Kinase Protein Research Articles

Screening and Activity Evaluation of Novel BCR-ABL/T315I Tyrosine Kinase Inhibitors.

Chronic myeloid leukemia (CML) is a kind of malignant tumor formed by the clonal proliferation of bone marrow hematopoietic stem cells. BCR-ABL fusion protein, found in more than 90% of patients, is a vital target for discovering anti-CML drugs. Up to date, imatinib is the first BCR-ABL tyrosine kinase inhibitor (TKI) approved by the FDA for treating CML. However, the drug resistance problems appeared for many reasons, especially the T135I mutation, a "gatekeeper" of BCR-ABL. Currently, there is no long-term effective and low side effect drug in clinical. This study intends to find novel TKIs targeting BCR-ABL with high inhibitory activity against T315I mutant protein by combining artificial intelligence technology and cell growth curve, cytotoxicity, flow cytometry and western blot experiments. The obtained compound was found to kill leukemia cells, which had good inhibitory efficacy in BaF3/T315I cells. Compound No 4 could induce cell cycle arrest, cause autophagy and apoptosis, and inhibit the phosphorylation of BCR-ABL tyrosine kinase, STAT5 and Crkl proteins. The results indicated that the screened compound could be used as a lead compound for further research to discover ideal chronic myeloid leukemia therapeutic drugs.

The impact of early-life exercise on CREB-signaling pathway and hippocampus neuroplasticity in diabetic adult male rats; the study of developmental model.

Childhood exercise enhances brain structure, while diabetes detrimentally affects it. This study examines early-life exercise's influence on adult diabetic rats' memory and neuroplasticity. Male Wistar pups were divided into Control, Diabetes, Exercise Training, and Diabetes exercise groups. Diabetes was induced on day 23 with Alloxan (200 mg/kg). A 3-week regimen included aerobic and resistance training thrice weekly. The aerobic intensity was 70%, and resistance varied from 50% to 100% of the maximal carrying capacity (MCC). Following the last training sessions, spatial memory and retrieval tests were performed in infancy, childhood, and emerging adulthood using the Morris Water Maze test (MWM). The hippocampus was excised to measure protein and gene expression of brain-derived neurotrophic factor (BDNF), calmodulin-dependent protein kinase (CAMKII), N-methyl-D-aspartate receptors (NMDAR), and cAMP-response element-binding protein (CREB) by western blotting and reverse transcription-polymerase-chain reaction (RT-PCR) methods. Blood samples were collected during each developmental stage to measure glucose levels, at the study's conclusion, to assess Interleukin-1β levels using the ELISA method. The Nissel staining assessed dead hippocampal cells in CA1. Post-natal exercise improved spatial memory (p < 0.05) and glucose levels (p < 0.05) in diabetic rats during adolescence and emerging adulthood. Despite reduced mRNA expression (NMDAR 40%, BDNF 62%, CREB 43%, CAMKII 66%), diabetic rats, by study end, showed increased BDNF, NMDARR, CAMKII, CREB protein/gene expression (p < 0.05) in emerging adulthood for both training groups. Early-life exercise influenced hippocampal BDNF/NMDAR-CAMKII/CREB pathways in a diabetic rat model, highlighting post-natal exercise's role in neuroplasticity memory enhancement and improved glucose level.

Decoding Arabidopsis thaliana CPK/SnRK Superfamily Kinase Client Signaling Networks Using Peptide Library and Mass Spectrometry

Members of the calcium-dependent protein kinase (CDPK/CPK) and SNF-related protein kinase (SnRK) superfamilies are commonly found in plants and some protists. Our knowledge of client specificity of the members of this superfamily is fragmentary. As this family is represented by over 30 members in Arabidopsis thaliana, the identification of kinase-specific and overlapping client relationships is crucial to our understanding the nuances of this large family of kinases as directed towards signal transduction pathways. Herein, we used the kinase client (KiC) assay—a relative, quantitative, high-throughput mass spectrometry-based in vitro phosphorylation assay—to identify and characterize potential CPK/SnRK targets of Arabidopsis. Eight CPKs (1, 3, 6, 8, 17, 24, 28, and 32), four SnRKs (subclass 1 and 2), and PPCK1 and PPCK2 were screened against a synthetic peptide library that contains 2095 peptides and 2661 known phosphorylation sites. A total of 625 in vitro phosphorylation sites corresponding to 203 non-redundant proteins were identified. The most promiscuous kinase, CPK17, had 105 candidate target proteins, many of which had already been discovered. Sequence analysis of the identified phosphopeptides revealed four motifs: LxRxxS, RxxSxxR, RxxS, and LxxxxS, that were significantly enriched among CPK/SnRK clients. The results provide insight into both CPK- and SnRK-specific and overlapping signaling network architectures and recapitulate many known in vivo relationships validating this large-scale approach towards discovering kinase targets.

