Protein Kinase Research Articles

L-aspartate ameliorates diet-induced obesity by increasing adipocyte energy expenditure.

Obesity always leads to profound perturbation of metabolome. Metabolome studies enrich the knowledge on associations between endogenous metabolites and obesity, potentially providing innovative strategies for the development of novel anti-obesity pharmacotherapy. This study aims to identify an endogenous metabolite that regulates energy expenditure and to explore its application for obesity treatment. C57BL/6 mice were fed with a high-fat and high-cholesterol (HFC) diet, comprising 60% fat and 1.2% cholesterol, for 12 weeks to induce obesity. Significant metabolites were identified in the livers of both health and obese mice through comparative hepatic metabolomics analysis. Correlation between serum or adipose L-aspartate level and body weight in obese mice, as well as human body mass index (BMI), was evaluated. In addition, saline or 200 mg/kg L-aspartate was orally administrated to HFC diet mice and HFC diet-induced obese mice for 6-7 weeks. Body weight, adipose tissue weight, glucose tolerance and liver damage were assessed to evaluate the effect on obesity prevention and treatment. Comprehensive lab animal monitoring system (CLAMS) and seahorse assay were employed to investigate the regulatory effect of L-aspartate on energy metabolism in vivo and in vitro, respectively. 3T3-L1 preadipocytes and murine white adipose tissue (WAT) were utilized to examine the impact of L-aspartate on adipocyte adipogenesis and lipogenesis and cellular signalling pathway in vitro and in vivo. L-aspartate, an approved drug for liver injury and chronic fatigue, was identified as an endogenous inducer of energy expenditure. Serum or adipose L-aspartate levels were found to be negatively correlated with the severity of obesity in both humans and mice. Administration of L-aspartate to HFC diet mice led to a significant reduction in body weight, with decreases of 14.5% in HFC diet mice and 8.5% in HFC diet-induced obese mice, respectively. In addition, the treatment improved related metabolic syndrome (Figure 2 and Figure S3). These therapeutics were associated with enhancements in whole-body energy expenditure and suppression of adipocyte adipogenesis along with activation of Adenosine 5'-monophosphate-activated protein kinase (AMPK) signalling pathway. L-aspartate may serve as a novel endogenous inducer of energy expenditure and suppressor of adipogenesis and lipogenesis along with activation of AMPK, thereby offering a promising therapeutic strategy for obesity prevention and treatment.

Mitogen-activated protein kinase dependent presynaptic potentiation in the lateral habenula mediates depressive-like behaviors in rats.

Emerging evidence suggests that the enhanced activity of lateral habenula (LHb) is involved in depressive disorders. This abnormal potentiation of LHb neurons was shown to originate from presynaptic alterations; however, the mechanisms underlying this presynaptic enhancement and physiological consequences are yet to be elucidated. Previously, we reported that presynaptic transmission in the LHb is temporally rhythmic, showing greater activity in the afternoon than in the morning. Here, we used a learned helpless rodent model of depression to show that exposure to a stressor or incubation with the stress hormone, corticosterone, abolished the presynaptic temporal variation in the LHb. In addition, selective inhibition of mitogen-activated protein kinase (MAPK) kinase (MAPKK, MEK) activity in the LHb restored the presynaptic alteration even after stress exposure. Moreover, we observed a slight increase in phosphorylated synapsin I after stress exposure. Finally, we found that a blockade of MAPK signaling before stress exposure successfully prevented the depression-like behaviors, including behavioral despair and helplessness, in an acute learned helpless animal model of depression. Our study delineates the cellular and molecular mechanisms responsible for the abnormal presynaptic enhancement of the LHb in depression, which may mediate depressive behaviors.

Viscosity regulates cell spreading and cell-extracellular matrix interactions.

