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Variants in seven genes (LRRK2, GBA1, PRKN, SNCA, PINK1, PARK7 and VPS35) have been formally adjudicated as causal contributors to Parkinsons disease; however, individuals with Parkinsons disease are often unaware of their genetic status since clinical testing is infrequently offered. As a result, genetic information is not incorporated into clinical care, and variant-targeted precision medicine trials struggle to enrol people with Parkinsons disease. Understanding the yield of genetic testing using an established gene panel in a large, geographically diverse North American population would help patients, clinicians, clinical researchers, laboratories and insurers better understand the importance of genetics in approaching Parkinsons disease. PD GENEration is an ongoing multi-centre, observational study (NCT04057794, NCT04994015) offering genetic testing with results disclosure and genetic counselling to those in the US (including Puerto Rico), Canada and the Dominican Republic, through local clinical sites or remotely through self-enrolment. DNA samples are analysed by next-generation sequencing including deletion/duplication analysis (Fulgent Genetics) with targeted testing of seven major Parkinsons disease-related genes. Variants classified as pathogenic/likely pathogenic/risk variants are disclosed to all tested participants by either neurologists or genetic counsellors. Demographic and clinical features are collected at baseline visits. Between September 2019 and June 2023, the study enrolled 10 510 participants across >85 centres, with 8301 having received results. Participants were: 59% male; 86% White, 2% Asian, 4% Black/African American, 9% Hispanic/Latino; mean age 67.4 ± 10.8 years. Reportable genetic variants were observed in 13% of all participants, including 18% of participants with one or more high risk factors for a genetic aetiology: early onset (

Estimates of the spectrum and frequency of pathogenic variants in Parkinsons disease (PD) in different populations are currently limited and biased. Furthermore, although therapeutic modification of several genetic targets has reached the clinical trial stage, a major obstacle in conducting these trials is that PD patients are largely unaware of their genetic status and, therefore, cannot be recruited. Expanding the number of investigated PD-related genes and including genes related to disorders with overlapping clinical features in large, well-phenotyped PD patient groups is a prerequisite for capturing the full variant spectrum underlying PD and for stratifying and prioritizing patients for gene-targeted clinical trials. The Rostock Parkinsons disease (ROPAD) study is an observational clinical study aiming to determine the frequency and spectrum of genetic variants contributing to PD in a large international cohort. We investigated variants in 50 genes with either an established relevance for PD or possible phenotypic overlap in a group of 12 580 PD patients from 16 countries [62.3% male; 92.0% White; 27.0% positive family history (FH+), median age at onset (AAO) 59 years] using a next-generation sequencing panel. Altogether, in 1864 (14.8%) ROPAD participants (58.1% male; 91.0% White, 35.5% FH+, median AAO 55 years), a PD-relevant genetic test (PDGT) was positive based on GBA1 risk variants (10.4%) or pathogenic/likely pathogenic variants in LRRK2 (2.9%), PRKN (0.9%), SNCA (0.2%) or PINK1 (0.1%) or a combination of two genetic findings in two genes (∼0.2%). Of note, the adjusted positive PDGT fraction, i.e. the fraction of positive PDGTs per country weighted by the fraction of the population of the world that they represent, was 14.5%. Positive PDGTs were identified in 19.9% of patients with an AAO ≤ 50 years, in 19.5% of patients with FH+ and in 26.9% with an AAO ≤ 50 years and FH+. In comparison to the idiopathic PD group (6846 patients with benign variants), the positive PDGT group had a significantly lower AAO (4 years, P = 9 × 10-34). The probability of a positive PDGT decreased by 3% with every additional AAO year (P = 1 × 10-35). Female patients were 22% more likely to have a positive PDGT (P = 3 × 10-4), and for individuals with FH+ this likelihood was 55% higher (P = 1 × 10-14). About 0.8% of the ROPAD participants had positive genetic testing findings in parkinsonism-, dystonia/dyskinesia- or dementia-related genes. In the emerging era of gene-targeted PD clinical trials, our finding that ∼15% of patients harbour potentially actionable genetic variants offers an important prospect to affected individuals and their families and underlines the need for genetic testing in PD patients. Thus, the insights from the ROPAD study allow for data-driven, differential genetic counselling across the spectrum of different AAOs and family histories and promote a possible policy change in the application of genetic testing as a routine part of patient evaluation and care in PD.

Emerged evidence indicated that stimulating hippocampal neurogenesis is a potential strategy for restoring cognition in AD. Mitogen‐activated protein kinases (MAPKs) play an essential role in neurogenesis. Meanwhile, the enzymatic power of the phosphatases is much greater than that of kinases. Dual‐specificity phosphatase 16 (DUSP16), known to as a phosphatase negatively regulate MAPKs, may be implicated in neural differentiation. Nevertheless, the effect of DUSP16 on cognitive disorders by stimulating neural progenitor cell (NPC) differentiation in AD mice remains unclear. Our study demonstrates an association between DUSP16 SNPs and clinical progression in individuals with mild cognitive impairment (MCI). Besides, increased DUSP16 expression was detected in both 3xTg and SAMP8 mouse models of AD, accompanied by NPC neural differentiation impairments. By silencing DUSP16, the induction of neural differentiation, synaptic transmission, and cognitive benefits were observed in both AD mice. Furthermore, DUSP16 was involved in the process of NPC differentiation through regulating c‐Jun N‐terminal kinase (JNK) phosphorylation and SOX2 expression. Moreover, ETS transcription factor (ELK1) was involved in the DUSP16 transcription, which resulted in the upregulation of DUSP16 at the state of AD. Our data uncovers a potential regulatory role for DUSP16 in adult hippocampal neurogenesis (AHN) and provides a possibility to find a novel strategy for AD intervention. [ABSTRACT FROM AUTHOR]

Background and Purpose Experimental Approach Key Results Conclusions and Implications Cannabis stimulates several G‐protein‐coupled‐receptors and causes bradycardia and hypotension upon sustained consumption. Moreover, in vitro studies suggest an interference of cannabinoid‐signalling with cardiomyocyte contractility and hypertrophy. We aimed at revealing a functional contribution of the cannabinoid‐sensitive receptor GPR55 to cardiomyocyte homeostasis and neurohumorally induced hypertrophy in vivo.Gpr55−/− and wild‐type (WT) mice were characterized after 28‐day angiotensin II (AngII; 1·μg·kg−1 min−1) or vehicle infusion. In isolated adult Gpr55−/− and WT cardiomyocytes, mitochondrial function was assessed under naïve conditions, while cytosolic Ca2+ handling was additionally determined following application of the selective GPR55 antagonist CID16020046.Gpr55 deficiency did not affect angiotensin II (AngII) mediated hypertrophic growth, yet, especially in females, it alleviated maladaptive pro‐hypertrophic and ‐inflammatory gene expression and improved inotropy and adrenergic responsiveness compared to WT. In‐depth analyses implied increased cytosolic Ca2+ concentrations and transient amplitudes, and accelerated sarcomere contraction kinetics in Gpr55−/− myocytes, which could be mimicked by GPR55 blockade with CID16020046 in female WT cells. Moreover, Gpr55 deficiency up‐regulated factors involved in glucose and fatty acid transport independent of the AngII challenge, accelerated basal mitochondrial respiration and reduced basal protein kinase (PK) A, G and C activity and phospholemman (PLM) phosphorylation.Our study suggests GPR55 as crucial regulator of cardiomyocyte hypertrophy and homeostasis presumably by regulating PKC/PKA‐PLM and PKG signalling, and identifies the receptor as potential target to counteract maladaptation, adrenergic desensitization and metabolic shifts as unfavourable features of the hypertrophied heart in females. [ABSTRACT FROM AUTHOR]

Protein kinases are critical components of a myriad biological processes and strongly associated with various diseases. While kinase research has been a point of focus in biomedical research for several decades, a large portion of the kinome is still considered understudied or "dark," because prior research is targeted towards a subset of kinases with well-established roles in cellular processes. We present an empirical and in-silico hybrid workflow to extend the functional knowledge of understudied kinases. Utilizing multiplex peptide activity arrays and robust in-silico analyses, we extended the functional knowledge of five dark tyrosine kinases (AATK, EPHA6, INSRR, LTK, TNK1) and explored their roles in schizophrenia, Alzheimer's dementia (AD), and major depressive disorder (MDD). Using this hybrid approach, we identified 195 novel kinase-substrate interactions with variable degrees of affinity and linked extended functional networks for these kinases to biological processes that are impaired in psychiatric and neurological disorders. Biochemical assays and mass spectrometry were used to confirm a putative substrate of EPHA6, an understudied dark tyrosine kinase. We examined the EPHA6 network and knowledgebase in schizophrenia using reporter peptides identified and validated from the multi-plex array with high affinity for phosphorylation by EPHA6. Identification and confirmation of putative substrates for understudied kinases provides a wealth of actionable information for the development of new drug treatments as well as exploration of the pathophysiology of disease states using signaling network approaches. [ABSTRACT FROM AUTHOR]

