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To explore the long-term influence of methamphetamine abuse on metabolomics character, with gas chromatography-mass spectrometry (GS-MS) technology, and the potential regulatory network using the bioinformatics method. Forty withdrawal methamphetamine abusers (WMA) were recruited from Shanghai Gaojing Forced Isolation Detoxification Institute. Forty healthy controls (HC) were recruited from society. GS-MS technology was used to detect metabolic products in serum. A bioinformatics method was used to build a regulatory network. Q-PCR was used to detect the candidate gene expressions, and ELISA was used to detect the regulatory enzyme expressions. Four pathways were significantly changed in the MA compared to the HC: (1) the arginine synthesis pathway, (2) alanine, aspartic acid and glutamate metabolic pathway, (3) cysteine and methionine metabolic pathway, and (4) the ascorbate and aldarate pathway (enrichment analysis Methamphetamine abuse influences the metabolic process in the long term, and Show less

The palmitoyl acyltransferase (PAT) ZDHHC14 is highly expressed in the hippocampus and is the only PAT predicted to bind Type-I PDZ domain-containing proteins. However, ZDHHC14's neuronal roles are unknown. Here, we identify the PDZ domain-containing Membrane-associated Guanylate Kinase (MaGUK) PSD93 as a direct ZDHHC14 interactor and substrate. PSD93, but not other MaGUKs, localizes to the axon initial segment (AIS). Using lentiviral-mediated shRNA knockdown in rat hippocampal neurons, we find that ZDHHC14 controls palmitoylation and AIS clustering of PSD93 and also of Kv1 potassium channels, which directly bind PSD93. Neurodevelopmental expression of ZDHHC14 mirrors that of PSD93 and Kv1 channels and, consistent with ZDHHC14's importance for Kv1 channel clustering, loss of ZDHHC14 decreases outward currents and increases action potential firing in hippocampal neurons. To our knowledge, these findings identify the first neuronal roles and substrates for ZDHHC14 and reveal a previously unappreciated role for palmitoylation in control of neuronal excitability. Show less

Nerve damage can lead to movement and sensory dysfunction, with high morbidity and disability rates causing severe burdens on patients, families, and society. DNA methylation is a kind of epigenetics, and a great number of previous studies have demonstrated that DNA methylation plays an important role in the process of nerve regeneration and remodeling. However, compared with the central nervous system, the peripheral nervous system shows stronger recovery after injury, which is related to the complex microenvironment and epigenetic changes occurring at the site of injury. Therefore, what common epigenetic changes between the central and peripheral nervous systems remain to be elucidated. We first screened differential methylation genes after spinal cord injury and sciatic nerve injury using whole-genome bisulfite sequencing and methylated DNA immunoprecipitation sequencing, respectively. Subsequently, a total of 16 genes had the same epigenetic changes after spinal cord injury and sciatic nerve injury. The Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were performed to identify the critical biological processes and pathways. Furthermore, a protein-protein interaction network analysis indicated that Dnm3, Ntrk3, Smurf1, Dpysl2, Kalrn, Shank1, Dlg2, Arsb, Reln, Bmp5, Numbl, Prickle2, Map6, and Htr7 were the core genes. These outcomes may provide novel insights into the molecular mechanism of the subacute phase of nerve injury. These verified genes can offer potential diagnostic and therapeutic targets for nerve injury. Show less

Clinical reports suggest a potential link between excess retinoids and development of depression. Although it has been shown that all-trans retinoic acid (ATRA) administration induces behavioral changes, further insight into how ATRA is involved is lacking. The hippocampus seems to be a major target of retinoids, and abnormal synaptic plasticity of the hippocampus is involved in depression. We examined two genes associated with synaptic function, discs large homolog 2 ( Show less

Ankylosing spondylitis (AS) is a common complex inflammatory disease; however, up to now distinct genes with monogenic pattern have not been reported for this disease. In the present study, we report a large Iranian family with several affected members with AS. DNAs of the three affected and two healthy cases were chosen for performing whole-exome sequencing (WES). After several filtering steps, candidate variants in the following genes were detected: RELN, DNMT1, TAF4β, MUC16, DLG2, and FAM208. However, segregation analysis confirmed the association of only one variant, c.7456A>G; p.(Ser2486Gly) in the RELN gene with AS in this family. In addition, in silico predictions supported the probable pathogenicity of this variant. In this study, for the first time, we report a novel variant in the RELN gene, c.7456A>G; p.(Ser2486Gly), which completely co-segregates with AS. This association suggests potential insights into the pathophysiological bases of AS and it could broaden horizons toward new therapeutic strategies. Show less

