In vitro and in vivo effects of Galectin-3 inhibitor TD139 on inflammation and ERK/JNK/p38 pathway in gestational diabetes mellitus.

This study aims to investigate the effects of the Galectin-3 (Gal-3) inhibitor TD139 on inflammation and the extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinase (JNK)/p38 pathway in gestational diabetes mellitus (GDM). Human placental tissues were treated with TD139 and TNF-α, assessing Gal-3, ERK/JNK/p38 activation, and inflammatory cytokines. GDM was induced in mice via subcutaneous injections of streptozotocin (STZ). After confirming GDM, mice were treated with 15 mg/kg TD139 on GD 10.5 12.5, 14.5, 16.5, and 18.5. Serum inflammatory cytokines were measured on GD 20.5, and post-delivery placental tissues were analyzed. Data were analyzed using one-way or two-way repeated measures ANOVA with post hoc tests. TD139 suppressed TNF-α-induced increases in Gal-3, IL-1ß, IL-6, MCP-1, and ERK/JNK/p38 activation in placental tissues. In STZ-induced GDM mice, TD139 reduced glucose levels, weight loss, and food and water intake. TD139 significantly lowered TNF-α, IL-1ß, IL-6, and MCP-1 in serum and placental tissues and inhibited the ERK/JNK/p38 pathway. TD139 improved pup numbers in GDM mice compared to untreated ones. TD139 reduces inflammation and inhibits the ERK/JNK/p38 pathway in TNF-α stimulated placental tissues and STZ-induced GDM mice, suggesting its therapeutic potential for managing GDM-related placental inflammation and improving pregnancy outcomes. The study used TNF-α to mimic GDM in placental tissues and an STZ-induced GDM mouse model, which may not fully represent human GDM complexity. Future research should explore alternative models, and broader signaling pathways, and thoroughly evaluate TD139's safety in pregnancy.

Exosomal miRNAs in patients with chronic heart failure and hyperuricemia and the underlying mechanisms.

Chronic heart failure (CHF) combined with hyperuricemia (HUA) is a comorbidity that is hard to diagnose by a single biomarker. Exosomal miRNAs are differentially expressed in cardiovascular diseases and are closely associated with regulating most biological functions. This study aimed to provide evidence for miRNA as a new molecular marker for precise diagnosis of the comorbidity of CHF with HUA and further analyze the potential targets of differentially expressed miRNA. This controlled study included 30 CHF patients combined with HUA (Group T) and 30 healthy volunteers (Group C). 6 peripheral blood samples from Group T and Group C were analyzed for exosomal miRNAs by high-throughput sequencing and then validated in the remaining 24 peripheral blood samples from Group T and Group C by applying real-time PCR (RT-PCR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using R software to predict the differential miRNAs' action targets. 42 differentially expressed miRNAs were detected (18 upregulated and 24 downregulated), in which miR-27a-5p was significantly upregulated (P<0.01), and miR-139-3p was significantly downregulated (P<0.01) in Group T. The combination of miR-27a-5p and miR-139-3p predicted the development of CHF combined with HUA with a maximum area under the curve (AUC) of 0.899 (95 % CI: 0.812-0.987, SEN=79.2 %, SPE=91.7 %, J value = 0.709). GO and KEGG enrichment analysis revealed that the differentially expressed miRNAs had a role in activating the AMPK-mTOR signaling pathway to activate the autophagic response. Collectively, our findings suggest that upregulated exosomal miR-27a-5p combined with downregulated exosomal miR-139-3p can be used as a novel molecular marker for precise diagnosis of CHF combined with HUA and enhanced autophagy by AMPK-mTOR signaling pathway may be one pathogenesis of the differentially expressed miRNAs.

Prenatal opioid exposure significantly impacts placental protein kinase C (PKC) and drug transporters, leading to drug resistance and neonatal opioid withdrawal syndrome.