UNC-89 is a giant sarcomeric M-line protein required for sarcomere organization and optimal muscle function. UNC-89 contains two protein kinase domains, PK1 and PK2, separated by an elastic region. Here we show that PK2 is a canonical kinase expected to be catalytically active. C. elegans expressing UNC-89 with a lysine to alanine (KtoA) mutation to inactivate PK2 have normally organized sarcomeres and SR, and normal muscle function. PK2 KtoA mutants have fragmented mitochondria, correlated with more mitochondrially-associated DRP-1. PK2 KtoA mutants have increased ATP levels, increased glycolysis and altered levels of electron transport chain complexes. Muscle mitochondria show increased complex I and decreased complex II basal respiration, each of which cannot be uncoupled. This suggests that mutant mitochondria are already uncoupled, possibly resulting from an increased level of the uncoupling protein, UCP-4. Our results suggest signaling from sarcomeres to mitochondria, to help match energy requirements with energy production. UNC-89, a giant protein localized to muscle sarcomeres, contains 2 protein kinase domains, one of which, PK2 is a canonical kinase. A missense mutation expected to inactivate PK2 results in worms with an unexpected phenotype suggesting signaling from sarcomeres to mitochondria. [ABSTRACT FROM AUTHOR]

ABSTRACT Fermented and aged mountain‐cultivated ginseng sprouts (FAMCGS) exhibit superior antioxidant and anti‐inflammatory properties compared to mountain‐cultivated ginseng sprouts (MCGS). However, the mechanisms behind these properties of FAMCGSE remain unclear. This study explores the anti‐inflammatory effects of FAMCGS extract (FAMCGSE) on LPS‐stimulated RAW 264.7 macrophages and the underlying mechanisms. The MTT assay confirmed that FAMCGSE (0 to 0.1%) maintained cell viability without inducing morphological changes. Pretreatment with FAMCGSE significantly mitigated LPS‐induced morphological alterations dose‐dependently. RT‐PCR and Western blot analyses showed that FAMCGSE significantly reduced the mRNA and protein levels of proinflammatory mediators such as TNF‐α, IL‐1β, IL‐6, iNOS, and COX‐2. Additionally, FAMCGSE decreased the production of TNF‐α, IL‐1β, IL‐6, nitric oxide, and PGE2 in LPS‐activated RAW264.7 cells. Mechanistically, FAMCGSE inhibited the phosphorylation of mitogen‐activated protein kinases (MAPKs; ERK, p38, and JNK) and prevented the LPS‐induced nuclear translocation of NF‐κB, with effects comparable to compound K (CK) or dexamethasone. Notably, FAMCGSE was particularly effective in inhibiting ERK and JNK activation, with less impact on p38, suggesting a specific inhibitory action on certain MAPK pathways. These findings highlight FAMCGSE's potential as an inhibitor of MAPK and NF‐κB pathways, indicating that FAMCGSE, including its main component CK, may be a promising therapeutic agent for inflammation‐related conditions. [ABSTRACT FROM AUTHOR]

Fungal pathogens must exhibit strong nutritional plasticity, effectively sensing and utilizing diverse nutrients to support virulence. How the signals generated by nutritional sensing are efficiently translated to the morphogenetic machinery for optimal growth and support of virulence remains incompletely understood. Here, we show that the conserved morphogenesis-related kinase, CotA, imparts isoform-specific control over Aspergillus fumigatus invasive growth in host-mimicking environments and during infection. CotA-mediated invasive growth is responsive to exogenous carbon source quality, with only preferred carbon sources supporting hyphal morphogenesis in a mutant lacking one of two identified protein isoforms. Strikingly, we find that the CotA protein does not regulate, nor is cotA gene expression regulated by, the carbon catabolite repression system. Instead, we show that CotA partially mediates invasive growth in specific carbon sources and virulence through the conserved downstream effector and translational repressor, SsdA. Therefore, A. fumigatus CotA accomplishes its conserved morphogenetic functions to drive pathogenic growth by translating host-relevant carbon source quality signals into morphogenetic outputs for efficient tissue invasive growth. Here, Martin-Vicente et al. show that the Aspergillus fumigatus protein kinase, CotA, controls invasive growth in an isoform dependent manner in response to carbon source diversity, partially mediated by the downstream effector, SsdA. [ABSTRACT FROM AUTHOR]

A delicate balance between neural stem cell (NSC) quiescence and proliferation is important for adult neurogenesis and homeostasis. Small ubiquitin-related modifier (SUMO)-dependent post-translational modifications cause rapid and reversible changes in protein functions. However, the role of the SUMO pathway during NSC reactivation and brain development is not established. Here, we show that the key components of the SUMO pathway play an important role in NSC reactivation and brain development in Drosophila. Depletion of SUMO/Smt3 or SUMO conjugating enzyme Ubc9 results in notable defects in NSC reactivation and brain development, while their overexpression leads to premature NSC reactivation. Smt3 protein levels increase with NSC reactivation, which is promoted by the Ser/Thr kinase Akt. Warts/Lats, the core protein kinase of the Hippo pathway, can undergo SUMO- and Ubc9-dependent SUMOylation at Lys766. This modification attenuates Wts phosphorylation by Hippo, leading to the inhibition of the Hippo pathway, and consequently, initiation of NSC reactivation. Moreover, inhibiting Hippo pathway effectively restores the NSC reactivation defects induced by SUMO pathway inhibition. Overall, our study uncovered an important role for the SUMO-Hippo pathway during Drosophila NSC reactivation and brain development. The balance of neural stem cell quiescence and proliferation is key for development and homeostasis, though how this balance is regulated is not fully understood. Here they show that SUMOylation of Wts, a core Hippo pathway kinase, results in its inactivation and induces NSC reactivation in Drosophila. [ABSTRACT FROM AUTHOR]

During exercise or stress, the sympathetic system stimulates cardiac contractility via β‐adrenergic receptor (β‐AR) activation, resulting in phosphorylation of the cardiac ryanodine receptor (RyR2). Three RyR2 phosphorylation sites have taken prominence in excitation–contraction coupling: S2808 and S2030 are described as protein kinase A specific and S2814 as a Ca2+/calmodulin kinase type‐2‐specific site. To examine the contribution of these phosphosites to Ca2+ signalling, we generated double knock‐in (DKI) mice in which Ser2808 and Ser2814 phosphorylation sites have both been replaced by alanine (RyR2‐S2808A/S2814A). These mice did not exhibit an overt phenotype. Heart morphology and haemodynamic parameters were not altered. However, they had a higher susceptibility to arrhythmias. We performed confocal Ca2+ imaging and electrophysiology experiments. Isoprenaline was used to stimulate β‐ARs. Measurements of Ca2+ waves and latencies in myocytes revealed an increased propensity for spontaneous Ca2+ releases in DKI myocytes, both in control conditions and during β‐AR stimulation. In DKI cells, waves were initiated from a lower threshold concentration of Ca2+ inside the sarcoplasmic reticulum, suggesting higher Ca2+ sensitivity of the RyRs. The refractoriness of Ca2+ spark triggering depends on the Ca2+ sensitivity of the RyR2. We found that RyR2‐S2808A/S2814A channels were more Ca2+ sensitive in control conditions. Isoprenaline further shortened RyR refractoriness in DKI cardiomyocytes. Together, our results suggest that ablation of both the RyR2‐Ser2808 and RyR2‐S2814 sites increases the propensity for pro‐arrhythmic spontaneous Ca2+ releases, as previously suggested for hyperphosphorylated RyRs. Given that the DKI cells present a full response to isoprenaline, the data suggest that phosphorylation of Ser2030 might be sufficient for β‐AR‐mediated sensitization of RyRs. Key points: Phosphorylation of cardiac sarcoplasmic reticulum Ca2+‐release channels (ryanodine receptors, RyRs) is involved in the regulation of cardiac function.Ablation of both the RyR2‐Ser2808 and RyR2‐Ser2814 sites increases the propensity for pro‐arrhythmic spontaneous Ca2+ releases, as previously suggested for hyperphosphorylated RyRs.The intra‐sarcoplasmic reticulum Ca2+ threshold for spontaneous Ca2+ wave generation is lower in RyR2‐double‐knock‐in cells.The RyR2 from double‐knock‐in cells exhibits increased Ca2+ sensitivity.Phosphorylation of Ser2808 and Ser2814 might be important for basal activity of the channel.Phosphorylation of Ser2030 might be sufficient for a β‐adrenergic response. [ABSTRACT FROM AUTHOR]

Background: Cold is a significant limiting factor in productivity, particularly in northwestern and eastern China. Calcium-Dependent Protein Kinases (CDPKs), a primary calcium signaling sensor in plants, play an important role in their response to cold. Snow lotus (Sasussured involucrata Kar L) is a plant that thrives in harsh climates and grows in northwest China. However, there were no reports on the transcriptome of OE-SikCDPK1 transgenic tobacco in response to cold. Results: When exposed to cold stress, OE-SikCDPK1 plants displayed a cold-tolerant phenotype compared to non-transgenic tobacco. Under cold conditions, relative water content reduced, relative conductivity increased, malondialdehyde levels rose, and cold-responsive gene expression increased. The OE-SikCDPK1 gene and non-transgenic tobacco were employed for research purposes. The transcriptome of leaves was sequenced using the HISAT2 sequencing platform, and the data were used to examine gene function annotation and differentially expressed genes (DEGs). 53,022 DEGs in tobacco leaves under cold treatment were obtained. The GO enrichment results revealed that it was enriched for biological-process, defense response and other processes under cold stress. The KEGG pathway enrichment analysis revealed that the metabolic pathways of significant enrichment of DEGs under cold stress mainly involved MAPK signaling pathway transduction. The transcription factor identification results showed that the transcription factors with the largest number of differential expressions under cold treatment were mainly from WRKY, AP2, MYB, bHLH, NAC and other transcription factor families. Conclusion: The cold tolerance mechanism of snow lotus SikCDPK1 was comprehensively analyzed at the transcriptional level for the first time using RNA-seq technology. This study demonstrates that SikCDPK1 can respond to cold by participating in the MAPK signaling pathway and regulating the expression levels of transcription factors, including WRKY, AP2, MYB, bHLH, and NAC. These results offer valuable insights for further exploration of the cold tolerance mechanism associated with SikCDPK1. [ABSTRACT FROM AUTHOR]