Robotic-assisted medial unicompartmental knee arthroplasty restored prearthritic alignment and led to superior functional outcomes compared with conventional techniques.

Robotic-assisted medial unicompartmental knee arthroplasty (UKA) can ensure precise preoperative planning, minimise soft tissue damage and restore native coronal alignment. However, few studies have investigated how these advantages translate into differences in early postoperative outcomes. This study aimed to compare differences in early outcomes between conventional UKA (C-UKA) and robotic-assisted UKA (R-UKA). This retrospective study investigated two groups of patients who underwent medial UKA: C-UKA group (n = 35) and R-UKA group (n = 35). We assessed (1) serum indicators (hemoglobin, creatine kinase and C-reactive protein) and pain visual analogue scale (VAS) at postoperative days (PODs) 1, 2, 4 and 6; (2) radiologic parameters including joint line height change and arithmetic and mechanical hip-knee-ankle angle (aHKA and mHKA); (3) patient-reported outcomes including Knee Society Scores, Western Ontario and Mcmaster Universities Arthritis Index (WOMAC) and Forgotten Joint Score-12 (FJS-12) at 1-year follow-up. Despite similar serum indicator results, pain VAS was lower in the R-UKA group than in the C-UKA group at PODs 2 (2.5 ± 1.3 vs. 3.6 ± 1.2, p = 0.02), 4 (2.4 ± 0.9 vs. 3.3 ± 1.0, p = 0.03) and 6 (1.9 ± 1.1 vs. 3.1 ± 1.1, p < 0.01). The joint line height change was significantly lower in the R-UKA group than in the C-UKA group (0.9 mm ± 0.6 mm vs. 2.0 mm ± 1.3 mm, p = 0.02). The equivalence test for preoperative aHKA and postoperative mHKA revealed equivalence in only the R-UKA group (p < 0.01). The R-UKA group showed better WOMAC and FJS-12 compared to C-UKA group at 1-year follow-up. R-UKA led to lower pain VAS in the early postoperative period compared with C-UKA. Additionally, R-UKA effectively restored the joint line and prearthritic lower limb alignment, resulting in superior functional outcomes at 1-year follow-up compared with C-UKA. Level III.

Linagliptin Mitigates TGF-β1 Mediated Epithelial-Mesenchymal Transition in Tacrolimus-Induced Renal Interstitial Fibrosis via Smad/ERK/P38 and HIF-1α/LOXL2 Signaling Pathways.

The dipeptidyl peptidase-4 (DPP-4) inhibitors, a novel anti-diabetic medication family, are renoprotective in diabetes, but a comparable benefit in chronic non-diabetic kidney diseases is still under investigation. This study aimed to elucidate the molecular mechanisms of linagliptin's (Lina) protective role in a rat model of chronic kidney injury caused by tacrolimus (TAC) independent of blood glucose levels. Thirty-two adult male Sprague Dawley rats were equally randomized into four groups and treated daily for 28 d as follows: The control group; received olive oil (1 mL/kg/d, subcutaneously), group 2; received Lina (5 mg/kg/d, orally), group 3; received TAC (1.5 mg/kg/d, subcutaneously), group 4; received TAC plus Lina concomitantly in doses as the same previous groups. Blood and urine samples were collected to investigate renal function indices and tubular injury markers. Additionally, signaling molecules, epithelial-mesenchymal transition (EMT), and fibrotic-related proteins in kidney tissue were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, immunohistochemical and histological examinations. Tacrolimus markedly induced renal injury and fibrosis as indicated by renal dysfunction, histological damage, and deposition of extracellular matrix (ECM) proteins. It also increased transforming growth factor β1 (TGF-β1), Smad4, p-extracellular signal-regulated kinase (ERK)1/2/ERK1/2, and p-P38/P38 mitogen-activated protein kinase (MAPK) protein levels. These alterations were markedly attenuated by the Lina administration. Moreover, Lina significantly inhibited EMT, evidenced by inhibiting Vimentin and α-smooth muscle actin (α-SMA) and elevating E-cadherin. Furthermore, Lina diminished hypoxia-related protein levels with a subsequent reduction in Snail and Twist expressions. We concluded that Lina may protect against TAC-induced interstitial fibrosis by modulating TGF-β1 mediated EMT via Smad-dependent and independent signaling pathways.

Treating myasthenia gravis beyond the eye clinic.

