Protein Kinase Research Articles

Hemozoin induces malaria via activation of DNA damage, p38 MAPK and neurodegenerative pathways in a human iPSC-derived neuronal model of cerebral malaria

Malaria caused by Plasmodium falciparum infection results in severe complications including cerebral malaria (CM), in which approximately 30% of patients end up with neurological sequelae. Sparse in vitro cell culture-based experimental models which recapitulate the molecular basis of CM in humans has impeded progress in our understanding of its etiology. This study employed healthy human induced pluripotent stem cells (iPSCs)-derived neuronal cultures stimulated with hemozoin (HMZ) - the malarial toxin as a model for CM. Secretome, qRT-PCR, Metascape, and KEGG pathway analyses were conducted to assess elevated proteins, genes, and pathways. Neuronal cultures treated with HMZ showed enhanced secretion of interferon-gamma (IFN-γ), interleukin (IL)1-beta (IL-1β), IL-8 and IL-16. Enrichment analysis revealed malaria, positive regulation of cytokine production and positive regulation of mitogen-activated protein kinase (MAPK) cascade which confirm inflammatory response to HMZ exposure. KEGG assessment revealed up-regulation of malaria, MAPK and neurodegenerative diseases-associated pathways which corroborates findings from previous studies. Additionally, HMZ induced DNA damage in neurons. This study has unveiled that exposure of neuronal cultures to HMZ, activates molecules and pathways similar to those observed in CM and neurodegenerative diseases. Furthermore, our model is an alternative to rodent experimental models of CM.

Oncogene Downregulation by Mahanine Suppresses Drug-Sensitive and Drug-Resistant Lung Cancer and Inhibits Orthotopic Tumor Progression

Background/Objectives: Lung cancer is one of the deadliest cancers, and drug resistance complicates its treatment. Mahanine (MH), an alkaloid from Murraya koenigii has been known for its anti-cancer properties. However, its effectiveness and mechanisms in treating non-small cell lung cancer (NSCLC) remain largely unexplored. The present study aimed to investigate MH’s effect on drug-sensitive and drug-resistant NSCLC and its potential mechanism of action. Methods: We isolated MH from M. koenigii leaves and the purity (99%) was confirmed by HPLC, LC-MS and NMR. The antiproliferative activity of MH was determined using MTT and colony formation assays against drug-sensitive (A549 and H1299) and Taxol-resistant lung cancer cells (A549-TR). Western blot analysis was performed to determine MH’s effects on various molecular targets. Anti-tumor activity of MH was determined against lung tumors developed in female NOD Scid mice injected with A549-Fluc bioluminescent cells (1.5 × 106) intrathoracically. Results: MH dose-dependently reduced the proliferation of all lung cancer cells (A549, H1299 and A549-TR), with IC50 values of 7.5, 5, and 10 µM, respectively. Mechanistically, MH arrested cell growth in the G0/G1 and G2/M phases of the cell cycle by inhibiting cyclin-dependent kinase 4/6 (CDK4/6) and cell division control 2 (CDC2) and induced apoptosis through the downregulation of B-cell leukemia/lymphoma 2 (BCL2) and B-cell lymphoma-extra large (BCL-XL). The apoptotic induction capacity of MH can also be attributed to its ability to inhibit pro-oncogenic markers, including mesenchymal–epithelial transition factor receptor (MET), phosphorylated protein kinase B (p-AKT), phosphorylated mammalian target of rapamycin (p-mTOR), survivin, rat sarcoma viral oncogene (RAS), myelocytomatosis oncogene (cMYC), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) levels. In vivo, MH (25 mg/kg b. wt.) significantly (p < 0.001) inhibited the growth of A549 lung cancer orthotopic xenografts in NOD Scid mice by 70%. Conclusions: Our study provides new mechanistic insights into MH’s therapeutic potential against NSCLC.

Metformin Mitigates Trimethyltin-Induced Cognition Impairment and Hippocampal Neurodegeneration

The neurotoxicant trimethyltin (TMT) triggers cognitive impairment and hippocampal neurodegeneration. TMT is a useful research tool for the study of Alzheimer's disease (AD) pathogenesis and treatment. Although the antidiabetic agent metformin has shown promising neuroprotective effects, however, its precise modes of action in neurodegenerative disorders need to be further elucidated. In this study, we investigated whether metformin can mitigate TMT cognition impairment and hippocampal neurodegeneration. To induce an AD-like phenotype, TMT was injected i.p. (8 mg/kg) and metformin was administered daily p.o. for 3 weeks at 200 mg/kg. Our results showed that metformin administration to the TMT group mitigated learning and memory impairment in Barnes maze, novel object recognition (NOR) task, and Y maze, attenuated hippocampal oxidative, inflammatory, and cell death/pyroptotic factors, and also reversed neurodegeneration-related proteins such as presenilin 1 and p-Tau. Hippocampal level of AMP-activated protein kinase (AMPK) as a key regulator of energy homeostasis was also improved following metformin treatment. Additionally, metformin reduced hippocampal acetylcholinesterase (AChE) activity, glial fibrillary acidic protein (GFAP)-positive reactivity, and prevented the loss of CA1 pyramidal neurons. This study showed that metformin mitigated TMT-induced neurodegeneration and this may pave the way to develop new therapeutics to combat against cognitive deficits under neurotoxic conditions.