The fidelity of signal transduction requires the binding of regulatory molecules to their cognate targets. However, the crowded cell interior risks off-target interactions between proteins that are functionally unrelated. How such off-target interactions impact fitness is not generally known. Here, we use Saccharomyces cerevisiae to inducibly express tyrosine kinases. Because yeast lacks bona fide tyrosine kinases, the resulting tyrosine phosphorylation is biologically spurious. We engineered 44 yeast strains each expressing a tyrosine kinase, and quantitatively analysed their phosphoproteomes. This analysis resulted in ~30,000 phosphosites mapping to ~3500 proteins. The number of spurious pY sites generated correlates strongly with decreased growth, and we predict over 1000 pY events to be deleterious. However, we also find that many of the spurious pY sites have a negligible effect on fitness, possibly because of their low stoichiometry. This result is consistent with our evolutionary analyses demonstrating a lack of phosphotyrosine counter-selection in species with tyrosine kinases. Our results suggest that, alongside the risk for toxicity, the cell can tolerate a large degree of non-functional crosstalk as interaction networks evolve. Synopsis: How do off-target interactions between proteins inside crowded cells affect cellular fitness? Expression of human tyrosine kinases in yeast, which lacks general tyrosine phosphorylation, shows that many individual spurious pY sites have no impact, but also a strong negative correlation between phosphorylation and fitness. 20% of pY sites are predicted to destabilise the substrate. Native pS/pT signalling in yeast is likely dysregulated. Many Y sites are likely phosphorylated at low stoichiometry. Spurious phosphorylation is widespread and not restricted to homologs of native substrates. Evolutionary selection to avoid spurious Y phosphorylation appears unlikely. Expression of human tyrosine kinases in yeast shows that while there is a strong negative correlation between phosphorylation and fitness, many individual pY sites have negligible impact. [ABSTRACT FROM AUTHOR]

AbstractBackgroundObjectiveMaterials and methodsResultsConclusionsAzithromycin is an antibacterial and anti-inflammatory drug widely used for the treatment of various diseases, including those caused by atypical pathogens, bacterial or viral infections, chronic sinusitis, and bronchial asthma, particularly in pediatric patients. However, concerns have emerged regarding its hepatotoxicity and its precise mechanism of action remains unclear.To investigate the molecular mechanisms responsible for azithromycin-induced acute liver injury to advance our understanding of the progression and pathogenesis of antibiotic-induced liver damage, and to improve prevention and treatment strategies.C57BL/6 mice, Nrf2-/- mice, and primary hepatocytes were used. Primary hepatocytes from mice were isolated using a two-step perfusion method and cultured in vitro via the ‘sandwich’ culture model.The exposure to azithromycin resulted in increased apoptosis and reactive oxygen species (ROS) levels. In mouse models, intraperitoneal administration of azithromycin at varying concentrations and time points substantially induced hepatic disarray, swelling, and dysfunction. Azithromycin markedly upregulated the mRNA and protein levels of phosphorylated adenosine-activated protein kinase (AMPK) while downregulating nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), and NADPH: quinone oxidoreductase 1 (NQO-1). Moreover, HO-1 and NQO-1 protein levels remained largely unaffected in primary hepatocytes co-cultured with azithromycin in Nrf2-/- mice.Our findings suggest that azithromycin-induced acute liver injury is mediated by suppression of Nrf2 activation and ROS production. This sheds light on the potential mechanisms involved in azithromycin-induced liver damage, underscoring the importance of exploring targeted interventions to mitigate the hepatotoxic effects. [ABSTRACT FROM AUTHOR]

SUMMARY Gametocidal (Gc) chromosomes have been widely utilized in genetic breeding due to their ability to induce chromosomal breakage and eliminate gametes that lack them. Long noncoding RNAs (lncRNAs) have various functional mechanisms in regulating pollen and anther development; however, their regulatory contributions to Gc action are still unknown. Here, we identified 2824 differentially expressed lncRNAs (DE‐lncRNAs) from the anther tissues of Triticum aestivum cv. Chinese Spring (CS) and Chinese Spring‐Gc 3C chromosome monosomic addition line (CS‐3C) through sequencing. In this study, we predicted 161 target mRNAs for 145 DE‐lncRNAs, including 104 cis‐regulatory, 60 trans‐regulatory, and three both cis‐regulatory and trans‐regulatory manner. Combined with our previous miRNA sequencing data, 241 DE‐lncRNAs functioned as potential endogenous target mimics (eTMs) for 84 differentially expressed microRNAs (DE‐miRNAs, including 12 novel miRNAs). The results of transient transformation in tobacco leaves indicated that L006278 could bind to MTCONS_00006277, which encoded a calcineurin CBL‐interacting protein kinase 19‐like, and suppress its expression. Furthermore, L117735 could function as an eTM for tae‐miR9657b‐3p, and L056972 could function as an eTM for gc‐m2240‐5p. To explore the function of lncRNAs in the process of Gc action, we transformed L006278, an up‐regulated lncRNA in CS‐3C, into rice to analyze its effect on pollen fertility. Overexpression of L006278 led to a reduction in rice pollen fertility. Overall, our findings indicate that lncRNAs can contribute to the regulation of pollen fertility during the process of Gc action by regulating the expression levels of target mRNAs and acting as eTMs for certain key miRNAs. [ABSTRACT FROM AUTHOR]

The protein kinases DYRK1A and DYRK1B are pivotal regulators of cell cycle progression by promoting cell cycle exit into quiescence. DYRK1B appears to play a more important role in cancer cell quiescence than DYRK1A, as evidenced by its overexpression or copy number variations in human tumour samples. Nonetheless, the stimuli driving DYRK1B upregulation and the potential divergence in expression patterns between DYRK1A and DYRK1B remain largely elusive. In the present study, we scrutinized the regulatory pathways modulating DYRK1B expression relative to DYRK1A in PANC-1 and A549 cancer cell lines across varying conditions. Serum deprivation, pharmacological mTOR inhibition and increased cell density resulted in the differential upregulation of DYRK1B compared to DYRK1A. We then aimed to assess the role of protein kinases MST1 and MST2, which are key transmitters of cell density dependent effects. Unexpectedly, exposure to the MST1/2 inhibitor XMU-MP-1 resulted in increased DYRK1B levels in A549 cells. Further investigation into the off-target effects of XMU-MP-1 unveiled the inhibition of Aurora kinases (AURKA and AURKB) as a potential causative factor. Consistently, AURK inhibitors VX-680 (tozasertib), MLN8237 (alisertib), AZD1152-HQPA (barasertib) resulted in the upregulation of DYRK1B expression in A549 cells. In summary, our findings indicate that the expression of DYRK1A and DYRK1B is differentially regulated in cancer cells and reveal that the kinase inhibitor XMU-MP-1 increases DYRK1B expression likely through off target inhibition of Aurora kinases. [ABSTRACT FROM AUTHOR]

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The RASopathies, collectively, are a spectrum of genetic syndromes caused by mutations in genes involved in the RAS/mitogen-activated protein kinase (MAPK) pathway, including but not limited to PTPN11, NRAS, KRAS, HRAS, BRAF, and MAP2K1. Recognized RASopathy conditions include neurofibromatosis type 1 (NF1), Noonan syndrome, capillary malformation-arteriovenous malformation syndrome, Costello syndrome, cardiofacio-cutaneous (CFC) syndrome, LEOPARD syndrome and Legius syndrome. The RASopathies often display overlapping clinical features, presumably owing to common RAS-MAPK signaling pathway activation driving dysregulated cell proliferation. Epidermal nevus syndromes (ENS) are described as the presence of epidermal nevi, in individuals also affected by extra-cutaneous organ system involvement, and there is recent recognition of mosaic RAS mutations as molecular drivers of ENS. Currently, no curative treatments exist for RASopathy driven conditions, but rather symptom-directed management is the currently accepted standard. Here, we detail a unique case of a child exhibiting diffuse spinal nerve root hypertrophy in the context of epidermal nevus syndrome driven by molecularly confirmed KRAS G12D mosaicism, treated with the MEK 1/2 inhibitor selumetinib. Herein, we report the response of this patient to targeted therapy of more than two years' duration, including stabilization of multilevel nerve root hypertrophy as well as significant improvement in epidermal nevi. While the effectiveness of MEK inhibitors such as selumetinib is established in NF1-associated inoperable plexiform neurofibromas, their use in managing hyperactive KRAS-driven epidermal nevi and hypertrophic neuropathy remains unproven, and this case, to our knowledge, is the first such case to be reported. Shared molecular dysregulation and overlapping clinical features between these conditions suggest potential for effective therapeutic application of MEK directed therapy to address a range of conditions resulting from germline and/or mosaic expression of aberrantly regulated RAS signaling. [ABSTRACT FROM AUTHOR]

