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This study explores the molecular underpinnings of neuropathic pain (NPP) and neuroinflammation, focusing on the role of TRIM28 in the regulation of autophagy and microglia ferroptosis. Leveraging transcriptomic data associated with NPP, we identified TRIM28 as a critical regulator of ferroptosis. Through comprehensive analysis, including Gene Ontology enrichment and protein-protein interaction network assessments, we unveiled GSK3B as a downstream target of TRIM28. Experimental validation confirmed the capacity of TRIM28 to suppress GSK3B expression and attenuate autophagic processes in microglia. We probed the consequences of autophagy and ferroptosis on microglia physiology, iron homeostasis, oxidative stress, and the release of proinflammatory cytokines. In a murine model, we validated the pivotal role of TRIM28 in NPP and neuroinflammation. Our analysis identified 20 ferroptosis regulatory factors associated with NPP, with TRIM28 emerging as a central orchestrator. Experimental evidence affirmed that TRIM28 governs microglial iron homeostasis and cell fate by downregulating GSK3B expression and modulating autophagy. Notably, autophagy was found to influence oxidative stress and proinflammatory cytokine release through the iron metabolism pathway, ultimately fueling neuroinflammation. In vivo experiments provided conclusive evidence of TRIM28-mediated pathways contributing to heightened pain sensitivity in neuroinflammatory states. The effect of TRIM28 on autophagy and microglia ferroptosis drives NPP and neuroinflammation. These findings offer promising avenues for identifying novel therapeutic targets to manage NPP and neuroinflammation. [ABSTRACT FROM AUTHOR]

Background: Women with diminished ovarian reserve (DOR) respond differently to gonadotropin medications. Objective: This study investigates the relationship between effective gene expression in the steroidogenesis pathway and gonadotropin responsiveness in DOR. Materials and Methods: In this case-control study, cumulus cells were obtained from women with DOR after gonadotropin administration (n = 20) and normal ovarian reserve (n = 20). They were divided into the following groups, oocyte number < 3 and oocyte number > 3. After RNA extraction and cDNA synthesis, quantitative polymerase chain reaction was performed to assess the expression levels of cytochrome P450 aromatase (CYP19A1), protein kinase A (PKA), and glycogen synthase kinase 3 beta (GSK3B) genes. Results: The women with DOR had statistically significant lower expression of CYP19A1 and PKA genes in their cumulus cells compared to control group (p = 0.04, and p < 0.001, respectively). There was also lower expression of the GSK3B gene in DOR compared to control group, but it was not significant. Although the expression of the CYP19A1, PKA, and GSK3B genes was lower in women with < 3 oocytes compared to women with more oocytes, this difference was not statistically significant. Conclusion: In conclusion, DOR may be associated with lower expression of CYP19A1 and PKA genes. Also, considering the decrease in the expression of these genes in people with DOR, the expression of these genes can be used as a tool to predict the treatment. [ABSTRACT FROM AUTHOR]

Brain‐derived neurotrophic factor (BDNF) content and signaling has been identified as one potential regulator of amyloid precursor protein (APP) processing. Recently published work has demonstrated that BDNF reduces BACE1 activity while also elevating the inhibition of GSK3β in the prefrontal cortex of male C57BL/6J mice. These results provide evidence that BDNF alters APP processing by reducing BACE1 activity, which may act through GSK3β inhibition. The purpose of this study was to further explore the role of GSK3β in BDNF‐induced regulation on BACE1 activity. We utilized a cell culture and an in vitro activity assay model to pharmacologically target BDNF and GSK3β signaling to confirm its involvement in the BDNF response. Treatment of differentiated SH‐SY5Y neuronal cells with 75 ng/mL BDNF resulted in elevated pTrkB content, pAkt content, pGSK3β content, and reduced BACE1 activity. An in vitro BACE1 activity assay utilizing mouse prefrontal cortex (n = 6/group) supplemented with BDNF, BDNF + ANA12 (Trkb antagonist), or BDNF + wortmannin (Akt inhibitor) demonstrated that BDNF reduced BACE1 activity; however, in the presence of TrkB or Akt inhibition, this effect was abolished. An in vitro ADAM10 activity assay utilizing mouse prefrontal cortex (n = 6/group) supplemented with BDNF, BDNF + ANA12 (Trkb antagonist), or BDNF + wortmannin (Akt inhibitor) demonstrated that BDNF did not alter ADAM10 activity. However, inhibiting BDNF signaling reduced ADAM10 activity. Collectively these studies suggest that GSK3β inhibition may be necessary for BDNF‐induced reductions in BACE1 activity. These findings will allow for the optimization of future therapeutic strategies by selectively targeting TrkB activation and GSK3β inhibition. [ABSTRACT FROM AUTHOR]

Abstract The aim of this study was to identify angiogenic microRNAs (miRNAs) that could be used in the treatment of hindlimb ischemic tissues. miRNAs contained in extracellular vesicles (EVs) deriving from the plasma were analyzed in C57BL/6 mice, which have ischemia tolerance, and in BALB/c mice without ischemia tolerance as part of a hindlimb ischemia model; as a result 43 angiogenic miRNA candidates were identified. An aortic ring assay was employed by using femoral arteries isolated from BALC/c mice and EVs containing miRNA; as a result, the angiogenic miRNA candidates were limited to 14. The blood flow recovery was assessed after injecting EVs containing miRNA into BALB/c mice with hindlimb ischemia, and miR-709 was identified as a promising angiogenic miRNA. miR-709-encapsulating EVs were found to increase the expression levels of the fibroblast growth factor 2 (FGF2) mRNA in the thigh tissues of hindlimb ischemia model BALB/c mice. miR-709 was also found to bind to the 3′UTR of glycogen synthase kinase 3 beta (GSK3B) in three places. GSK3B-knockdown human artery-derived endothelial cells were found to express high levels of FGF2, and were characterized by increased cell proliferation. These findings indicate that miR-709 induces an upregulation of FGF2 through the downregulation of GSK3B.

