Myosin Light Chain Research Articles

MRCK-1 activates non-muscle myosin for outgrowth of a unicellular tube in Caenorhabditis elegans.

The formation and patterning of unicellular biological tubes is essential for metazoan development. It is well established that vascular tubes and neurons use similar guidance cues to direct their development, but the downstream mechanisms that promote the outgrowth of biological tubes are not well characterized. We show that the conserved kinase MRCK-1 and its substrate the regulatory light chain of non-muscle myosin, MLC-4, are required for outgrowth of the unicellular excretory canal in C. elegans. Ablation of MRCK-1 or MLC-4 in the canal causes severe truncations with unlumenized projections of the basal membrane. Structure-function analysis of MRCK-1 indicates that the kinase domain, but not the small GTPase-binding CRIB domain, is required for canal outgrowth. Expression of a phosphomimetic form of MLC-4 rescues canal truncations in mrck-1 mutants and shows enrichment at the growing canal tip. Moreover, our work reveals a novel function for non-muscle myosin downstream of MRCK-1 in excretory canal outgrowth that may be conserved in the development of seamless tubes in other organisms.

Effects of Licorice Functional Components Intakes on Blood Pressure: A Systematic Review with Meta-Analysis and NETWORK Toxicology

Objective: To investigate the effects of licorice functional ingredient intake on blood pressure, explore its potential mechanisms of action, and provide safety information for personalized nutritional interventions in special populations and for the application of licorice-derived functional foods. Methods: PubMed, Cochrane Library, Medline, Embase, EBSCO, ScienceDirect, and Web of Science databases were searched from inception to 31 August 2024. Randomized controlled trials (RCTs) investigating the intake of licorice or its functional components were included. The range of continuous variables was assessed using the weighted mean difference (WMD) with 95% confidence intervals. Genes associated with hypertension were screened using an online database. Machine learning, receiver operating characteristic(ROC) curve analysis, molecular docking, and gene set enrichment analysis (GSEA) were employed to explore the potential mechanisms underlying licorice-induced blood pressure fluctuations. Results: Eight RCTs (541 participants) were included in the meta-analysis, which indicated interventions containing glycyrrhizic acid (GA) as the main component increased systolic blood pressure (SBP) and diastolic blood pressure (DBP) (SBP: WMD [95% CI] = 3.48 [2.74, 4.21], p < 0.001; DBP: WMD [95% CI] = 1.27 [0.76, 1.78], p < 0.001). However, interventions dominated by licorice flavonoids(LF) had no significant effect on SBP or DBP (SBP: WMD [95% CI] = 0.58 [−1.15, 2.31], p = 0.511; DBP: WMD [95% CI] = 0.17 [−1.53, 1.88], p = 0.843). Three machine learning algorithms identified five biomarkers associated with hypertension: calmodulin 3 (CALM3), cluster of differentiation 9 (CD9), growth factor independence 1B transcriptional repressor (GFI1B), myosin light chain kinase (MYLK), and Ras suppressor-1 (RSU1). After removing biomarkers with lower validity and reliability, GFI1B, MYLK, and RSU1 were selected for subsequent analysis. The network toxicology results suggested that GA and its metabolite glycyrrhetinic acid may act on GFI1B, MYLK, and RSU1, influencing blood pressure fluctuations by modulating nitrogen metabolism signaling pathways. Conclusions: There were distinct differences in the effects of licorice functional components on blood pressure. Functional constituents dominated by GA were shown to increase both SBP and DBP, whereas those dominated by LF did not exhibit significant effects on blood pressure. The hypertensive mechanism of GA may involve the modulation of GFI1B, MYLK, and RSU1 to regulate nitrogen metabolic pathways.

Improvement on gel properties of chicken myofibrillar protein with electron beam irradiation: Based on protein structure, gel quality, water state

This study aimed to investigate the effects of electron beam (E-beam) irradiation at different doses (0–15 kGy) on the solubility, rheological properties, emulsification characteristics, and moisture distribution of chicken myofibrillar proteins (MPs). Irradiation treatment notably increased the solubility, surface hydrophobicity, emulsification properties, and apparent viscosity of MPs, based on conformational changes caused by irradiation-induced oxidative denaturation of proteins. However, high doses of irradiation (15 kGy) induced in excessive cross-linking and aggregation of proteins, reducing the solubility, emulsification properties, and shear stress. Degradation of myosin heavy and light chains in irradiated MPs increased the content of β-turns and random coils. Additionally, the initial relaxation times of T21 and T22 in irradiated protein gels were reduced, and the peak value of P21 was increased, which improved the water-capturing ability of protein gels. Altogether, these results findings suggest that electron beam irradiation can be applied as a potential technique for modifying muscle proteins.