Calcium-Dependent Protein Kinases (CDPKs) are a class of serine/threonine protein kinases encoded by several gene families that play key roles in biotic and abiotic stresses response and plant growth and development. However, snow lotus (Saussurea involucrata kar L.) CDPKs has rarely been reported. In this study, 20 CDPK genes in snow lotus were identified based on transcriptome data and classified into four groups (I-IV) based on their structural features and phylogenetic analyses. Among them, the transcript levels of SikCDPK1 were significantly induced by low temperature and multiple hormone treatments, and SikCDPK1 gene was found to have different expression in snow lotus seeds, leaves, stems and roots. The full-length promoter activity of SikCDPK1 gene was higher than that of the 5’ end deletion fragment, and the promoter fragment containing the low temperature inducing element had increased activation after low temperature treatment. The promoter activity of SikCDPK1 gene was mainly expressed in roots and rosette leaves. In addition, overexpressing plants of SikCDPK1 were more tolerant compared to the wild type after being subjected to low temperature stress. Physiological analyses indicated that SikCDPK1 improved plant tolerance to low temperature stress by maintaining cell membrane stability and reducing the accumulation of reactive oxygen species (ROS). These findings provided insights into CDPK gene families in snow lotus and broaden our understanding of the biological role of SikCDPK1 and the mechanism of low temperature stress tolerance in snow lotus. [ABSTRACT FROM AUTHOR]

The Aurora Kinases (AURKs) are a family of serine-threonine protein kinases critical for cell division. Somatic cells express only AURKA and AURKB. However, mammalian germ cells and some cancer cells express all three isoforms. A major question in the field has been determining the molecular and cellular changes when cells express three instead of two aurora kinases. Using a systematic genetic approach involving different Aurora kinase oocyte-specific knockout combinations, we completed an oocyte-AURK genetic interaction map and show that one genomic copy of Aurka is necessary and sufficient to support female fertility and oocyte meiosis. We further confirm that AURKB and AURKC alone cannot compensate for AURKA. These results highlight the importance of AURKA in mouse oocytes, demonstrating that it is required for spindle formation and proper chromosome segregation. Surprisingly, a percentage of oocytes that lack AURKB can complete meiosis I, but the quality of those eggs is compromised, suggesting a role in AURKB to regulate spindle assembly checkpoint or control the cell cycle. Together with our previous studies, we wholly define the genetic interplay among the Aurora kinases and reinforce the importance of AURKA expression in oocyte meiosis. [ABSTRACT FROM AUTHOR]

Cancer, the world’s second leading cause of death after cardiovascular diseases, is characterized by hallmarks such as uncontrolled cell growth, metastasis, angiogenesis, hypoxia, and resistance to therapy. Autophagy, a cellular process that can both support and inhibit cancer progression, plays a critical role in cancer development and progression. This process involves the formation of autophagosomes that ultimately fuse with lysosomes to degrade cellular components. A key regulator of this process is Sirtuin 1 (SIRT1), which significantly influences autophagy. This review delves into the role of SIRT1 in modulating autophagy and its broader impacts on carcinogenesis. SIRT1 regulates crucial autophagy mediators, such as AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR), effectively promoting or suppressing autophagy. Beyond its direct effects on autophagy, SIRT1’s regulatory actions extend to other cell death processes, including apoptosis and ferroptosis, thereby influencing tumor cell proliferation, metastasis, and chemotherapy responses. These insights underscore the complex interplay between SIRT1 and autophagy, with significant implications for cancer therapy. Targeting SIRT1 and its associated pathways presents a promising strategy to manipulate autophagy in cancer treatment. This review underscores the potential of SIRT1 as a therapeutic target, opening new avenues for enhancing cancer treatment efficacy. [ABSTRACT FROM AUTHOR]

Background: Linarine is a natural chemical component widely found in Buddleja officinalis Maxim., Chrysanthemum indicum L., Mentha canadensis L., and other medicinal plants. Modern pharmacological studies have shown that linarine with good anti-inflammatory and antioxidant activities can inhibit the proliferation and induce apoptosis of many kinds of tumor cells. Moreover, linarine showed protective effect on the liver, kidneys, and other organs. Methods: Inflammation model of human corneal epithelial cell (HCEC) was constructed using NaCl induction, and cytotoxicity was detected by the CCK8 assay. The levels of inflammatory factors tumor necrosis factor-a (TNF-a) and interleukin 1b (IL-1b) were measured using Enzyme-linked immunoassay (ELISA). Chronic painful stimulation (tail clamping) in combination with Benzalkonium Chloride Solution drops in a desiccator established a mouse model of dry eye disease (DED). The following parameters were recorded: body mass, anal temperature, tear secretion, tear film rupture time, and corneal fluorescein staining. The levels of inflammatory factors mitogen activated protein kinase (MAPK), nuclear factor kappa-B (NF-kB), c-Jun N-terminal kinase (JNK), IL-1b, Interleukin 18(IL-18), A2A, A3, P2X4, P2X7, P2Y1 were measured by using immunofluorescence (IF) staining. Results: Linarine can inhibit the secreation of TNF-a, and IL-1b in HCECs. Linarine prolonged tear film rupture time, promoted tear secretion, repaired corneal damage, and reduced the levels of inflammatory factors of MAPK, NF-kB, JNK, IL-1b, IL-18, and modulated the levels of the purinergic receptor. Conclusions: Linarine is effective in treating dry eye in mice by inhibiting purinergic receptors-mediated inflammatory response. [ABSTRACT FROM AUTHOR]

Common single-nucleotide variants (SNVs) of eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2AK3) slightly increase the risk of disorders in the periphery and the central nervous system. EIF2AK3 encodes protein kinase RNA-like endoplasmic reticulum kinase (PERK), a key regulator of ER stress. Three exonic EIF2AK3 SNVs form the PERK-B haplotype, which is present in 28% of the global population. Importantly, the precise impact of these SNVs on PERK activity remains elusive. In this study, we demonstrate that PERK-B SNVs do not alter PERK expression or basal activity in vitro and in the novel triple knock-in mice expressing the exonic PERK-B SNVs in vivo. However, the kinase activity of PERK-B protein is higher than that of PERK-A in a cell-free assay and in mouse liver homogenates. Pancreatic tissue in PERK-B/B mice also exhibit increased susceptibility to apoptosis under acute ER stress. Monocyte-derived macrophages from PERK-B/B mice exhibit higher PERK activity than those from PERK-A/A mice, albeit with minimal functional consequences at acute timepoints. The subtle PERK-B-driven effects observed in liver and pancreas during acute stress implicate PERK as a contributor to disease susceptibility. The novel PERK-B mouse model provides valuable insights into ER stress-induced PERK activity, aiding the understanding of the genetic basis of disorders associated with ER stress. [ABSTRACT FROM AUTHOR]

Non‐small cell lung cancer comprises up to 85% of lung cancer cases and has a poor prognosis. At present, there are still no effective treatments for this illness. Evidence suggests that the prostaglandin [cyclooxygenase‐2 (COX‐2)] and leukotriene [lipoxygenase‐5 (5‐LOX)] pathways are involved in lung cancer carcinogenesis. Therefore, novel agents that target COX‐2 and 5‐LOX may have therapeutic potential. In the present study, we examined the role of asiatic acid (AA), a triterpenoid saponin, in targeting the protein kinases responsible for lung cancer proliferation and mobility. The experimental data revealed that AA inhibited the growth of lung cancer cells (> 50%) and it significantly impeded the proliferation of lung cancer cells by inhibiting COX‐2, which results in downregulation of the phosphotidyl inositol‐3 kinase/protein kinase B/mammalian target of rapamycin signaling pathway, leading to an induction of cytotoxic autophagy‐mediated apoptosis. Mechanistically, the expression of mitogen‐activated protein kinase/extracellular signal‐regulated kinase, hypoxia‐inducible factor‐1 and vascular endothelial growth factor is downregulated by AA, thereby reducing cell mobility and invasion. It also shows negative osmotic fragility on healthy human erythrocytes. It is concluded that AA may be a viable therapeutic drug for non‐small cell lung cancer treatment, which opens new opportunities for synthesizing analogues. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a debilitating progressively neurodegenerative disease. The best-characterized hallmark of AD, which is marked by behavioral alterations and cognitive deficits, is the aggregation of deposition of amyloid-beta (Aβ) and hyper-phosphorylated microtubule-associated protein Tau. Despite decades of experimental progress, the control rate of AD remains poor, and more precise deciphering is needed for potential therapeutic targets and signaling pathways involved. In recent years, phosphoinositide 3-kinase (PI3K) and Akt have been recognized for their role in the neuroprotective effect of various agents, and glycogen synthase kinase 3 (GSK3), a downstream enzyme, is also crucial in the tau phosphorylation and Aβ deposition. An overview of the function of PI3K/Akt pathway in the pathophysiology of AD is provided in this review, along with a discussion of recent developments in the pharmaceuticals and herbal remedies that target the PI3K/Akt signaling pathway. In conclusion, despite the challenges and hurdles, cumulative findings of novel targets and agents in the PI3K/Akt signaling axis are expected to hold promise for advancing AD prevention and treatment. [ABSTRACT FROM AUTHOR]