Diabetes mellitus is a persistent metabolic condition marked by elevated blood glucose levels due to compromised insulin secretion or functionality. The search for natural antidiabetic agents has gained attention due to their potential effectiveness and safety profiles. Sessuvium portulacastrum, a coastal plant, has been traditionally used for various medicinal purposes. This study investigates the antidiabetic potential of Sessuvium portulacastrum aqueous extract by analyzing its inhibitory effects on key enzymes involved in carbohydrate metabolism and exploring its molecular interactions with critical target proteins. The aqueous extract of Sessuvium portulacastrum was prepared and used for in vitro analysis. The reduced activity of the extract against α-amylase and α-glucosidase enzymes, crucial in glucose absorption and postprandial hyperglycemia, was assessed. Molecular docking techniques were employed to explore the potential interactions between active compounds in the extract and diabetes-related proteins, including BAX, GSK3β, and CADH. The study revealed significant inhibition of both alpha-amylase and alpha-glucosidase enzymes by Sessuvium portulacastrum aqueous extract, indicating its potential to reduce glucose absorption and postprandial hyperglycemia. Moreover, the molecular docking analysis demonstrated strong binding interactions between active compounds in the extract and key proteins involved in diabetes-related pathways, namely apoptotic pathways, glycogen synthesis, and cell adhesion. The findings of this study highlight the promising antidiabetic potential of Sessuvium portulacastrum aqueous extract. Upcoming research should get an attention on isolating and characterizing the active compounds responsible for these effects on antidiabetic therapies from natural sources.

The aim of this study was to identify angiogenic microRNAs (miRNAs) that could be used in the treatment of hindlimb ischemic tissues. miRNAs contained in extracellular vesicles (EVs) deriving from the plasma were analyzed in C57BL/6 mice, which have ischemia tolerance, and in BALB/c mice without ischemia tolerance as part of a hindlimb ischemia model; as a result 43 angiogenic miRNA candidates were identified. An aortic ring assay was employed by using femoral arteries isolated from BALC/c mice and EVs containing miRNA; as a result, the angiogenic miRNA candidates were limited to 14. The blood flow recovery was assessed after injecting EVs containing miRNA into BALB/c mice with hindlimb ischemia, and miR-709 was identified as a promising angiogenic miRNA. miR-709-encapsulating EVs were found to increase the expression levels of the fibroblast growth factor 2 (FGF2) mRNA in the thigh tissues of hindlimb ischemia model BALB/c mice. miR-709 was also found to bind to the 3′UTR of glycogen synthase kinase 3 beta (GSK3B) in three places. GSK3B-knockdown human artery-derived endothelial cells were found to express high levels of FGF2, and were characterized by increased cell proliferation. These findings indicate that miR-709 induces an upregulation of FGF2 through the downregulation of GSK3B. [ABSTRACT FROM AUTHOR]

Doxorubicin (DOX), widely used as a chemotherapeutic agent for various cancers, is limited in its clinical utility by its cardiotoxic effects. Despite its widespread use, the precise mechanisms underlying DOX-induced cardiotoxicity at the cellular and molecular levels remain unclear, hindering the development of preventive and early detection strategies. To characterize the cytotoxic effects of DOX on isolated ventricular cardiomyocytes, focusing on the expression of specific microRNAs (miRNAs) and their molecular targets associated with endogenous cardioprotective mechanisms such as the ATP-sensitive potassium channel (KATP), Sirtuin 1 (SIRT1), FOXO1, and GSK3β. We isolated Guinea pig ventricular cardiomyocytes by retrograde perfusion and enzymatic dissociation. We assessed cell morphology, Reactive Oxygen Species (ROS) levels, intracellular calcium, and mitochondrial membrane potential using light microscopy and specific probes. We determined the miRNA expression profile using small RNAseq and validated it using stem-loop qRT-PCR. We quantified mRNA levels of some predicted and validated molecular targets using qRT-PCR and analyzed protein expression using Western blot. Exposure to 10 µM DOX resulted in cardiomyocyte shortening, increased ROS and intracellular calcium levels, mitochondrial membrane potential depolarization, and changes in specific miRNA expression. Additionally, we observed the differential expression of KATP subunits (ABCC9, KCNJ8, and KCNJ11), FOXO1, SIRT1, and GSK3β molecules associated with endogenous cardioprotective mechanisms. Supported by miRNA gene regulatory networks and functional enrichment analysis, these findings suggest that DOX-induced cardiotoxicity disrupts biological processes associated with cardioprotective mechanisms. Further research must clarify their specific molecular changes in DOX-induced cardiac dysfunction and investigate their diagnostic biomarkers and therapeutic potential. [ABSTRACT FROM AUTHOR]

Diabetes mellitus is a persistent metabolic condition marked by elevated blood glucose levels due to compromised insulin secretion or functionality. The search for natural antidiabetic agents has gained attention due to their potential effectiveness and safety profiles. Sessuvium portulacastrum, a coastal plant, has been traditionally used for various medicinal purposes. This study investigates the antidiabetic potential of Sessuvium portulacastrum aqueous extract by analyzing its inhibitory effects on key enzymes involved in carbohydrate metabolism and exploring its molecular interactions with critical target proteins. The aqueous extract of Sessuvium portulacastrum was prepared and used for in vitro analysis. The reduced activity of the extract against a-amylase and a-glucosidase enzymes, crucial in glucose absorption and postprandial hyperglycemia, was assessed. Molecular docking techniques were employed to explore the potential interactions between active compounds in the extract and diabetes-related proteins, including BAX, GSK3ß, and CADH. The study revealed significant inhibition of both alpha-amylase and alpha-glucosidase enzymes by Sessuvium portulacastrum aqueous extract, indicating its potential to reduce glucose absorption and postprandial hyperglycemia. Moreover, the molecular docking analysis demonstrated strong binding interactions between active compounds in the extract and key proteins involved in diabetes-related pathways, namely apoptotic pathways, glycogen synthesis, and cell adhesion. The findings of this study highlight the promising antidiabetic potential of Sessuvium portulacastrum aqueous extract. Upcoming research should get an attention on isolating and characterizing the active compounds responsible for these effects on antidiabetic therapies from natural sources. [ABSTRACT FROM AUTHOR]