Preventive effects of matrine on LPS-induced inflammation in RAW 264.7 cells and intestinal damage in mice through the TLR4/NF-κB/MAPK pathway

BackgroundMatrine is a tetracyclic quinolizidine alkaloid with diverse bioactive properties, including anti-inflammatory and neuroprotective properties. However, the underlying anti-inflammatory mechanisms remain unclear. PurposeThis study aimed to explore how matrine reduces Lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 cells and to assess its protective effects against LPS-induced intestinal damage. MethodsThe effect of matrine on cell viability was assessed using the cell counting kit-8 (CCK-8) assay. Additionally, its impact on inflammatory cytokines and macrophage polarization was assessed using enzyme-linked immunosorbent assay (ELISA), flow cytometry, and quantitative real-time polymerase chain reaction (qRT-PCR) analyses. The effects on intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), nitric oxide (NO) production, and oxidative stress were evaluated using 2′,7′-dichlorodihydrofluorescein diacetate staining and JC-1 and Griess assays. Immunofluorescence staining was used to observe the translocation of the NF-κB p65 subunit. Western blotting (WB) and qRT-PCR were employed to analyze the expression levels of proteins related to the toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB)/mitogen-activated protein kinase (MAPK) pathway. An LPS-induced mouse model was established to study the intestinal inflammation and barrier injury. Mouse feces characteristics, colon length, and disease activity index (DAI) were recorded. Hematoxylin-eosin (H&E) and alcian blue/periodic acid schiff (AB/PAS) staining were used to observe morphological changes and barrier damage in the duodenum, jejunum, ileum, and colon and to measure villus length, crypt depth, goblet cell count, and positive areas in the duodenum, jejunum, and ileum. The content of short-chain fatty acids (SCFAs) in the colon was determined using gas chromatography (GC). ResultsMatrine inhibited LPS-induced inflammatory cytokine levels, suppressed macrophage M1 polarization, and promoted M2 macrophage polarization. Matrine reduced LPS-induced increases in ROS and NO levels and regulates oxidative stress. Additionally, matrine inhibited the nuclear translocation of the NF-κB p65 subunit and exerted anti-inflammatory effects by suppressing the activation of the TLR4/NF-κB/MAPK pathway. In vivo experiments indicated that matrine significantly alleviated LPS-induced diarrhea, increased DAI, and shortened the colon. Matrine reduced the production of the pro-inflammatory cytokine interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α and the pro-inflammatory mediator NO in mouse intestinal tissues while promoting the content of the anti-inflammatory cytokine IL-10. Furthermore, it improved intestinal tissue structure and alleviated LPS-induced intestinal barrier damage. Finally, matrine increased the SCFA levels in the intestine. ConclusionMatrine exerted its anti-inflammatory effects and protects against intestinal injury through the TLR4/NF-κB/MAPK signaling pathway.

The involvement of lidocaine in amyloid-β1-42-dependent mitochondrial dysfunction and apoptosis in hippocampal neurons via nerve growth factor-protein kinase B pathway.

This project is conceived to reveal the role of lidocaine in the process of Alzheimer's disease (AD) and its possible downstream targets. After the employment of AD cell model in mice hippocampal neuronal HT-22 cells in the presence of amyloid-β1-42 (Aβ1-42), Cell Counting Kit-8 method investigated cell viability. Oxidative damage was assayed based on a dichloro-dihydro-fluorescein diacetate fluorescent probe and commercially available kits. The 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide fluorescent probe estimated mitochondrial function. Terminal-deoxynucleotidyl transferase mediated nick end labeling, western blotting, and immunofluorescence appraised the apoptotic level. Western blot also ascertained the alternations of nerve growth factors (NGF)-protein kinase B (Akt) pathway-related proteins. Aβ1-42 concentration dependently triggered the viability loss, oxidative damage, and apoptosis in HT-22 cells. Lidocaine promoted the viability and reduced the mitochondrial impairment and mitochondria-dependent apoptosis in Aβ1-42-treated HT-22 cells in a concentration-dependent manner. Besides, lidocaine activated the NGF-Akt pathway and NGF absence blocked NGF-Akt pathway, aggravated mitochondrial dysfunction as well as mitochondria-dependent apoptosis in lidocaine-administrated HT-22 cells in response to Aβ1-42. Altogether, these observations concluded that lidocaine might stimulate NGF-Akt pathway to confer protection against mitochondrial impairment and apoptosis in Aβ1-42-mediated cellular model of AD.