Myasthenia gravis (MG) is one of the most well characterised autoimmune disorders affecting the neuromuscular junction with autoantibodies targeting the acetylcholine receptor (AChR) complex. The vast majority of patients present with ocular symptoms including double vision and ptosis, but may progress on to develop generalised fatiguable muscle weakness. Severe involvement of the bulbar muscles can lead to dysphagia, dysarthria and breathing difficulties which can progress to myasthenic crisis needing ventilatory support. Given the predominant ocular onset of the disease, it is important that ophthalmologists are aware of the differential diagnosis, investigations and management including evolving therapies. When the disease remains localised to the extraocular muscles (ocular MG) IgG1 and IgG3 antibodies against the AChR (including clustered AChR) are present in nearly 50% of patients. In generalised MG this is seen in nearly 90% patients. Other antibodies include those against muscle specific tyrosine kinase (MuSK) and lipoprotein receptor related protein 4 (LRP4). Even though decremental response on repetitive nerve stimulation is the most well recognised neurophysiological abnormality, single fibre electromyogram (SFEMG) in experienced hands is the most sensitive test which helps in the diagnosis. Initial treatment should be using cholinesterase inhibitors and then proceeding to immunosuppression using corticosteroids and steroid sparing drugs. Patients requiring bulbar muscle support may need rescue therapies including plasma exchange and intravenous immunoglobulin (IVIg). Newer therapeutic targets include those against the B lymphocytes, complement system, neonatal Fc receptors (FcRn) and various other elements of the immune system.

Mild endoplasmic reticulum stress alleviates FB1-triggered intestinal pyroptosis via the Sec62-PERK pathway

Fumonisin B1 (FB1), a water-soluble mycotoxin released by Fusarium moniliforme Sheld, is widely present in corn and its derivative products, and seriously endangers human life and health. Recent studies have reported that FB1 can lead to pyroptosis, however, the mechanisms by which FB1-induced pyroptosis remain indistinct. In the present study, we aim to investigate the mechanisms of pyroptosis in intestinal porcine epithelial cells (IPEC-J2) and the relationship between FB1-induced endoplasmic reticulum stress (ERS) and pyroptosis. Our experimental results showed that the pyroptosis protein indicators in IPEC-J2 were significantly increased after exposure to FB1. The ERS markers, including glucose-regulated Protein 78 (GRP78), PKR-like ER kinase protein (PERK), and preprotein translocation factor (Sec62) were also significantly increased. Using small interfering RNA silencing of PERK or Sec62, the results demonstrated that upregulation of Sec62 activates the PERK pathway, and activation of the PERK signaling pathway is upstream of FB1-induced pyroptosis. After using the ERS inhibitor 4-PBA reduced the FB1-triggered intestinal injury by the Sec62-PERK pathway. In conclusion, we found that FB1 induced pyroptosis by upregulating Sec62 to activate the PERK pathway, and mild ERS alleviates FB1-triggered damage. It all boils down to one fact, the study provides a new perspective for further, and improving the toxicological mechanism of FB1.Graphical

Patterns of alteration in boar semen quality from 9 to 37 months old and improvement by protocatechuic acid

BackgroundComprehending the patterns of alteration in boar semen quality and identifying effective nutritional interventions are crucial for enhancing the productivity of commercial pig systems. This study aimed to examine the alteration in semen quality in boars, and assess the impact of protocatechuic acid (PCA) on semen quality during the phase of declining semen quality.MethodsIn Exp. 1, a total of 38 Pig Improvement Company (PIC) boars were selected and their semen quality data were recorded from the age of 9 to 37 months. In Exp. 2, 18 PIC boars (28 months old) were randomly assigned into three groups (n = 6) and fed a basal diet, a basal diet containing 500 or 1,000 mg/kg PCA, respectively. The experiment lasted for 12 weeks.ResultsThe semen volume, concentration, and total number of spermatozoa in boars exhibited an increase from 9 to 19 months old and showed a significant linear decreased trend in 28, 24, and 22 months old. Sperm motility displayed an upward trajectory, reaching its peak at 20 months of age, and showed a significant linear decreased trend at 20 months old. Dietary supplementation of PCA demonstrated an effect to mitigate the decrease in semen volume, concentration of spermatozoa, total number of spermatozoa (P > 0.05), and significantly increased the sperm motility (P < 0.05). Moreover, supplementation of 1,000 mg/kg PCA significantly increased the sperm viability (P < 0.05). Analysis on cellular signaling pathways revealed that PCA restored serum testosterone levels and alleviated oxidative damage by upregulating the expression of HO-1, SOD2, and NQO1 in testicular stromal cells. Notably, PCA can enhance phosphorylation by selectively binding to AMP-activated protein kinase (AMPK) protein, thereby improving sperm mitochondrial function and augmenting sperm motility via PGC-1/Nrf1.ConclusionsThese data elucidated the pattern of semen quality variation in boars within the age range of 9 to 37 months old, and PCA has the potential to be a natural antioxidant to enhance sperm quality through modulation of the AMPK/PGC-1/Nrf1 signaling pathway.