Abstract Brain‐derived neurotrophic factor (BDNF) content and signaling has been identified as one potential regulator of amyloid precursor protein (APP) processing. Recently published work has demonstrated that BDNF reduces BACE1 activity while also elevating the inhibition of GSK3β in the prefrontal cortex of male C57BL/6J mice. These results provide evidence that BDNF alters APP processing by reducing BACE1 activity, which may act through GSK3β inhibition. The purpose of this study was to further explore the role of GSK3β in BDNF‐induced regulation on BACE1 activity. We utilized a cell culture and an in vitro activity assay model to pharmacologically target BDNF and GSK3β signaling to confirm its involvement in the BDNF response. Treatment of differentiated SH‐SY5Y neuronal cells with 75 ng/mL BDNF resulted in elevated pTrkB content, pAkt content, pGSK3β content, and reduced BACE1 activity. An in vitro BACE1 activity assay utilizing mouse prefrontal cortex (n = 6/group) supplemented with BDNF, BDNF + ANA12 (Trkb antagonist), or BDNF + wortmannin (Akt inhibitor) demonstrated that BDNF reduced BACE1 activity; however, in the presence of TrkB or Akt inhibition, this effect was abolished. An in vitro ADAM10 activity assay utilizing mouse prefrontal cortex (n = 6/group) supplemented with BDNF, BDNF + ANA12 (Trkb antagonist), or BDNF + wortmannin (Akt inhibitor) demonstrated that BDNF did not alter ADAM10 activity. However, inhibiting BDNF signaling reduced ADAM10 activity. Collectively these studies suggest that GSK3β inhibition may be necessary for BDNF‐induced reductions in BACE1 activity. These findings will allow for the optimization of future therapeutic strategies by selectively targeting TrkB activation and GSK3β inhibition.

Abstract Objective Anesthetics have been linked to cognitive alterations, particularly in the elderly. The current research delineates how Fibroblast Growth Factor 2 (Fgf2) modulates tau protein phosphorylation, contributing to cognitive impairments in aged rats upon sevoflurane administration. Methods Rats aged 3, 12, and 18 months were subjected to a 2.5% sevoflurane exposure to form a neurotoxicity model. Cognitive performance was gauged, and the GEO database was employed to identify differentially expressed genes (DEGs) in the 18-month-old cohort post sevoflurane exposure. Bioinformatics tools, inclusive of STRING and GeneCards, facilitated detailed analysis. Experimental validations, both in vivo and in vitro, examined Fgf2’s effect on tau phosphorylation. Results Sevoflurane notably altered cognitive behavior in older rats. Out of 128 DEGs discerned, Fgf2 stood out as instrumental in regulating tau protein phosphorylation. Sevoflurane exposure spiked Fgf2 expression in cortical neurons, intensifying tau phosphorylation via the PI3K/AKT/Gsk3b trajectory. Diminishing Fgf2 expression correspondingly curtailed tau phosphorylation, neurofibrillary tangles, and enhanced cognitive capacities in aged rats. Conclusion Sevoflurane elicits a surge in Fgf2 expression in aging rats, directing tau protein phosphorylation through the PI3K/AKT/Gsk3b route, instigating cognitive aberrations.

Abstract Iron metabolism disorders are implicated in the pathogenesis of Alzheimer’s disease (AD). It was previously reported that transferrin receptor (TFR1) expression was upregulated in AD mouse model. However, the precise biological functions of TFR1 in AD progression remains unclear. Herein, we observed a gradual increase in TFR1 protein expression during the differentiation of AD patient-derived induced pluripotent stem cells (AD-iPS). TFR1 knockdown inhibited the protein expression of ferritin and ferritin heavy chain 1 (FTH1), enhanced the expression of ferroportin 1 (FPN1), and decreased intracellular levels of total iron, labile iron, and reactive oxygen species (ROS). Moreover, TFR1 knockdown improved mitochondrial membrane potential (MMP), increased adenosine triphosphate (ATP) content, downregulated mitochondrial fission proteins, and upregulated mitochondrial fusion proteins. TFR1 knockdown alleviated iron overload and mitochondrial dysfunction in neural cells differentiated from AD-iPS, while TFR1 overexpression showed the opposite results. Additionally, TFR1interacted with glycogen synthase kinase 3 beta (GSK3B) and promoted GSK3B expression. GSK3B overexpression reversed the inhibitory effects of TFR1 knockdown on iron overload and mitochondrial dysfunction in AD-iPS differentiated neural cells. In conclusion, TFR1 knockdown alleviated iron overload and mitochondrial dysfunction in neural cells differentiated from AD-iPS by promoting GSK3B expression. Our findings provide a potential therapeutic target for the treatment of AD. Graphical Abstract

Glycogen synthase kinase-3 beta (GSK3β) is a serine/threonine kinase that plays key roles in glycogen metabolism, Wnt/β-catenin signaling cascade, synaptic modulation, and multiple autophagy-related signaling pathways. GSK3β is an attractive target for drug discovery since its aberrant activity is involved in the development of neurodegenerative diseases such as Alzheimer's and Parkinson's disease. In the present study, multiple machine learning models aimed at identifying novel GSK3β inhibitors were developed and evaluated for their predictive reliability. The most powerful models were combined in a consensus approach, which was used to screen about 2 million commercial compounds. Our consensus machine learning-based virtual screening led to the identification of compounds G1 and G4, which showed inhibitory activity against GSK3β in the low-micromolar and sub-micromolar range, respectively. These results demonstrated the reliability of our virtual screening approach. Moreover, docking and molecular dynamics simulation studies were employed for predicting reliable binding modes for G1 and G4, which represent two valuable starting points for future hit-to-lead and lead optimization studies. [ABSTRACT FROM AUTHOR]