Fluid viscosity and osmolarity are among some of the underappreciated mechanical stimuli that cells can detect. Abnormal changes of multiple fluidic factors such as viscosity and osmolarity have been linked with diseases such as cystic fibrosis, cancer, and coronary heart disease. Changes in viscosity have been recently suggested as a regulator of cell locomotion. These novel studies focus on cell migration and spreading on glass substrates and through microchannels, and it remains a question whether viscosity impacts the cellular remodeling of extracellular matrices (ECMs). Here, we demonstrate that elevated viscosity induces cellular remodeling of collagen substrates and enhances cell spreading on ECM-mimetic substrates. Our results expand on recent work showing that viscosity induces increased cellular forces and demonstrates that viscosity can drive local ECM densification. Our data further show that microtubules, Ras-related C3 botulinum toxin substrate 1 (Rac1), actin-related protein 2/3 (Arp2/3) complex, Rho-associated protein kinase 1 (ROCK), and myosin are important regulators of viscosity-induced ECM remodeling. In the context of viscosity-induced cell spreading, cells cultured on glass and collagen substrates exhibit markedly different responses to pharmacological treatments, indicating that microtubules, Rac1, and Arp2/3 play distinct roles in regulating cellular spreading depending on the substrate. In addition, our results demonstrate that high osmotic pressures override viscosity-induced cell spreading by suppressing membrane ruffling. Our results demonstrate viscosity as a regulator of ECM remodeling and cell spreading in a fibrillar microenvironment. We also reveal a complex interplay between viscosity and osmolarity. We anticipate that our research can pave the way for future investigations into the crucial roles played by viscosity in both physiological and pathological conditions.

TMET-34. PYROPTOSIS IS AN ACQUIRED VULNERABILITY OF BRAIN METASTASES

Abstract Metastasis, the spread of cancer cells from one part of the body to another, is a leading cause of death among cancer patients. Three particularly dangerous forms of metastases are the spread of lung, skin, and breast cancers to the brain. On average survival time of all brain metastasis (BM) patients is around 4 months, which is accompanied by a failing and undefined standard of care. We have strived to bridge the gap between novel safe therapeutics and brain metastasis patients by discovering and targeting metabolic vulnerabilities in brain metastases. Here, we conducted comparative metabolomic screening in patient-derived BM lines versus normal brain cells to identify dihydroorotate dehydrogenase (DHODH) as a cancer-selective targetable vulnerability. DHODH is an essential enzyme that is located within the inner membrane of the mitochondria which has a primary role in producing pyrimidine metabolites through the de novo uridine synthesis pathway and also contributes to the proper functioning of the electron transport chain. Through this, DHODH has been implicated in the essential functioning of energy metabolism, DNA and RNA synthesis, and most recently in the modulation of the immune system. In our studies we have elucidated the significant effect DHODH inhibitors and the induction of DHODH CRISPR knock-out has against the cell viability, sphere formation, and proliferation of patient derived BM samples in vitro and in vivo. Furthermore, through metabolomics we have discovered that following DHODH perturbation, there is a significant increase in a signaling lipid (D-ethryo-Dihydrosphingosine) that specifically inhibits Protein Kinase C (PKC) and initiates Gasdermin E (GSDME) mediated pyroptosis. By uncovering this mechanism we have discovered GSDME mediated pyroptosis is an aquired vulnerability of brain metastases. Overall, this study highlights the translational potential of DHODH inhibitors in BM and elucidates novel mechanistic underpinnings of nucleotide deprivation within the context of cell death.

DNAR-10. PHASE I CLINICAL TRIAL OF PEPOSERTIB PLUS RADIOTHERAPY IN ADULTS WITH NEWLY-DIAGNOSED MGMT-UNMETHYLATED GLIOBLASTOMA