Kinase Inhibitors and Kinase-Targeted Cancer Therapies: Recent Advances and Future Perspectives

Over 120 small-molecule kinase inhibitors (SMKIs) have been approved worldwide for treating various diseases, with nearly 70 FDA approvals specifically for cancer treatment, focusing on targets like the epidermal growth factor receptor (EGFR) family. Kinase-targeted strategies encompass monoclonal antibodies and their derivatives, such as nanobodies and peptides, along with innovative approaches like the use of kinase degraders and protein kinase interaction inhibitors, which have recently demonstrated clinical progress and potential in overcoming resistance. Nevertheless, kinase-targeted strategies encounter significant hurdles, including drug resistance, which greatly impacts the clinical benefits for cancer patients, as well as concerning toxicity when combined with immunotherapy, which restricts the full utilization of current treatment modalities. Despite these challenges, the development of kinase inhibitors remains highly promising. The extensively studied tyrosine kinase family has 70% of its targets in various stages of development, while 30% of the kinase family remains inadequately explored. Computational technologies play a vital role in accelerating the development of novel kinase inhibitors and repurposing existing drugs. Recent FDA-approved SMKIs underscore the importance of blood–brain barrier permeability for long-term patient benefits. This review provides a comprehensive summary of recent FDA-approved SMKIs based on their mechanisms of action and targets. We summarize the latest developments in potential new targets and explore emerging kinase inhibition strategies from a clinical perspective. Lastly, we outline current obstacles and future prospects in kinase inhibition.

Subtractive Proteomics and Reverse-Vaccinology Approaches for Novel Drug Target Identification and Chimeric Vaccine Development against Bartonella henselae Strain Houston-1.

Bartonella henselae is a Gram-negative bacterium causing a variety of clinical symptoms, ranging from cat-scratch disease to severe systemic infections, and it is primarily transmitted by infected fleas. Its status as an emerging zoonotic pathogen and its capacity to persist within host erythrocytes and endothelial cells emphasize its clinical significance. Despite progress in understanding its pathogenesis, limited knowledge exists about the virulence factors and regulatory mechanisms specific to the B. henselae strain Houston-1. Exploring these aspects is crucial for targeted therapeutic strategies against this versatile pathogen. Using reverse-vaccinology-based subtractive proteomics, this research aimed to identify the most antigenic proteins for formulating a multi-epitope vaccine against the B. henselae strain Houston-1. One crucial virulent and antigenic protein, the PAS domain-containing sensor histidine kinase protein, was identified. Subsequently, the identification of B-cell and T-cell epitopes for the specified protein was carried out and the evaluated epitopes were checked for their antigenicity, allergenicity, solubility, MHC binding capability, and toxicity. The filtered epitopes were merged using linkers and an adjuvant to create a multi-epitope vaccine construct. The structure was then refined, with 92.3% of amino acids falling within the allowed regions. Docking of the human receptor (TLR4) with the vaccine construct was performed and demonstrated a binding energy of -1047.2 Kcal/mol with more interactions. Molecular dynamic simulations confirmed the stability of this docked complex, emphasizing the conformation and interactions between the molecules. Further experimental validation is necessary to evaluate its effectiveness against B. henselae.

SRC inhibition enables formation of a growth suppressive MAGI1-PP2A complex in isocitrate dehydrogenase-mutant cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive bile duct malignancy that frequently exhibits isocitrate dehydrogenase (IDH1/IDH2) mutations. Mutant IDH (IDHm) ICC is dependent on SRC kinase for growth and survival and is hypersensitive to inhibition by dasatinib, but the molecular mechanism underlying this sensitivity is unclear. We found that dasatinib reduced p70 S6 kinase (S6K) and ribosomal protein S6 (S6), leading to substantial reductions in cell size and de novo protein synthesis. Using an unbiased phosphoproteomic screen, we identified membrane-associated guanylate kinase, WW, and PDZ domain containing 1 (MAGI1) as an SRC substrate in IDHm ICC. Biochemical and functional assays further showed that SRC inhibits a latent tumor-suppressing function of the MAGI1-protein phosphatase 2A (PP2A) complex to activate S6K/S6 signaling in IDHm ICC. Inhibiting SRC led to activation and increased access of PP2A to dephosphorylate S6K, resulting in cell death. Evidence from patient tissue and cell line models revealed that both intrinsic and extrinsic resistance to dasatinib is due to increased phospho-S6 (pS6). To block pS6, we paired dasatinib with the S6K/AKT inhibitor M2698, which led to a marked reduction in pS6 in IDHm ICC cell lines and patient-derived organoids in vitro and substantial growth inhibition in ICC patient-derived xenografts in vivo. Together, these results elucidated the mechanism of action of dasatinib in IDHm ICC, revealed a signaling complex regulating S6K phosphorylation independent of mTOR, suggested markers for dasatinib sensitivity, and described a combination therapy for IDHm ICC that may be actionable in the clinic.