Background: Neonatal Opioid Withdrawal Syndrome (NOWS) is a consequence of in-utero exposure to prenatal maternal opioids, resulting in the manifestation of symptoms like irritability, feeding problems, tremors, and withdrawal signs. Opioid use disorder (OUD) during pregnancy can profoundly impact both mother and fetus, disrupting fetal brain neurotransmission and potentially leading to long-term neurological, behavioral, and vision issues, and increased infant mortality. Drug resistance complicates OUD and NOWS treatment, with protein kinase regulation of drug transporters not fully understood. Methods: DNA methylation levels of ATP-binding cassette (ABC) and solute carrier (SLC) drug transporters, along with protein kinase C (PKC) genes, were assessed in 96 placental samples using the Illumina Infinium MethylationEPIC array (850K). Samples were collected from three distinct groups: 32 mothers with infants prenatally exposed to opioids who needed pharmacological intervention for NOWS, 32 mothers with prenatally opioid-exposed infants who did not necessitate NOWS treatment, and 32 mothers who were not exposed to opioids during pregnancy. Results: We identified 69 significantly differentially methylated SLCs, with 24 hypermethylated and 34 hypomethylated, and 11 exhibiting both types of methylation changes including SLC13A3, SLC15A2, SLC16A11, SLC16A3, SLC19A2, and SLC26A1. We identified methylation changes in 11 ABC drug transporters (ABCA1, ABCA12, ABCA2, ABCB10, ABCB5, ABCC12, ABCC2, ABCC9, ABCE1, ABCC7, ABCB3): 3 showed hypermethylation, 3 hypomethylation, and 5 exhibited both. Additionally, 7 PKC family genes (PRKCQ, PRKAA1, PRKCA, PRKCB, PRKCH, PRKCI, and PRKCZ) showed methylation changes. These genes are associated with 13 pathways involved in NOWS, including ABC transporters, bile secretion, pancreatic secretion, insulin resistance, glutamatergic synapse, and gastric acid secretion. Conclusion: We report epigenetic changes in PKC-related regulation of drug transporters, which could improve our understanding of clinical outcomes like drug resistance, pharmacokinetics, drug-drug interactions, and drug toxicity, leading to maternal relapse and severe NOWS. Novel drugs targeting PKC pathways and transporters may improve treatment outcomes for OUD in pregnancy and NOWS.

Kinase Drug Discovery: Impact of Open Science and Artificial Intelligence.

Given their central role in signal transduction, protein kinases (PKs) were first implicated in cancer development, caused by aberrant intracellular signaling events. Since then, PKs have become major targets in different therapeutic areas. The preferred approach to therapeutic intervention of PK-dependent diseases is the use of small molecules to inhibit their catalytic phosphate group transfer activity. PK inhibitors (PKIs) are among the most intensely pursued drug candidates, with currently 80 approved compounds and several hundred in clinical trials. Following the elucidation of the human kinome and development of robust PK expression systems and high-throughput assays, large volumes of PK/PKI data have been produced in industrial and academic environments, more so than for many other pharmaceutical targets. In addition, hundreds of X-ray structures of PKs and their complexes with PKIs have been reported. Substantial amounts of PK/PKI data have been made publicly available in part as a result of open science initiatives. PK drug discovery is further supported through the incorporation of data science approaches, including the development of various specialized databases and online resources. Compound and activity data wealth compared to other targets has also made PKs a focal point for the application of artificial intelligence (AI) in pharmaceutical research. Herein, we discuss the interplay of open and data science in PK drug discovery and review exemplary studies that have substantially contributed to its development, including kinome profiling or the analysis of PKI promiscuity versus selectivity. We also take a close look at how AI approaches are beginning to impact PK drug discovery in light of their increasing data orientation.

Inhibition of miR-194-5p avoids DUSP9 downregulation thus limiting sepsis-induced cardiomyopathy.