This study aimed to investigate the role of circ0106714-miR-942-5p-discs large homolog 2 (DLG2), a novel interactome, in colorectal cancer (CRC). Circ0106714 was found to be the most significantly downregulated circular RNA in CRC using a bioinformatics method, and we researched whether the ability of circ0106714 to sponge miR-942-5p and release DLG2 could affect CRC development via Hippo-YES-associated protein (YAP) signaling. We first employed qRT-PCR and immunoblotting to detect messenger RNA (mRNA) and protein expression, respectively. Live imaging of mice tumor xenografts was then conducted to study the effect of circ0106714 on tumor progression in vivo. Reporter gene assays were subsequently conducted to verify the predicted targeting relationship between circ0106714, miR-942-5p, and DLG2 mRNA in SW480 and HCT116 cell lines. As well as using flow cytometry for both apoptosis and cell cycle profile analyses, CCK-8 and clone foci formation assays were performed to assess cell survival. Wound healing assay and transwell invasion assay were later carried out to evaluate the migration and invasion of the cell lines. Findings revealed that circ0106714 and DLG2 were significantly downregulated, while miR-942-5p was significantly upregulated in human CRC tissues and cell lines. However, circ0106714 upregulation significantly suppressed tumor progression in vivo and inhibited the malignancy phenotypes of tumor cells in vitro by targeting miR-942-5p. Also discovered in this research was that miR-942-5p could directly target DLG2 mRNA, thus enhancing the malignancy phenotypes of CRC cells. We even found that DLG2 overexpression resulted in enhanced phosphorylation of YAP, a critical downstream effector of DLG2. This downstream effector was demonstrated to have a tumor-suppressive capacity in CRC cell lines. In sum, circ0106714 could suppress CRC by sponging miR-942-5p and releasing DLG2, thus promoting YAP phosphorylation. Show less

DLG2, also known as postsynaptic density protein-93 (PSD-93) or chapsyn-110, is an excitatory postsynaptic scaffolding protein that interacts with synaptic surface receptors and signaling molecules. A recent study has demonstrated that mutations in the DLG2 promoter region are significantly associated with autism spectrum disorder (ASD). Although DLG2 is well known as a schizophrenia-susceptibility gene, the mechanisms that link DLG2 gene disruption with ASD-like behaviors remain unclear. Mice lacking exon 14 of the Dlg2 gene (Dlg2 Dlg2 These results suggest that homozygous Dlg2 deletion in mice leads to ASD-like behavioral phenotypes, including social deficits and increased repetitive behaviors, as well as reductions in excitatory synaptic input onto dorsolateral spiny projection neurons, implying that the dorsal striatum is one of the brain regions vulnerable to the developmental dysregulation of DLG2. Show less

Venous thrombo-embolism (VTE) is multi-factorial disease involving several genetic and acquired risk factors responsible for its onset. It may occur spontaneously upon climbing at High Altitude (HA). Several studies demonstrated that hypoxic conditions prevailing at HA pose an independent risk factor for VTE; however, molecular mechanism remains unknown. Present study aims to identify genes associated with HA-induced VTE pathophysiology using real time TaqMan Low-Density Array (TLDA) of known candidate genes. Gene expression of total 93 genes were studied and analyzed in patients of VTE from HA (HA-VTE) and from sea level (SL-VTE) in comparison to respective controls. Both HA-VTE and SL-VTE patients showed up-regulation of 37 genes involved in blood coagulation cascade, clot formation, platelet formation, endothelial response, angiogenesis, cell adhesion and calcium channel activity. Seven genes including ACE, EREG, C8A, DLG2, USF1, F2 and PCDHA7 were up-regulated in both HA-controls and VTE patients (both HA-VTE and SL-VTE) indicating their role during VTE event and also upon HA exposure. Ten genes; CDH18, FGA, EDNBR, GATA2, MAPK9, BCAR1, FRK, F11, PCDHA1 and ST8SIA4 were uniquely up-regulated in HA-VTE. The differentially expressed genes from the present study could be determining factors for HA-VTE susceptibility and provide insights into VTE occurrence at HA. Show less