Casein kinase 1δ (CK1δ) controls essential biological processes including circadian rhythms and wingless-related integration site (Wnt) signaling, but how its activity is regulated is not well understood. CK1δ is inhibited by autophosphorylation of its intrinsically disordered C-terminal tail. Two CK1 splice variants, δ1 and δ2, are known to have very different effects on circadian rhythms. These variants differ only in the last 16 residues of the tail, referred to as the extreme C termini (XCT), but with marked changes in potential phosphorylation sites. Here, we test whether the XCT of these variants have different effects in autoinhibition of the kinase. Using NMR and hydrogen/deuterium exchange mass spectrometry, we show that the δ1 XCT is preferentially phosphorylated by the kinase and the δ1 tail makes more extensive interactions across the kinase domain. Mutation of δ1-specific XCT phosphorylation sites increases kinase activity both in vitro and in cells and leads to changes in the circadian period, similar to what is reported in vivo. Mechanistically, loss of the phosphorylation sites in XCT disrupts tail interaction with the kinase domain. δ1 autoinhibition relies on conserved anion-binding sites around the CK1 active site, demonstrating a common mode of product inhibition of CK1δ. These findings demonstrate how a phosphorylation cycle controls the activity of this essential kinase. [ABSTRACT FROM AUTHOR]

Mitochondria undergo fragmentation in response to bioenergetic stress, mediated by dynamin-related protein 1 (DRP1) recruitment to the mitochondria. The major pro-fission DRP1 receptor is mitochondrial fission factor (MFF), and mitochondrial dynamics proteins of 49 and 51 kilodaltons (MiD49/51), which can sequester inactive DRP1. Together, they form a trimeric DRP1-MiD-MFF complex. Adenosine monophosphate-activated protein kinase (AMPK)-mediated phosphorylation of MFF is necessary for mitochondrial fragmentation, but the molecular mechanisms are unclear. Here, we identify MFF as a target of small ubiquitin-like modifier (SUMO) at Lys151, MFF SUMOylation is enhanced following AMPK-mediated phosphorylation and that MFF SUMOylation regulates the level of MiD binding to MFF. The mitochondrial stressor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) promotes MFF SUMOylation and mitochondrial fragmentation. However, CCCP-induced fragmentation is impaired in MFF-knockout mouse embryonic fibroblasts expressing non-SUMOylatable MFF K151R. These data suggest that the AMPK-MFF SUMOylation axis dynamically controls stress-induced mitochondrial fragmentation by regulating the levels of MiD in trimeric fission complexes. [ABSTRACT FROM AUTHOR]

PIM1, the proviral integration site for Moloney murine leukemia virus, is a member of the serine/threonine protein kinase family. It is involved in many biological events, such as cell survival, cell cycle progression, cell proliferation, and cell migration, and has been widely studied in malignant diseases. However, recent studies have shown that PIM1 plays a prominent role in immunoinflammatory diseases, including autoimmune uveitis, inflammatory bowel disease, asthma, and rheumatoid arthritis. PIM1 can function in inflammatory signal transduction by phosphorylating multiple inflammatory protein substrates and mediating macrophage activation and T lymphocyte cell specification, thus participating in the development of multiple immunoinflammatory diseases. Moreover, the inhibition of PIM1 has been demonstrated to ameliorate certain immunoinflammatory disorders. Based on these studies, we suggest PIM1 as a potential therapeutic target for immunoinflammatory diseases and a valid candidate for future research. Herein, for the first time, we provide a detailed review that focuses on the roles of PIM1 in the pathogenesis of immunoinflammatory diseases. [ABSTRACT FROM AUTHOR]

Tetrapod endogenous retroelements (ERE) encode proteins that have been exapted to perform many roles in development and also in innate immunity, including GAG (group specific antigen) proteins from the ERE long terminal repeat (LTR) family, some of which can assemble into viral-like capsids (VLCs) and transmit mRNA across synapses. The best characterized member of this family is ARC (activity-regulated cytoskeletal gene), that is involved in memory formation. Other types of EREs, such as LINES and SINES (long and short interspersed elements), have instead been exapted for immune defenses against infectious agents. These immune EREs identify host transcripts by forming the unusual left-handed Z-DNA and Z-RNA conformations to enable self/nonself discrimination. Elevated levels of immune EREs in the brain are associated with neurodegenerative disease. Here I address the question of how pathways based on immune EREs are relate to the memory EREs that mediate neural plasticity. I propose that during infection and in other inflammatory states, ERE encoded GAG capsids deliver interferon-induced immune EREs that rapidly inhibit translation of viral RNAs in the dendritic splines by activation of protein kinase R (PKR). The response limits transmission of viruses and autonomously replicating elements, while protecting bystander cells from stress-induced cell death. Further, the PKR-dependent phosphorylation of proteins, like tau, disrupts the endocytic pathways exploited by viruses to spread to other cells. The responses come at a cost. They impair memory formation and can contribute to pathology by increasing the deposition of amyloid beta. [ABSTRACT FROM AUTHOR]

Salinization poses a significant challenge in agriculture, exacerbated by anthropogenic global warming. Biostimulants, derived from living microorganisms or natural extracts, have emerged as valuable tools for conventional and organic agriculture. However, our understanding of the molecular mechanisms underlying the effects of biostimulants is very limited, especially in crops under real cultivation conditions. In this study, we adopted an integrative approach to investigate the effectiveness of the combined application of plant growth‐promoting bacterium (Bacillus megaterium strain BM08) and a non‐microbial biostimulant under control conditions (normal watering) and salt stress. After confirming the yield increase under both conditions, we investigated the molecular mechanisms underlying the observed effect by measuring a number of physiological parameters (i.e., lipid peroxidation, antioxidants, chlorophylls, total phenolics and phytohormone content), as well as RNA sequencing and primary metabolite analyses. Our findings reveal that the combined effect of the microbial and non‐microbial biostimulants led to a decrease in the antioxidant response and an up‐regulation of genes involved in cytokinin biosynthesis under salt stress conditions. This, in turn, resulted in a higher concentration of the bioactive cytokinin, isopentenyladenosine, in roots and leaves and an increase in γ‐aminobutyric acid, a non‐proteic amino acid related to abiotic stress responses. In addition, we observed a decrease in malic acid, along with an abscisic acid (ABA)‐independent up‐regulation of SR‐kinases, a family of protein kinases associated with abiotic stress responses. Furthermore, we observed that the single application of the non‐microbial biostimulant triggers an ABA‐dependent response under salt stress; however, when combined with the microbial biostimulant, it potentiated the mechanisms triggered by the BM08 bacterial strain. This comprehensive investigation shows that the combination of two biostimulants is able to elicit a cytokinin‐dependent response that may explain the observed yield increase under salt stress conditions. [ABSTRACT FROM AUTHOR]

Protein S‐acylation or palmitoylation is a reversible post‐translational modification that influences many proteins encoded in plant genomes. Exciting progress in the past 3 years demonstrates that S‐acylation modulates subcellular localization, interacting profiles, activity, or turnover of substrate proteins in plants, participating in developmental processes and responses to abiotic or biotic stresses. In this review, we summarize and discuss the role of S‐acylation in the targeting of substrate proteins. We highlight complex roles of S‐acylation in receptor signaling. We also point out that feedbacks of protein S‐acyl transferase by signaling initiated from their substrate proteins may be a recurring theme. Finally, the reversibility of S‐acylation makes it a rapid and efficient way to respond to environmental cues. Future efforts on exploring these important aspects of S‐acylation will give a better understanding of how plants enhance their fitness under ever changing and often harsh environments. [ABSTRACT FROM AUTHOR]

A novel electrochemiluminescence (ECL) method was developed for determination of protein kinase A (PKA) ultra-sensitively based on amidated nano-titanium (NH2-TiO2) embellished carbon dots (Mg@N-CDs) fluorescent probe, which integrated the target recognition and ECL signal enhancement. The Cys-labeled kemptides were employed to build a serine-rich synthetic substrate-heptapeptide (Cys-kemptide) on the Au-electrode surface. Then, the PKA-induced biosensor was triggered as a signal switch to introduce the large amounts of TiO2 decorated Mg@N-CD nanohybrid (Ti@NMg-CDs) into AuE/Cys-phosphopeptides for signal output. In particular, the presence of PKA could induce the formation of Cys-phosphopeptides by the catalytic reaction between specific substrate (kemptide) and PKA, which acts as an initiator to link the Ti@NMg-CDs according to the bridge interactions Ti–O-P. In this way, multiple Cys-phosphopeptides were adsorbed onto a single Ti@NMg-CDs, and the Ti@NMg-CDs not only provided high specific selectivity but also large surface area, as well as unprecedented high ECL efficiency. Using this PKA-induced enhanced sensor, the limit of detection of the PKA was 4.89 × 10−4 U/mL (S/N = 3). The proposed ECL biosensor was also universally applicable for the screening of PKA inhibitors and determining of other kinases activity. Our sensing system has excellent performance of specificity and the screening of kinase inhibitors, as well as it will inspire future effort in clinical diagnostics and new drug discovery. [ABSTRACT FROM AUTHOR]