Mouse liver blood flow is regulated by hepatic stellate cells in response to the sympathetic neurotransmitter norepinephrine

BackgroundThere is no clear information on the regulation of liver blood flow by the autonomic nervous system. We conducted this study to investigate whether quiescent hepatic stellate cells (qHSCs) regulate liver blood flow in response to the sympathetic neurotransmitter norepinephrine (NE). MethodsqHSCs isolated from mice were cultured in Dulbecco's modified Eagle medium without fetal bovine serum for 1 day on collagen gel. NE-induced qHSC contraction was evaluated using the quantitative single-cell contraction measurement method that we had developed previously. For the measurement of liver perfusion pressure in situ, a buffer solution was perfused from the portal vein in mice. ResultsNE-induced a reversible contraction of qHSCs. This contraction was suppressed by the nonmuscle myosin II inhibitor blebbistatin, the myosin light chain kinase inhibitor ML-9, the Rho kinase inhibitor H-1152, the calmodulin inhibitor W-7, the store-operated calcium channel inhibitor YM-58483, and the IP3 receptor inhibitor xestospongin C. In contrast, the transient receptor potential C channel inhibitor SKF96365 did not affect the NE-induced contraction. ConclusionThese results suggest that qHSCs contract in response to NE. New & noteworthyThe present study provides direct evidence for the first time that norepinephrine (NE) induces a reversible contraction of isolated single quiescent hepatic stellate cells (qHSCs) and further suggests that the NE-mediated qHSC contraction participates in the regulation of liver blood flow in vivo.

PDGFRB promotes dedifferentiation and pulmonary metastasis through rearrangement of cytoskeleton under hypoxic microenvironment in osteosarcoma

BackgroundOsteosarcoma (OS) cells commonly suffer from hypoxia and dedifferentiation, resulting in poor prognosis. We plan to identify the role of hypoxia on dedifferentiation and the associated cellular signaling. MethodsWe performed sphere formation assays and determined spheroid cells as dedifferentiated cells by detecting stem cell-like markers. RNAi assay was used to explore the relationship between hypoxia inducible factor 1 subunit alpha (HIF1A) and platelet derived growth factor receptor beta (PDGFRB). We obtained PDGFRB knockdown and overexpression cells through lentiviral infection experiments and detected the expression of PDGFRB, p-PDGFRB, focal adhesion kinase (FAK), p-FAK, phosphorylated myosin light chain 2 (p-MLC2), and ras homolog family member A (RhoA) in each group. The effects of PDGFRB on cytoskeleton rearrangement and cell adhesion were explored by immunocytochemistry. Wound-healing experiments, transwell assays, and animal trials were employed to investigate the effect of PDGFRB on OS cell metastasis both in vitro and in vivo. ResultsDedifferentiated OS cells were found to exhibit high expression of HIF1A and PDGFRB, and HIF1A upregulated PDGFRB, subsequently activated RhoA, and increased the phosphorylation of MLC2. PDGFRB also enhanced the phosphorylation of FAK. The OS cell morphology and vinculin distribution were altered by PDGFRB. PDGFRB promoted cell dedifferentiation and had a significant impact on the migration and invasion abilities of OS cells in vitro. In addition, PDGFRB increased pulmonary metastasis of OS cells in vivo. ConclusionOur results demonstrated that HIF1A up-regulated PDGFRB under hypoxic conditions, and PDGFRB regulated the actin cytoskeleton, a process likely linked to the activation of RhoA and the phosphorylation of, thereby promoting OS dedifferentiation and pulmonary metastasis.

VRAC Complex Modulates Mechano-Electrical Signal Responses in Human Airway Smooth Muscle Shortening.

