Activation Of Kinase Research Articles

Activation of spleen tyrosine kinase (SYK) contributes to neuronal pyroptosis and cognitive impairment in diabetic mice via the NLRP3/Caspase-1/GSDMD signaling pathway.

Diabetes mellitus (DM) patients are at increased risk of cognitive impairment. The precise mechanisms underlying the association between DM and cognitive impairment remain unclear. Spleen tyrosine kinase (SYK), a crucial regulator of signal transduction, has been implicated in microglial pyroptosis in experimental ischemic stroke models. The present study investigated the potential role of SYK in DM-associated cognitive impairment. Diabetes was induced by streptozotocin (STZ) in C57BL/6 mice, and cognitive function and cerebral injury were assessed 12weeks later using the Morris water maze (MWM), TUNEL assay and Western blotting. In vitro, the inhibition of SYK was investigated in a mouse hippocampal neuronal cell line cultured with high glucose. Compared with control mice, DM mice presented impaired spatial learning and memory. Additionally, SYK activation was linked to neuronal pyroptosis, as evidenced by increases in the number of TUNEL-positive cells and protein levels of NLRP3, ASC, procaspase-1, caspase-1, GSDMD, the GSDMD N-terminal fragment, pro-IL-1β, and IL-1β in the hippocampus of DM mice. Compared with no treatment, SYK knockdown markedly attenuated cognitive impairment and histologic and ultrastructural pathological changes in the hippocampus of DM mice. The increased expression of pyroptosis-associated proteins and the increased number of TUNEL-positive cells were also significantly reduced. In vitro, high glucose significantly activated SYK to trigger the canonical pyroptotic pathway in cultured HT22 cells. The inhibition of SYK with a small interfering RNA or specific inhibitor significantly ameliorated the neuronal pyroptosis mediated by high glucose. Our findings demonstrate that SYK activation plays a pivotal role in promoting the cognitive impairment associated with DM. This effect is mediated by triggering neuronal pyroptosis through the canonical NLRP3/Caspase-1/GSDMD pathway. These results suggest that SYK may serve as a potential target for preventing or mitigating cognitive impairment in patients with DM.

Current Concepts in the Molecular Mechanisms and Management of Diabetic Neuropathy by Pharmacotherapeutics and Natural Compounds.

One of the most crippling effects of diabetes mellitus is diabetic neuropathy, which can cause discomfort, loss of movement, and even amputation. Diabetic neuropathy manifests in a variety of ways, ranging from pain to death. Diagnosing diabetic neuropathy can be challenging since it often goes unnoticed for many years following the onset of diabetes. In addition to oxidative stress in neurons, hyperglycemia activates a number of metabolic pathways that are important sources of damage and possible targets for treatment in diabetic neuropathy. Downstream metabolic cascades caused by prolonged hyperglycemia include activation of protein kinase C, increased production of advanced glycation end products, excessive release of cytokines, increased oxidative stress, and injury to peripheral nerves. Despite the fact that these metabolic anomalies are considered the main cause of diabetes-related microvascular issues, the diverse mechanistic processes of neuropathy are characterized by organ-specific histological and biochemical features. Although the symptoms of diabetic neuropathy can be treated, there are few options to correct the underlying problem. Diabetic neuropathy exerts a tremendous financial, psychological, and physical burden on society, emphasizing the need for efficient and focused treatment. The major goal of this review is to shed light on the multiple mechanisms and pathways that contribute to the onset of diabetic neuropathy and to provide readers with a comprehensive understanding of emerging therapeutic strategies to postpone or reverse various forms of diabetic neuropathy. The article discusses available medications and provides the latest guidelines for the treatment of pain and distal symmetric polyneuropathy, including diabetic autonomic neuropathy, which may help the patients control pain well and assess alternatives for treatment that might be more successful in preventing or delaying the course of a disease.