Abstract BACKGROUND DNA-dependent protein kinase (DNA-PK) is an attractive target in GBM because it promotes unwanted DNA repair and renders cancer cells less vulnerable to DNA damaging agents, such as radiotherapy (RT). Peposertib, a small molecule inhibitor of DNA-PK, has demonstrated pre-clinical efficacy in sensitizing GBM to RT, resulting in tumor regression. METHODS 21 patients with newly-diagnosed MGMT-unmethylated GBM received peposertib plus 6-weeks of RT to determine the maximum-tolerated dose (MTD) based on the Bayesian Optimal Interval design. The dose-limiting toxicity (DLT) period was 10-weeks. Patients received at least 6-cycles of adjuvant temozolomide (NCT04555577). Single-nuclei RNA-sequencing (snRNA-Seq) and spatial transcriptomics (Visium, 10x Genomics) were performed on matched pair primary-recurrent tumors and controls, incorporating tumor cell state and cell type definitions by automated cluster classification and differential expression of marker genes. RESULTS The MTD of peposertib in combination with RT was 300mg per RT fraction day. One DLT (G3 radiation necrosis @300mg) was observed. After a median follow-up of 15.4 months (interquartile-range 8.9-18.5), mPFS was 10.8 months (95%CI 8.33-NR) and mOS was 22.9 months (95%CI 16-NR). 6/21 (29%) patients have undergone re-resection to date. snRNA-Seq on 102,709 cells from 12 primary and 3 recurrent tumors revealed that pre-treatment tumor cell states were progenitor-like, while treatment with peposertib plus RT was associated with differentiated and inflammatory malignant cell states, along with macrophage infiltration. Spatial transcriptomics of 8 matched primary-recurrent tumors (20,793 transcriptomes) confirmed malignant cell differentiation and enrichment of pro-inflammatory macrophages after inhibition of DNA-PK. 5 patients will be enrolled into a window-of-opportunity expansion cohort to evaluate intratumoral drug concentration. CONCLUSION The combination of peposertib and intracranial RT was safe. Survival data for this MGMT-unmethylated GBM population was promising. Single nuclei and spatial transcriptomic analyses revealed enrichment of pro-inflammatory macrophages in the tumor microenvironmentafter DNA-PK inhibition. The study was supported by EMD-Serono (CrossRef Funder ID: 10.13039/100004755).

CSIG-22. PHARMACOLOGIC INHIBITION AND KNOCKDOWN OF CAMK2 IMPAIR GROWTH OF PATIENT-DERIVED GLIOBLASTOMA CULTURES

Abstract INTRODUCTION Glioblastoma (GBM), a primary brain malignancy with a median survival of 15-18 months, presents a pressing challenge in neuro-oncology. Patient-derived GBM cultures (PDGCs) exhibit spontaneous calcium (Ca2+) waves, which are thought to drive tumor growth and represent a therapeutic target. The mechanisms underlying the generation and the effectors of these Ca2+ waves remain elusive. We hypothesize CAMK2, a Ca2+/calmodulin-dependent protein kinase expressed as four distinct isoforms, is a key effector of such Ca2+ waves by phosphorylating substrates that promote tumor growth. METHODS Using multiple PDGCs, CAMK2 isoform-specific mRNA levels were assessed via qRT-PCR both at baseline and after shRNA-mediated knockdown (KD). Tumorsphere formation and WST-8 assays were performed to assess PDGC clonogenic potential after either KD or pharmacologic inhibition with the cell-permeable CAMK2 inhibitor KN93. RESULTS The relative mRNA levels of CAMK2 isoforms across three PDGC lines were, in decreasing order: CAMK2D, CAMK2G, CAMK2B, and CAMK2A. Expression of CAMK2D- and CAMK2B-specific shRNAs significantly reduced tumorsphere growth compared to control shRNA. CAMK2D KD reduced sphere formation by 55.13 ± 3.54% (p=0.0001) and 47.72 ± 9.55% (p=0.008) in separate PDGC lines, while CAMK2B KD reduced sphere formation by 41.15 ± 11.51% (p=0.021) and sphere size by 41.78% ± 10.50% (p=0.049) (n=3/experiment). KN93 treatment reduced tumorsphere formation by 59.23 ± 1.40% (p=0.0006) and 42.94 ± 13.0% (p=0.032) in two PDGC lines (n=3) and PDGC viability, while CAMK2-inactive analogue KN92 and DMSO minimally impacted viability (p<0.0001, two-way ANOVA). CONCLUSION CAMK2D and CAMK2B KD, as well as KN93 treatment, reduce tumorsphere formation, suggesting CAMK2 has a tumorigenic role in GBM. Further investigation of CAMK2 isoforms and in vivo validation are underway. Future aims include directly testing the link between spontaneous Ca2+ waves and CAMK2 activation and identifying CAMK2 phosphorylation targets that mediate tumor growth.