Heat stress causes chromatin accessibility and related gene expression changes in crown tissues of barley (Hordeum vulgare).

Plant responses to stress caused by high temperatures involve changes occurring at the molecular, metabolic, and physiological levels. Understanding the mechanisms by which plants recognize signals to activate this response is a prerequisite for identifying key genes and signaling pathways and for obtaining heat-tolerant plants. We demonstrated the first implementation of an assay for transposase-accessible chromatin to identify open chromatin regions (OCRs) in crown tissues of barley using three genotypes carrying different allelic forms of the sdw1 gene encoding gibberellin 20-oxidase subjected to elevated temperatures. In parallel, we performed gene expression analysis, which allowed us to relate changes in chromatin state to changes in transcriptional activity. The obtained data revealed that the hypersensitive chromatin regions within the genes were more repeatable than those outside the gene intervals. We observed that prolonged exposure to high temperatures increased chromatin accessibility. Genes with OCRs in their regulatory regions were involved in stress signaling and tolerance, including calcium-dependent protein kinase, mitogen-activated protein kinase (MAPK3), receptor-like cytoplasmic kinase (RLK), TIFY domain-containing transcriptional regulator, bZIP transcription factor, and regulatory protein NPR1. The effect of genotype on gene expression was not as pronounced as that of temperature. By combining results from the differential analysis of chromatin accessibility and expression profiles, we identified genes with high temperature-induced changes in chromatin accessibility associated with expression alterations. Importantly, our data revealed a relationship between the loss of chromatin accessibility in response to heat and the downregulation of genes related to gibberellin signaling.

Generation and Characterization of Three Novel Mouse Mutant Strains Susceptible to Audiogenic Seizures

The mechanisms of epileptogenesis after brain injury, ischemic stroke, or brain tumors have been extensively studied. As a result, many effective antiseizure drugs have been developed. However, there are still many patients who are resistant to therapy. The molecular and genetic bases regarding such drug-resistant seizures have been poorly elucidated. In many cases, heavy seizures are instigated by brain development malformations and often caused by gene mutations. Such malformations can be demonstrated in mouse models by generating mutant strains. One of the most potent mutagens is ENU (N-ethyl-N-nitrosourea). In the present study, we describe three novel mutant strains generated by ENU-directed mutagenesis. Two of these strains present a very strong epileptic phenotype triggered by audiogenic stimuli (G9-1 and S5-1 strains). The third mouse strain is characterized by behavioral disorders and hyperexcitation of neuronal networks. We identified changes in the expression of those genes encoding neurotransmission proteins in the cerebral cortexes of these mice. It turned out that the G9-1 strain demonstrated the strongest disruptions in the expression of those genes encoding plasma membrane channels, excitatory glutamate receptors, and protein kinases. On the other hand, the number of GABAergic neurons was also affected by the mutation. All three lines are characterized by increased anxiety, excitability, and suppressed motor and orientational–exploratory activities. On the other hand, the strains with an epileptic phenotype—G9-1 and S5-1ave reduced learning ability, and the A9-2 mice line retains high learning ability.

Exploring the Effect of Active Components in Oil Tree Peony Seed Meal on Swine Disease Resistance and its Potential Mechanisms Based on Network Pharmacology and Molecular Docking.