Sepsis-induced cardiomyopathy (SIC) is described as a reversible myocardial depression that occurs in patients with septic shock. Increasing evidence shows that microRNA-194-5p (miR-194-5p) participates in the regulation of oxidative stress, mitochondrial dysfunction, and apoptosis and its expression is associated with the occurrence and progression of cardiovascular disease; however, the effects of miR-194-5p in SIC are still unclear. This study explores whether miR-194-5p could modulate SIC by affecting oxidative stress, mitochondrial function, and apoptosis. Experimental septic mice were induced by intraperitoneal injection of lipopolysaccharide (LPS) in C57BL/6J mice. The biological role of miR-194-5p in SIC in vivo was investigated using cardiac echocardiography, ELISA, western blot, qRT-PCR, transmission electron microscopy, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, bioinformatics analysis, and dual-luciferase reporter gene assay. Our major finding is that miR-194-5p antagomir mitigates sepsis-induced cardiac dysfunction, inflammation, oxidative stress, apoptosis and mitochondrial dysfunction in the hearts of septic mice, while miR-194-5p agomir triggers the opposite effects. Furthermore, dual-specificity phosphatase 9 (DUSP9) is a direct target of miR-194-5p and the cardioprotective effects of miR-194-5p antagomir on cardiac dysfunction, inflammation, apoptosis, mitochondrial dysfunction and oxidative stress are abolished through inhibiting DUSP9. Therefore, miR-194-5p inhibition could mitigate SIC via DUSP9 in vivo and the novel miR-194-5p/DUSP9 axis might be the potential treatment targets for SIC patients.

Assessing the antitumor effects of metformin on ovarian clear cell carcinoma.

Developing novel therapies that outperform the existing chemotherapeutic treatments is required for treatment-resistant ovarian clear cell carcinoma. We investigated the antitumor effect of metformin on ovarian clear cell carcinoma, enhancement of the antitumor effect by its combination with chemotherapy, and its molecular regulatory mechanism. First, we evaluated the viability of ovarian clear cell carcinoma lines using the water-soluble tetrazolium-1 assay and found that metformin suppressed cell viability. Cell viability was significantly suppressed by co-treatment with cisplatin and metformin. In contrast, co-treatment with paclitaxel and metformin showed no significant difference in viability compared with the group without metformin. Western blot analysis showed increased phosphorylation of AMP-activated protein kinase in some cell lines and suppressed phosphorylation of the mammalian target of rapamycin in a particular cell line. Flow cytometry analysis revealed a significant increase in the rate of apoptosis in the metformin-treated group and rate of cell cycle arrest at the G2/M phase in a particular cell line. These results indicated that metformin may be effective against cultured ovarian clear cell carcinoma cells, particularly in combination with cisplatin.

Acquired Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) Resistance of Human Colorectal Cancer Cells Is Linked to Histone Acetylation and Is Synergistically Ameliorated by Combination with HDAC Inhibitors.

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an attractive target for the treatment of various malignancies; however, its therapeutic potential is limited because of the frequent occurrence of tumor cell resistance. In this study, we determined whether TRAIL resistance acquired by repeated administration could be overcome by HDAC inhibition in human colorectal cancer cells. METHODS: TRAIL-resistant HCT116 human colorectal cancer cells (HCT116-TR) were generated by repeated treatment with 10 and 25 ng/mL TRAIL twice weekly for 28 days. RESULTS: The resulting TRAIL-resistant cells were noncross-resistant to other chemotherapeutic agents. The levels of histone acetylation-related proteins, such as ac-histone H4 and HDAC1, were altered in HCT116-TR cells compared with the parental HCT116 cell line. The combined treatment with TRAIL and HDAC inhibitors significantly increased apoptosis in HCT116-TR cells and indicated a synergistic effect. The mechanism by which HDAC inhibition sensitizes HCT116-TR cells to TRAIL is dependent on the intrinsic pathway. In addition, we found that HDAC inhibition enhanced the sensitivity of cells to TRAIL through mitogen-activated protein kinases/CCAAT/enhancer-binding protein homologs of protein-dependent upregulation of death receptor 5. CONCLUSION: These results suggest that histone acetylation is responsible for acquired TRAIL resistance after repeated exposure and acquired resistance to TRAIL may be overcome by combination therapies with HDAC inhibitors.