Chronic pain is one of the serious conditions that affect human health and remains cure still remains a serious challenge as the molecular mechanism remains largely unclear. Here, we used label-free proteomics to identify potential target proteins that regulate peripheral inflammatory pain and reveal its mechanism of action. Inflammation in peripheral tissue was induced by injecting complete Freund's adjuvant (CFA) into rat hind paw. A proteomic method was adopted to compare the spinal dorsal horn (SDH) in peripheral inflammatory pain (PIP) model rats with controls. Differential proteins were identified in SDH proteome by label-free quantification. The role of screened target proteins in the PIP was verified by small interfering RNA (siRNA). A total of 3072 and 3049 proteins were identified in CFA and normal saline (NS) groups, respectively, and 13 proteins were identified as differentially expressed in the CFA group. One of them, neurexin-2, was validated for its role in the inflammatory pain. Neurexin-2 was up-regulated in the CFA group, which was confirmed by quantitative PCR. Besides, intrathecal siRNA-mediated knock-down of neurexin-2 attenuated CFA-induced mechanical and thermal hyperalgesia and reduced the expression of SDH membrane glutamate receptors (eg mGlu receptor 1, AMPA receptor) and postsynaptic density (eg PSD-95, DLG2). These findings increased the understanding of the role of neurexin-2 in the inflammatory pain, implicating that neurexin-2 acts as a potential regulatory protein of inflammatory pain through affecting synaptic plasticity in the SDH of rats. Show less

Neuroblastoma (NB) is a childhood neural crest tumor. There are two groups of aggressive NBs, one with MYCN amplification, and another with 11q chromosomal deletion; these chromosomal aberrations are generally mutually exclusive. The DLG2 gene resides in the 11q-deleted region, thus makes it an interesting NB candidate tumor suppressor gene. We evaluated the association of DLG2 gene expression in NB with patient outcomes, stage and MYCN status, using online microarray data combining independent NB patient data sets. Functional studies were also conducted using NB cell models and the fruit fly. Using the array data we concluded that higher DLG2 expression was positively correlated to patient survival. We could also see that expression of DLG2 was inversely correlated with MYCN status and tumor stage. Cell proliferation was lowered in both 11q-normal and 11q-deleted NB cells after DLG2 over expression, and increased in 11q-normal NB cells after DLG2 silencing. Higher level of DLG2 increased the percentage of cells in the G2/M phase and decreased the percentage of cells in the G1 phase. We detected increased protein levels of Cyclin A and Cyclin B in fruit fly models either over expressing dMyc or with RNAi-silenced dmDLG, indicating that both events resulted in enhanced cell cycling. Induced MYCN expression in NB cells lowered DLG2 gene expression, which was confirmed in the fly; when dMyc was over expressed, the dmDLG protein level was lowered, indicating a link between Myc over expression and low dmDLG level. We conclude that low DLG2 expression level forces cell cycle progression, and that it predicts poor NB patient survival. The low DLG2 expression level could be caused by either MYCN-amplification or 11q-deletion. Video Abstract. Show less

Impaired function of gonadotropin-releasing hormone (GnRH) neurons can cause a phenotypic spectrum ranging from delayed puberty to isolated hypogonadotropic hypogonadism (IHH). We sought to identify a new genetic etiology for these conditions. Exome sequencing was performed in an extended family with autosomal dominant, markedly delayed puberty. The effects of the variant were studied in a GnRH neuronal cell line. Variants in the same gene were sought in a large cohort of individuals with IHH. We identified a rare missense variant (F900V) in DLG2 (which encodes PSD-93) that cosegregated with the delayed puberty. The variant decreased GnRH expression in vitro. PSD-93 is an anchoring protein of NMDA receptors, a type of glutamate receptor that has been implicated in the control of puberty in laboratory animals. The F900V variant impaired the interaction between PSD-93 and a known binding partner, Fyn, which phosphorylates NMDA receptors. Variants in DLG2 that also decreased GnRH expression were identified in three unrelated families with IHH. The findings indicate that variants in DLG2/PSD-93 cause autosomal dominant delayed puberty and may also contribute to IHH. The findings also suggest that the pathogenesis involves impaired NMDA receptor signaling and consequently decreased GnRH secretion. Show less