This study aims to explore the feasibility of using network pharmacology and molecular docking technology to predict the effects of active components from oil tree peony seed meal (PSM) on swine diseases. Ten active components of PSM were screened Screening through literature search and network pharmacology standards, including Betulinic acid, Quercetin, Kaempferol, Luteolin, Isorhamnetin, Hydroxygenkwanin, Hederagenin, Benzoyl Paeoniflorin, Albiflorin, Paeoniflorin. Ten types of swine diseases were selected, including African Swine Fever, Aftosa, Swine Vesicular Disease, Transmissible Gastroenteritis, Swine Streptococcal Infection, Blue Aural Disease, Swine Infectious Atrophic Rhinitis, Swine Influenza, Swine Erysipelas, Swine Epidemic Encephalitis. The results showed that the average number of cross genes between the potential target genes of PSM active components and each swine disease target gene accounted for 7.64 % of the total number of swine disease target genes. The GO enrichment analyses showed that putative targets exist in endosomes, lysosomes, cell membranes, nerves, growth factor activity, receptor tyrosine kinase binding, enzyme binding, growth factor binding, transcription coactivator binding, oxidoreductase activity, prostaglandin E receptor activity and insulin receptor substrate binding. The KEGG enrichment analysis results showed that these putative genes were involved in various cancer progression pathways, signaling pathways, and hormone regulatory pathways. A total of 8 core targets were obtained through protein-protein interaction networks analysis, including Protein Kinase CAMP-Activated Catalytic Subunit Alpha (PRKACA), Non-Receptor Tyrosine Kinase (SRC), Mitogen-Activated Protein Kinase 1 (MAPK1), E1A Binding Protein P300 (EP300), Hypoxia Inducible Factor 1 Subunit Alpha (HIF1A), Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Beta (PIK3CB), C-X-C chemokine receptor type 4 (CXCR4) and Estrogen Receptor 2 (ESR2). The HIF-1 signaling pathway was found to be associated with all 10 selected swine diseases. The PD-L1 expression, and PD-1 checkpoint pathway in cancer, and thyroid hormone signaling pathway were not only enriches the core target with a quantity of 7, but also associated with 9 Swine diseases. In addition, the molecular docking results indicate that the core ingredients have strong affinity with hub genes. The research suggests that the active components of PSM may intervene in swine diseases through multiple components, targets, and pathways.

The impact of trauma relevant concentrations of prostaglandin E2 on the anti-microbial activity of the innate immune system

BackgroundThe mechanisms underlying the state of systemic immune suppression that develops following major trauma are poorly understood. A post-injury increase in circulating levels of prostaglandin E2 (PGE2) has been proposed as a contributory factor, yet few studies have addressed how trauma influences PGE2 biology.MethodsBlood samples from 95 traumatically-injured patients (injury severity score ≥8) were collected across the pre-hospital (≤2 hours), acute (4-12 hours) and subacute (48-72 hours) post-injury settings. Alongside ex vivo assessments of lipopolysaccharide (LPS)-induced cytokine production by monocytes, neutrophil reactive oxygen species production and phagocytosis, serum concentrations of PGE2 and its scavenger albumin were measured, and the expression of enzymes and receptors involved in PGE2 synthesis and signalling analysed. Leukocytes from trauma patients were treated with cyclooxygenase (COX) inhibitors (indomethacin or NS-398), or the protein kinase A inhibitor H89, to determine whether injury-induced immune suppression could be reversed by targeting the PGE2 pathway. The effect that trauma relevant concentrations of PGE2 had on the anti-microbial functions of neutrophils, monocytes and monocyte-derived macrophages (MDMs) from healthy controls (HC) was examined, as was the effect of PGE2 on efferocytosis. To identify factors that may trigger PGE2 production post-trauma, leukocytes from HC were treated with mitochondrial-derived damage associated molecular patterns (mtDAMPs) and COX-2 expression and PGE2 generation measured.ResultsPGE2 concentrations peaked in blood samples acquired ≤2 hours post-injury and coincided with significantly reduced levels of albumin and impaired LPS-induced cytokine production by monocytes. Significantly higher COX-2 and phospholipase A2 expression was detected in neutrophils and/or peripheral blood mononuclear cells isolated from trauma patients. Treatment of patient leukocytes with indomethacin, NS-398 or H89 enhanced LPS-induced cytokine production and neutrophil extracellular trap generation. Exposure to physiological concentrations of PGE2 suppressed the anti-microbial activity of monocytes, neutrophils and MDMs of HC, but did not influence efferocytosis. In a formyl-peptide receptor-1 dependent manner, mtDAMP treatment significantly increased COX-2 protein expression in neutrophils and monocytes, which resulted in increased PGE2 production.ConclusionsPhysiological concentrations of PGE2 suppress the anti-microbial activities of neutrophils, monocytes and MDMs. Targeting the PGE2 pathway could be a therapeutic approach by which to enhance innate immune function post-injury.

Computational Study of Coumarin Compounds as Potential Inhibitors of Casein Kinase 2: DFT, 2D-QSAR, ADMET and Molecular Docking Investigations

Casein kinase 2 (CK2) is an ubiquitous, essential, and highly pleiotropic protein kinase whose abnormally high constitutive activity is suspected to underlie its pathogenic potential in neoplasia and other diseases. Recently, there has been a notable increase in interest in the use of casein kinase 2 (CK2) inhibitors to improve the treatment of a specific form of cancer while minimizing the risk of undesirable side effects. Recently, using different virtual screening approaches, we have identified several novel CK2 inhibitors. In particular, we have discovered that the coumarin moiety can be considered an attractive CK2 inhibitor scaffold. In the present work, quantitative structure-activity relationship (QSAR) analysis has been employed to envisage the inhibitory effects of 32 coumarin derivatives on the CK2 protein. The most efficient model is found by using multiple linear regression (MLR). Its capability is considered by the external and internal validation values found (R2 = 0.884, Q2cv = 0.822, R2pred = 0.821, and R2p = 0.811), which aligned well with Tropsha and Golbraikh’s approach. The highest docking score founded for the newly designed coumarins is −7.50 kcal mol-1, which indicates that candidates can bind to the CK2 receptor with greater affinity. Based on the results of the ADMET properties and drug similarity analyses, a DFT investigation was conducted to confirm the stability of the newly explored compounds. It appears that the most stable complexes are those of compound with the highest binding affinity with a lower risk of toxicité.