FDA-approved small molecule kinase inhibitors for cancer treatment (2001-2015): Medical indication, structural optimization, and binding mode Part I.

The dysregulation of kinases has emerged as a major class of targets for anticancer drug discovery given its node roles in the etiology of tumorigenesis, progression, invasion, and metastasis of malignancies, which is validated by the FDA approval of 28 small molecule kinase inhibitor (SMKI) drugs for cancer treatment at the end of 2015. While the preclinical and clinical data of these drugs are widely presented, it is highly essential to give an updated review on the medical indications, design principles and binding modes of these anti-tumor SMKIs approved by the FDA to offer insights for the future development of SMKIs with specific efficacy and safety.

Understanding the Cytomegalovirus Cyclin-Dependent Kinase Ortholog pUL97 as a Multifaceted Regulator and an Antiviral Drug Target.

Herpesviral protein kinases, such as the therapy-relevant pUL97 of human cytomegalovirus (HCMV), are important for viral replication efficiency as well as pathogenesis, and represent key antiviral drug targets. HCMV pUL97 is a viral cyclin-dependent kinase (CDK) ortholog, as it shares functional and structural properties with human CDKs. Recently, the formation of vCDK/pUL97-cyclin complexes and the phosphorylation of a variety of viral and cellular substrate proteins has been demonstrated. Genetic mapping and structural modeling approaches helped to define two pUL97 interfaces, IF1 and IF2, responsible for cyclin binding. In particular, the regulatory importance of interactions between vCDK/pUL97 and host cyclins as well as CDKs has been highlighted, both as determinants of virus replication and as a novel drug-targeting option. This aspect was substantiated by the finding that virus replication was impaired upon cyclin type H knock-down, and that such host-directed interference also affected viruses resistant to existing therapies. Beyond the formation of binary interactive complexes, a ternary pUL97-cyclin H-CDK7 complex has also been described, and in light of this, an experimental trans-stimulation of CDK7 activity by pUL97 appeared crucial for virus-host coregulation. In accordance with this understanding, several novel antiviral targeting options have emerged. These include kinase inhibitors directed to pUL97, to host CDKs, and to the pUL97-cyclin H interactive complexes. Importantly, a statistically significant drug synergy has recently been reported for antiviral treatment schemes using combinations of pharmacologically relevant CDK7 and vCDK/pUL97 inhibitors, including maribavir. Combined, such findings provide increased options for anti-HCMV control. This review focuses on regulatory interactions of vCDK/pUL97 with the host cyclin-CDK apparatus, and it addresses the functional relevance of these key effector complexes for viral replication and pathogenesis. On this basis, novel strategies of antiviral drug targeting are defined.

The molecular signature of the peripheral cannabinoid receptor 1 antagonist AM6545 in adipose, liver and muscle tissue.

The endocannabinoid system plays an important role in the regulation of metabolism, growth and regeneration of peripheral tissues, including liver, adipose and muscle tissue. Studies in cells, rodents and humans showed that cannabinoid receptor 1 (CB1) antagonist treatment is an effective strategy to improve features of metabolic health such as substrate metabolism, at least in models of metabolic dysregulation. However, acute signaling events that might induce these metabolic adaptations are not understood. It is not clear whether, and to which extent, a single treatment with a CB1 antagonist induces acute effects in peripheral, metabolic tissues. Therefore, the present study compared the phosphorylation status of signaling pathways and metabolic markers in liver, adipose and muscle tissue of mice treated with the peripherally restricted CB1 antagonist AM6545 and vehicle-treated mice. Protein kinase A phosphorylation was downregulated in white and brown adipose tissue, whereas the mitogen-activated protein kinase, phospho-extracellular signal-regulated kinase, was higher in liver, white adipose and muscle tissue of AM6545-treated mice. Additionally, Akt-mammalian target of rapamycin activation was higher in all tissues of AM6545-treated mice, whereas the phosphorylation status of metabolic markers remained unaffected. These data indicate that acute CB1 antagonism is effective to induce phosphorylation events of signaling cascades and metabolic markers in metabolic tissues of healthy, lean mice within a 90-min time window. The observed adaptations to AM6545 treatment do not fully align with earlier in vitro and in vivo findings, which could be ascribed to differences in cell type, exposure intensity (dose and time), health status and species.