Inhibition of peritendinous adhesion through targeting JAK2-STAT3 signaling pathway: The therapeutic potential of AG490

Peritendinous adhesion is a common complication following tendon injury repair, posing a significant clinical challenge that requires urgent attention. The primary cause of peritendinous adhesion is the excessive deposition of collagen matrix due to the abnormal proliferation of fibroblasts in an inflammatory state. Janus kinase2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) are key signaling molecules involved in cell proliferation and fibrosis development in various organs. However, the role of the JAK-2 and STAT3 signaling pathways in peritendinous adhesion fibrosis remains unclear. In our study, we first observed upregulation of p-JAK2 and p-STAT3 proteins in human peritendinous adhesion specimens and rat peritendinous adhesion models. In vitro, the JAK2/STAT3 pathway inhibitor AG490 effectively inhibited TGF-β1-induced fibroblast proliferation. Wound healing and transwell assays demonstrated that AG490 suppressed TGF-β1-induced fibroblast migration. Furthermore, we found that AG490 decreased the expression of pro-inflammatory factors, including IL-1β and TNF-α, as well as extracellular matrix (ECM) proteins in fibroblasts under TGF-β1 stimulation. In vivo, histological staining showed that AG490 prevented fibrous tissue formation in a rat model of tendon injury. Moreover, AG490 inhibited the overexpression of pro-inflammatory factors IL-1β and TNF-α, as well as ECM in the peritendinous adhesions. In conclusion, AG490 inhibited fibrosis and inflammation in injured tendons by targeting the JAK2-STAT3 signaling pathway, presenting a promising strategy for the prophylaxis of peritendinous adhesions.

The OXI1 kinase regulates plant immunity by linking microbial pattern-induced ROS burst to MAPK activation.

Plant cell-surface-localized pattern recognition receptors (PRRs) recognize microbial patterns and activate pattern-triggered immunity (PTI). Typical PTI responses include reactive oxygen species (ROS) burst controlled by the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOGUE D (RbohD) and activation of the mitogen-activated protein kinase (MAPK) cascade composed of MAPKKK3/5-MKK4/5-MPK3/6. However, the mechanisms through which PRRs regulate and coordinate these immune responses are not fully understood. Here, we showed that Arabidopsis thaliana OXIDATIVE SIGNAL-INDUCIBLE1 (OXI1), a kinase known to be activated by ROS, is involved in the LYK5-CERK1 receptor complex, which recognizes fungal cell wall-derived chitin. The oxi1 mutant exhibits enhanced susceptibility to various pathogens and reduced chitin-induced MAPK activation and ROS burst. We showed that chitin induces the phosphorylation of OXI1 in an RbohD-dependent manner. H2O2 and chitin treatment causes the oxidation of OXI1 at Cys104 and Cys205, which is essential for the kinase activity of OXI1. These oxidation sites are required for chitin-induced MAPK activation and disease resistance. Activated OXI1 directly phosphorylates MAPKKK5 to regulate MAPK activation. Additionally, OXI1 phosphorylates RbohD, suggesting that it may activate RbohD to promote ROS burst to further enhance the long-term MAPK activation. Together, our findings reveal a pathway linking PRR-mediated ROS production to MAPK activation through OXI1.

Inactivation of checkpoint kinase 1 (Chk1) during parvovirus minute virus of mice (MVM) infection inhibits cellular homologous recombination repair and facilitates viral genome replication.

During infection, the autonomous parvovirus minute virus of mice (MVM) induces cellular DNA breaks and localizes to such sites, which presumably affords an environment beneficial for genome replication. MVM replication also benefits from the DNA damage response (DDR) mediated by the ataxia-telangiectasia mutated (ATM) kinase, while the ataxia telangiectasia and Rad-3 related (ATR) arm of the DDR is disabled, which prevents activation of its primary target, checkpoint kinase 1 (Chk1). We find here that Chk1 inactivation strongly correlates with dephosphorylation of one of its targets, RAD51, known to play a pivotal role in homologous recombination repair (HRR), thus leading to substantial inhibition of DNA repair in infected cells. We demonstrate colocalization of replicating MVM DNA with cellular double-strand breaks (DSBs) during infection, and show that an agent that exogenously induces cellular DSBs significantly increases viral DNA replication levels, establishing a role for cellular genome damage in facilitating virus DNA replication. Additionally, overexpression of active Chk1 during MVM infection was found to re-establish the activating phosphorylation of RAD51 Thr 309, significantly suppress infection-induced reduction of HRR efficiency with a concomitant increase in cellular genome DSBs, and reduce viral DNA replication levels. Thus, we conclude that during infection, MVM inhibition of Chk1 activation enhances viral replication, at least in part, by inhibiting cellular HRR.IMPORTANCEThe autonomous parvovirus minute virus of mice (MVM) has a compact DNA genome encoding a minimum number of proteins. During infection, it induces cellular DNA damage and both utilizes and modifies the subsequent cellular DNA damage response (DDR) in various ways to facilitate its replication. One of MVM's activities in this regard is to inhibit one of the primary arms of the DDR, the ataxia telangiectasia and Rad-3 related (ATR) pathway, which prevents activation of checkpoint kinase 1 (Chk1), a key protein involved in controlling the cellular DDR and preserving genome integrity. We show that prevention by MVM of Chk1 activation leads to inhibition of homologous recombination repair (HRR) of cellular DNA, which helps sustain viral replication. This work illuminates another way in which autonomous parvoviruses adjust the cellular environment for their replicative advantage.