Comprehensive analysis of VEGF/VEGFR inhibitor-induced immune-mediated hypertension: integrating pharmacovigilance, clinical data, and preclinical models

IntroductionThis study aimed to elucidate the differential immunological mechanisms and characteristics of hypertension induced by VEGF inhibitors (VEGFi) and VEGF receptor inhibitors (VEGFRi), with the goal of optimizing monitoring strategies and treatment protocols.MethodsWe investigated the risk of immune-related adverse events associated with VEGFi/VEGFRi-induced hypertension by analyzing the FDA Adverse Event Reporting System (FAERS) database. Findings were corroborated with blood pressure characteristics observed in clinical patients and preclinical models exposed to various VEGF/VEGFRi. Clinical and preclinical studies were conducted to compare immunological responses and hypertension profiles between inhibitor classes. An integrative analysis across cancer types and species was performed, focusing on key signaling pathways.ResultsAnalysis of FAERS data, in conjunction with clinical observations, revealed that both VEGFi and VEGFRi significantly elevated the risk of immune-mediated, blood pressure-related adverse events (ROR=7.75, 95% CI: 7.76-7.95). Subsequent clinical and preclinical studies demonstrated differential immunological responses and hypertension profiles between inhibitor classes. VEGFRi exhibited a more rapid onset, greater blood pressure elevation, and higher incidence of immune-mediated adverse events compared to VEGFi (Systolic BP: ROR=0 for VEGFi vs. ROR=12.25, 95% CI: 6.54-22.96 for VEGFRi; Diastolic BP: ROR=5.09, 95% CI: 0.60-43.61 for VEGFi vs. ROR=12.90, 95% CI: 3.73-44.55 for VEGFRi). Integrative analysis across cancer types and species, focusing on key signaling pathways, revealed that VEGF/VEGFRi-induced blood pressure elevation was associated with immunomodulation of the mitogen activated protein kinase (MAPK) pathway (R=-0.379, P=0.0435), alterations in triglyceride metabolism (R=-0.664, P=0.0001), modulation of myo-inositol 1,4,5-trisphosphate-sensitive calcium release channel activity (R=0.389, P=0.0378), and dysregulation of nitric oxide eNOS activation and metabolism (R=-0.439, P=0.0179).DiscussionThe temporal dynamics of these effects demonstrated greater significance than dose-dependent responses. Both VEGFi and VEGFRi significantly augmented the risk of immune-mediated, blood pressure-related adverse events, with VEGFRi inducing a more rapid and pronounced onset of blood pressure elevation and a higher incidence of immune-related, blood pressure-associated adverse events compared to VEGFi.

Functional bias of contractile control in mouse resistance arteries

Constrictor agonists set arterial tone through two coupling processes, one tied to (electromechanical), the other independent (pharmacomechanical) of, membrane potential (VM). This dual arrangement raises an intriguing question: is the contribution of each mechanism (1) fixed and proportionate, or (2) variable and functionally biased. Examination began in mouse mesenteric arteries with a vasomotor assessment to a classic Gq/11 (phenylephrine) or Gq/11/G12/13 (U46619) agonist, in the absence and presence of nifedipine, to separate among the two coupling mechanisms. Each constrictor elicited a concentration response curve that was attenuated and rightward shifted by nifedipine, findings consistent with functional bias. Electromechanical coupling preceded pharmacomechanical, the latter’s importance rising with agonist concentration. In this regard, ensuing contractile and phosphorylation (CPI-17 & MYPT1 (T-855 & T-697)) measures revealed phenylephrine-induced pharmacomechanical coupling was tied to protein kinase C (PKC) activity, while that enabled by U46619 to PKC and Rho-kinase. A complete switch to pharmacomechanical coupling arose when agonist superfusion was replaced by pipet application to a small portion of artery. This switch was predicted, a priori, by a computer model of electromechanical control and supported by additional measures of VM and cytosolic Ca2+. We conclude that the coupling mechanisms driving agonist-induced constriction are variable and functionally biased, their relative importance set in accordance with agonist concentration and manner of application. These findings have important implications to hemodynamic control in health and disease, including hypertension and arterial vasospasm.