Vasopressin controls water permeability in the renal collecting duct by regulating the water channel protein, aquaporin-2 (AQP2). Phosphoproteomic studies have identified multiple proteins that undergo phosphorylation changes in response to vasopressin. The kinases responsible for the phosphorylation of most of these sites have not been identified. Here, we use large-scale Bayesian data integration to predict the responsible kinases for 51 phosphoproteomically identified vasopressin-regulated phosphorylation sites in the renal collecting duct. To do this, we applied Bayes' rule to rank the 515 known mammalian protein kinases for each site. Bayes' rule was applied recursively to integrate each of the seven independent datasets, each time using the posterior probability vector of a given step as the prior probability vector of the next step. In total, 30 of the 33 phosphorylation sites that increase with vasopressin were predicted to be phosphorylated by protein kinase A (PKA) catalytic subunit-α, consistent with prior studies implicating PKA in vasopressin signaling. Eighteen of the vasopressin-regulated phosphorylation sites were decreased in response to vasopressin and all but three of these sites were predicted to be targets of extracellular signal-regulated kinases, ERK1 and ERK2. This result implies that ERK1 and ERK2 are inhibited in response to vasopressin V2 receptor occupation, secondary to PKA activation. The six phosphorylation sites not predicted to be phosphorylated by PKA or ERK1/2 are potential targets of other protein kinases previously implicated in aquaporin-2 regulation, including cyclin-dependent kinase 18 (CDK18), calmodulin-dependent kinase 2δ (CAMK2D), AMP-activated kinase catalytic subunit-α-1 (PRKAA1) and CDC42 binding protein kinase β (CDC42BPB). NEW & NOTEWORTHY: Vasopressin regulates water transport in the renal collecting duct in part through phosphorylation or dephosphorylation of proteins that regulate aquaporin-2. Prior studies have identified 51 vasopressin-regulated phosphorylation sites in 45 proteins. This study uses Bayesian data integration techniques to combine information from multiple prior proteomics and transcriptomics studies to predict the protein kinases that phosphorylate the 51 sites. Most of the regulated sites were predicted to be phosphorylated by protein kinase A or ERK1/ERK2. [ABSTRACT FROM AUTHOR]

Bacteria adapt the biosynthesis of their envelopes to specific growth conditions and prevailing stress factors. Peptidoglycan (PG) is the major component of the cell wall in Gram‐positive bacteria, where PASTA kinases play a central role in PG biosynthesis regulation. Despite their importance for growth, cell division and antibiotic resistance, the mechanisms of PASTA kinase activation are not fully understood. ReoM, a recently discovered cytosolic phosphoprotein, is one of the main substrates of the PASTA kinase PrkA in the Gram‐positive human pathogen Listeria monocytogenes. Depending on its phosphorylation, ReoM controls proteolytic stability of MurA, the first enzyme in the PG biosynthesis pathway. The late cell division protein GpsB has been implicated in PASTA kinase signalling. Consistently, we show that L. monocytogenes prkA and gpsB mutants phenocopied each other. Analysis of in vivo ReoM phosphorylation confirmed GpsB as an activator of PrkA leading to the description of structural features in GpsB that are important for kinase activation. We further show that ReoM phosphorylation is growth phase‐dependent and that this kinetic is reliant on the protein phosphatase PrpC. ReoM phosphorylation was inhibited in mutants with defects in MurA degradation, leading to the discovery that MurA overexpression prevented ReoM phosphorylation. Overexpressed MurA must be able to bind its substrates and interact with ReoM to exert this effect, but the extracellular PASTA domains of PrkA or MurJ flippases were not required. Our results indicate that intracellular signals control ReoM phosphorylation and extend current models describing the mechanisms of PASTA kinase activation. [ABSTRACT FROM AUTHOR]

Microtubule affinity‐regulating kinase 2 (MARK2) is a Ser/Thr protein kinase that regulates cell polarity and immune responses. Here, we report that Orf9b, one of the accessory proteins encoded in the SARS‐CoV‐2 genome, increases MARK2 activity via interaction with the autoinhibitory KAI domain. We found that co‐expression of Orf9b enhances the kinase activity of MARK2 in HEK293 cells. Orf9b does not bind to or enhance the activity of the mutant form of MARK2 lacking the KA1 domain. Orf9b lowers inhibitory phosphorylation of MARK2 at T595 while mutation experiments indicate that this site is dispensable for Orf9b‐mediated enhancement of MARK2 activity. Our results suggest that Orf9b enhances MARK2 activity by binding the autoinhibitory KA1 domain, which closely interacts with the kinase domain. [ABSTRACT FROM AUTHOR]

Protein kinases have essential responsibilities in controlling several cellular processes, and their abnormal regulation is strongly related to the development of cancer. The implementation of protein kinase inhibitors has significantly transformed cancer therapy by modifying treatment strategies. These inhibitors have received substantial FDA clearance in recent decades. Protein kinases have emerged as primary objectives for therapeutic interventions, particularly in the context of cancer treatment. At present, 69 therapeutics have been approved by the FDA that target approximately 24 protein kinases, which are specifically prescribed for the treatment of neoplastic illnesses. These novel agents specifically inhibit certain protein kinases, such as receptor protein-tyrosine kinases, protein-serine/threonine kinases, dual-specificity kinases, nonreceptor protein-tyrosine kinases, and receptor protein-tyrosine kinases. This review presents a comprehensive overview of novel targets of kinase inhibitors, with a specific focus on cyclin-dependent kinases (CDKs) and epidermal growth factor receptor (EGFR). The majority of the reviewed studies commenced with an assessment of cancer cell lines and concluded with a comprehensive biological evaluation of individual kinase targets. The reviewed articles provide detailed information on the structural features of potent anticancer agents and their specific activity, which refers to their ability to selectively inhibit cancer-promoting kinases including CDKs and EGFR. Additionally, the latest FDA-approved anticancer agents targeting these enzymes were highlighted accordingly. [ABSTRACT FROM AUTHOR]

The complement system is critically involved in the pathogenesis of sepsis. In particular, complement anaphylatoxin C5a is generated in excess during sepsis, leading to cellular dysfunction. Recent studies have shown that excessive C5a impairs adrenomedullary catecholamine production release and induces apoptosis in adrenomedullary cells. Currently, the mechanisms by which C5a impacts adrenal cell function are poorly understood. The PC12 cell model was used to examine the cellular effects following treatment with recombinant rat C5a. The levels of caspase activation and cell death, protein kinase signaling pathway activation, and changes in inflammatory protein expression were examined following treatment with C5a. There was an increase in apoptosis of PC12 cells following treatment with high-dose C5a. Ten inflammatory proteins, primarily involved in apoptosis, cell survival, and cell proliferation, were upregulated following treatment with high-dose C5a. Five inflammatory proteins, involved primarily in chemotaxis and anti-inflammatory functions, were downregulated. The ERK/MAPK, p38/MAPK, JNK/MAPK, and AKT protein kinase signaling pathways were upregulated in a C5aR-dependent manner. These results demonstrate an apoptotic effect and cellular signaling effect of high-dose C5a. Taken together, the overall data suggest that high levels of C5a may play a role in C5aR-dependent apoptosis of adrenal medullary cells in sepsis. [ABSTRACT FROM AUTHOR]

In this study, a mixture of Platycodon grandiflorum, Pyrus serotina, Chaenomeles sinensis, and Raphanus sativus (PPCRE) was investigated for their immuno-enhancing effects, as well as the molecular mechanism of PPCRE in RAW264.7 cells. PPCRE dramatically increased nitric oxide (NO) and prostaglandin E2 (PGE2) generation depending on the concentration while exhibiting no cytotoxicity. PPCRE markedly upregulated the mRNA and protein expression of immune-related cytotoxic factors such as cyclooxygenase (COX)-1, COX-2, and inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α), as well as the mRNA level of IL-4. PPCRE increased the mitogen-activated protein kinase (MAPK) signaling pathway by upregulating the phosphorylation of extracellular signal-regulated kinase (ERK), stress-activated protein kinase/Jun N-terminal-kinase (SAPK/JNK), and p38. Furthermore, PPCRE considerably activated the nuclear factor kappa B (NF-κB) signaling pathway by increasing phosphorylation of NF-κB-p65. PPCRE-stimulated RAW264.7 cells increased macrophage phagocytic capacity. In conclusion, our study found that PPCRE improved immune function by modulating inflammatory mediators and regulating the MAPK and NF-κB pathway of signaling in macrophages. [ABSTRACT FROM AUTHOR]

The effects of trehalose, an autophagy-inducing disaccharide with neuroprotective properties, on the neurotoxicity of parkinsonian mimetics 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpiridinium (MPP+) are poorly understood. In our study, trehalose suppressed 6-OHDA-induced caspase-3/PARP1 cleavage (detected by immunoblotting), apoptotic DNA fragmentation/phosphatidylserine externalization, oxidative stress, mitochondrial depolarization (flow cytometry), and mitochondrial damage (electron microscopy) in SH-SY5Y neuroblastoma cells. The protection was not mediated by autophagy, autophagic receptor p62, or antioxidant enzymes superoxide dismutase and catalase. Trehalose suppressed 6-OHDA-induced activation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and AMP-activated protein kinase (AMPK), as revealed by immunoblotting. Pharmacological/genetic inhibition of JNK, p38 MAPK, or AMPK mimicked the trehalose-mediated cytoprotection. Trehalose did not affect the extracellular signal-regulated kinase (ERK) and mechanistic target of rapamycin complex 1 (mTORC1)/4EBP1 pathways, while it reduced the prosurvival mTORC2/AKT signaling. Finally, trehalose enhanced oxidative stress, mitochondrial damage, and apoptosis without decreasing JNK, p38 MAPK, AMPK, or AKT activation in SH-SY5Y cells exposed to MPP+. In conclusion, trehalose protects SH-SY5Y cells from 6-OHDA-induced oxidative stress, mitochondrial damage, and apoptosis through autophagy/p62-independent inhibition of JNK, p38 MAPK, and AMPK. The opposite effects of trehalose on the neurotoxicity of 6-OHDA and MPP+ suggest caution in its potential development as a neuroprotective agent. [ABSTRACT FROM AUTHOR]