TBK1-associated adapters TANK and AZI2 protect mice against TNF-induced cell death and severe autoinflammatory diseases.

The cytokine TNF can trigger highly proinflammatory RIPK1-dependent cell death. Here, we show that the two adapter proteins, TANK and AZI2, suppress TNF-induced cell death by regulating the activation of TBK1 kinase. Mice lacking either TANK or AZI2 do not show an overt phenotype. Conversely, animals deficient in both adapters are born in a sub-Mendelian ratio and suffer from severe multi-organ inflammation, excessive antibody production, male sterility, and early mortality, which can be rescued by TNFR1 deficiency and significantly improved by expressing a kinase-dead form of RIPK1. Mechanistically, TANK and AZI2 both recruit TBK1 to the TNF receptor signaling complex, but with distinct kinetics due to interaction with different complex components. While TANK binds directly to the adapter NEMO, AZI2 is recruited later via deubiquitinase A20. In summary, our data show that TANK and AZI2 cooperatively sustain TBK1 activity during different stages of TNF receptor assembly to protect against autoinflammation.

Anti-Inflammatory Effects of Honeysuckle Leaf Against Lipopolysaccharide-Induced Neuroinflammation on BV2 Microglia

Background/Objectives: Neurodegenerative disorders have emerged as a major global public health concern, and the burden is predicted to increase over time. Modulating neuroinflammation and microglial activity is considered a promising target for improving neurodegenerative disorders. The leaf of honeysuckle (LH), which has anti-inflammatory properties, has long been collected, regardless of the season, and used for medicinal purposes. However, research on its effects on neuroinflammation is scarce. In this study, we determined the neuroprotective effects of LH water extract by inhibiting microglial activation induced by lipopolysaccharide (LPS). Methods: The production or secretion of pro-inflammatory mediators was examined in LPS-exposed BV2 cells to ascertain the efficacy of LH water extract in improving neuroinflammation. In addition, the phosphorylation of mitogen-activated protein kinases (MAPKs) and the degradation of inhibitory κBα (IκBα) were analyzed to elucidate the regulatory mechanisms of LH water extract. Ultra-performance liquid chromatography (UPLC) analysis was conducted to identify the active component of the LH. Results: LH water extract suppressed the formation of inducible nitric oxide synthase (iNOS), nitric oxide (NO), and pro-inflammatory cytokines, including interleukin (IL)-1β and tumor necrosis factor (TNF)-α, in LPS-activated BV2 cells. LH impeded the activation of c-Jun N-terminal kinase (JNK). Moreover, chlorogenic acid was found in LH. Conclusions: The above findings suggest that LH water extract could improve neuroinflammation.

Synapse-to-nucleus ERK-CREB transcriptional signaling requires dendrite-to-soma Ca2+propagation mediated by L-type voltage-gated Ca2+ channels.