PAK4 is Required for Meiotic Resumption, Spindle Assembly, and Cortical Migration in Mouse Oocytes During Meiotic Maturation.

Oocyte meiotic errors can cause infertility, miscarriage, and birth defects. Here the role and the underlying mechanism of p21 activated kinase 4 (PAK4) in mouse oocyte meiosis is evaluated. It is found that PAK4 expression and its phosphorylation are detected in high level at germinal vesicle (GV) stage, and gradually decreased after meiotic resumption in oocytes. PAK4 has direct physical interaction with both mitogen-activated protein kinases 1/2 (MEK1/2) and Paxillin, they are colocalized on the spindle structure during metaphases I and II. Phospho-PAK4 is distributed beneath the cytoplasmic membrane and on the chromosomes, and colocalized with the microtubule organizing center (MTOC) proteins, Pericentrin and γ-tubulin, as well as phosphor-MEK1/2 and phosphor-Paxillin on spindle poles. PAK4 inhibition by chemical inhibitor LCH-7749944, specific Pak4 morpholino oligo or the dominant negative mutant Pak4K350, 351M influence the meiotic resumption, spindle assembly and its cortical migration, and associated with the downregulation in the dephosphorylation of cyclin dependent kinase 1 (CDK1) and the levels of Cyclin B1, MEK1/2, Paxillin, g-tubulin, acetylated a-tubulin, Arp3, and Cofilin phosphorylation in oocytes. In sum, PAK4 functions to sustain the rational levels of Cyclin B1, MEK1/2, Paxillin, y-tubulin, acetylated a-tubulin, Arp3, and phosphor-Cofilin in mouse oocytes, thereby promotes the meiotic resumption, spindle assembly, and migration during meiotic maturation.

Impairment of neuronal tyrosine phosphatase STEP worsens post-ischemic inflammation and brain injury under hypertensive condition

Hypertension is associated with poor outcome and higher mortality in patients with ischemic stroke. The impairment of adaptive vascular mechanisms under hypertensive condition compromises collateral blood flow after arterial occlusion in patients with acute ischemic stroke resulting in hypoperfusion. The increased oxidative stress caused by hypoperfusion is thought to be a trigger for the rapid evolution of ischemic infarct volume under hypertensive condition. However, the cellular factors and pathways that contribute to the exacerbation of ischemic brain injury under hypertensive condition is not yet understood. The current study reveals that predisposition to hypertension leads to basal loss of function of the neuron-specific tyrosine phosphatase STEP, which plays a crucial role in neuroprotection against excitotoxic insult. The findings further show that a mild ischemic insult in hypertensive rats triggers an early onset and sustained activation of the neuronal extracellular signal regulated kinase (ERK MAPK), a member of the mitogen activated protein kinase family and a substrate of STEP. This leads to rapid increase in the activation of neuronal NF-κB, expression of neuronal cyclooxygenase-2 and subsequent biosynthesis of the pro-inflammatory mediator prostaglandin E2, resulting in rapid morphological transformation of microglia to the pro-inflammatory state and subsequent exacerbation of ischemic brain injury. Restoration of STEP signaling with intravenous administration of a STEP-derived peptide mimetic reduces the pro-inflammatory response in neurons, activation of microglia, and ischemic brain injury. The findings suggest that the basal loss of STEP function under hypertensive condition contributes to the exacerbation of ischemic brain injury by enhancing post-ischemic inflammatory response. The study not only presents a novel role of STEP in regulating neuroimmune communication but also highlights the therapeutic potential of a STEP-mimetic in mitigating ischemic brain damage under hypertensive condition.

Weighted Gene Co-Expression Network Analysis Uncovers Critical Genes and Pathways Involved in Soybean Response to Soybean Mosaic Virus