mTOR Dysregulation, Insulin Resistance, and Hypertension.

Worldwide, diabetes mellitus (DM) and cardiovascular diseases (CVDs) represent serious health problems associated with unhealthy diet and sedentarism. Metabolic syndrome (MetS) is characterized by obesity, dyslipidemia, hyperglycemia, insulin resistance (IR) and hypertension. The mammalian target of rapamycin (mTOR) is a serine/threonine kinase with key roles in glucose and lipid metabolism, cell growth, survival and proliferation. mTOR hyperactivation disturbs glucose metabolism, leading to hyperglycemia and further to IR, with a higher incidence in the Western population. Metformin is one of the most used hypoglycemic drugs, with anti-inflammatory, antioxidant and antitumoral properties, having also the capacity to inhibit mTOR. mTOR inhibitors such as rapamycin and its analogs everolimus and temsirolimus block mTOR activity, decrease the levels of glucose and triglycerides, and reduce body weight. The link between mTOR dysregulation, IR, hypertension and mTOR inhibitors has not been fully described. Therefore, the main aim of this narrative review is to present the mechanism by which nutrients, proinflammatory cytokines, increased salt intake and renin-angiotensin-aldosterone system (RAAS) dysregulation induce mTOR overactivation, associated further with IR and hypertension development, and also mTOR inhibitors with higher potential to block the activity of this protein kinase.

Fine-tuning of the dual-role transcription factor WRKY8 via differential phosphorylation for robust broad-spectrum plant immunity.

Plants utilize plasma membrane-localized pattern recognition receptors (PRRs) to perceive pathogen-associated molecular patterns (PAMPs) to activate broad-spectrum pattern-triggered immunity (PTI). However, the regulatory mechanism ensuring robust broad-spectrum plant immunity remains largely unknown. Here, we reveal the dual roles of the transcription factor WRKY8 in transcriptional regulation of PRR genes: repressing the nlp20/nlp24 receptor gene RLP23 whereas promoting the chitin receptor gene CERK1. Remarkably, SsNLP1 and SsNLP2, two nlp24 type PAMPs in the destructive fungal pathogen Sclerotinia sclerotiorum, activate two calcium-elicited kinases, CPK4 and CPK11 to phosphorylate WRKY8 and consequently release its inhibition on RLP23 expression to accumulate RLP23. Meanwhile, SsNLPs activate a RLCK type kinase, PBL19 to phosphorylate WRKY8 and consequently enhance the accumulation of CERK1. Intriguingly, RLP23 is repressed at late stage by PBL19-mediated phosphorylation of WRKY8, to avoid excessive immunity for normal growth. Our findings unveil a "killing two birds with one stone" strategy employed by plants to elicit robust broad-spectrum immunity, which is based on PAMP-triggered fine-tuning of a dual-role transcription factor to simultaneously amplify two PRRs recognizing PAMPs well conserved in a wide range of pathogens. Moreover, our results reveal a novel plant strategy based on fine-tuning of multiple PRR gene expression to balance the trade-off between growth and immunity.

Recent Molecular Targets and Their Ligands for the Treatment of Alzheimer Disease.