The cAMP-response element binding protein (CREB) transcription factor controls the expression of the neuronal immediate early genes c-Fos, Arc, and Bdnf and is essential for long-lasting synaptic plasticity underlying learning and memory. Despite this critical role, there is still ongoing debate regarding the synaptic excitation-transcription (E-T) coupling mechanisms mediating CREB activation in the nucleus. Here we employed optical uncaging of glutamate to mimic synaptic excitation of distal dendrites in conjunction with simultaneous imaging of intracellular Ca2+ dynamics and transcriptional reporter gene expression to elucidate CREB E-T coupling mechanisms in hippocampal neurons cultured from both male and female rats. Using this approach, we found that CREB-dependent transcription was engaged following dendritic stimulation of N-methyl, D-aspartate receptors (NMDARs) only when Ca2+ signals propagated to the soma via subsequent activation of L-type voltage-gated Ca2+ channels resulting in activation of Extracellular signal-Regulated Kinase (ERK) MAP kinase signaling to sustain CREB phosphorylation in the nucleus. In contrast, dendrite-restricted Ca2+ signals generated by NMDARs failed to stimulate CREB-dependent transcription. Furthermore, Ca2+-CaM-dependent kinase (CaMK)-mediated signaling pathways that may transiently contribute to CREB-phosphorylation following stimulation were ultimately dispensable for downstream CREB-dependent transcription and c-Fos induction. These findings emphasize the essential role that L-type Ca2+ channels play in rapidly relaying signals over long distances from synapses located on distal dendrites to the nucleus to control gene expression.Significance Statement The transcription factor CREB controls gene expression programs required for long-lasting synaptic plasticity and learning and memory, yet the synapse-to-nucleus signaling mechanisms mediating CREB activation are still unclear. Using glutamate uncaging to mimic synaptic input to dendrites, this study shows that Ca2+ signals propagated to the soma by L-type voltage-gated Ca2+ channels engage the ERK MAP kinase cascade to mediate CREB phosphorylation and CREB-dependent transcription. In contrast, dendrite-restricted Ca2+ signals generated primarily by NMDARs failed to effectively engage this signaling pathway or CREB-dependent transcription. In addition, we found that while ERK and CaMK pathways may both contribute to increased CREB phosphorylation immediately following neuronal stimulation, sustained ERK signaling to CREB was necessary to effectively drive CREB-dependent transcription.

PP2C phosphatase Pic6 suppresses MAPK activation and disease resistance in tomato.

Type 2C protein phosphatases (PP2Cs) are essential for regulating plant immune responses to pathogens. Our study focuses on the tomato PP2C-immunity associated candidate 6 (Pic6), elucidating its role in negatively regulating pattern-triggered immunity (PTI) signaling pathways in tomato. Using reverse transcription quantitative polymerase chain reaction (RT-qPCR), we observed that treatment with microbe-associated molecular patterns (MAMPs)- flg22 and flgII-28-significantly increased Pic6 mRNA levels in wild-type (RG-PtoR) tomato plants. Pic6 features a conserved N-terminal kinase-interacting motif (KIM) and a C-terminal PP2C domain. We produced variants of Pic6 with mutations in these regions, demonstrating their involvements in negatively regulating tomato immunity. Agrobacterium-mediated transient overexpression of Pic6 resulted in enhanced growth of the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) strain DC3000ΔhopQ1-1 compared to a YFP control. Additionally, Pic6 overexpression inhibited mitogen-activated protein kinase (MAPK) activation in response to flg22 and flgII-28 treatments. Importantly, Pic6 exhibited phosphatase activity and interacted with tomato Mkk1/Mkk2 proteins and dephosphorylated them in a KIM-dependent manner. Furthermore, we generated RG-pic6 loss-of-function mutants by CRISPR/Cas9, revealing that the absence of Pic6 heightened MAPK activity and increased resistance to Xanthomonas euvesicatoria strain 85-10 (Xe 85-10) when compared with the wild-type (RG-PtoR) plants. Transcript analyses showed that after flg22/flgII-28 treatment, PTI-reporter genes NAC and osmotin were significantly upregulated in RG-pic6 mutants in comparison to the wild-type (RG-PtoR) plants. Overall, our findings indicate that Pic6 acts as a negative regulator of MAPK signaling and playing a pivotal role in modulating tomato immunity against bacterial pathogens.

CD40 agonist on patient-derived xenograft mice for the treatment of B-cell acute lymphoblastic leukemia.