Head and neck squamous cell carcinomas (HNSCC) are among the most common malignancies in men worldwide. Nevertheless, their clinical management is hampered by the limited availability of reliable predictive and prognostic biomarkers. Protein kinase D (PKD) isoforms contribute to major cellular processes. However, their potential role in HNSCC has not been studied systematically, which is the focus of this study. A total of 63 therapy-naive patients with squamous cell carcinoma were consecutively enrolled. Tissue microarray duplicate cores from each case were tested in situ for PKD1, PKD2, and PKD3 expression using immunohistochemistry, and the results were correlated with clinicopathological parameters. We found a high frequency of PKD1/PKD2 positive cases in oropharyngeal and PKD2 positive cases in laryngeal localizations. Only high PKD2 levels were statistically linked to elevated tumor grades, more advanced TNM (3–4) tumor stages, and p16INK4a expression, while elevated PKD3 levels were associated with favorable disease-specific survival. Both PKD2 and PKD3 have been proposed to promote tumor cell proliferation, migration/invasion, and angiogenesis. However, the role of PKD3 was elusive in some cancers. Our findings suggest that testing for PKD isotypes with immunohistochemistry may support the diagnostic estimation of tumor progression and prognosis in HNSCC with a potential therapeutic relevance. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is a chronic degenerative disease characterized by subchondral osteosclerosis, mainly due to osteoblast activity. This research investigates the function of Sik1, a member of the AMP-activated protein kinase family, in OA. Proteomic analysis was conducted on clinical samples from 30 OA patients, revealing a negative correlation between Sik1 expression and OA. In vitro experiments utilized BMSCs to examine the effect of Sik1 on osteogenic differentiation. BMSCs were cultured and induced toward osteogenesis with specific media. Sik1 overexpression was achieved through lentiviral transfection, followed by analysis of osteogenesis-associated proteins using Western blotting, RT-qPCR, and alkaline phosphate staining. In vivo experiments involved destabilizing the medial meniscus in mice to establish an OA model, assessing the therapeutic potential of Sik1. The CT scans and histological staining were used to analyze subchondral bone alterations and cartilage damage. The findings show that Sik1 downregulation correlates with advanced OA and heightened osteogenic differentiation in BMSCs. Sik1 overexpression inhibits osteogenesis-related markers in vitro and reduces cartilage damage and subchondral osteosclerosis in vivo. Mechanistically, Sik1 modulates osteogenesis and subchondral bone changes through Runx2 activity regulation. The research emphasizes Sik1 as a promising target for treating OA, suggesting its involvement in controlling bone formation and changes in the subchondral osteosclerosis. [ABSTRACT FROM AUTHOR]

Simple Summary: Cancer chemotherapy is dominated by cytotoxic drugs that non-selectively kill proliferating cells by blocking DNA replication and inducing apoptosis. Some natural anticancer products are less toxic to normal, slow-replicating cells but kill aggressive tumor cells. The combination of two structural isomers of pentacyclic triterpenes, oleanolic acid and ursolic acid, showed potent cytotoxic activity in human breast cancer cells in vitro by inducing cytotoxic autophagy at a significantly lower dose than previously reported. They were nontoxic to breast epithelial cells. Their activity was mainly due to the inhibition of AKT-mediated cell survival and the inhibition of the mammalian target of rapamycin (mTOR) signaling pathway, a central regulator of cell metabolism, growth, and proliferation. The compounds were equally effective against ER+ and triple-negative breast cancer cells. Combining low doses of nontoxic inhibitors of PI3k kinase increased the activities of these compounds. In contrast, inhibition of autophagy by 3-methyl adenine annulled their activity, demonstrating that cytotoxic autophagy may be the dominant mechanism of cell death. These compounds have potential for cancer therapy in neoadjuvant or adjuvant settings. Background: Oleanolic acid (OA) and Ursolic acid (UA) are bioactive triterpenoids. Reported activities vary with the dose used for testing their activities in vitro. Studies using doses of ≥20 µM showed apoptosis activities in cancer cells. However, reported drug levels in circulation achieved by oral administration of UA and OA are ≤2 µM, thus limiting their use for treatment or delivering a combination treatment. Materials and Methods: The present report demonstrates the efficacy of OA, UA, and OA + UA on tumor cell-specific cytotoxicity at low doses (5 µM to 10 µM) in breast cancer (BrCa) cell lines MCF7 and MDA-MB231. Results: The data show that both OA and UA killed BrCa cells at low doses, but were significantly less toxic to MCF-12A, a non-tumorigenic cell line. Moreover, OA + UA at ≤10 µM was lethal to BrCa cells. Mechanistic studies unraveled the significant absence of apoptosis, but their cytotoxicity was due to the induction of excessive autophagy at a OA + UA dose of 5 µM each. A link to drug-induced cytotoxic autophagy was established by demonstrating a lack of their cytotoxicity by silencing the autophagy-targeting genes (ATGs), which prevented OA-, UA-, or OA + UA-induced cell death. Further, UA or OA + UA treatment of BrCa cells caused an inhibition of PI3 kinase-mediated phosphorylation of Akt/mTOR, the key pathways that regulate cancer cell survival, metabolism, and proliferation. Discussion: Combinations of a PI3K inhibitor (LY294002) with OA, UA, or OA + UA synergistically inhibited BrCa cell survival. Therefore, the dominance of cytotoxic autophagy by inhibiting PI3K-mediated autophagy may be the primary mechanism of PTT-induced anticancer activity in BrCa cells. Conclusion: These results suggest it would be worthwhile testing combined OA and UA in clinical settings. [ABSTRACT FROM AUTHOR]

Early fluctuations in blood glucose levels increased susceptibility to macrophage dysfunction. However, the underlying pathological mechanisms linking glucose variations and macrophage dysregulation remains elusive. In current study, we established an animal model of transient intermittent hyperglycaemia (TIH) to simulate early fluctuations in blood glucose levels. Our findings revealed that both TIH and diabetic group exhibited more severe periodontal lesions and increased secretion of pro‐inflammatory cytokines compared to healthy controls. In immortalized bone marrow–derived macrophages (iBMDMs), phagocytosis and chemotaxis were impaired with transient and lasting hyperglycaemia, accompanied by enhanced glycolysis. We also found that TIH activated pyruvate kinase M2 (PKM2) through the phosphorylation of extracellular regulated protein kinase (ERK) in vivo, particularly at dimeric levels. In macrophage cultured with TIH, PKM2 translocated into the nucleus and involved in the regulating inflammatory genes, including TNF‐α, IL‐6 and IL‐1β. PKM2 translocation and secretion of inflammatory cytokines were attenuated by PD98059, while PKM2 tetramer activator TEPP‐46 prevented the formation of dimeric PKM2 in macrophages. Moreover, inhibition of glycolysis alleviated the TIH‐induced pro‐inflammatory cytokines. In conclusion, our manuscript provides a rationale for understanding how TIH modulates metabolic rewiring and dysfunction in macrophages via ERK‐dependent PKM2 nuclear translocation. [ABSTRACT FROM AUTHOR]

Background: Receptor tyrosine kinase (RTK) fusions of RET, NTRK1/3, and ALK are enriched among pediatric thyroid cancer patients with metastatic and persistent disease, and their oncoproteins represent attractive drug targets. Methods: We performed RNA-sequencing in a papillary thyroid cancer (PTC) lacking other frequent driver alterations. Results: We report a novel RTK fusion, TG-insulin-like growth factor 1 receptor gene (IGF1R), in a 17-year-old female patient with angioinvasive follicular variant PTC. The in-frame fusion protein preserves the cholinesterase‐like domain of TG with dimerization properties and the transmembrane and kinase domain of IGF1R. The tumor sample shows increased IGF1R mRNA expression and tyrosine kinase phosphorylation, augmentation of Mitogen activated protein kinase (MAPK) transcriptional output genes, and decreased NIS levels. Conclusions: We reveal a novel targetable kinase fusion oncogene in thyroid cancer which is not incorporated in different thyroid-specific sequencing panels. The integration of IGF1R fusion screening in the next versions of thyroid-specific targeted next-generation sequencing panels may be beneficial to thyroid cancer patients. [ABSTRACT FROM AUTHOR]