Alzheimer's disease is a multifaceted neurodegenerative disease. Cholinergic dysfunction, amyloid ß toxicity, tauopathies, oxidative stress, neuroinflammation are among the main pathologies of the disease. Ligands targeting more than one pathology, multi-target directed ligands, attract attention in the recent years to tackle Alzheimer's disease. In this review, we aimed to cover different biochemical pathways, that are revealed in recent years for the pathology of the disease, as druggable targets such as cannabinoid receptors, matrix metalloproteinases, histone deacetylase and various kinases including, glycogen synthase kinase-3, mitogen-activated protein kinase and c-Jun N-terminal kinase, and their ligands for the treatment of Alzheimer's disease in the hope of providing more realistic insights into the field.

UBA2 SUMOylates NQO1 and promotes the proliferation of hepatocellular carcinoma by modulating the MAPK pathway.

In our previous study, we found that small ubiquitin-related modifier (SUMO)-activating enzyme ubiquitin-associated-2 domain (UBA2) was upregulated in hepatocellular carcinoma (HCC) patients who were insensitive to chemoembolization. In this study, we aimed to investigate the role of UBA2 in HCC progression. Three cohorts were used to evaluate the efficacy of UBA2 as a prognostic factor for HCC. Our results indicated that UBA2 was associated with aggressive clinical behaviors and was a strong indicator of poor prognosis in HCC. In vitro experiments demonstrated that UBA2 accelerated cell growth, invasion, and migration. These results were further supported by in vivo experiments. RNA-sequencing analysis indicated NQO1 as a target of UBA2, with its levels altering following UBA2 manipulation. The results were verified by western blotting (WB) and quantitative PCR. The SUMOplot Analysis Program predicted lysine residue K240 as a modification target of UBA2, which was confirmed by immunoprecipitation (IP) assays. Subsequent mutation of NQO1 at K240 in HCC cell lines and functional assays revealed the significance of this modification. In addition, the oncogenic effect of UBA2 could be reversed by the SUMO inhibitor ML792 in vivo and in vitro. In conclusion, our study elucidated the regulatory mechanism of UBA2 in HCC and suggested that the SUMO inhibitor ML792 may be an effective combinatory treatment for patients with aberrant UBA2 expression.

The Autophagic and Apoptotic Death of Forebrain Neurons of Rats with Global Brain Ischemia Is Diminished by the Intranasal Administration of Insulin: Possible Mechanism of Its Action.

Insulin is a promising neuroprotector. To better understand the mechanism of insulin action, it was important to show its ability to diminish autophagic neuronal death in animals with brain ischemic and reperfusion injury. In forebrain ischemia and reperfusion, the number of live neurons in the hippocampal CA1 region and frontal cortex of rats decreased to a large extent. Intracerebroventricular administration of the autophagy and apoptosis inhibitors to ischemic rats significantly increased the number of live neurons and showed that the main part of neurons died from autophagy and apoptosis. Intranasal administration of 0.5 IU of insulin per rat (before ischemia and daily during reperfusion) increased the number of live neurons in the hippocampal CA1 region and frontal brain cortex. In addition, insulin significantly diminished the level of autophagic marker LC3B-II in these forebrain regions, which markedly increased during ischemia and reperfusion. Our studies demonstrated for the first time the ability of insulin to decrease autophagic neuronal death, caused by brain ischemia and reperfusion. Insulin administered intranasally activated the Akt-kinase (activating the mTORC1 complex, which inhibits autophagy) and inhibited the AMP-activated protein kinase (which activates autophagy) in the hippocampus and frontal cortex of rats with brain ischemia and reperfusion.

The role of serine/threonine protein kinases in cardiovascular disease and potential therapeutic methods.