Background: Soybean mosaic virus (SMV) is a globally prevalent and detrimental virus that belongs to the Potyvirus genus. Pathogenic viruses of this genus are typically linear in shape, with dimensions ranging between 630 and 750 nm, and are composed of single-stranded RNA and proteins. We have developed an SMV-resistant soybean line, Dongnong 93-046, which has no significant changes in disease resistance identification in the adult plants and has neat grains with no obvious brown or black markings. To explore the defense mechanisms of soybean against SMV, we performed comparative transcriptomic sequencing of the leaves between the Dongnong 93-046 inoculated with SMV at 8 h (T) and the non-inoculated control (C) on the HiSeq2000 platform. In addition, we performed non-targeted metabolomic sequencing of leaves from the treatment and control groups. Results: We identified a total of 41,189 differentially expressed genes (DEGs). A total of 9809 differentially expressed genes (DEGs) met the criteria of |Log2FC (Fold Change)| ≥ 1 and adjusted p-value ≤ 0.001. Among the 41,189 DEGs identified, 9196 exhibited FPKM values greater than 10. KEGG pathway enrichment analysis of the 9809 DEGs revealed significant enrichment of genes involved in resistance-related pathways such as plant–pathogen interaction, linoleic acid metabolism, mitogen-activated protein kinase (MAPK) signaling pathway, and plant hormone signaling transduction. Functional analysis using MapMan software identified multiple DEGs that were associated with pathways such as jasmonate synthesis and phenylpropanoid biosynthesis. Weighted gene co-expression network analysis (WGCNA) using the differential metabolites and the 9196 DEGs revealed a strong correlation between gene clusters within the Turquoise module and the content of jasmonate-related metabolites. Further functional enrichment analysis of the 894 genes within the gene clusters showed a significant and repeated enrichment of pathways related to plant–pathogen interaction, linoleic acid metabolism, and plant hormone signaling transduction. Subsequent focused pathway analysis identified key genes involved in plant hormone signaling transduction pathways, such as the jasmonate ZIM domain protein Glyma.16G010000, the gene Glyma.01G235600 encoding the essential diterpene reductase required for jasmonate synthesis in the jasmonate biosynthesis pathway, and the transcription factor Glyma.02G232600 involved in the plant–pathogen interaction pathway, among others. This study provides a theoretical framework for understanding the resistance mechanism of soybean cultivar Dongnong 93-046 against the SMV N1 strain, offers potential gene resources for breeding soybean varieties with resistance to SMV, and paves the way for new strategies to control SMV infection, enhance resistance, and improve crop yield and quality.

Tubulin-Targeted Therapy in Melanoma Increases the Cell Migration Potential by Activation of the Actomyosin Cytoskeleton─An In Vitro Study.

One of the most dangerous aspects of cancers is their ability to metastasize, which is the leading cause of death. Hence, it holds significance to develop therapies targeting the eradication of cancer cells in parallel, inhibiting metastases in cells surviving the applied therapy. Here, we focused on two melanoma cell lines─WM35 and WM266-4─representing the less and more invasive melanomas. We investigated the mechanisms of cellular processes regulating the activation of actomyosin as an effect of colchicine treatment. Additionally, we investigated the biophysical aspects of supplement therapy using Rho-associated protein kinase (ROCK) inhibitor (Y-27632) and myosin II inhibitor ((-)-blebbistatin), focusing on the microtubules and actin filaments. We analyzed their effect on the proliferation, migration, and invasiveness of melanoma cells, supported by studies on cytoskeletal architecture using confocal fluorescence microscopy and nanomechanics using atomic force microscopy (AFM) and microconstriction channels. Our results showed that colchicine inhibits the migration of most melanoma cells, while for a small cell population, it paradoxically increases their migration and invasiveness. These changes are also accompanied by the formation of stress fibers, compensating for the loss of microtubules. Simultaneous administration of selected agents led to the inhibition of this compensatory effect. Collectively, our results highlighted that colchicine led to actomyosin activation and increased the level of cancer cell invasiveness. We emphasized that a cellular pathway of Rho-ROCK-dependent actomyosin contraction is responsible for the increased invasive potential of melanoma cells in tubulin-targeted therapy.

β-catenin-inhibited Sumoylation modification of LKB1 and fatty acid metabolism is critical in renal fibrosis

Liver kinase B1 (LKB1) is a serine/threonine kinase controlling cell homeostasis. Among post-translational modification, Sumoylation is vital for LKB1 activating adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK), the key regulator in energy metabolism. Of note, AMPK-regulated fatty acid metabolism is highly involved in maintaining normal renal function. However, the regulative mechanisms of LKB1 Sumoylation remain elusive. In this study, we demonstrated that β-catenin, a notorious signal in renal fibrosis, inhibited the Sumoylation of LKB1, thereby disrupting fatty acid oxidation in renal tubular cells and triggering renal fibrosis. Mechanically, we found that Sumo3 was the key mediator for LKB1 Sumoylation in renal tubular cells, which was transcriptionally inhibited by β-catenin/Transcription factor 4 (TCF4) signaling. Overexpression of Sumo3, not Sumo1 or Sumo2, restored β-catenin-disrupted fatty acid metabolism, and retarded lipid accumulation and fibrogenesis in the kidney. In vivo, conditional knockout of β-catenin in tubular cells effectively preserved fatty acid oxidation and blocked lipid accumulation by maintaining LKB1 Sumoylation and AMPK activation. Furthermore, ectopic expression of Sumo3 strongly inhibited Wnt1-aggravated lipid accumulation and fibrogenesis in unilateral ischemia-reperfusion mice. In patients with chronic kidney disease, we found a loss of Sumo3 expression, and it was highly related to LKB1 repression. This contributes to fatty acid metabolism disruption and lipid accumulation, resulting in renal fibrosis. Overall, our study revealed a new mechanism in fatty acid metabolism dysfunction and provided a new therapeutic target pathway for regulating Sumo modification in renal fibrosis.