Cluster of differentiation 40 (CD40) is expressed on B-cell acute lymphoblastic leukemia (B-ALL) cases. However, the effect of CD40 activation on B-ALL cells has never been tested in vivo. The aim of our preclinical study was to investigate the therapeutic potential of a CD40 agonist in the treatment of B-ALL using patient-derived xenograft (PDX) mouse models. Intravenous administration of the CD40 agonist significantly impeded B-ALL cell proliferation and growth in vivo, accompanied by rapid activation of the extracellular signal-regulated kinase (ERK) pathway, leading to the induction of apoptosis and disruption of cell cycle progression. Co-treatment with a specific inhibitor of ERK further demonstrated that CD40 stimulation induced the pro-apoptosis of B-ALL cells in an ERK-dependent manner. Proteomic analysis revealed alterations in key signaling pathways associated with B-ALL expansion and maintenance. Moreover, the CD40 agonist markedly reduced the frequency of leukemia-initiating cells and leukemia development in PDX mice. Our study showed that the CD40 agonist can be associated with chemotherapeutic agents such as vincristine and dexamethasone, and this combination showed improved effectiveness. Additionally, the CD40 agonist was more effective on pre-B-ALL (EGIL B-III) that expressed CD40, than on common B-ALL (EGIL B-II) that lacked CD40 expression. These findings suggest that CD40 agonists are promising immunotherapeutic candidates for pediatric B-ALL, warranting further clinical investigations to improve patient outcomes in CD40-expressing B-ALL.

Amyloid beta-activated alpha-1-syntrophin has ramifications on Rac1 activation, ROS production and neuronal cell death.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the presence of β-amyloid (Aβ)-containing extracellular neuritic plaques and phosphorylated tau-containing intracellular neurofibrillary tangles. It remains the primary neuropathological criteria for the diagnosis of AD. Additionally, several other processes are currently being recognized as significant risk factors for AD development, including the brain's susceptibility to reactive oxygen species (ROS). The ROS production is among the early signs in the progression of AD. However, the underlying mechanisms behind increased ROS production in AD remain poorly understood. We have observed SNTA1 plays critical role in regulating ROS levels in different pathological conditions. Here, we wanted to gain further insight into the role of SNTA1 in the development of AD by using IMR32 cell line. Our results show that the accumulation of Aβ plaques in Alzheimer's model neuroblastoma cells significantly increases the expression and activation of SNTA1 and MKK6 kinase. The activation of MKK6 results in the phosphorylation of SNTA1, creating a binding site for Rac1, leading to its activation and subsequent production of ROS. Excessive ROS production leads to cell cycle arrest in the G2/M phase, a hallmark of AD. Our study provides new insight into the mechanism of Aβ-mediated cell death in AD and suggests that MKK6-mediated activation of alpha-1-syntrophin promotes ROS production in neuronal cells, resulting in cell death. This study presents a mechanistic insight into Aβ-mediated cell death and could serve as a paradigm for reducing neuronal cell death in AD.

Early-Life Stress Influences the Transcriptional Activation of Alpha-2A Adrenergic Receptor and Associated Protein Kinase A Signaling Molecules in the Frontal Cortex of Rats.

Early life is a highly sensitive period associated with profound changes in brain structure and function. Adverse experiences of early-life stress (ELS) are prominent risk factors for the precipitation of major depressive disorder (MDD). In recent years, dysfunction of the central noradrenergic (NA) system and subsequent deficits in norepinephrine (NE) signaling have gained increasing attention in the pathophysiology of MDD. However, the role of the α-2A adrenergic receptor and its downstream second messenger signaling system has not been investigated in connection to early-life stress-induced depression, limiting valuable insights into neurobiological mechanisms underlying this disorder. In this study, we used maternal separation (MS) as a rodent model of ELS to investigate whether ELS-induced depressive behavior is related to the α-2A adrenergic receptor and its associated second messenger signaling cascade. To do so, we studied expression levels of the α-2A adrenergic receptor (Adra2a), G alpha proteins (stimulatory subunit-Gαs [Gnas] and inhibitory subunit-Gαi [Gnai1 and Gnai2]), and downstream protein kinase A (PKA) catalytic [Prkarcα and Prkarcβ] and regulatory subunits [Prkar1α, Prkar1β, Prkar2α, and Prkar2β]) in the frontal cortex (FC) of MS rats. We found reduced sucrose preference in MS animals, along with reduced transcript levels of Adra2a, Gnai2, Prkar1β, and Prkarcβ. These findings suggest that ELS exposure may contribute to depression symptomatology via alterations in the expression of key genes involved in the NA system, highlighting potential mechanisms underlying ELS-induced depressive behavior.

Mitapivat for Acquired Pyruvate Kinase Deficiency.