Transcriptomic analysis of fludioxonil resistance mechanisms in Botrytis cinerea

Botrytis cinerea is a destructive plant pathogen that infects a wide range of economically important crops. Limiting pathogen infection during production and post-harvest is largely dependent on fungicide applications; fungicide resistant isolates of B. cinerea have been recovered from various hosts. Resistance of B. cinerea to fludioxonil has been associated with overexpression of transporter genes (BcatrB and mfsM2) and mutations on histidine kinase proteins (Bos1, Bchhk2, Bchhk17). To identify possible mechanisms associated with fludioxonil resistance, genomic expression of three sensitive and three low-resistant isolates were studied. Overexpression of BcatrB was observed when comparing low-resistant and sensitive isolates, but was not specific to the fludioxonil treatment. Seven amino acid substitutions and one deletion were identified in the transcription factor Bcmrr1 in low-resistant isolates, associated with overexpression of BcatrB. The L497 deletion, previously associated with highly resistance isolates (MDR1h), was observed in two low-resistant isolates. Other differentially expressed genes associated with transmembrane transport, oxidoreductase activity and lipid metabolic processes could be key in understanding the fungicidal mechanism(s) of fludioxonil. Expression profiles were isolate-specific. Following fludioxonil exposure, two sensitive isolates of B. cinerea sensu stricto showed a change in gene expression levels associated with cell membrane and peroxidase activity. In one low-resistant isolate of B. cinerea group S, fludioxonil exposure resulted in the overexpression of stress response genes and MFS transporter Bcstl1; one sensitive and two low-resistant isolates showed no significant changes in gene expression profiles. This work provides insight into the effect of fludioxonil on B. cinerea and potential fungicide resistance mechanisms.

CARMIL1 regulates liver cancer cell proliferation by activating the ERK/mTOR pathway through the TRIM27/p53 axis

Capping protein regulatory factor and myosin 1 linker 1 is termed CARMIL1. CARMIL1 is involved in several physiological processes; it forms an actin filament network and plasma membrane-bound cellular projection tissues and positively regulates the cellular components and tissues. CARMIL1 exhibits important biological functions in cancer; nonetheless, these functions have not been completely explored. We aimed to investigate the novel functions of CARMIL1 in liver cancer, particularly in cell proliferation. The cell counting kit-8, 5-ethynyl-2′-deoxyuridine, Component A experiments, and subcutaneous tumor formation model suggest that CARMIL1 is central to the proliferation of liver cancer cells both in vivo and in vitro. We extracted CARMIL1 samples from The Cancer Genome Atlas Program and analyzed its enrichment. CARMIL1 regulated the pathway activity by affecting the expression of star molecular proteins of the extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR). Moreover, it influenced the proliferation ability of liver cancer cells. Western blotting suggested that CARMIL1 downregulation could affect ERK and mTOR phosphorylation. Results of the co-immunoprecipitation demonstrated that CARMIL1 binds to tripartite motif (TRIM)27, which in turn binds to p53. Subsequently, CARMIL1 can regulate p53 stability and promote its degradation through TRIM27. Additionally, CARMIL1 inhibition enhanced the sensitivity of liver cancer cells to sorafenib. Tumor growth was significantly inhibited in the group treated with sorafenib and CARMIL1, compared with the group treated with CARMIL1 alone. Sorafenib is a first-line targeted chemotherapeutic drug for hepatocellular carcinoma treatment. It increases the long-term survival of hepatocellular carcinoma by 44%. In this study, downregulated CARMIL1 combined with sorafenib significantly reduced the tumor volume and weight of the mouse subcutaneous tumor model, indicating the potential possibility of combining CARMIL1 with sorafenib in hepatocellular carcinoma treatment. In summary, CARMIL1 promotes liver cancer cell proliferation by regulating the TRIM27/p53 axis and activating the ERK/mTOR pathway.