Numerous studies have considered galectin-3 or Glycogen synthase kinase 3 beta (GSK3B) as a potential prognosis marker for various cancers. However, the correlation between the protein expression of galectin-3/GSK3B and the clinical parameters of astrocytoma has not been reported. This study aims to validate the correlation between the clinical outcomes and protein expression of galectin-3/GSK3B in astrocytoma. Immunohistochemistry staining was performed to detect galectin-3/GSK3B protein expression in patients with astrocytoma. The Chi-square test, Kaplan−Meier evaluation, and Cox regression analysis were used to determine the correlation between clinical parameters and galectin-3/GSK3B expression. Cell proliferation, invasion, and migration were compared between a non-siRNA group and a galectin-3/GSK3B siRNA group. Protein expression in galectin-3 or GSK3B siRNA-treated cells was evaluated using western blotting. Galectin-3 and GSK3B protein expression were significantly positively correlated with the World Health Organization (WHO) astrocytoma grade and overall survival time. Multivariate analysis revealed that WHO grade, galectin-3 expression, and GSK3B expression were independent prognostic factors for astrocytoma. Galectin-3 or GSK3B downregulation induced apoptosis and decreased cell numbers, migration, and invasion. siRNA-mediated gene silencing of galectin-3 resulted in the downregulation of Ki-67, cyclin D1, VEGF, GSK3B, p-GSK3B Ser9 (p-GSK3B S9), and β-catenin. In contrast, GSK3B knockdown only decreased Ki-67, VEGF, p-GSK3B S9, and β-catenin protein expression but did not affect cyclin D1 and galectin-3 protein expression. The siRNA results indicated that GSK3B is downstream of the galectin-3 gene. These data support that galectin-3 mediated tumor progression by upregulating GSK3B and β-catenin protein expression in glioblastoma. Therefore, galectin-3 and GSK3B are potential prognostic markers, and their genes may be considered to be anticancer targets for astrocytoma therapy.

Background: We intended to investigate the role and regulatory mechanism of EPS8L3 in increase the development of pancreatic cancer (PC). Methods: In order to analyze the relationship between EPS8L3 level and clinicopathological indicators of PC patients, qRT-PCR was used to detect the expression of EPS8L3 in tumor specimens of 40 PC patients. EPS8L3 knockdown models were then constructed in PC cell lines. Furthermore, the effect of EPS8L3 on PC cell function was analyzed by CCK-8 and Transwell assay. Dual luciferase reporter gene assay and recovery assay were used to further investigate the underlying mechanism. Results: qRT-PCR results indicated that EPS8L3 was highly expressed in PC tissues compared with adjacent ones. At the same time, the incidence of distant metastasis was higher in PC patients with high EPS8L3 level. In vitro analysis such as CCK-8 and Transwell experimentations indicated that knockdown of EPS8L3 markedly inhibited the proliferative and metastatic ability. Bio-informatics together with luciferase report assay proposing that EPS8L3 can target GSK3B. Western Blot results revealed that knockdown of EPS8L3 markedly reduced the GSK3B expression in PC cells, and there was a positively associated between the two in PC cells. In addition, the recovery experimentation proved that EPS8L3 and GSK3B have a mutual regulation effect. Overexpression of GSK3B can reversal the prohibitive effect of EPS8L3 knockdown on the malignant development of PC cells, thereby jointly regulating the occurrence and development of PC. Conclusions: EPS8L3 promotes the development of PC by regulating GSK3B, suggesting that EPS8L3 can be used as a biomarker for early diagnosis and treatment of PC.

This study aimed to investigate the influence of genetic variants in neuroplasticity-related genes on antidepressant treatment phenotypes. The BDNF-TrkB signaling pathway, as well as the downstream kinases Akt and ERK and the mTOR pathway, have been implicated in depression and neuroplasticity. However, clinicians still struggle with the unpredictability of antidepressant responses in depressed patients. We genotyped 26 polymorphisms in BDNF, NTRK2, NGFR, CREB1, GSK3B, AKT, MAPK1, MTOR, PTEN, ARC, and SYN1 in 80 patients with major depressive disorder treated according to the Texas Medical Algorithm for 27 months at Hospital Magalhães Lemos, Porto, Portugal. Our results showed that BDNF rs6265, PTEN rs12569998, and SYN1 rs1142636 SNP were associated with treatment-resistant depression (TRD). Additionally, MAPK1 rs6928 and GSK3B rs6438552 gene polymorphisms were associated with relapse. Moreover, we found a link between the rs6928 MAPK1 polymorphism and time to relapse. These findings suggest that the BDNF, PTEN, and SYN1 genes may play a role in the development of TRD, while MAPK1 and GSK3B may be associated with relapse. GO analysis revealed enrichment in synaptic and trans-synaptic transmission pathways and glutamate receptor activity with TRD-associated genes. Genetic variants in these genes could potentially be incorporated into predictive models of antidepressant response. [ABSTRACT FROM AUTHOR]

Background: We intended to investigate the role and regulatory mechanism of EPS8L3 in increase the development of pancreatic cancer (PC). Methods: In order to analyze the relationship between EPS8L3 level and clinicopathological indicators of PC patients, qRT-PCR was used to detect the expression of EPS8L3 in tumor specimens of 40 PC patients. EPS8L3 knockdown models were then constructed in PC cell lines. Furthermore, the effect of EPS8L3 on PC cell function was analyzed by CCK-8 and Transwell assay. Dual luciferase reporter gene assay and recovery assay were used to further investigate the underlying mechanism. Results: qRT-PCR results indicated that EPS8L3 was highly expressed in PC tissues compared with adjacent ones. At the same time, the incidence of distant metastasis was higher in PC patients with high EPS8L3 level. In vitro analysis such as CCK-8 and Transwell experimentations indicated that knockdown of EPS8L3 markedly inhibited the proliferative and metastatic ability. Bio-informatics together with luciferase report assay proposing that EPS8L3 can target GSK3B. Western Blot results revealed that knockdown of EPS8L3 markedly reduced the GSK3B expression in PC cells, and there was a positively associated between the two in PC cells. In addition, the recovery experimentation proved that EPS8L3 and GSK3B have a mutual regulation effect. Overexpression of GSK3B can reversal the prohibitive effect of EPS8L3 knockdown on the malignant development of PC cells, thereby jointly regulating the occurrence and development of PC. Background: We intended to investigate the role and regulatory mechanism of EPS8L3 in increase the development of pancreatic cancer (PC). Methods: In order to analyze the relationship between EPS8L3 level and clinicopathological indicators of PC patients, qRT-PCR was used to detect the expression of EPS8L3 in tumor specimens of 40 PC patients. EPS8L3 knockdown models were then constructed in PC cell lines. Furthermore, the effect of EPS8L3 on PC cell function was analyzed by CCK-8 and Transwell assay. Dual luciferase reporter gene assay and recovery assay were used to further investigate the underlying mechanism. Results: qRT-PCR results indicated that EPS8L3 was highly expressed in PC tissues compared with adjacent ones. At the same time, the incidence of distant metastasis was higher in PC patients with high EPS8L3 level. In vitro analysis such as CCK-8 and Transwell experimentations indicated that knockdown of EPS8L3 markedly inhibited the proliferative and metastatic ability. Bio-informatics together with luciferase report assay proposing that EPS8L3 can target GSK3B. Western Blot results revealed that knockdown of EPS8L3 markedly reduced the GSK3B expression in PC cells, and there was a positively associated between the two in PC cells. In addition, the recovery experimentation proved that EPS8L3 and GSK3B have a mutual regulation effect. Overexpression of GSK3B can reversal the prohibitive effect of EPS8L3 knockdown on the malignant development of PC cells, thereby jointly regulating the occurrence and development of PC. [ABSTRACT FROM AUTHOR]