Leucine-rich repeat containing 8A (LRRC8A) is an obligatory constituent of the volume-regulated anion channel (VRAC) that is fundamental to a wide range of biological processes, including regulating cell size, proliferation, and migration. Here we explored the physiological role for VRAC in excitation-contraction (E-C) coupling and shortening of human airway smooth muscle (HASM). In HASM cells, pharmacological inhibition of VRAC with DCPIB (0.1-10 μM) markedly attenuated swell-activated Cl- conductance and contractile agonist (histamine or carbachol)-induced cellular stiffening as measured by single-cell patch clamp and optical magnetic twisting cytometry, respectively. In addition, HASM cells treated with DCPIB or transfected with LRRC8A-targeting siRNA showed reduced agonist-induced phosphorylation of protein kinase B (AKT), paxillin, myosin phosphatase target subunit 1 (MYPT1), and myosin light chain (MLC). Consistent with the changes of these E-C coupling effectors, DCPIB appreciably decreased agonist-induced small airways narrowing in human precision-cut lung slices (hPCLS). Taken together, our findings shed a new light on the mechanistic link between HASM shortening and regulatory volume decrease via LRRC8A, revealing a previously unrecognized nodal point for modulation of the E-C coupling and acute airways constriction.

Alterations in Hurler-Scheie Syndrome Revealed by Mass Spectrometry-Based Proteomics and Phosphoproteomics Analysis.

Hurler-Scheie syndrome (MPS IH/S), also known as mucopolysaccharidosis type I-H/S (MPS IH/S), is a lysosomal storage disorder caused by deficiency of the enzyme alpha-L-iduronidase (IDUA) leading to the accumulation of glycosaminoglycans (GAGs) in various tissues, resulting in a wide range of symptoms affecting different organ systems. Postgenomic omics technologies offer the promise to understand the changes in proteome, phosphoproteome, and phosphorylation-based signaling in MPS IH/S. Accordingly, we report here a large dataset and the proteomic and phosphoproteomic analyses of fibroblasts derived from patients with MPS IH/S (n = 8) and healthy individuals (n = 8). We found that protein levels of key lysosomal enzymes such as cathepsin D, prosaposin, arylsulfatases (arylsulfatase A and arylsulfatase B), and IDUA were downregulated. We identified 16,693 unique phosphopeptides, corresponding to 4,605 proteins, in patients with MPS IH/S. We found that proteins related to the cell cycle, mitotic spindle assembly, apoptosis, and cytoskeletal organization were differentially phosphorylated in MPS IH/S. We identified 12 kinases that were differentially phosphorylated, including hyperphosphorylation of cyclin-dependent kinases 1 and 2, hypophosphorylation of myosin light chain kinase, and calcium/calmodulin-dependent protein kinases. Taken together, the findings of the present study indicate significant alterations in proteins involved in cytoskeletal changes, cellular dysfunction, and apoptosis. These new observations significantly contribute to the current understanding of the pathophysiology of MPS IH/S specifically, and the molecular mechanisms involved in the storage of GAGs in MPS more generally. Further translational clinical omics studies are called for to pave the way for diagnostics and therapeutics innovation for patients with MPS IH/S.

Protective effect of betaine supplementation in low-met diet against Aeromonas hydrophila-induced intestinal injury in grass carp (Ctenopharyngodon idella)

This study evaluated the effect of supplemental betaine in low-methionine (Met) diet concerning the regulatory role of bacteria-induced intestinal injury in fish. 540 grass carp (Ctenopharyngodon Idella) (210 ± 0.68 g) were selected and randomly divided into 6 groups, which contained a reference group fed with the diet supplemented with DL-Met (3.1 g kg−1 diet) and 5 groups fed on the diets added with increasing levels of betaine (0–6.4 g kg−1 diet) for 60 days. Then, a 14-day Aeromonas hydrophila challenge was conducted. Compared with BET0 group, results indicated that appropriate betaine supplementation (1) enhanced antioxidant enzymes activities (except glutathione S-transferase pi-1 (GSTP1) and copper zinc superoxide dismutase (CuZnSOD) and glutathione (GSH) contents, and NF-E2-related-factor 2 (Nrf2) protein nuclear translocation, while reduced reactive oxygen species (ROS), and representative oxidative damage markers; (2) up-regulated the transcription abundance of anti-apoptosis-related molecules while reduced DNA-ladder occurrence and the transcription levels of pro-apoptosis-related genes; (3) down-regulated the transcription of tight junction-related proteins (such as zonula occludens-1 (ZO-1) etc.) in fish intestines. Furthermore, correlation analysis revealed that feeding betaine ameliorating the oxidative damage, apoptosis and tight junction breakdown was possibly related to intestinal Nrf2, p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinases (JNK), and myosin light chain kinase (MLCK) signaling, respectively. According to the regression analysis for intestinal ROS abundance, claudin-b and caspase-2 transcription, the appropriate betaine supplementations were estimated to be 3.82, 3.98, and 3.62 g kg−1 diet, respectively. Notably, betaine has the potential to spare part Met and the efficacy of betaine relative to Met was 206.67 % based on intestinal ROS contents in grass carp (210–776 g).