HUCMSCs Regulate Bile Acid Metabolism to Prevent Heart Failure–Induced Intestinal Injury by Inhibiting the Activation of the STAT3/NF‐κB/MAPK Signaling Pathway via TGR5

ABSTRACTThe protective effects of human umbilical cord mesenchymal stem cells (hUCMSCs) on heart failure (HF)‐induced intestinal injury have not been fully understood. Flow cytometry and immunofluorescence analysis revealed that hUCMSCs renewed themselves, grew, and transformed into various cell types. Meanwhile, hUCMSCs safeguarded against intestinal damage, regulated imbalances in the intestinal flora and bile acid metabolism, and enhanced the levels of hyodeoxycholic acid (HDCA) in pigs with HF. HDCA protected against HF‐induced intestinal injury in mice through Takeda G protein–coupled receptor 5 (TGR5). Protein analysis showed that HDCA exerted protective effects on the intestines via the signal transducer and activator of transcription 3 (STAT3)/nuclear factor kappa B (NF‐κB)/mitogen‐activated protein kinase (MAPK) signaling pathway. Mouse experiments revealed that HDCA bound to TGR5 to inhibit MAPK and NF‐κB signaling pathway activation, which relies on the STAT3 signaling pathway. Moreover, hUCMSCs protected against intestinal injury in the pig model of HF by suppressing the activation of the STAT3/NF‐κB/MAPK signaling pathway via TGR5.

Exploring phytochemical inhibitors of protein kinase C alpha for therapeutic targeting of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by neurodegeneration linked to amyloid-β (Aβ) plaques and tau protein tangles. Protein kinase C alpha (PKCα) plays a crucial role in modulating amyloid-β protein precursor (AβPP) processing, potentially mitigating AD progression. Consequently, PKCα stands out as a promising target for AD therapy. Despite the identification of numerous inhibitors, the pursuit of more effective and precisely targeted PKCα inhibitors remains crucial. In this study, we employed an integrated virtual screening approach of molecular docking and molecular dynamics (MD) simulations to identify phytochemical inhibitors of PKCα from the IMPPAT database. Molecular docking screening via InstaDock identified compounds with strong binding affinities to PKCα. Subsequent ADMET and PASS analyses filtered out compounds with favorable pharmacokinetic profiles. Interaction analysis using Discovery Studio Visualizer and PyMOL further elucidated binding conformations of selected compounds with PKCα. Top hits underwent 200 ns MD simulations using GROMACS to validate stability of the interactions. Finally, we propose two phytochemicals, Kammogenin and Imperialine, with appreciable drug-likeliness and binding potential with PKCα. Taken together, the findings suggest Kammogenin and Imperialine as potential PKCα inhibitors, highlighting their therapeutic promise for AD after further validation.

Knockdown of hepatic mitochondrial calcium uniporter mitigates MASH and fibrosis in mice

BackgroundMitochondrial calcium uniporter (MCU) plays pleiotropic roles in cellular physiology and pathology that contributes to a variety of diseases, but the role and potential mechanism of MCU in the pathogenesis of metabolic dysfunction-associated steatohepatitis (MASH) remain poorly understood.Methods and resultsHere, hepatic knockdown of MCU in C57BL/6J mice was achieved by tail vein injection of AAV8-mediated the CRISPR/Cas9. Mice were fed a Choline-deficient, L-amino acid-defined high-fat diet (CDAHFD) for 8 weeks to induce MASH and fibrosis. We find that expression of MCU enhanced in MASH livers of humans and mice. MCU knockdown robustly limits lipid droplet accumulation, steatosis, inflammation, and hepatocyte apoptotic death during MASH development both in vivo in mice and in vitro in cellular models. MCU-deficient mice strikingly mitigate MASH-related fibrosis. Moreover, the protective effects of MCU knockdown against MASH progression are accompanied by a reduced level of mitochondrial calcium, limiting hepatic oxidative stress, and attenuating mitochondrial dysfunction. Mechanically, RNA sequencing analysis and protein immunoblotting indicate that knockdown MCU inhibited the Hippo/YAP pathway activation and restored the AMP-activated protein kinase (AMPK) activity during MASH development both in vitro and in vivo.ConclusionsMCU is up-regulated in MASH livers in humans and mice; and hepatic MCU knockdown protects against diet-induced MASH and fibrosis in mice. Thus, targeting MCU may represent a novel therapeutic strategy for MASH and fibrosis.Graphical

Mechanistic Insights into Influenza A Virus-Induced Cell Death and Emerging Treatment Strategies