FAM129B is one of Niban-like proteins described in neoplastic cells and implicated in melanoma cell invasion, but no reports have been published on FAM129B and cell differentiation. We show that FAM129B is early and transiently expressed and crucial for 3T3-F442A adipogenesis. Fam129b is expressed downstream of the early genes Cebpb , Klf4 , Klf5 and Srebf1a , but upstream of Pparg2 since knockdown of Fam129b blocked Pparg2 expression and adipose differentiation. Glycogen synthase kinase 3 beta activity, a crucial kinase for adipogenesis, and the ERK1/2 are involved in FAM129B phosphorylation as part of the adipogenic program. Phosphorylated FAM129B is crucial for Pparg2 expression and the lipogenic gene expression downstream of Pparg2 , and hence for adipogenesis. Fam129b knockdown reduced adipocyte cluster formation and size, regulating commitment and clonal amplification. In vivo, BAT, inguinal and epidydimal fat expressed Fam129b, suggesting a role in adipose tissue development. We conclude that FAM129B is a cooperative protein that regulates differentiation during the early stages of adipogenesis. • FAM129B is a novel cooperative protein that regulates early stages of adipogenesis. • Fam129b knockdown blocked Pparg2 expression and adipose differentiation. • Fam129b is expressed downstream of Cebpb , Klf4 , Klf5 and Srebf1a but upstream of Pparg2. • GSK3β and ERK1/2 are involved in FAM129B phosphorylation as part of the adipogenic program. • This is the first report involving FAM129B with cell differentiation processes. [ABSTRACT FROM AUTHOR]

Abstract Glucose is the main brain fuel in fed conditions, while astrocytic glycogen is used as supplemental fuel when the brain is stimulated. Brain glycogen levels are decreased shortly after induced seizures in rodents, but little is known about how glycogen levels are affected interictally in chronic models of epilepsy. Reduced glutamine synthetase activity has been suggested to lead to increased brain glycogen levels in humans with chronic epilepsy. Here, we used the mouse pilocarpine model of epilepsy to investigate whether brain glycogen levels are altered, both acutely and in the chronic stage of the model. One day after pilocarpine‐induced convulsive status epilepticus (CSE), glycogen levels were higher in the hippocampal formation, cerebral cortex, and cerebellum. Opposite to expected, this was accompanied by elevated glutamine synthetase activity in the hippocampus but not the cortex. Increased interictal glycogen amounts were seen in the hippocampal formation and cerebral cortex in the chronic stage of the model (21 days post‐CSE), suggesting long‐lasting alterations in glycogen metabolism. Glycogen solubility in the cerebral cortex was unaltered in this epilepsy mouse model. Glycogen synthase kinase 3 beta (Gsk3b) mRNA levels were reduced in the hippocampal formations of mice in the chronic stage, which may underlie the elevated brain glycogen content in this model. This is the first report of elevated interictal glycogen levels in a chronic epilepsy model. Increased glycogen amounts in the brain may influence seizure susceptibility in this model, and this warrants further investigation.

Abstract Allergic rhinitis (AR) is a chronic inflammatory disease of the nasal mucosa. M2 macrophage polarization can reduce inflammation and repair tissue injury during AR development. Studies have substantiated the involvement of miRNAs in AR pathogenesis. Herein, the molecular mechanism of miR‐214‐3p in AR development was explored. To mimic the AR environment, ovalbumin (OVA) was used to treat macrophages. MiR‐214‐3p and glycogen synthase kinase 3 beta (GSK3B) expression in nasal mucus tissues and macrophages was assessed by RT‐qPCR. The M2 phenotypic signature of CD206 in macrophages was assessed by flow cytometry. The protein expression of GSK3B and M2 macrophage markers (ARG‐1 and IL‐10) was evaluated by western blotting. The correlation between miR‐214‐3p and GSK3B was validated by a luciferase reporter assay. We found that miR‐214‐3p was overexpressed in macrophages and nasal mucus tissues from AR patients. MiR‐214‐3p facilitated M2 polarization of macrophages upon OVA stimulation. Mechanistically, miR‐214‐3p targeted the GSK3B 3′ untranslated region in macrophages. In addition, GSK3B was downregulated in macrophages and nasal mucus tissues from AR patients. In rescue assays, GSK3B downregulation reversed the inhibitory effects of miR‐214‐3p silencing on M2 polarization of macrophages treated with OVA. Overall, miR‐214‐3p facilitates M2 macrophage polarization by targeting GSK3B.

Alzheimer’s disease (AD) remains one of the most common dementias of neurodegenerative disease-related diseases. Nucleosome assembly protein 1-like 5 (NAP1L5) belongs to the NAP1L protein family, which acts as a histone chaperone. However, the function and mechanism of NAP1L5 in AD are still unclear. Bioinformatics analysis, RT-qPCR, and Western blotting results showed that NAP1L5 was downregulated in the brain tissues of AD patients and a mouse cell model of AD. NAP1L5 overexpression alleviated (Amyloid-β precursor protein) APP metabolism and Tau phosphorylation. We further demonstrated that NAP1L5 regulated the AD-like pathological characteristics through the GSK3B/Wnt/β-Catenin signaling pathway. Moreover, we showed that the Wnt/β-Catenin signaling pathway, regulated by NAP1L5, was mediated by AQP1-mediated mechanism in N2a-APP695sw cell. In sum, these results suggested that NAP1L5 overexpression has neuroprotective effects and might act as potential biomarker and target for the diagnosis and treatment of AD. [ABSTRACT FROM AUTHOR]