Epigenetic modifications in transdifferentiation of adipose tissue-derived stromal cells into spontaneously beating cardiomyocyte-like cells

Abstract Background Adipose tissue-derived stromal vascular fraction (adipose SVF) contains pluripotent mesenchymal stem cells and rarely transdifferentiate into beating cardiomyocytes. We developed a simple culture protocol under which the adult murine inguinal adipose SVF reproductively transdifferentiates into beating cardiomyocyte-like cells (beating CM) without inductions in the primary culture. We also reported that Mef2c is a crucial factor in this conversion. However, the characteristics of beating CM and the factors that regulate the differentiation of adipose SVF toward the cardiac lineage are unknown. Purpose To investigate sequential changes in global gene expression profiles of adipose SVF in primary culture and determine the mechanism of differentiation into beating CM. Methods Adipose SVF cells were isolated from adult mice's inguinal subcutaneous fat pad and cultured using the previously established beating CM induction method (Sci Rep. 2021). At 6 time points (days 0, 3, 7, 14, 21, and 28) in primary culture, total RNA was extracted, and RNA-sequencing was performed (n = 3). The data was analyzed using Subio Platform and MetaCore software. To assess the candidate gene and proteins, adipose SVF cells were transduced with the gene using a lentivirus vector and supplemented with the specific inhibitors. The cardiac differentiation was evaluated by quantitative PCR on day 28. Results The beating CM was confirmed on days 14, 21, and 28 in the primary culture. Among prefiltered 14,574 genes, the expression level of 749 genes were significantly changed (153 genes upregulated vs. 596 genes downregulated) between days 7 and 14 (2-fold, P < 0.05), in which beating CM appeared. The GO molecular functions analysis showed that O-GluNAC transferase and histone deacetylase (HDAC)-associated genes were expressed dominantly before day 7, and KLF4-associated genes were expressed prominently after day 14 in primary culture. The expression of cardiac troponin T (Tnnt2) and myosin light chain ventricular type (Myl2) increased on day 3 and day 14, respectively. The HDAC7, an epigenetic regulator, decreased the expression on day 14 or later. Treatment with the HDAC inhibitor (150 nmol/L) increased the expression of Tnnt2 (5-fold vs. control) on day 28 in adipose SVF. Furthermore, treatment of Mef2c-transduced adipose SVFs with HDAC inhibitors increased expression of cardiac troponin T (29-fold vs. control, P < 0.05). Conclusion The time course analysis demonstrated that a remarkable change occurred in the regulation of transcription on day 14 in adipose SVF primary culture. Alterations in epigenetic modifications increased beating CM in adipose SVFs. Adipose SVF could be applied to new cardiac regeneration therapies by increasing the efficiency of introduction into the beating CM.

Carvedilol suppresses coronary artery spasm in A-kinase anchoring protein 150 knockout mouse

Abstract Backgrounds A-kinase anchoring proteins (AKAPs) act as scaffold to regulate activation of protein kinases and subsequent downstream signaling. AKAP 150 interacts with protein kinase A and protein kinase C, and has a critical role in regulating L-type calcium channel activity. We previously reported enhanced phospholipase C activity as an important mechanism of coronary spastic angina (CSA), known as Prinzmetal’s angina. Coronary artery spasm was induced by intravenous ergometrine injection in mouse model of vascular smooth muscle cell (VSMC)-specific enhanced phospholipase C activity concomitantly with enhanced intracellular Ca2+ concentration ([Ca2+]i). Then, we hypothesized that coronary artery spasm would be inhibited by AKAP 150 knockout, however, coronary artery spasm was unexpectedly induced in AKAP 150 knockout (AKAP-KO) mice without affecting [Ca2+]i, suggesting that AKAP-KO mouse is an unique model of CSA independent of [Ca2+]i. Previous report showed that AKAP-KO mice have a high Ca2+/calmodulin-dependent protein kinase (CaMK) II activity, and carvedilol, a non-selective beta-adrenergic receptor antagonist, inhibits CaMK II activity. Aim We tested the hypothesis that carvedilol could prevent coronary artery spasm via inhibiting CaMK II activity, which is a key molecule to regulate VSMC contraction, in AKAP-KO mice. Methods and Results We pretreated 14-18-weeks-old AKAP-KO mice with intraperitoneal injection of carvedilol (2.5mg/kg), propranolol (20mg/kg), or vehicle (control). Coronary artery spasm, evaluated by ST-segment elevation on lead II of body surface electrocardiogram, was induced by intravenous ergometrine injection (30mg/kg) in all controls (5/5, 100%) and all propranolol-treated mice (5/5, 100%), whereas it tended to be inhibited in carvedilol-treated mice (3/6, 50%) (p=0.08 vs control). In isolated VSMCs obtained from the aorta of AKAO-KO mice, the changes in [Ca2+]i from baseline in response to acetylcholine (ACh, 100μM) did not differ among the groups of pretreatment with carvedilol (10μM), propranolol (10μM), or vehicle, indicating that coronary artery spasm in AKAP-KO mice was induced by Ca2+-independent mechanism. In response to ACh, the phosphorylation of CaMK II was tended to be decreased (p=0.07), and the phosphorylation of myosin light chain was decreased (p<0.05) in VSMCs treated with carvedilol compared with control. Conclusions Carvedilol may inhibit coronary artery spasm probably via inhibiting CaMK II activity in AKAP-KO mice. Our results may provide a new paradigm into the pathogenesis of CSA as a model of enhanced Ca2+ sensitivity and important clinical implications for the mechanism of calcium channel antagonist-refractory coronary artery spasm.