Pyruvate kinase (PK) activation is emerging as a promising treatment modality for numerous congenital hemolytic anemias of diverse pathophysiology, and one agent, mitapivat, is already licensed to treat patients with congenital PK deficiency. However, PK deficiency may also be acquired in the setting of clonal myeloid disorders and other pathologies, where it may result in severe hemolytic anemia and remains without known therapies. This case report describes the novel application of mitapivat therapy in a patient with acquired PK deficiency causing red cell transfusion dependence, liberating the patient from transfusions and resulting in marked improvement in symptoms and quality of life.

Abstract 4136847: Administration of the Recombinant Activated Protein C Rescues the Cardiac Vulnerability to Ischemic Insults in Aging through Modulating Inflammatory Response during Ischemia and Reperfusion

Introduction: Activated protein C (APC) is an endogenous vitamin-K-dependent serine protease and exhibits therapeutic potential for ischemic heart disease. The APC derivatives with anticoagulant and/or cytoprotective properties were produced through chemical engineering to characterize the functional domain of APC for limiting ischemia/reperfusion (I/R) injury. Hypothesis: The thromboinflammatory regulation by APC ameliorates ischemic insults caused by I/R through the receptor EPCR/PAR1. Methods: Young (3 months)/aged (24 months) wild type C57BL/6J mice and PAR1 R46Q/R46Q (block cleavage by APC) knock in mutant (3 months, C57BL/6J) mice were subjected to ligation of left anterior descending coronary artery with 45 min of ischemia. 5 min prior to reperfusion, wild type APC, cytoprotective selective APC-2Cys, or anticoagulant selective APC-E170A (0.2 μg/kg i.v.) was administered followed by 24 hr of reperfusion. The left ventricles of hearts were used for immunoblotting and flow cytometry analysis. Results: APC and APC-2Cys but not APC-E170A reduced I/R-induced myocardial infarction. Echocardiography showed that APC and APC-2Cys but not APC-E170A can rescue systolic dysfunction by I/R in young/aged WT but not in PAR1 R46Q/R46Q mice. Biochemical analysis showed that administration of APC and APC-2Cys inhibited activation of inflammation signaling c-Jun N-terminal protein kinase (JNK) and NF-κB by I/R and reduced mRNA levels of the proinflammatory cytokines TNFα and IL-6 in young/aged WT but not in PAR1 R46Q/R46Q hearts. The plasma cytokine array demonstrated that APC and APC2-Cys reduced potency of pro-inflammatory cytokines and apoptotic signaling cytokines during I/R. Moreover, I/R-triggered inflammasome NLRP3 was reduced by APC and APC-2Cys in young/aged WT but not in PAR1 R46Q/R46Q hearts. The flow cytometry showed that APC and APC-2Cys increased lymphocyte and decreased myeloid cell abundance and increased the recruitment of inflammation-mediating CD8a + T-cells during I/R in young/aged WT but not PAR1 R46Q/R46Q hearts, indicating that the APC/EPCR/PAR1 axis mediates APC's immune regulation under I/R. Moreover, APC and APC-2Cys reduced I/R-triggered macrophage abundance in young/aged WT hearts, although the proportion of M1 and M2 macrophages was unchanged. Conclusions: The cytoprotective domain of APC is critical for its cardioprotection against ischemic insults. EPCR/PAR1 receptor complex mediates APC's immune regulation in aging during I/R conditions.

Regulatory Role of eIF2αK4 in Amino Acid Transporter Expression in Mouse Brain Capillary Endothelial Cells.

Amino acid transporters are expressed in the brain capillary endothelial cells that form the blood-brain barrier (BBB), and their expression levels change during the neonatal period. This study aimed to investigate the molecular mechanisms regulating amino acid transporter levels in mouse brain capillary endothelial cells. Capillaries were isolated from the brains of neonatal and adult mice. Activation of eukaryotic translation initiation factor 2α kinase 4 (eIF2αK4) was analyzed in MBEC4 (mouse brain capillary endothelial) cells under amino acid-depleted conditions. Protein expression was determined using western blotting and proteomic analyses. Phosphorylation of eIF2α, a downstream target of eIF2αK4, was induced in the brain capillaries of neonates compared to adults. In vitro experiments using MBEC4 cells revealed that amino acid depletion induced eIF2α phosphorylation and expression of the amino acid transporter, solute carrier (Slc)-7a1. The eIF2αK4 inhibitor, GCN2iB, inhibited these inductions. Proteomic analysis revealed arginine depletion-dependent induction of amino acid transporters Slc1a4, Slc3a2, Slc7a1, Slc7a5, and Slc38a1. These effects were also inhibited by GCN2iB, suggesting the involvement of eIF2αK4 activation. In contrast, the expression of Slc2a1, Slc16a1, Abcb1b, Abcg2, transferrin receptor, insulin receptor, claudin-1, ZO-1, and Jam1 was not suppressed by the GCN2iB treatment. Overall, the eIF2αK4 pathway plays a regulatory role in amino acid transporter expression in brain capillary endothelial cells and facilitates the maintenance of amino acid homeostasis in the brain. This study provides new insights into the regulatory mechanisms underlying nutrient transport across the BBB.