The sheer complexity of the brain has complicated our ability to understand the cellular and molecular mechanisms underlying its function in health and disease. Genome-wide association studies have uncovered genetic variants associated with specific neurological phenotypes and diseases. In addition, single-cell transcriptomics have provided molecular descriptions of specific brain cell types and the changes they undergo during disease. Although these approaches provide a giant leap forward towards understanding how genetic variation can lead to functional changes in the brain, they do not establish molecular mechanisms. To address this need, we developed a 3D co-culture system termed iAssembloids (induced multi-lineage assembloids) that enables the rapid generation of homogenous neuron-glia spheroids. We characterize these iAssembloids with immunohistochemistry and single-cell transcriptomics and combine them with large-scale CRISPRi-based screens. In our first application, we ask how glial and neuronal cells interact to control neuronal death and survival. Our CRISPRi-based screens identified that GSK3β inhibits the protective NRF2-mediated oxidative stress response in the presence of reactive oxygen species elicited by high neuronal activity, which was not previously found in 2D monoculture neuron screens. We also apply the platform to investigate the role of APOE- ε 4, a risk variant for Alzheimer's Disease, in its effect on neuronal survival. We find that APOE- ε 4-expressing astrocytes may promote more neuronal activity as compared to APOE- ε 3-expressing astrocytes. This platform expands the toolbox for the unbiased identification of mechanisms of cell-cell interactions in brain health and disease.

Glucose is the main brain fuel in fed conditions, while astrocytic glycogen is used as supplemental fuel when the brain is stimulated. Brain glycogen levels are decreased shortly after induced seizures in rodents, but little is known about how glycogen levels are affected interictally in chronic models of epilepsy. Reduced glutamine synthetase activity has been suggested to lead to increased brain glycogen levels in humans with chronic epilepsy. Here, we used the mouse pilocarpine model of epilepsy to investigate whether brain glycogen levels are altered, both acutely and in the chronic stage of the model. One day after pilocarpine‐induced convulsive status epilepticus (CSE), glycogen levels were higher in the hippocampal formation, cerebral cortex, and cerebellum. Opposite to expected, this was accompanied by elevated glutamine synthetase activity in the hippocampus but not the cortex. Increased interictal glycogen amounts were seen in the hippocampal formation and cerebral cortex in the chronic stage of the model (21 days post‐CSE), suggesting long‐lasting alterations in glycogen metabolism. Glycogen solubility in the cerebral cortex was unaltered in this epilepsy mouse model. Glycogen synthase kinase 3 beta (Gsk3b) mRNA levels were reduced in the hippocampal formations of mice in the chronic stage, which may underlie the elevated brain glycogen content in this model. This is the first report of elevated interictal glycogen levels in a chronic epilepsy model. Increased glycogen amounts in the brain may influence seizure susceptibility in this model, and this warrants further investigation. [ABSTRACT FROM AUTHOR]

Allergic rhinitis (AR) is a chronic inflammatory disease of the nasal mucosa. M2 macrophage polarization can reduce inflammation and repair tissue injury during AR development. Studies have substantiated the involvement of miRNAs in AR pathogenesis. Herein, the molecular mechanism of miR‐214‐3p in AR development was explored. To mimic the AR environment, ovalbumin (OVA) was used to treat macrophages. MiR‐214‐3p and glycogen synthase kinase 3 beta (GSK3B) expression in nasal mucus tissues and macrophages was assessed by RT‐qPCR. The M2 phenotypic signature of CD206 in macrophages was assessed by flow cytometry. The protein expression of GSK3B and M2 macrophage markers (ARG‐1 and IL‐10) was evaluated by western blotting. The correlation between miR‐214‐3p and GSK3B was validated by a luciferase reporter assay. We found that miR‐214‐3p was overexpressed in macrophages and nasal mucus tissues from AR patients. MiR‐214‐3p facilitated M2 polarization of macrophages upon OVA stimulation. Mechanistically, miR‐214‐3p targeted the GSK3B 3′ untranslated region in macrophages. In addition, GSK3B was downregulated in macrophages and nasal mucus tissues from AR patients. In rescue assays, GSK3B downregulation reversed the inhibitory effects of miR‐214‐3p silencing on M2 polarization of macrophages treated with OVA. Overall, miR‐214‐3p facilitates M2 macrophage polarization by targeting GSK3B. [ABSTRACT FROM AUTHOR]

Brain metastases represent a major cause of mortality among patients with breast cancer, and few effective targeted treatment options are currently available. Development of new biomarkers and therapeutic targets for breast cancer brain metastases (BCBM) is therefore urgently needed. In this study, we compared the gene expression profiles of the brain metastatic cell line MDA-MB-231-BR (231-BR) and its parental MDA-MB-231, and identified a total of 84 genes in the primary screening through a series of bioinformatic analyses, including construction of protein-protein interaction (PPI) networks by STRING database, identification of hub genes by applying of MCODE and Cytohubba algorithms, identification of leading-edge subsets of Gene Set Enrichment Analysis (GSEA), and identification of most up-regulated genes. Eight genes were identified as candidate genes due to their elevated expression in brain metastatic 231-BR cells and prognostic values in patients with BCBM. Then we knocked down the eight individual candidate genes in 231-BR cells and evaluated their impact on cell migration through a wound-healing assay, and four of them (KRT19, FKBP10, GSK3B and SPANXB1) were finally identified as key genes. Furthermore, the expression of individual key genes showed a correlation with the infiltration of major immune cells in the brain tumor microenvironment (TME) as analyzed by Tumor Immune Estimation Resource (TIMER) and Gene Expression Profiling Interactive Analysis (GEPIA), suggesting possible roles of them in regulation of the tumor immune response in TME. Therefore, the present work may provide new potential biomarkers for BCBM. Additionally, using GSEA, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Enrichment Analysis, we determined the top enriched cellular functions or pathways in 231-BR cells, which may help better understand the biology governing the development and progression of BCBM. [ABSTRACT FROM AUTHOR]