Influenza A virus (IAV) infection initiates a complex interplay of cell death modalities, including apoptosis, necroptosis, pyroptosis, and their integration, known as PANoptosis, which significantly impacts host immune responses and tissue integrity. These pathways are intricately regulated by viral proteins and host factors, contributing to both viral clearance and pathogenesis-related tissue damage. This review comprehensively explores the molecular mechanisms underlying these cell death processes in influenza infection. We highlight the roles of key regulatory proteins, such as ZBP1 (Z-DNA binding protein 1) and RIPK3 (receptor-interacting protein kinase 3), in orchestrating these responses, emphasizing the dual roles of cell death in both antiviral defense and tissue injury. Furthermore, we discuss emerging therapeutic strategies targeting these pathways, aiming to enhance antiviral efficacy while minimizing collateral tissue damage. Future research should focus on targeted approaches to modulate cell death mechanisms, aiming to reduce tissue damage and improve clinical outcomes for patients with severe influenza.

Emerging Functions of Protein Tyrosine Phosphatases in Plants

Reversible protein phosphorylation, known as the “switch” of the cell, is controlled by protein kinases (PKs) and protein phosphatases (PPs). Based on substrate specificity, PPs are classified into protein serine/threonine phosphatases and protein tyrosine phosphatases (PTPs). PTPs can dephosphorylate phosphotyrosine and phosphoserine/phosphothreonine. In plants, PTPs monitor plant physiology, growth, and development. This review summarizes an overview of the PTPs’ classification and describes how PTPs regulate various plant processes, including plant growth and development, plant hormone responses, and responses to abiotic and biotic stresses. Then, future research directions on the PTP family in plants are discussed. This summary will serve as a reference for researchers studying PTPs in plants.

Establishment of potential lncRNA-related hub genes involved competitive endogenous RNA in lung adenocarcinoma

Long non-coding RNAs (lncRNAs) have a notable role in the diagnosis and prognosis of cancer. However, the associations between lncRNA-related hub genes (LRHGs) expression and the corresponding outcomes have not been fully understood in lung adenocarcinoma (LUAD). Here, a total of 71 patients diagnosed with LUAD and 60 healthy volunteers at The First Affiliated Hospital of Huzhou University from April, 2023 to December, 2023 were enrolled in the present study. A LRHGs model was established using least absolute shrinkage and selection operator analyses of The Cancer Genome Atlas-LUAD datasets. The underlying mechanisms of the LRHGs were investigated via Gene Set Enrichment Analysis and Gene Set Variation Analysis. Additionally, the diagnostic role of serum HOXD cluster antisense RNA 2 (HOXD-AS2) was assessed by receiver operating characteristic (ROC) curve analysis. Lastly, TCGA-LUAD samples were divided into high- and low-HOXD-AS2 expression groups based on the median expression. The associations between HOXD-AS2 expression and miR-4538 as well as Calmodulin-Dependent Protein Kinase Type II subunit Beta (CAMK2B) levels were conducted through Pearson correlation analysis. A comprehensive analysis identified 141 differentially expressed lncRNAs between 539 LUAD tissues and 59 normal samples. A prognostic marker for overall survival was established by constructing a predictive signature consisting of 9 LRHGs. Subsequently, 474 LUAD samples were categorized into a high or low-risk group based on the median of the risk score. An independent prognostic model was constructed to confirm the validity of this categorization. Further comparisons of the clinicopathological features and LRHG-related pathways were performed between the two groups. Examinations of LRHG expression in two LUAD clusters and of the association between LRHG expression and immune infiltration were also conducted. HOXD-AS2 expression was shown to be elevated in LUAD tissues compared with matched normal tissues, and the serum HOXD-AS2 level was also notably increased in LUAD samples compared with healthy controls. The results of the ROC analysis indicated that the sensitivity and specificity of HOXD-AS2 were higher than that of cytokeratin-19 fragment (CYFRA21-1), which is a serum marker for LUAD. Pearson analyses indicated that miR-4538 level was negatively associated with HOXD-AS2 expression, but CAMK2B level showed positive correlation in LUAD. The results of the present study therefore indicated that the constructed LRHG model, particularly HOXD-AS2, could independently diagnose and predict the prognosis of LUAD, which suggested the underlying mechanism of the HOXD-AS2/miR-4538/CAMK2B, and might offer efficient strategies for LUAD treatment.

Genome-wide identification and biochemical characterization of glycoside hydrolase gene family members in Tilletia Horrida.