Loss of myosin light chain kinase induces the cellular senescence associated secretory phenotype to promote breast epithelial cell migration

Overexpression or activation of oncogenes or loss of tumor-suppressor genes can induce cellular senescence as a defense mechanism against tumor development, thereby maintaining cellular homeostasis. However, cancer cells can circumvent this senescent state and continue to spread. Myosin light chain kinase (MLCK) is downregulated in many breast cancers. Here we report that downregulation of MLCK in normal breast epithelial cells induces a senescence-associated secretory phenotype and stimulates migration. The reduction of MLCK results in increased p21Cip1 expression, dependent on p53 and the AKT-mammalian target of rapamycin pathway. Subsequently, p21Cip1 promotes the secretion of soluble ICAM-1, IL-1α, IL-6 and IL-8, thereby enhancing collective cell migration in a non-cell-autonomous manner. These findings provide new mechanistic insights into the role of MLCK in cellular senescence and cancer progression.

MYL9 binding with MYO19 suppresses epithelial-mesenchymal transition in non-small cell lung cancer.

The elusive function of myosin light chain 9 (MYL9) in cancer is an area ripe for further investigation. Bioinformatics was utilized to compare the expression levels of MYL9 in non-small cell lung cancer (NSCLC) and normal tissues. Gene set enrichment analysis (GSEA) was employed to investigate the pathways associated with MYL9. BioGRID database was utilized to screen for potential targets of MYL9. The expression of MYL9 and myosin 19 (MYO19) mRNA was quantified using quantitative reverse transcriptase PCR. Cell migration was assessed using a scratch wound healing assay. Protein levels of MYL9, MYO19, and epithelial-mesenchymal transition (EMT) biomarkers were examined using western blot (WB). EpCAM expression in different cell groups was profiled using flow cytometry analysis. Co-immunoprecipitation assays were performed to determine the binding affinity between MYL9 and MYO19. Additionally, direct protein interaction between MYL9 and MYO19 was explored using a GST-pull-down assay. In NSCLC patients, MYL9 was significantly downregulated in vivo and in cell cultures, showing high enrichment in the EMT pathway. Scratch assays indicated its inhibitory effect on cell migration. Western blotting revealed that MYL9 suppresses EMT marker protein expression in NSCLC cells. Flow cytometry showed that MYL9 reduced EpCAM levels on the cell surface. MYO19 was identified as a potential target of MYL9 through CoIP and GST-pull-down assays. Rescue experiments demonstrated that MYO19 enhances cell migration, EMT marker expression, and EpCAM levels, but these effects were countered by MYL9 overexpression. MYL9 impedes the migration and EMT in NSCLC cells by binding to MYO19.

Loss of Cdc42 causes abnormal optic cup morphogenesis and microphthalmia in mouse.