Protein phosphorylation is an important link in a variety of signaling pathways, and most of the important life processes in cells involve protein phosphorylation. Based on the amino acid residues of phosphorylated proteins, protein kinases can be categorized into the following families: serine/threonine protein kinases, tyrosine-specific protein kinases, histidine-specific protein kinases, tryptophan kinases, and aspartate/glutamyl protein kinases. Of all the protein kinases, most are serine/threonine kinases, where serine/threonine protein kinases are protein kinases that catalyze the phosphorylation of serine or threonine residues on target proteins using ATP as a phosphate donor. The current socially accepted classification of serine/threonine kinases is to divide them into seven major groups: protein kinase A, G, C (AGC), CMGC, Calmodulin-dependent protein kinase (CAMK), Casein kinase (CK1), STE, Tyrosine kinase (TKL) and others. After decades of research, a preliminary understanding of the specific classification and respective functions of serine/threonine kinases has entered a new period of exploration. In this paper, we review the literature of the previous years and introduce the specific signaling pathways and related therapeutic modalities played by each of the small protein kinases in the serine/threonine protein kinase family, respectively, in some common cardiovascular system diseases such as heart failure, myocardial infarction, ischemia-reperfusion injury, and diabetic cardiomyopathy. To a certain extent, the current research results, including molecular mechanisms and therapeutic methods, are fully summarized and a systematic report is made for the prevention and treatment of cardiovascular diseases in the future.

Melatonin-mediated low-temperature tolerance of cucumber seedlings requires Ca2+/CPKs signaling pathway.

Melatonin (Mel) is recognized as a prominent plant growth regulator. This study investigated the alleviating effect of Mel pretreatment on growth inhibition caused by low-temperature (LT) stress (10 °C/6 °C) in cucumber seedlings and explored the role of the Ca2+/Calcium-dependent protein kinases (CPKs) signaling pathway in Mel-regulated LT tolerance. The main results are as follows: compared to LT treatment alone, 100 µM Mel increased both the content of Ca2+ (highest about 42.01%) and the expression levels of Ca2+ transporter and cyclic nucleotide-gated channel (CNGC) genes under LT. Similarly, Mel enhanced the content of CPKs (highest about 27.49%) and the expression levels of CPKs family genes in cucumber leaves under LT. Additionally, pretreatment with 100 µM Mel for three days strengthened the antioxidant defense and photosynthesis of seedlings under LT. Genes in the ICE-CBF-COR pathway and the MAPK cascade were upregulated by Mel, with maximum upregulations reaching approximately 2.5-fold and 1.9-fold, respectively, thus conferring LT tolerance to cucumber seedlings. However, the above beneficial effects of Mel were weakened by co-treatment with calcium signaling blockers (LaCl3 or EGTA) or CPKs inhibitors (TFP or W-7), suggesting that the Ca2+/CPKs pathway is involved in the Mel-mediated regulation of LT tolerance. In conclusion, this study revealed that Mel can alleviate growth inhibition in cucumber seedlings under LT stress and demonstrated that the Ca2+/CPKs signaling pathway is crucial for the Mel-mediated enhancement of LT tolerance. The findings hold promise for providing theoretical insights into the application of Mel in agricultural production and for investigating its underlying mechanisms of action.

Docosahexaenoic acid supplementation during porcine oocyte in vitro maturation improves oocyte quality and embryonic development by enhancing the homeostasis of energy metabolism.

Although supplementation with docosahexaenoic acid (DHA) during porcine oocyte IVM is well-established, the available data are limited due to the lack of consistency. Moreover, to our knowledge, the anti-oxidant effects of DHA on porcine oocytes have not been reported. Hence, this study aimed to examine the effects of DHA supplementation on the regulation of energy metabolism during porcine oocyte maturation to improve oocyte maturation and embryonic development. By supplementing the IVM medium with various DHA concentrations, 25 µM DHA was identified as the optimal concentration which improved intraoocyte glutathione content and enhanced embryonic development after parthenogenesis. Compared to embryos derived from the control group, those derived from SCNT or IVF showed significantly improved blastocyst formation upon DHA supplementation during IVM. In addition, various transcription factors associated with oocyte development and apoptosis in mature oocytes were beneficially regulated in the DHA-treated oocytes. Moreover, DHA improved the AMP-activated protein kinase (AMPK)-regulatory ability of porcine oocytes and ameliorated nuclear maturation and embryonic development, which were decreased by artificially downregulating AMPK. To our knowledge, this is the first study to examine the effects of DHA as an AMPK regulator on oocyte maturation and embryo development in pigs. Furthermore, DHA addition to the IVM medium upregulated the relative expression of genes associated with mitochondrial potential and lipid metabolism. Therefore, the membrane potential of mitochondria (evaluated based on the JC-1 aggregate/JC-1 monomer ratio) and the levels of fatty acids and lipid droplets in matured oocytes increased, resulting in increased ATP synthesis. In conclusion, the DHA treatment of porcine oocytes with 25 µM DHA during IVM enhances the homeostasis of energy metabolism by improving mitochondrial function and lipid metabolism, leading to improved quality of matured oocytes and enhanced embryonic developmental potential of in vitro produced (IVP) embryos. Thus, 25 µM DHA supplementation could serve as a tool for improving the quality of IVP embryos. The study findings provide a basis for further research on improving the production efficiency of cloned animals by securing high-quality matured oocytes and enhancing energy metabolism in mammalian oocytes, including those of pigs.