Rice kernel smut, caused by Tilletia horrida, is becoming an increasingly serious disease in hybrid rice planting, leading to production losses and quality decline of male-sterile rice varieties. Successful infection requires an efficient energy source that the pathogen obtains from rice plants, such as carbohydrates. Glycoside hydrolases (GHs), one of the largest sub-families in the cell wall-degrading enzyme family, play a key role in the infection progress of pathogens. To investigate their roles in facilitating infection, in this study, we identified and characterized genes encoding GH family proteins of T. horrida and further explored the functions and structures of these genes. Through genome-wide sequencing and bioinformatics analyses, 52 GH genes were identified from T. horrida, named ThGhd_1 to ThGhd_52. The subcellular location, conserved motifs, and structures of ThGhds were identified by bioinformatics analyses. Phylogenetic analysis revealed that ThGhds with similar domains clustered together, although some proteins clustered in different branches, which might reflect functional diversity. Protein-protein interaction network analysis revealed that ThGhds interact with partner proteins involved in reactive oxygen species signaling, protein kinase activity, and plant hormone signal transduction pathways. RNA-sequencing analysis showed that the expression of ThGhd genes responded differently at different infection time points, with dynamic changes detected during the T. horrida infection process, indicating that these genes are involved in interactions with rice and have potential roles in pathogenic mechanisms. The results of this study provide valuable resources for the structure elucidation of GH family proteins of T. horrida and can help to further elucidate their roles in pathogenesis.

Gastrodin ameliorates diabetic nephropathy by activating the AMPK/Nrf2 pathway.

Diabetic nephropathy (DN) is a leading cause of end-stage kidney failure, contributing to elevated morbidity and mortality rates in individuals with diabetes. Despite its potential renoprotective effects, the molecular mechanism by which gastrodin (GSTD) impacts DN remains unclear. To investigate this, mice were initially induced with DN via intraperitoneal streptozotocin (STZ) injection (50mg/kg) and subsequently treated with varying doses of GSTD (5, 10, 20mg/kg). Furthermore, the potential molecular mechanism of GSTD in mitigating DN was explored in vivo in conjunction with compound C, an inhibitor of 5'-AMP-activated protein kinase (AMPK). Subsequently, the blood weight, fasting blood glucose levels, and renal injury markers of DN-afflicted mice were assessed. Additionally, renal tissues were subjected to quantitative reverse-transcriptase-polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) to evaluate inflammatory factor levels, colorimetric assays to measure renal malondialdehyde (MDA) levels, and immunoblotting analysis to examine AMPK/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. The results demonstrated that a 6-week GSTD regimen effectively improved metabolic manifestations associated with DN, including reductions in fasting blood glucose levels, 24-hour urine output, renal indices, amelioration of glomerular histopathological abnormalities, diminished glycogen accumulation, and fibrosis. Furthermore, DN-afflicted renal tissues exhibited decreased MDA levels and elevated expression of AMPK/Nrf2 pathway-associated proteins. The beneficial effects of GSTD on DN and its protein modulation were reversed upon co-intervention with compound C. Together, our findings imply that GSTD improves DN by activating the AMPK/Nrf2 pathway, thereby mitigating STZ-induced renal damage, inflammatory responses, and oxidative stress.

Cannabidiol Enhances Mitochondrial Metabolism and Antioxidant Defenses in Human Intestinal Epithelial Caco-2 Cells

Background: The reintroduction of hemp production has resulted in increased consumption of cannabidiol (CBD) products, particularly CBD oil, yet their effects on intestinal health are not fully understood. Proper mitochondrial function and antioxidant defenses are vital for maintaining the intestinal epithelial barrier. AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor gamma coactivator (PGC)1α are key mediators of mitochondrial metabolism. Methods & Results: Using Caco-2 cells, we found that CBD oil promoted AMPK phosphorylation, upregulated differentiation markers, and enhanced PGC1α/SIRT3 mitochondrial signaling. CBD oil reduced reactive oxygen species production and increased antioxidant enzymes. Moreover, CBD oil also increased levels of citrate, malate, and succinate—key metabolites of the tricarboxylic acid cycle—alongside upregulation of pyruvate dehydrogenase and isocitrate dehydrogenase 1. Similarly, pure CBD induced metabolic and antioxidant signaling. Conclusions: CBD enhances mitochondrial metabolic activity and antioxidant defense in Caco-2 cells, making it a promising candidate for treating intestinal dysfunction.

