suggesting a more adhesive phenotype for cells with high isoform A expression. Compared to normal tissue, prostate cancer samples had also reduced numbers of CD24- and CD38-positive cells. Our data suggest that a high level of myosin 1C isoform A is a specific marker both for prostate cancer cells and prostate cancer cell lines. High expression of isoform A is associated with less activated (CD24/CD38 low) and more adhesive (CD29/CD54 high) surface phenotype compared to benign prostate tissue.

Lineage Commitment, Signaling Pathways, and the Cytoskeleton Systems in Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) from adult tissues are promising candidates for personalized cell therapy and tissue engineering. Significant progress was achieved in our understanding of the regulation of MSCs proliferation and differentiation by different cues during the past years. Proliferation and differentiation of MSCs are sensitive to the extracellular matrix (ECM) properties, physical cues, and chemical signaling. Sheath stress, matrix stiffness, surface adhesiveness, and micro- and nanotopography define cell shape and dictate lineage commitment of MSCs even in the absence of specific chemical signals. We discuss mechanotransduction as the major route from ECM through the cytoskeleton toward signaling pathways and gene expression. All components of the cytoskeleton from primary cilium and focal adhesions (FAs) to actin, microtubules (MTs), and intermediate filaments (IFs) are involved in the mechanotransduction. Differentiation of MSCs is regulated via the complex network of interrelated signaling pathways, including RhoA/ROCK, Akt/Erk, and YAP/TAZ effectors of Hippo pathway. These pathways could be regulated both by chemical and mechanical stimuli. Attenuation of these pathways in MSCs results in specific changes in FAs and actin cytoskeleton. Besides, differentiation of MSCs affects MTs and IFs. Recent findings highlight the role of intranuclear actin in the regulation of transcription factors in response to mechanical environmental stimuli. Alterations of cytoskeletal components reflect the MSC senescence state and their migratory capacity. In this review, we discuss the relationships between the molecular interactions in signaling pathways and morphological response of cytoskeletal components and reveal the complex interrelations between cytoskeleton systems and signaling pathways during lineage commitment of MSCs. Impact Statement This review describes the complex network of relationships between mechanical and biochemical stimuli in mesenchymal stem cells (MSC) and their balance which defines the morphological changes of cell shape due to rearrangement of cytoskeletal systems during lineage commitment of MSCs.

Non-linear Dose Response of Lymphocyte Cell Lines to Microtubule Inhibitors.

Microtubule (MT) inhibitors show anti-cancer activity in a wide range of tumors in vitro and demonstrate high clinical efficacy. To date they are routinely included into many chemotherapeutic regimens. While the mechanisms of MT inhibitors' interactions with tubulin have been well-established, the relationship between their concentration and effect on neoplastic cells is not completely understood. The common notion is that tumor cells are most vulnerable during division and all MT inhibitors block them in mitosis and induce mitotic checkpoint-associated cell death. At the same time multiple evidence of more subtle effects of lower doses of MT inhibitors on cell physiology exist. The extent of efficacy of the low-dose MT inhibitor treatment and the mechanisms of resulting cell death currently present a critical issue in oncology. The prospect of MT inhibitor dose reduction is promising as protocols at higher concentration have multiple side effects. We assessed cell cycle changes and cell death induced by MT inhibitors (paclitaxel, nocodazole, and vinorelbine) on human lymphoid B-cell lines in a broad concentration range. All inhibitors had similar accumulation effects and demonstrated "trigger" concentrations that induce cell accumulation in G2/M phase. Concentrations slightly below the "trigger" promoted cell accumulation in sub-G1 phase. Multi-label analysis of live cells showed that the sub-G1 population is heterogeneous and may include cells that are still viable after 24 h of treatment. Effects observed were similar for cells expressing Tat-protein. Thus cell cycle progression and cell death are differentially affected by high and low MT inhibitor concentrations.

Specific and reliable detection of Myosin 1C isoform A by RTqPCR in prostate cancer cells.

BACKGROUND: Prostate cancer (PC) diagnostics and treatment often present a challenging task due to cancer subtype heterogeneity and differential disease progression in patient subgroups. Hence, the critical issue is finding a reliable and sensitive diagnostic and prognostic PC marker, especially for cases of biopsies with low percentages of cancer cells. Isoform A of myosin 1C was shown to be expressed in PC cells and responsible for their invasive properties, however, its feasibility for diagnostic purposes remains to be elucidated. METHODS: To verify the role of myosin 1C isoform A mRNA expression as a putative prostate cancer marker we performed RT qPCR normalized by three reference genes (GAPDH, YWHAZ, HPRT1) on PC3, RWPE-1, LNCaP and 22Rv1 cell lines. Myosin 1C isoform A detection specificity was confirmed by immunofluorescence staining, cancer and non-cancer prostate cell lines were immunophenotyped by flow cytometry. RESULTS: Median normalized mRNA expression level of myosin 1C isoform A in PC cells (PC3 and 22Rv1) is two orders of magnitude higher compared to RWPE-1 cells, which functionally correspond to benign prostate cells. Myosin 1C isoform A expression allows PC cell detection even at a dilution ratio of 1:1000 cancer to non-cancer cells. At the protein level, the mean fluorescence intensity of myosin 1C isoform A staining in PC3 nuclei was only twice as high as in RWPE-1, while the immunophenotypes of both cell lines were similar (CD44+/CD90-/CD133-/CD57-/CD24+-). CONCLUSIONS: We report a distinct difference in myosin 1C isoform A mRNA levels in malignant (PC3) and benign (RWPE-1) prostate cell lines and suggest a combination of three reference genes for accurate data normalization. For the first time we provide an immunophenotype comparison of RWPE-1 and PC3 cells and demonstrate that RT qPCR analysis of MYO 1C A using appropriate reference genes is sufficient for PC detection even in low-abundance cancer specimens.