Brain metastases represent a major cause of mortality among patients with breast cancer, and few effective targeted treatment options are currently available. Development of new biomarkers and therapeutic targets for breast cancer brain metastases (BCBM) is therefore urgently needed. In this study, we compared the gene expression profiles of the brain metastatic cell line MDA-MB-231-BR (231-BR) and its parental MDA-MB-231, and identified a total of 84 genes in the primary screening through a series of bioinformatic analyses, including construction of protein-protein interaction (PPI) networks by STRING database, identification of hub genes by applying of MCODE and Cytohubba algorithms, identification of leading-edge subsets of Gene Set Enrichment Analysis (GSEA), and identification of most up-regulated genes. Eight genes were identified as candidate genes due to their elevated expression in brain metastatic 231-BR cells and prognostic values in patients with BCBM. Then we knocked down the eight individual candidate genes in 231-BR cells and evaluated their impact on cell migration through a wound-healing assay, and four of them (KRT19, FKBP10, GSK3B and SPANXB1) were finally identified as key genes. Furthermore, the expression of individual key genes showed a correlation with the infiltration of major immune cells in the brain tumor microenvironment (TME) as analyzed by Tumor Immune Estimation Resource (TIMER) and Gene Expression Profiling Interactive Analysis (GEPIA), suggesting possible roles of them in regulation of the tumor immune response in TME. Therefore, the present work may provide new potential biomarkers for BCBM. Additionally, using GSEA, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Enrichment Analysis, we determined the top enriched cellular functions or pathways in 231-BR cells, which may help better understand the biology governing the development and progression of BCBM.

Generating stable livestock pluripotent stem cells (PSCs) can be used for complex genome editing, cellular agriculture, gamete generation, regenerative medicine and in vitro breeding schemes. Over the past decade, significant progress has been made in characterizing pluripotency markers for livestock species. In this study, we investigated embryo development and gene expression of the core pluripotency triad (OCT4 , NANOG , SOX2) and cell lineage commitment markers (REX1 , CDX2 , GATA4) in the presence of three small molecules and their combination [PD0325901 (FGF inhibitor), SB431542 (TGFβ inhibitor), and CHIR99021 (GSK3B inhibitor)] from day 2–7 post-insemination in goat. Significant reduction in rate of blastocyst formation was observed when SB was used along with PD or CHIR and their three combinations had more sever effect. SB and CHIR decreased the expression of SOX2 while increasing the GATA4 expression. PD decrease the relative expression of NANOG , OCT4 and GATA4 , while increased the expression of REX1. Among the combination of two molecules, only SB + CHIR combination significantly decreased the expression of GATA4 , while the combination of the three molecules significantly decreases the expression of NANOG , SOX2 and CDX2. According to these results, the inhibition of the FGF signaling pathway, by PD may lead to blocking the hypoblast formation as observed by reduction of GATA4. OCT4 and NANOG expressions did not show signs of maintenance pluripotency. GATA4 , NANOG and OCT4 in the PD group were downregulated and REX1 as EPI-marker was upregulated thus REX1 may be considered as a marker of EPI/ICM in goat. • In goat, we particularly showed that inhibition of TGFβ signaling with SB431542 along with PD0325901 and CHIR99021 lead to significant decrease in blastocyst formation. • SB431542 decreased the expression of SOX2 and this effect could have been mediated through inhibition of trophoblast formation. • The combination of the three molecules (SB431542, PD0325901, and CHIR99021) significantly decreases the expression of NANOG, SOX2 and CDX2. [ABSTRACT FROM AUTHOR]

[Display omitted] • XLR-11 increases the expression of VEGF , ANG1 , and ANG2 in human brain microvascular endothelial cells. • The VEGF , ANG1 , and ANG2 secretion from human brain microvascular endothelial cells was elevated after XLR-11 treatment. • Western blot analysis revealed that the expression of VEGF , ANG1 , and ANG2 increased in XLR-11 treated cells. • The phosphorylation at Ser9 residue in GSK3B enhanced after XLR-11 treatment. The endocannabinoid system receptors, cannabinoid receptors type-1 (CBR-1) and −2 (CBR-2), are implicated in several behavioral and cognitive processes. Many studies have indicated a correlation between cannabinoid receptors and angiogenesis. The current study aims to reveal the possible molecular signaling involved in brain angiogenesis induced by the activation of CBR-1 and CBR-2. We investigated whether the synthetic cannabinoid XLR-11, an agonist of CBR-1 and CBR-2, influences the mRNA and protein expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (ANG1) and −2 (ANG2) in human brain microvascular endothelial cells (hBMVEs). Furthermore, we determined the phosphorylation of glycogen synthase kinase 3 beta (GSK3B) expression. Treatment of hBMVEs cells with XLR-11 elevated the mRNA levels of VEGF , ANG1 , and ANG2. The secretion of these proangiogenic factors was increased in the media. Furthermore, the intracellular expression of VEGF , ANG1 , ANG2 , and GSK3B was significantly increased. This current research provides a new possible approach by targeting the cannabinoid receptors to control and regulate brain angiogenesis for treating a variety of angiogenesis-related diseases. This could be achived by using different agonists or antagonists of the cannabinoid receptors based on the nature of the diseases. [ABSTRACT FROM AUTHOR]

Numerous studies have considered galectin-3 or Glycogen synthase kinase 3 beta (GSK3B) as a potential prognosis marker for various cancers. However, the correlation between the protein expression of galectin-3/GSK3B and the clinical parameters of astrocytoma has not been reported. This study aims to validate the correlation between the clinical outcomes and protein expression of galectin-3/GSK3B in astrocytoma. Immunohistochemistry staining was performed to detect galectin-3/GSK3B protein expression in patients with astrocytoma. The Chi-square test, Kaplan-Meier evaluation, and Cox regression analysis were used to determine the correlation between clinical parameters and galectin-3/GSK3B expression. Cell proliferation, invasion, and migration were compared between a non-siRNA group and a galectin-3/GSK3B siRNA group. Protein expression in galectin-3 or GSK3B siRNA-treated cells was evaluated using western blotting. Galectin-3 and GSK3B protein expression were significantly positively correlated with the World Health Organization (WHO) astrocytoma grade and overall survival time. Multivariate analysis revealed that WHO grade, galectin-3 expression, and GSK3B expression were independent prognostic factors for astrocytoma. Galectin-3 or GSK3B downregulation induced apoptosis and decreased cell numbers, migration, and invasion. siRNA-mediated gene silencing of galectin-3 resulted in the downregulation of Ki-67, cyclin D1, VEGF, GSK3B, p-GSK3B Ser9 (p-GSK3B S9), and β-catenin. In contrast, GSK3B knockdown only decreased Ki-67, VEGF, p-GSK3B S9, and β-catenin protein expression but did not affect cyclin D1 and galectin-3 protein expression. The siRNA results indicated that GSK3B is downstream of the galectin-3 gene. These data support that galectin-3 mediated tumor progression by upregulating GSK3B and β-catenin protein expression in glioblastoma. Therefore, galectin-3 and GSK3B are potential prognostic markers, and their genes may be considered to be anticancer targets for astrocytoma therapy.