A Tympanic Piezo-Bioreactor Modulates Ion Channel-Associated Mechanosignaling to Stabilize Phenotype and Promote Tenogenesis in Human Tendon-Derived Cells.

Preserving the function of human tendon-derived cells (hTDCs) during cell expansion is a significant challenge in regenerative medicine. In this study, a non-genetic approach is introduced to control the differentiation of hTDCs using a newly developed tympanic bioreactor. The system mimics the functionality of the human tympanic membrane, employing a piezoelectrically tuned acoustic diaphragm made of polyvinylidene fluoride-co-trifluoroethylene and boron nitride nanotubes. The diaphragm is vibrationally actuated to deliver targeted electromechanical stimulation to hTDCs. The results demonstrate that the system effectively maintains the tendon-specific phenotype of hTDCs, even under conditions that typically induce nonspecific differentiation, such as osteogenesis. This stabilization is achieved by modulating integrin-mediated mechanosignaling via ion channel-regulated calcium activity, potentially by TREK-1 and PIEZO1, yet targeted studies are required for confirmation. Finally, the system sustains the activation of key differentiation pathways (bone morphogenetic protein, BMP) while downregulating osteogenesis-associated (mitogen-ctivated protein kinase, MAPK and wingless integrated, WNT) pathways and upregulating Focal Adhesion Kinase (FAK) signaling. This approach offers a finely tunable, dose-dependent control over hTDC differentiation, presenting significant potential for non-genetic approaches in cell therapy, tendon tissue engineering, and the regeneration of other mechanosensitive tissues.

Cloperastine Reduces IL-6 Expression via Akt/GSK3/Nrf2 Signaling in Monocytes/Macrophages and Ameliorates Symptoms in a Mouse Sepsis Model Induced by Lipopolysaccharide.

Cloperastine (CLP) is a drug with a central antitussive effect that is used to treat bronchitis. Therefore, we have attempted to examine the anti-inflammatory effects of CLP. CLP reduced the secretion of interleukin (IL)-6, a pro-inflammatory cytokine, from RAW264.7 monocyte/macrophage-linage cells treated with lipopolysaccharide (LPS). IL-6 is a biomarker of sepsis and has been suggested to exacerbate its symptoms. We found that the intraperitoneal administration of CLP reduced IL-6 levels in the lungs and also improved hypothermia in mice with LPS-induced sepsis. CLP ameliorated kidney pathologies such as congestion and increased the survival rate of mice administered with a lethal dose of LPS. To reveal the mechanisms underlying the anti-inflammatory function of CLP, we analysed the intracellular signaling in LPS-treated RAW264.7 cells. CLP induced the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase 3 (GSK3) and also increased the amount of nuclear factor erythroid-2-related factor 2 (Nrf2) in RAW264.7 cells with/without LPS. Wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), reduced the upregulated phosphorylation levels of Akt and GSK3 and the increased amount of Nrf2. It also halted the reduction of IL-6 secretion caused by CLP. These results suggest that CLP has an anti-inflammatory function via Akt/GSK3/Nrf2 signaling and could be a candidate drug for the treatment of inflammatory diseases, including sepsis.

EIF4F controls ERK MAPK signaling in melanomas with BRAF and NRAS mutations

The eIF4F translation initiation complex plays a critical role in melanoma resistance to clinical BRAF and MEK inhibitors. In this study, we uncover a function of eIF4F in the negative regulation of the rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) signaling pathway. We demonstrate that eIF4F is essential for controlling ERK signaling intensity in treatment-naïve melanoma cells harboring BRAF or NRAS mutations. Specifically, the dual-specificity phosphatase DUSP6/MKP3, which acts as a negative feedback regulator of ERK activity, requires continuous production in an eIF4F-dependent manner to limit excessive ERK signaling driven by oncogenic RAF/RAS mutations. Treatment with small-molecule eIF4F inhibitors disrupts the negative feedback control of MAPK signaling, leading to ERK hyperactivation and EGR1 overexpression in melanoma cells in vitro and in vivo. Furthermore, our quantitative analyses reveal a high spare signaling capacity in the ERK pathway, suggesting that eIF4F-dependent feedback keeps the majority of ERK molecules inactive under normal conditions. Overall, our findings highlight the crucial role of eIF4F in regulating ERK signaling flux and suggest that pharmacological eIF4F inhibitors can disrupt the negative feedback control of MAPK activity in melanomas with BRAF and NRAS activating mutations.