Antimicrobial Peptide Pro10-1D Exhibits Anti-Allergic Activity: A Promising Therapeutic Candidate

Although antimicrobial peptides (AMPs) exhibit a range of biological functions, reports on AMPs with therapeutic effects in allergic disorders are limited. In this study, we investigated the anti-allergic effects of Pro10-1D, a 10-meric AMP derived from insect defensin protaetiamycine. Our findings demonstrate that Pro10-1D effectively inhibits antigen-induced degranulation of mast cells (MCs) with IC50 values of approximately 11.6 μM for RBL-2H3 cells and 2.7 μM for bone marrow-derived MCs. Furthermore, Pro10-1D suppressed the secretion of cytokines with IC50 values of approximately 2.8 μM for IL-4 and approximately 8.6 μM for TNF-α. Mechanistically, Pro10-1D inhibited the Syk-LAT-PLCγ1 signaling pathway in MCs and decreased the activation of mitogen-activated protein kinases (MAPKs). Pro10-1D demonstrated a dose-dependent reduction in IgE-mediated passive cutaneous anaphylaxis in mice with an ED50 value of approximately 7.6 mg/kg. Further investigation revealed that Pro10-1D significantly reduced the activity of key kinases Fyn and Lyn, which are critical in the initial phase of the FcεRI-mediated signaling pathway, with IC50 values of approximately 22.6 μM for Fyn and approximately 1.5 μM for Lyn. Collectively, these findings suggest that Pro10-1D represents a novel therapeutic candidate for the treatment of IgE-mediated allergic disorders by targeting the Lyn/Fyn Src family kinases in MCs.

Clotting promotes glioma growth and infiltration through activation of focal adhesion kinase.

The tumor architecture of high-grade gliomas is shaped by tumor cell necrosis, invasive growth and the leakage of a fibrin-rich edema from poorly organized tumor blood vessels. Here, we demonstrate a marked upregulation of clot formation in the interstitial spaces of tumor tissues from patients with glioblastoma while tumor-free brain is essentially devoid of fibrin. The accumulation of fibrin in tumor interstitial spaces is functionally relevant as we demonstrate increased infiltration and growth of primary glioblastoma cells after embedding in a 3-dimensional matrix made of fibrin ex vivo. Additionally, we detected accelerated tumor growth after implanting glioblastoma cells together with clotted plasma in brains of immune deficient mice while glioblastoma development in clotting-deficient hemophilia mice was delayed. Glioblastoma growth correlated with the outgrowth of invadopodia and their adhesive interactions with the 3-dimensional clot matrix, which was mediated by integrins β1 and β3 and their common downstream target focal adhesion kinase (FAK). Knocking down FAK with CRISPR Cas9 caused an upregulation of p21/p27 cell cycle inhibitors, strong growth inhibition in cultured glioblastoma cells and sustained anti-tumorigenic effects in orthotopic glioblastoma xenografts in vivo. These results go hand in hand with genomic data from The Cancer Genome Atlas that indicate increased clotting activity and reduced patient survival in glioma subgroups with high integrin β1 and β3 expression. We therefore conclude that clotting in glioma interstitial spaces provides tumor cells with a potent proliferative stimulus that can be reversed by targeting the adhesive machinery of glioblastoma cells via inhibition of FAK.