EIF4A3 (eukaryotic translation initiation factor 4A3) is an RNA helicase and core component of the exon junction complex. While this RNA-binding protein (RBP) is well-characterized for its crucial roles in splicing, RNA trafficking and nonsense-mediated decay, its role in the regulation of metabolic signaling pathways remains elusive. In a recent study, we describe a new role for EIF4A3 as a negative regulator of macroautophagy/autophagy. Mechanistically, we report that EIF4A3, through its ability to safeguard splicing, can maintain low basal levels of autophagy through the cytosolic retention of the key autophagy transcription factor TFEB. Upon EIF4A3 depletion, the shuttling of TFEB to the nucleus results in an integrated transcriptional response, which induces both early and late steps of the autophagy pathway and enhances autophagic flux. We further report the upregulation of EIF4A3 across multiple cancer types and highlight the relevance of this newly identified EIF4A3-TFEB signaling axis in human tumors. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) remains one of the most common dementias of neurodegenerative disease-related diseases. Nucleosome assembly protein 1-like 5 (NAP1L5) belongs to the NAP1L protein family, which acts as a histone chaperone. However, the function and mechanism of NAP1L5 in AD are still unclear. Bioinformatics analysis, RT-qPCR, and Western blotting results showed that NAP1L5 was downregulated in the brain tissues of AD patients and a mouse cell model of AD. NAP1L5 overexpression alleviated (Amyloid-β precursor protein) APP metabolism and Tau phosphorylation. We further demonstrated that NAP1L5 regulated the AD-like pathological characteristics through the GSK3B/Wnt/β-Catenin signaling pathway. Moreover, we showed that the Wnt/β-Catenin signaling pathway, regulated by NAP1L5, was mediated by AQP1-mediated mechanism in N2a-APP695sw cell. In sum, these results suggested that NAP1L5 overexpression has neuroprotective effects and might act as potential biomarker and target for the diagnosis and treatment of AD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Liu and Sun.)

• rs230529 was significantly associated with regional gray matter density and NFKB1 expression in multiple brain regions, including cerebellum. • Regulatory targets of NFKB1 (108/305) are enriched in SZ susceptibility genes, including DRD2, AKT1, GSK3B, and IL6. • A substantial proportion of NFKB1 target genes were subject to combinatorial regulation by NFKB1 and miRNAs, constituting a hybrid NFKB1 -miRNA-gene regulatory network, which showed a disease status-dependent pattern. • Multiple NFKB1 -related molecules are shown to be implicated in the treatment of SZ, making NFKB1 a potential new target for anti-SZ drug development. NFKB1 was associated with treatment-refractory schizophrenia (SZ) and response to antipsychotics; however, the underlying mechanisms through which NFKB1 confers its risk for SZ are largely unknown. We aimed to investigate the potential role of NFKB1 in SZ. In the present study, we investigated the association of the risk SNP rs230529 of NFKB1 with gray matter density and with NFKB1 mRNA levels in various human brain regions. The spatiotemporal expression pattern of NFKB1 in human brains was explored. We constructed a miRNA- NFKB1 -target gene regulatory network and analyzed its druggability through targeting NFKB1 for SZ treatment. NFKB1 showed the highest expression levels in the cerebellum, in which these levels were stratified by genotypes of rs230529. Interestingly, the allelic state of rs230529 was significantly associated with regional gray matter density in multiple brain regions (including the cerebellum), which also differed between patients with schizophrenia and controls. Furthermore, regulatory targets of NFKB1 were enriched among SZ susceptibility genes. A substantial proportion of NFKB1 target genes were subject to combinatorial regulation by NFKB1 and miRNAs, constituting a hybrid NFKB1- miRNA-gene regulatory network. Some components of this network showed expression changes relevant to both the disease and the treatment. Finally, we detected the dynamic changes of NFKB1-miR-155-5p-GSK3B and NFKB1-miR-155-5p/let-7a-5p-IL6 networks in course of the treatment of SZ. Taken together, our findings support the involvement of NFKB1 -mediated dysregulation in the development of SZ. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality worldwide. The current TNM (Tumor, Node, and Metastasis) classification approach is suboptimal in determining the prognosis of CRC patients. The prognosis for CRC is affected by a variety of features that are present at the initial diagnosis. Herein, we performed a systematic exploration and established a novel five-panel gene signature as a prognostic and early diagnosis biomarker after performing differential gene expression analyses in five independent in silico CRCs cohort and independently validating it in one clinical cohort, using immunohistochemistry. Four genes (BDNF, PTGS2, GSK3B, and CTNNB1) were significantly upregulated and one gene (HPGD) was significantly downregulated in primary tumor tissues compared with adjacent normal tissues throughout all the five in silico datasets. The univariate CoxPH analysis yielded a five-gene signature that accurately predicted overall survival (OS) and recurrence-free survival (RFS) in the in silico training (AUC = 0.73 and 0.69, respectively) and one independent in silico validation cohort (AUC = 0.69 and 0.74, respectively). This five-gene signature demonstrated significant associations with poor OS in independent clinical validation cohorts of colon cancer (CC) patients (AUC = 0.82). Intriguingly, a risk stratification model comprising of the five-gene signature together with TNM stage and gender status achieved an even superior AUC of 0.89 in the clinical cohorts. On the other hand, the circulating mRNA expression of the upregulated four-gene signature achieved a robust AUC = 0.83 with high sensitivity and specificity as a diagnosis marker in plasma from CRC patients. We have identified a novel, five-gene signature as an independent predictor of OS, which in combination with TNM stage and gender offers an easy-to-translate and facile assay for the personalized risk-assessment in CRC patients. [ABSTRACT FROM AUTHOR]