Recent Advances in IRAK1: Pharmacological and Therapeutic Aspects

Interleukin receptor-associated kinase (IRAK) proteins are pivotal in interleukin-1 and Toll-like receptor-mediated signaling pathways. They play essential roles in innate immunity and inflammation. This review analyzes and discusses the physiological functions of IRAK1 and its associated diseases. IRAK1 is involved in a wide range of diseases such as dry eye, which highlights its potential as a therapeutic target under various conditions. Various IRAK1 inhibitors, including Pacritinib and Rosoxacin, show therapeutic potential against malignancies and inflammatory diseases. The covalent IRAK1 inhibitor JH-X-119-01 shows promise in B-cell lymphomas, emphasizing the significance of covalent bonds in its activity. Additionally, the emergence of selective IRAK1 degraders, such as JNJ-101, provides a novel strategy by targeting the scaffolding function of IRAK1. Thus, the evolving landscape of IRAK1-targeted approaches provides promising avenues for increasingly safe and effective therapeutic interventions for various diseases.

Target prediction and potential application of dihydroartemisinin on hepatocarcinoma treatment.

With high incidence of hepatocarcinoma and limited effective treatments, most patients suffer in pain. Antitumor drugs are single-targeted, toxicity, causing adverse side effects and resistance. Dihydroartemisinin (DHA) inhibits tumor through multiple mechanisms effectively. This study explores and evaluates safety and potential mechanism of DHA towards human hepatocarcinoma based on network pharmacology in a comprehensive way. Adsorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of DHA were evaluated with pkCSM, SwissADME, and ADMETlab. Potential targets of DHA were obtained from SwissTargetPrediction, Drugbank, TargetNET, and PharmMapper. Target gene of hepatocarcinoma was obtained from OMIM, GeneCards, and DisGeNET. Overlapping targets and hub genes were identified and analyzed for Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathway. Molecular docking was utilized to investigate the interactions sites and hydrogen bonds. Cell counting kit-8 (CCK8), wound healing, invasion, and migration assays on HepG2 and SNU387 cell proved DHA inhibits malignant biological features of hepatocarcinoma cell. DHA is safe and desirable for clinical application. A total of 131 overlapping targets were identified. Biofunction analysis showed targets were involved in kinase activity, protein phosphorylation, intracellular reception, signal transduction, transcriptome dysregulation, PPAR pathway, and JAK-STAT signaling axis. Top 9 hub genes were obtained using MCC (Maximal Clique Centrality) algorithm, namely CDK1, CCNA2, CCNB1, CCNB2, KIF11, CHEK1, TYMS, AURKA, and TOP2A. Molecular docking suggests that all hub genes form a stable interaction with DHA for optimal binding energy were all less than - 5kcal/mol. Dihydroartemisinin might be a potent and safe anticarcinogen based on its biological safety and effective therapeutic effect.

TAZ is involved in breast cancer cell migration via regulating actin dynamics.

Cancer metastasis is dependent on cell migration. Several mechanisms, including epithelial-to-mesenchymal transition (EMT) and actin fiber formation, could be involved in cancer cell migration. As a downstream effector of the Hippo signaling pathway, transcriptional coactivator with PDZ-binding motif (TAZ) is recognized as a key mediator of the metastatic ability of breast cancer cells. We aimed to examine whether TAZ affects the migration of breast cancer cells through the regulation of EMT or actin cytoskeleton. MCF-7 and MDA-MB-231 cells were treated with siRNA to attenuate TAZ abundance. Transwell migration assay and scratch wound healing assay were performed to study the effects of TAZ knockdown on cancer cell migration. Fluorescence microscopy was conducted to examine the vinculin and phalloidin. Semiquantitative immunoblotting and quantitative real-time PCR were performed to study the expression of small GTPases and kinases. Changes in the expression of genes associated with cell migration were examined through next-generation sequencing. TAZ-siRNA treatment reduced TAZ abundance in MCF-7 and MDA-MB-231 breast cancer cells, which was associated with a significant decrease in cell migration. TAZ knockdown increased the expression of fibronectin, but it did not exhibit the typical pattern of EMT progression. TGF-β treatment in MDA-MB-231 cells resulted in a reduction in TAZ and an increase in fibronectin levels. However, it paradoxically promoted cell migration, suggesting that EMT is unlikely to be involved in the decreased migration of breast cancer cells in response to TAZ suppression. RhoA, a small Rho GTPase protein, was significantly reduced in response to TAZ knockdown. This caused a decrease in the expression of the Rho-dependent downstream pathway, i.e., LIM kinase 1 (LIMK1), phosphorylated LIMK1/2, and phosphorylated cofilin, leading to actin depolymerization. Furthermore, myosin light chain kinase (MLCK) and phosphorylated MLC2 were significantly decreased in MDA-MB-231 cells with TAZ knockdown, inhibiting the assembly of stress fibers and focal adhesions. TAZ knockdown inhibits the migration of breast cancer cells by regulating the intracellular actin cytoskeletal organization. This is achieved, in part, by reducing the abundance of RhoA and Rho-dependent downstream kinase proteins, which results in actin depolymerization and the disassembly of stress fibers and focal adhesions.