Inflammation and oxidative stress (OS) are the major pathogenic characteristics of acute kidney injury (AKI). Studies have shown that Schisandrin (Sch) could regulate inflammatory disease. However, the function and mechanism of Sch in AKI progression are still unknown. Here, we investigated Sch's potential effects and mechanism on mice's renal damage and macrophages induced by lipopolysaccharide (LPS). Sch decreased LPS‐induced inflammatory factor production while increasing the activity of related antioxidant enzymes in macrophages and mouse kidney tissues. Hematoxylin and eosin staining revealed that Sch may have the ability to profoundly inhibit inflammatory cell invasion and tissue damage caused by LPS in renal tissue. Furthermore, Western blot and immunohistochemical studies showed that Sch exerted its effects mainly through up‐regulation of nuclear factor erythroid 2‐related factor 2/heme oxygenase‐1 and inhibition of Toll‐like receptor 4‒mitogen‐activated protein kinases/nuclear factor‐kappa B pathways. Collectively, this study illustrates that Sch suppresses LPS‐stimulated AKI by descending inflammation and OS, illuminating prospective AKI treatment options. [ABSTRACT FROM AUTHOR]

Objectives: Galectin‐9 (Gal‐9) is an immune checkpoint ligand for T‐cell immunoglobulin and mucin domain 3. Although the roles of Gal‐9 in regulating immune responses have been well investigated, their biological roles have yet to be fully documented. This study aimed to analyse the expression of Gal‐9 bone marrow (BM) cells in C57BL/6J (B6) mice. Furthermore, the co‐expression of Gal‐9 with the mammalian target of rapamycin (mTOR) and AMP‐activated protein kinase (AMPK) was investigated. Methods: The BM cells in adult C57BL/6J (B6) mice were collected and analysed in vitro. Results: In a flow cytometric analysis of BM cells, Gal‐9 was highly expressed in c‐KithiSca‐1−CD34−CD71+ erythroid progenitors (EPs), whereas it was downregulated in more differentiated c‐KitloCD71+TER119+ cells. Subsequently, a negative selection of CD3−B220−Sca‐1−CD34−CD41−CD16/32− EPs was performed. This resulted in substantial enrichment of KithiCD71+Gal‐9+ cells and erythroid colony‐forming units (CFU‐Es), suggesting that the colony‐forming subset of EPs are included in the KithiCD71+Gal‐9+ population. Furthermore, we found that EPs had lower mTOR and AMPK expression levels in Gal‐9 knockout B6 mice than in wild‐type B6 mice. Conclusions: These results may stimulate further investigation of the role of Gal‐9 in haematopoiesis. [ABSTRACT FROM AUTHOR]

Cardiovascular disease is a leading cause of morbidity and mortality, and exercise-training (TRN) is known to reduce risk factors and protect the heart from ischemia and reperfusion injury. Though the cardioprotective effects of exercise are well-documented, underlying mechanisms are not well understood. This review highlights recent findings and focuses on cardiac factors with emphasis on K+ channel control of the action potential duration (APD), β-adrenergic and adenosine regulation of cardiomyocyte function, and mitochondrial Ca2+ regulation. TRN-induced prolongation and shortening of the APD at low and high activation rates, respectively, is discussed in the context of a reduced response of the sarcolemma delayed rectifier potassium channel (IK) and increased content and activation of the sarcolemma KATP channel. A proposed mechanism underlying the latter is presented, including the phosphatidylinositol-3kinase/protein kinase B pathway. TRN induced increases in cardiomyocyte contractility and the response to adrenergic agonists are discussed. The TRN-induced protection from reperfusion injury is highlighted by the increased content and activation of the sarcolemma KATP channel and the increased phosphorylated glycogen synthase kinase-3β, which aid in preventing mitochondrial Ca2+ overload and mitochondria-triggered apoptosis. Finally, a brief section is presented on the increased incidences of atrial fibrillation associated with age and in life-long exercisers. [ABSTRACT FROM AUTHOR]

Interactions between protein kinases and their substrates are critical for the modulation of complex signaling pathways. Currently, there is a large amount of information available about kinases and their substrates in disparate public databases. However, these data are difficult to interpret in the context of cellular systems, which can be facilitated by examining interactions among multiple proteins at once, such as the network of interactions that constitute a signaling pathway. We present KiNet, a user-friendly web portal that integrates and shares information about kinase-substrate interactions from multiple databases of post-translational modifications. KiNet enables the visual exploration of these interactions in systems contexts, such as pathways, domain families, and custom protein set inputs, in an interactive fashion. We expect KiNet to be useful as a knowledge discovery tool for kinase-substrate interactions, and the aggregated KiNet dataset to be useful for protein kinase studies and systems-level analyses. The portal is available at https://kinet.kinametrix.com/. [ABSTRACT FROM AUTHOR]

Fermented red ginseng (FRG) enhances the bioactivity and bioavailability of ginsenosides, which possess various immunomodulatory, antiaging, anti-obesity, and antidiabetic properties. However, the effects of FRG extract on muscle atrophy and the underlying molecular mechanisms remain unclear. This study aimed to elucidate the effects of FRG extract on muscle atrophy using both in vitro and in vivo models. In vitro experiments used dexamethasone (DEX)-induced C2C12 myotubes to assess cell viability, myotube diameter, and fusion index. In vivo experiments were conducted on hind limb immobilization (HI)-induced mice to evaluate grip strength, muscle mass, and fiber cross-sectional area (CSA) of the gastrocnemius (GAS), quadriceps (QUA), and soleus (SOL) muscles. Molecular mechanisms were investigated through the analysis of key signaling pathways associated with muscle protein synthesis, energy metabolism, and protein degradation. FRG extract treatment enhanced viability of DEX-induced C2C12 myotubes and restored myotube diameter and fusion index. In HI-induced mice, FRG extract improved grip strength, increased muscle mass and CSA of GAS, QUA, and SOL muscles. Mechanistic studies revealed that FRG extract activated the insulin-like growth factor 1/protein kinase B (Akt)/mammalian target of rapamycin signaling pathway, promoted muscle energy metabolism via the sirtuin 1/peroxisome proliferator-activated receptor gamma-coactivator-1α pathway, and inhibited muscle protein degradation by suppressing the forkhead box O3a, muscle ring-finger 1, and F-box protein (Fbx32) signaling pathways. FRG extract shows promise for ameliorating muscle atrophy by modulating key molecular pathways associated with muscle protein synthesis, energy metabolism, and protein degradation, offering insights for future drug development. [ABSTRACT FROM AUTHOR]

This study investigated the antiobesity effects of black ginger extract (BGE) in high-fat diet (HFD)-induced obese mice. Mice were divided into six groups: normal diet control (NC, AIN-93G normal diet), 60% HFD control (HFD), HFD containing metformin at 250 mg/kg b.w. (Met, positive control), and HFD containing BGE at 5, 10, or 20 mg/kg b.w. for 15 weeks. BGE administration significantly prevented HFD-induced increases in weight gain, organ weight, and adipose tissue mass. Furthermore, it resulted in decreased adipogenesis and lipogenesis-related factors, including phosphorylated mitogen-activated protein kinase, peroxisome proliferator-activated receptor gamma, CCAAT/enhancer-binding proteins, sterol regulatory element-binding protein 1, phosphorylated cAMP response element-binding protein, glucose-6-phosphate dehydrogenase, fatty acid synthase, dephosphorylated ATP-citrate lyase, dephosphorylated acetyl-CoA carboxylase, and lipoprotein lipase, in white adipose tissues. Moreover, BGE administration enhanced lipolysis in white adipose tissue, as evidenced by elevated levels of adipose triglyceride lipase, phosphorylated hormone-sensitive lipase, and protein kinase A, along with reduced levels of perilipin and phosphodiesterase 3B. BGE induced thermogenesis in brown adipose tissues, as reflected by the increased expression of AMP-activated protein kinase, uncoupling protein 1, and carnitine palmitoyltransferase 1 and decreased levels of fatty acid-binding protein 4. In conclusion, this study provides comprehensive evidence supporting the antiobesity effects of BGE, elucidating the underlying molecular mechanisms involved in preventing weight gain, suppressing adipogenesis, promoting lipolysis, and stimulating thermogenesis. These findings suggest the potential therapeutic utility of BGE in combating obesity and associated metabolic disorders (KHGASP-2023-034). [ABSTRACT FROM AUTHOR]

Aryl hydrocarbon receptor (AhR) plays an important role in inflammation and immunity as a new therapeutic target for infectious disease and sepsis. Punicalagin (PUN) is a Chinese herbal monomer extract of pomegranate peel that has beneficial anti-inflammatory, antioxidant and anti-infective effects. However, whether PUN is a ligand of AhR, its effect on AhR expression, and its signaling pathway remain poorly understood. In this study, we found that PUN was a unique polyphenolic compound that upregulated AhR expression at the transcriptional level, and regulated the AhR nongenomic pathway. AhR expression in lipopolysaccharide-induced macrophages was upregulated by PUN in vitro and in vivo in a time- and dose-dependent manner. Using specific inhibitors and siRNA, induction of AhR by PUN depended on sequential phosphorylation of 90-kDa ribosomal S6 kinase (p90RSK), which was activated by the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and phosphoinositide-dependent protein kinase (PDK)1 pathways. PUN promoted p90RSK-mediated activator protein-1 (AP-1) activation. AhR knockout or inhibitors reversed suppression of interleukin (IL)-6 and IL-1β expression by PUN. PUN decreased Listeria load and increased macrophage survival via AhR upregulation. In conclusion, we identified PUN as a novel selective AhR modulator involved in AhR expression via the MEK/ERK and PDK1 pathways targeting p90RSK/AP-1 in inflammatory macrophages, which inhibited macrophage inflammation and promoted bactericidal activity. [ABSTRACT FROM AUTHOR]