Congenital ocular malformations originate from defective morphogenesis during early eye development and cause 25% of childhood blindness. Formation of the eye is a multi-step, dynamic process; it involves evagination of the optic vesicle, followed by distal and ventral invagination, leading to the formation of a two-layered optic cup with a transient optic fissure. These tissue folding events require extensive changes in cell shape and tissue growth mediated by cytoskeleton mechanics and intercellular adhesion. We hypothesized that the Rho GTPase Cdc42 may be an essential, convergent effector downstream of key regulatory factors required for ocular morphogenesis. CDC42 controls actin remodeling, apicobasal polarity, and junction assembly. Here we identify a novel essential function for Cdc42 during eye morphogenesis in mouse; in Cdc42 mutant eyes expansion of the ventral optic cup is arrested, resulting in microphthalmia and a wide coloboma. Our analyses show that Cdc42 is required for expression of the polarity effector proteins PRKCZ and PARD6, intercellular junction protein tight junction protein 1, β-catenin, actin cytoskeleton F-actin, and contractile protein phospho myosin light chain 2. Expression of RPE fate determinants OTX2 and MITF, and formation of the RPE layer are severely affected in the temporal domain of the proximal optic cup. EdU incorporation is significantly downregulated. In addition, mitotic retinal progenitor cells mis-localized deeper, basal regions, likely contributing to decreased proliferation. We propose that morphogenesis of the ventral optic cup requires Cdc42 function for coordinated optic cup expansion and establishment of subretinal space, tissue tension, and differentiation of the ventral RPE layer.

Evaluation of STK17B as a cancer immunotherapy target utilizing highly potent and selective small molecule inhibitors.

The serine/threonine kinase 17B (STK17B) is involved in setting the threshold for T cell activation and its absence sensitizes T cells to suboptimal stimuli. Consequently, STK17B represents an attractive potential target for cancer immunotherapy. To assess the potential of STK17B as an immuno-oncology target, we developed potent and selective tool compounds from starting points in Blueprint Medicines Corporation's proprietary kinase inhibitor library. To characterize these molecules, enzyme and cellular assays for STK17A and STK17B were established to drive chemistry optimization. Mass spectrometry-based phosphoproteomics profiling with tool inhibitors led to the identification of Ser19 on myosin light chain 2 as STK17B substrate, which is then developed into a flow cytometry-based pharmacodynamic readout of STK17B inhibition both in vitro and in vivo. In a mouse T cell activation assay, STK17B inhibitors demonstrated the ability to enhance interleukin-2 (IL-2) production. Similarly, treatment with STK17B inhibitors resulted in stronger cytokine secretion in human T cells activated using a T cell bispecific antibody. Subsequent chemistry optimization led to the identification of a highly selective and orally bioavailable tool compound, BLU7482. In vivo, STK17B inhibition led to dose-dependent modulation of myosin light chain 2 phosphorylation and enhanced priming of naïve T cells, as determined by upregulation of CD69, IL-2 and interferon-γ secretion. In line with increased T cell activation, treatment with STK17B inhibitor enhanced antitumor activity of anti-PD-L1 antibody in the MCA205 model. In summary, we successfully identified and optimized STK17B kinase inhibitors which led to increased T cell responses in vitro and in vivo. This allowed us to evaluate the potential of STK17B inhibition as an approach for cancer immunotherapy.

DRAK2 regulates myosin light chain phosphorylation in T cells.

Death-associated protein kinase-related apoptosis-inducing kinase-2 (DRAK2 or STK17B) is a serine/threonine kinase expressed in T cells. Drak2-deficient (Drak2-/-) mice respond effectively to tumors and pathogens while displaying resistance to T cell-mediated autoimmune disease. However, the molecular mechanisms by which DRAK2 impacts T cell function remain unclear. Gaining further insight into the function of DRAK2 in T cells will shed light on differentially regulated pathways in autoreactive and pathogen-specific T cells, which is critical for improving autoimmune therapies. Here, we demonstrate that DRAK2 contributes to activation of myosin light chain (MLC) in both murine and human T cells. In the absence of Drak2, the amount of polymerized actin was decreased, suggesting that DRAK2 modulates actomyosin dynamics. We further show that myosin-dependent T cell functions, such as migration, T cell receptor microcluster accumulation, and conjugation to antigen presenting cells are decreased in the absence of Drak2. These findings reveal that DRAK2 plays an important role in regulating MLC activation within T cells.

Impact and mechanisms of drag-reducing polymers on shear stress regulation in pulmonary hypertension.