Design, Synthesis, and Evaluation of EA-Sulfonamides and Indazole-Sulfonamides as Promising Anticancer Agents: Molecular Docking, ADME Prediction, and Molecular Dynamics Simulations

New EA-sulfonamides and indazole-sulfonamides were synthesized, characterized, and evaluated for their anticancer activities. The target compound structures were elucidated using various spectroscopic techniques such as NMR-{1H and 13C}, infrared spectroscopy, and high-resolution mass spectrometry. The anticancer activities of the novel compounds were evaluated against four human cancer cell lines, namely A-549, MCF-7, Hs-683, and SK-MEL-28 as well as the normal cell line HaCaT, using 5-fluorouracil and etoposide as reference drugs. Among the tested compounds, 9, 10, and 13 exhibited potent anticancer activities which are better than or similar to the reference compounds 5-fluorouracil and etoposide, against the A-549, MCF-7, and Hs-683 cancer cell lines, with IC50 values ranging from 0.1 to 1 μM. Molecular docking studies of compounds 9, 10, and 13 showed a strong binding with selected protein kinase targets, which are linked to the tested cancer types. Furthermore, the analysis of the molecular dynamics simulation results demonstrated that compound 9 exhibits significant stability when bound to both JAK3 and ROCK1 kinases. This new compound has the potential to be developed as a novel therapeutic agent against various cancers.

Halloysite Nanotube-Based Delivery of Pyrazolo[3,4-d]pyrimidine Derivatives for Prostate and Bladder Cancer Treatment

Background/Objectives: The development of therapies targeting unregulated Src signaling through selective kinase inhibition using small-molecule inhibitors presents a significant challenge for the scientific community. Among these inhibitors, pyrazolo[3,4-d]pyrimidine heterocycles have emerged as potent agents; however, their clinical application is hindered by low solubility in water. To overcome this limitation, some carrier systems, such as halloysite nanotubes (HNTs), can be used. Methods: Herein, we report the development of HNT-based nanomaterials as carriers for pyrazolo[3,4-d]pyrimidine molecules. To achieve this objective, the clay was modified by two different approaches: supramolecular loading into the HNT lumen and covalent grafting onto the HNT external surface. The resulting nanomaterials were extensively characterized, and their morphology was imaged by high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM). In addition, the kinetic release of the molecules supramolecularly loaded into the HNTs was also evaluated. QSAR studies were conducted to elucidate the physicochemical and pharmacokinetic properties of these inhibitors, and structure-based virtual screening (SBVS) was performed to analyze their binding poses in protein kinases implicated in cancer. Results: The characterization methods demonstrate successful encapsulation of the drugs and the release properties under physiological conditions. Furthermore, QSAR studies and SBVS provide valuable insights into the physicochemical, pharmacokinetic, and binding properties of these inhibitors, reinforcing their potential efficacy. Conclusions: The cytotoxicity of these halloysite-based nanomaterials, and of pure molecules for comparison, was tested on RT112, UMUC3, and PC3 cancer cell lines, demonstrating their potential as effective agents for prostate and bladder cancer treatment.

Representing and Quantifying Conformational Changes of Kinases and Phosphatases Using the TSR-Based Algorithm

Protein kinases and phosphatases are key signaling proteins and are important drug targets. An explosion in the number of publicly available 3D structures of proteins has been seen in recent years. Three-dimensional structures of kinase and phosphatase have not been systematically investigated. This is due to the difficulty of designing structure-based descriptors that are capable of quantifying conformational changes. We have developed a triangular spatial relationship (TSR)-based algorithm that enables a unique representation of a protein’s 3D structure using a vector of integers (keys). The main objective of this study is to provide structural insight into conformational changes. We also aim to link TSR-based structural descriptors to their functions. The 3D structures of 2527 kinases and 505 phosphatases are studied. This study results in several major findings as follows: (i) The clustering method yields functionally coherent clusters of kinase and phosphatase families and their superfamilies. (ii) Specific TSR keys are identified as structural signatures for different types of kinases and phosphatases. (iii) TSR keys can identify different conformations of the well-known DFG motif of kinases. (iv) A significant number of phosphatases have their own distinct DFG motifs. The TSR keys from kinases and phosphatases agree with each other. TSR keys are successfully used to represent and quantify conformational changes of CDK2 upon the binding of cyclin or phosphorylation. TSR keys are effective when used as features for unsupervised machine learning and for key searches. If discriminative TSR keys are identified, they can be mapped back to atomic details within the amino acids involved. In conclusion, this study presents an advanced computational methodology with significant advantages in not only representing and quantifying conformational changes of protein structures but also having the capability of directly linking protein structures to their functions.