Contribution of inwardly rectifying potassium channel 4.1 in orofacial neuropathic pain: Regulation of pannexin 3 via the reactive oxygen species-activated P38 MAPK signal pathway.

The involvement of inwardly rectifying potassium channel 4.1 (Kir4.1) in neuropathic pain has been established. However, there is limited understanding of the downstream mechanism through which Kir4.1 contributes to orofacial neuropathic pain. The objective of this study was to examine the regulation of Kir4.1 on the expression of pannexin 3 (Panx3) in the trigeminal ganglion (TG) and the underlying mechanism in the context of orofacial neuropathic pain caused by chronic constriction injury of the infraorbital nerve (CCI-ION). The study observed a significant increase in Panx3 expression in the TG of mice with CCI-ION. Inhibition of Panx3 in the TG of CCI-ION mice resulted in alleviation of orofacial mechanical allodynia. Furthermore, conditional knockdown (CKD) of Kir4.1 in the TG of both male and female mice led to mechanical allodynia and upregulation of Panx3 expression. Conversely, overexpression of Kir4.1 decreased Panx3 levels in the TG and relieved mechanical allodynia in CCI-ION mice. In addition, silencing Kir4.1 in satellite glial cells (SGCs) decreased Panx3 expression and increased the phosphorylation of P38 MAPK. Moreover, silencing Kir4.1 in SGCs increased the levels of reactive oxygen species (ROS). The elevated phosphorylation of P38 MAPK resulting from Kir4.1 silencing was inhibited by using a superoxide scavenger known as the tempol. Silencing Panx3 in the TG in vivo attenuated the mechanical allodynia caused by Kir4.1 CKD. In conclusion, these findings suggest that the reduction of Kir4.1 promotes the expression of Panx3 by activating the ROS-P38 MAPK signalling pathway, thus contributing to the development of orofacial neuropathic pain.

Regulatory complexity of cellular differentiation in Candida albicans revealed through systematic screening of protein kinase mutants.

A recent study in mBio reports the construction and preliminary screening of a library containing mutants of 99 of the 119 predicted protein kinases in Candida albicans (the majority of the remaining 20 are probably essential) (J. Kramara, M.-J. Kim, T. L. Ollinger, L. C. Ristow, et al., mBio e01249-24, 2024, https://doi.org/10.1128/mbio.01249-24). Using a quantitative competition assay in 10 conditions that represent nutritional, osmotic, cell wall, and pH stresses that are considered to model various aspects of the host environment allowed them to phenotypically cluster kinases, which highlight both the integration and specialization of signaling pathways, suggesting novel functions for many kinases. In addition, they tackle two complex and partially overlapping differentiation events, hyphal morphogenesis and biofilm formation. They find that a remarkable 88% of the viable kinase mutants in C. albicans affect hyphal growth, illustrating how integrated morphogenesis is in the overall biology of this organism, and begin to dissect the regulatory relationships that control this key virulence trait.