Pulmonary hypertension (PH) is a refractory disease characterized by elevated pulmonary artery pressure and resistance. Drag-reducing polymers (DRPs) are blood-soluble macromolecules that reduce vascular resistance by altering the blood dynamics and rheology. Our previous work indicated that polyethylene oxide (PEO) can significantly reduce the medial wall thickness and vascular resistance of the pulmonary arteries, but the specific mechanism is still unclear. This study was designed to investigate the role and mechanism of PEO on intracellular calcium [Ca2+] i and cytoskeletal proteins of endothelial cells (ECs) induced by low shear stress (LSS) in PH. Primary Pulmonary Artery Endothelial Cells (PAECs) were subjected to steady LSS (1 dyn/cm2) or physiological shear stress (SS) (10 dyn/cm2) for 20 h in a BioFlux 200 flow system. Calcium influx assays were conducted to evaluate the mechanisms of PEO on [Ca2+] i. Subsequently, taking the key protein that induces cytoskeletal remodeling, the regulatory light chain (RLC) phosphorylation, as the breakthrough point, this study focused on the two key pathways of PEO that regulate phosphorylation of RLC: Myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK) pathways. Our current research revealed that PEO at LSS (1 dyn/cm2) significantly suppressed LSS-induced [Ca2+] i and the expression level of transient receptor potential channel 1(TRPC1). In addition, ECs convert LSS stimuli into the upregulation of cytoskeletal proteins, including filamentous actin (F-actin), MLCK, ROCK, p-RLC, and pp-RLC. Further experiments using pharmacological inhibitors demonstrated that PEO at the LSS downregulated cytoskeleton-related proteins mainly through the ROCK and MLCK pathways. This study considered intracellular calcium and cytoskeleton rearrangement as entry points to study the application of PEO in the biomedical field, which has important theoretical significance and practical application value for the treatment of PH.

Unraveling potential gene biomarkers for dengue infection through RNA sequencing.

Dengue virus hijacks host cell mechanisms and immune responses in order to replicate efficiently. The interaction between the host and the virus affects the host's gene expression, which remains largely unexplored. This pilot study aimed to profile the host transcriptome as a potential strategy for identifying specific biomarkers for dengue prediction and detection. High-throughput RNA sequencing (RNA-seq) was employed to generate host transcriptome profiles in 16 dengue patients and 10 healthy controls. Differentially expressed genes (DEGs) were identified in patients with severe dengue and those with dengue with warning signs compared to healthy individuals. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to elucidate the functions of upregulated and downregulated genes. Compared to healthy controls, 6466 genes were significantly differentially expressed (p < 0.05) in the dengue with warning signs group and 3082 genes in the severe dengue group, with over half being upregulated. The major KEGG pathways implicated included transport and catabolism (14.4%-16.3%), signal transduction (6.6%-7.3%), global and overview maps (6.7%-7.1%), viral diseases (4.6%-4.8%), and the immune system (4.4%-4.6%). Several genes exhibited consistent and significant upregulation across all dengue patients, regardless of severity: Interferon alpha inducible protein 27 (IFI27), Potassium Channel Tetramerization Domain Containing 14 (KCTD14), Syndecan 1 (SDC1), DCC netrin 1 receptor (DCC), Ubiquitin C-terminal hydrolase L1 (UCHL1), Marginal zone B and B1 cell-specific protein (MZB1), Nestin (NES), C-C motif chemokine ligand 2 (CCL2), TNF receptor superfamily member 17 (TNFSF17), and TNF receptor superfamily member 13B (TNFRSF13B). Further analysis revealed potential biomarkers for severe dengue prediction, including TNF superfamily member 15 (TNFSF15), Plasminogen Activator Inhibitor-2 (SERPINB2), motif chemokine ligand 7 (CCL7), aconitate decarboxylase 1 (ACOD1), Metallothionein 1G (MT1G), and Myosin Light Chain Kinase (MYLK2), which were expressed 3.5 times, 2.9 times, 2.3 times, 2.1 times, 1.7 times, and 1.4 times greater, respectively, than dengue patients exhibiting warning signs. The identification of these host biomarkers through RNA-sequencing holds promising implications and potential to augment existing dengue detection algorithms, contributing significantly to improved diagnostic and prognostic capabilities.