Rational modification of melatonin for broad-spectrum antifungal agents discovery.

Natural products, known for their environmental safety, are regarded as a significant basis for the modification and advancement of fungicides. Melatonin, as a low-cost natural indole, exhibits diverse biological functions, including antifungal activity. However, its potential as an antifungal agent has not been fully explored. In this study, a series of melatonin derivatives targeting the mitogen-activated protein kinase (Mps1) protein of fungal pathogens were synthesized based on properties of melatonin, among which the trifluoromethyl-substituted derivative Mt-23 exhibited antifungal activity against seven plant pathogenic fungi, and effectively reduced the severity of crop diseases, including rice blast, Fusarium head blight of wheat and gray mold of tomato. In particular, its EC50 (5.4 µM) against the rice blast fungus Magnaporthe oryzae is only one-fourth that of isoprothiolane (22 µM), a commercial fungicide. Comparative analyzes revealed that Mt-23 simultaneously targets the conserved protein kinase Mps1 and lipid protein Cap20. Surface plasmon resonance assays showed that Mt-23 directly binds to Mps1 and Cap20. In this study, we provide a strategy for developing antifungal agents by modifying melatonin, and the resultant melatonin derivative Mt-23 is a commercially valuable, eco-friendly and broad-spectrum antifungal agent to combat crop disease.

Macula densa mTORC1 is an important regulator of cellular and aging-dependent kidney functions

Macula densa (MD) cells are major regulators of nephron, kidney, and whole body functions, including the control of renal blood flow, glomerular filtration rate (GFR), renin release and thus the maintenance of body fluid and electrolyte balance. Our recent studies identified the high rate of MD protein synthesis regulated by the mTORC1 complex of the mechanistic target of rapamycin (mTOR) as a novel component of MD cell and glomerular hemodynamic regulatory functions. Raptor is an essential player in mTORC1’s key roles to regulate cell energy homeostasis, growth, protein synthesis, mitochondrial function, apoptosis, and autophagy via phosphorylation of substrates including ribosomal protein S6 kinase (S6K) and eukaryotic translation-initiation factor 4E-binding proteins (4E-BPs). mTORC1 signaling controls the aging process, and deficient mTORC1 activity extends health and lifespan. Therefore, we hypothesized that MD cell-specific mTOR loss of function (lof) prevents aging-related kidney function decline. The present study aimed to demonstrate the key importance of MD cell mTOR activity in renal physiology and aging by characterizing the phenotype of a newly generated genetic MD-mTOR lof mouse model. MD-mTOR lof mice were generated by crossing Nos1-CreERT2 and Raptor floxed mice. Tamoxifen induction (at 3 weeks of age) of Nos1-CreERT2+/- Raptor−/− mice resulted in conditional and inducible Raptor deletion in cells of the Nos1 lineage, which in the kidney is entirely specific to MD cells. Kidney structure/function phenotyping was performed using intravital multiphoton microscopy (MPM), the Medibeacon transdermal GFR measurement system, and tissue harvest, immunohistochemistry, and the OPP incorporation assay of protein synthesis. High and MD-specific Raptor, pS6K, and p4EBP1 immunofluorescence labeling was observed in WT kidney sections. The immunolabeling of these mTOR component and targets were absent specifically in MD cells in MD-mTOR lof mouse kidneys, validating the MD cell-specific genetic approach. The intensity of incorporated OPP labeling was highest in MD cells among all renal cell types in WT. In contrast, MD OPP labeling was significantly reduced in MD-mTOR lof mice indicating diminished, mTORC1-dependent global MD cell protein synthesis. Importantly, in 15-month old aged mice the GFR was significantly higher (preserved) in MD-mTOR lof (1794±197 uL/min/100g BW) compared to WT (1117±61) and MD-mTOR gof (929±61) mice (n=6-18 mice in each group). Altogether, these studies identified mTORC1 as an essential regulator of MD cell biology, and the importance of MD cells in aging-related kidney function decline. MD cells may be important new targets in future anti-aging therapies. DK064234, DK123564, DK135290. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