The Knob Domain of the Fiber-1 Protein Affects the Replication of Fowl Adenovirus Serotype 4

Fowl adenovirus serotype 4 (FAdV-4) outbreaks have caused significant economic losses in the Chinese poultry industry since 2015. The relationships among viral structural proteins in infected hosts are relatively unknown. To explore the role of different parts of the fiber-1 protein in FAdV-4-infected hosts, we truncated fiber-1 into fiber-1-Δ1 (73–205 aa) and fiber-1-Δ2 (211–412 aa), constructed pEF1α-HA-fiber-1-Δ1 and pEF1α-HA-fiber-1-Δ2 and then transfected them into leghorn male hepatocyte (LMH) cells. After FAdV-4 infection, the roles of fiber-1-Δ1 and fiber-1-Δ2 in the replication of FAdV-4 were investigated, and transcriptome sequencing was performed. The results showed that the fiber-1-Δ1 and fiber-1-Δ2 proteins were the shaft and knob domains, respectively, of fiber-1, with molecular weights of 21.4 kDa and 29.6 kDa, respectively. The fiber-1-Δ1 and fiber-1-Δ2 proteins were mainly localized in the cytoplasm of LMH cells. Fiber-1-Δ2 has a greater ability to inhibit FAdV-4 replication than fiber-1-Δ1, and 933 differentially expressed genes (DEGs) were detected between the fiber-1-Δ1 and fiber-1-Δ2 groups. Functional analysis revealed these DEGs in a variety of biological functions and pathways, such as the phosphoinositide 3-kinase–protein kinase b (PI3K–Akt) signaling pathway, the mitogen-activated protein kinase (MAPK) signaling pathway, cytokine–cytokine receptor interactions, Toll-like receptors (TLRs), the Janus tyrosine kinase–signal transducer and activator of transcription (Jak–STAT) signaling pathway, the nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) signaling pathway, and other innate immune pathways. The mRNA expression levels of type I interferons (IFN-α and INF-β) and proinflammatory cytokines (IL-1β, IL-6 and IL-8) were significantly increased in cells overexpressing the fiber-1-Δ2 protein. These results demonstrate the role of the knob domain of the fiber-1 (fiber-1-Δ2) protein in FAdV-4 infection and provide a theoretical basis for analyzing the function of the fiber-1 protein of FAdV-4.

Intercellular junction-driven stromal cell stacking in a confined 3D microcavity.

Understanding the detailed mechanisms driving fibroblast migration within native tissue settings during pathophysiological events presents a critical research challenge. In this study, we elucidate how stromal cells migrate and contribute to the development of three-dimensional (3D) cellular aggregates within confined microcavities. Integrin α5β1 and β-catenin (β-cat) are central in guiding this collective migration and achieving optimal filling of the microcavity. When β-cat is suppressed, cells tend to migrate more sporadically, leading to less efficient cellular organization. Furthermore, we also detail the pivotal roles of Cx43 and N-cadherin (N-cad) in orchestrating collective migration and in shaping efficient cellular stacking. Suppressing gap junctions, especially Cx43, significantly impacts the extracellular matrix expression, integrin α5 and β1, and other elements in the 3D construct, emphasizing the importance of physicochemical cell-cell interactions. The distribution patterns of N-cad and focal adhesion kinase (FAK) further corroborate the essential roles in forming cell-cell junctions and FAK in establishing the foundational layer that underpins the cell stacking within the microcavity. Interestingly, neither Rho-associated protein kinase (ROCK) nor RhoA significantly alter the cell migration pattern toward microcavity. These findings provide fresh perspectives on fibroblast activities in 3D space, enriching our understanding and offering implications for advancements in wound healing and tissue engineering.