Calcium wave dynamics in the embryonic mouse gut mesenchyme: impact on smooth muscle differentiation

Intestinal smooth muscle differentiation is a complex physico-biological process involving several different pathways. Here, we investigate the properties of Ca2+ waves in the developing intestinal mesenchyme using GCamp6f expressing mouse embryos and investigate their relationship with smooth muscle differentiation. We find that Ca2+ waves are absent in the pre-differentiation mesenchyme and start propagating immediately following α-SMA expression. Ca2+ waves are abrogated by CaV1.2 and gap-junction blockers, but are independent of the Rho pathway. The myosine light-chain kinase inhibitor ML-7 strongly disorganized or abolished Ca2+ waves, showing that perturbation of the contractile machinery at the myosine level also affected the upstream Ca2+ handling chain. Inhibiting Ca2+ waves and contractility with CaV1.2 blockers did not perturb circular smooth muscle differentiation at early stages. At later stages, CaV1.2 blockers abolished intestinal elongation and differentiation of the longitudinal smooth muscle, leading instead to the emergence of KIT-expressing interstitial cells of Cajal at the gut periphery. CaV1.2 blockers also drove apoptosis of already differentiated, CaV1.2-expressing smooth muscle and enteric neural cells. We provide fundamental new data on Ca2+ waves in the developing murine gut and their relation to myogenesis in this organ.

Seminal plasma uterine priming alters uterine transcriptomics and negatively impacts embryo growth and uterine artery resistance but not offspring liver transcriptomics in beef cattle.

Seminal plasma uterine priming is important for pregnancy and offspring phenotype in mice and swine; however, impacts on the uterus of the dam and her offspring in cattle are unknown. We sought to determine the effects of seminal plasma uterine priming at estrus on uterine transcriptomics, early gestation (d 35, 40, and 45) embryo morphometrics, mid- to late-gestation (d 140 to 220) uterine artery hemodynamics, birth morphometrics, and liver transcriptomics in offspring at 30 d of age. Multiparous Angus-based commercial beef cows were randomly assigned to receive treatment at estrus: 0.5mL pooled seminal plasma in the uterine body (n = 31, seminal plasma primed) or no treatment (n = 31, control). Seven d later a subset of cows (n = 4/treatment) underwent uterine biopsies, and the remaining cows underwent embryo transfer. Embryo crown-rump length and uterine artery hemodynamics were measured during gestation using ultrasonography. Morphometrics of the calf were collected within 24h of parturition. Liver biopsies were collected at 30 d of age. Data were analyzed by ANOVA in a completely randomized design for the effect of treatment. Myosin heavy chain I (JSP.1) was downregulated [Benjamin-Hochberg adj P (BH) <= 0.05] and ABO alpha 1-3-N-acetylgalactosaminyltransferase and alpha 1-3-galactosyltransferase (ABO) was upregulated (BH adj P <= 0.05) in the uterus of seminal plasma primed cows 7 d after treatment. Embryo crown-rump length was less (P < 0.05) in seminal plasma primed cows. Mid- to late-gestation (d 140 to 220) uterine artery resistance was increased (P < 0.05) in seminal plasma primed cows. Seminal plasma priming did not alter birth weights or curve-crown-rump length, but heart girth was increased (P < 0.05) in offspring from seminal plasma primed cows. There were no differentially expressed genes (BH adj P <= 0.05) in offspring liver at 30 d of age; however, myosin light chain, phosphorylatable, fast skeletal muscle (MYLPF) was absent in all liver samples from calves from seminal plasma primed cows. In contrast, vomeronasal 1 receptor bosTauV1R414 (BOSTAUV1R414) was present in 6 of the 7 liver samples from calves from seminal plasma primed cows. Seminal plasma uterine priming alters uterine transcriptomics, negatively impacts early gestation embryo growth and mid- to late-gestation uterine artery resistance suggesting downstream vascular anomalies. However, these in utero conditions did not impact offspring from birth to 30 d of age.

Tandem mass tag-based quantitative proteomics analysis of modified atmosphere packaging in large yellow croaker (Pseudosciaena crocea) fillets during refrigerated storage

A tandem mass tagging-labeled proteomic approach was employed to explore the relationship between quality parameters and protein changes in large yellow croaker fillets refrigerated under carbon dioxide, oxygen, and nitrogen atmospheres. After 96 h, fillets stored in carbon dioxide and nitrogen showed improved texture, water-holding capacity, and color, compared to those stored in oxygen. Functional analysis respectively identified 117 and 65 differentially expressed proteins in carbon dioxide and nitrogen, including key proteins such as troponin, myosin light chain, actin, and collagen types IV and XIII, that were linked to extracellular adhesion, cytoskeleton integrity, energy metabolism, and membrane functions. Carbon dioxide and nitrogen were detrimental to the growth and reproduction of aerobic microorganisms. High concentrations of carbon dioxide can inhibit microbial activity, while nitrogen preserves freshness by regulating the pressure balance within the packaging. These findings provided a theoretical basis for the optimized gas-conditioned preservation of large yellow croaker fillets.