Prunella vulgaris Extract Ameliorates Testosterone-Induced Benign Prostatic Hyperplasia by Regulating Androgen Levels, Cell Proliferation, and Apoptosis

Background/Objectives: Benign prostatic hyperplasia (BPH) is a prevalent urological condition affecting elderly men. Prunella vulgaris L. (PV), a perennial herbaceous plant native to Europe and Asia, has anti-inflammatory, antioxidant, and antimicrobial effects. In this study, we determined the effect of PV extract on the development of BPH. Methods: Rats were treated via a daily hypodermic injection of testosterone propionate (TP; 3 mg/kg) for 4 weeks. Groups of BPH rats were treated with or without PV (60 or 80 mg/kg) by oral gavage. Results: In BPH model rats, PV considerably reduced their relative prostate weight and serum concentrations of dihydrotestosterone (DHT) and testosterone. The TP-induced increases in epithelial thickness in the prostate, proliferating cell nuclear antigen (PCNA) expression, and cyclin D1 expression were remarkably reduced, whereas terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells and cleaved caspase-3 levels were increased, in PV-treated rats compared to BPH rats. The mRNA expression levels of growth factors, such as transforming growth factor-β (TGF-β), fibroblast growth factor (FGF), and insulin-like growth factor (IGF-2), were significantly reduced in PV-treated rats. Mechanistically, the TP-induced activation of c-Jun N-terminal kinase (JNK) was reduced by PV administration. Conclusions: These results designate that PV effectively ameliorates the development of testosterone-induced BPH through anti-androgenic, anti-proliferative, and pro-apoptotic activities, suggesting that it could be a potential therapeutic substance for BPH.

PATH-68. THE CLINICAL AND PROTEOMIC CLASSIFICATION FOR THE PRECISION TREATMENT STRATEGY OF CHORDOMA

Abstract Chordoma is a rare and heterogeneous mesenchymal malignancy, with distinct clinical and biological behaviors. Till now, its comprehensive clinical-molecular characteristics and accurate molecular classification remain obscure, leading to numerous diagnostic and therapeutic challenges. In this research, we enroll a large cohort of chordoma (102 patients) and describe their clinical, imageological and histopathological features. Through tandem mass tag-based proteomic analysis and nonnegative matrix factorization clustering, we classify chordoma into the following three molecular subtypes: bone microenvironment dominant, mesenchymal derived, and mesenchymal to epithelial transition mediated pattern. The three subtypes exhibit discrete clinical prognosis and distinct biological attributes of osteoclastogenesis and immunogenicity, oxidative phosphorylation, and receptor tyrosine kinase activation, suggesting targeted therapeutic strategies of Denosumab, S-Gboxin and Anlotinib, respectively. Notably, these approaches demonstrate positive treatment outcomes for each subtype in vivo and in vitro, as well as in patient-derived xenograft models. Altogether, this work sheds light on the clinical-proteomic characteristics of chordoma that deepens our understanding of this rare disease and provides candidate precision treatment strategy for chordoma according to molecular classification, underscoring their potential for clinical application.

Avenanthramides Ameliorate Insulin Resistance by Modulating Gluconeogenesis and Glycogen Synthesis in HepG2 Cells.

Diabetes mellitus (DM) is a multifaceted metabolic condition, mainly defined by elevated blood glucose levels. A feature of type 2 DM includes insulin resistance (IR), which involves impairments within the insulin signaling pathways. Avenanthramides (AVNs) are phenolic alkaloids found in Avena sativa L. The major AVNs are AVN A, AVN B, and AVN C. They have been reported to offer benefits in preventing inflammation, cancer, and cardiovascular diseases. However, the effects of AVNs on the liver glucose metabolism pathways remain unknown. This study examined the effects and underlying mechanisms through which AVNs alleviate IR induced by free fatty acid (FFA) in HepG2 cells. The results indicated that FFA treatment significantly decreased glucose consumption by 34.54% compared to the control. However, treatments with AVN A, B, and C at 100 μM increased glucose uptake by 57.93%, 58.28%, and 53.10%, respectively, compared to FFA treatment alone. This effect occurs through the increased expression of glucose transporter 4. Furthermore, AVNs significantly enhanced the glycogen content. AVNs induced increased phosphorylation of insulin receptor substrate-1 (IRS-1), phosphatidylinositol-3-kinase (PI3K), and protein kinase B (Akt). AVNs treatment decreased the levels of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase in HepG2 cells. This effect was attributed to AMP-activated protein kinase activation and inhibition of forkhead box protein O1. Collectively, these results suggest that AVNs regulate glucose metabolism by activating the IRS-1/PI3K/Akt pathway, which is related to glycogen synthesis, and by inhibiting key molecules that promote gluconeogenesis.