BDNF/TrkB Induces pCREBS133 Phosphorylation and Activates Mitochondrial Biogenesis in Rat Phrenic Motor Neurons

Neural control of the diaphragm muscle (DIAm) is essential to sustain breathing. During breathing, fatigue resistant DIAm motor units are recruited comprising smaller phrenic motor neurons (PhMNs) and type I and IIa muscle fibers. Larger PhMNs comprise more fatigable DIAm motor units that are infrequently active during expulsive behaviors. The difference in activation history is reflected in the higher mitochondrial volume density in smaller PhMNs. In PhMNs, brain-derived neurotrophic factor (BDNF) signals through its high-affnity receptor, tropomyosin receptor kinase B (TrkB.FL). Previously, we found that intrathecal BDNF treatment induces CREB phosphorylation at Serine 133 (pCREBs133). We hypothesized that pCREBs133 binds to the PGC1a promoter, transcriptionally targeting PGC1a expression, mediating downstream increased expression of NRF1, NRF2, and TFAM and mitochondrial biogenesis. To test this hypothesis, we used a chemosensitive TrkBF616 rat model with a knock-in allele sensitive to 1NMPP1 in that TrkB kinase is inhibited following 1NMPP1, a small molecule derivative of the protein kinase inhibitor protein phosphatase 1 that plays a significant role inhibiting the TrkB phosphorylation and activation. Systemic treatment of 1NMPP1 in TrkBF616 rats inhibits TrkB kinase activity. A bioinformatic analysis was performed to identify a putative binding site for pCREBs133 in the PGC1a promoter sequence in rats. Using a chromatin immunoprecipitation (ChIP) assay we found that pCREBS133 binds to the PGC1a promoter in rat cervical ventral horn. We showed that intrathecal BDNF treatment increased pCREBs133 phosphorylation, which was blocked by 1NMPP1 treatment in TrkBF616 rats. PGC1a, NRF1/2, and TFAM protein was compared in cervical ventral horn lysates from BDNF- and BDNF+1NMPP1-treated TrkBF616 rats by qRT-PCR and Western Blot. Relative mtDNA copy number (normalized to nuclear DNA) was quantified by qPCR to assess the mitochondrial biogenesis. All results were analyzed by paired t-test. Following 1NMPP1-induced inhibition of TrkB kinase, PGC1a, NRF1/2, and TFAM mRNA and protein expression was reduced. Consistent with these findings, mtDNA copy number was decreased following TrkB kinase inhibition by 1NMPP1. We conclude that inhibition of TrkB kinase by 1NMPP1 reduces PGC1a-mediated mitochondrial biogenesis in rat cervical ventral horn. Supported by NIH grants AG44615 and HL146114. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The IL-17 pathway intertwines with neurotrophin and TLR/IL-1R pathways since its domain shuffling origin

The IL-17 pathway displays remarkably diverse functional modes between different subphyla, classes, and even orders, yet its driving factors remains elusive. Here, we demonstrate that the IL-17 pathway originated through domain shuffling between a Toll-like receptor (TLR)/IL-1R pathway and a neurotrophin-RTK (receptor-tyrosine-kinase) pathway (a Trunk-Torso pathway). Unlike other new pathways that evolve independently, the IL-17 pathway remains intertwined with its donor pathways throughout later evolution. This intertwining not only influenced the gains and losses of domains and components in the pathway but also drove the diversification of the pathway's functional modes among animal lineages. For instance, we reveal that the crustacean female sex hormone, a neurotrophin inducing sex differentiation, could interact with IL-17Rs and thus be classified as true IL-17s. Additionally, the insect prothoracicotropic hormone, a neurotrophin initiating ecdysis in Drosophila by binding to Torso, could bind to IL-17Rs in other insects. Furthermore, IL-17R and TLR/IL-1R pathways maintain crosstalk in amphioxus and zebrafish. Moreover, the loss of the Death domain in the pathway adaptor connection to IκB kinase and stress-activated protein kinase (CIKSs) dramatically reduced their abilities to activate nuclear factor-kappaB (NF-κB) and activator protein 1 (AP-1) in amphioxus and zebrafish. Reinstating this Death domain not only enhanced NF-κB/AP-1 activation but also strengthened anti-bacterial immunity in zebrafish larvae. This could explain why the mammalian IL-17 pathway, whose CIKS also lacks Death, is considered a weak signaling activator, relying on synergies with other pathways. Our findings provide insights into the functional diversity of the IL-17 pathway and unveil evolutionary principles that could govern the pathway and be used to redesign and manipulate it.