Reduced insulin secretion results in hyperglycaemia and diabetes involving a complex aetiology that is yet to be fully elucidated. Genetic susceptibility is a key factor in beta cell dysfunction and hyperglycaemia but the responsible genes have not been defined. The Collaborative Cross (CC) is a recombinant inbred mouse panel with diverse genetic backgrounds allowing the identification of complex trait genes that are relevant to human diseases. The aim of this study was to identify and characterise genes associated with hyperglycaemia. Using an unbiased genome-wide association study, we examined random blood glucose and insulin sensitivity in 53 genetically unique mouse strains from the CC population. The influences of hyperglycaemia susceptibility quantitative trait loci (QTLs) were investigated by examining glucose tolerance, insulin secretion, pancreatic histology and gene expression in the susceptible mice. Expression of candidate genes and their association with insulin secretion were examined in human islets. Mechanisms underlying reduced insulin secretion were studied in MIN6 cells using RNA interference. Wide variations in blood glucose levels and the related metabolic traits (insulin sensitivity and body weight) were observed in the CC population. We showed that elevated blood glucose in the CC strains was not due to insulin resistance nor obesity but resulted from reduced insulin secretion. This insulin secretory defect was demonstrated to be independent of abnormalities in islet morphology, beta cell mass and pancreatic insulin content. Gene mapping identified the E2f8 (p = 2.19 × 10 Collectively, these findings suggest that E2F transcription factor 8 (E2F8) and discs large homologue 2 (DLG2) regulate insulin secretion. The CC resource enables the identification of E2f8 and Dlg2 as novel genes associated with hyperglycaemia due to reduced insulin secretion in pancreatic beta cells. Taken together, our results provide better understanding of the molecular control of insulin secretion and further support the use of the CC resource to identify novel genes relevant to human diseases. Show less

Numerous single nucleotide polymorphisms (SNPs), which have been identified as susceptibility factors for Parkinson's disease (PD) as per genome-wide association studies, have not been fully characterized for PD patients in China. This study aimed to replicate the relationship between 12 novel SNPs of 12 genes and PD risk in southern Chinese population. Twelve SNPs of 12 genes were detected in 231 PD patients and 249 controls, using the SNaPshot technique. Meta-analysis was used to assess heterogeneity of effect sizes between this study and published data. The impact of SNPs on gene expression was investigated by analysing the SNP-gene association in the expression quantitative trait loci (eQTL) data sets. rs8180209 of SNCA (allele model: P = .047, OR = 0.77; additive model: P = .047, OR = 0.77), rs2270968 of MCCC1 (dominant model: P = .024, OR = 1.52), rs7479949 of DLG2 (recessive model; P = .019, OR = 1.52), rs10748818 of GBF1 (additive model: P < .001, OR = 0.37), and rs4771268 of MBNL2 (recessive model: P = .003, OR = 0.48) were replicated to be significantly associated with the increased risk of PD. Noteworthy, a meta-analysis of previous studies suggested rs8180209, rs2270968, rs7479949 and rs4771268 were in line with those of our cohort. Our study replicated five novel functional SNPs in SNCA, MCCC1, DLG2, GBF1 and MBNL2 could be associated with increased risk of PD in southern Chinese population. Show less

Postsynaptic density protein-93 (PSD-93) plays an important role in ischemic brain injury through N-methyl-D-aspartate receptor (NMDAR)-triggered neurotoxicity. GTPase-activating protein for Ras (SynGAP) is a GAP specifically expressed in the central nervous system to regulate nerve development and synaptic plasticity. However, the link between PSD-93 and SynGAP and their role in ischemic brain injury remain elusive. Here, we showed that PSD-93 interacted with SynGAP and mediated SynGAP ubiquitination and degradation following ischemic brain injury. Proteasome inhibitor MG-132 could reverse the decrease of SynGAP protein level in wild-type mice following cerebral ischemia reperfusion through inhibiting SynGAP ubiquitination. Furthermore, NMDA receptor inhibitor MK801 could increase SynGAP protein level in wild-type mice following cerebral ischemia reperfusion. However, in PSD-93 knockout mice, MG-132 or NMDAR inhibitor had no significant effect on SynGAP expression. Both MG-132 and PSD-93 knockout reduced infarct volume and improved neurological deficit in mice at different time points after cerebral ischemia reperfusion. Furthermore, we identified that 670-685 amino acid sequence of SynGAP was essential to the binding of SynGAP to PSD-93, and designed a fusion peptide Tat-SynGAP (670-685aa) that could attenuate ischemic brain damage in wild-type mice. In conclusion, we provide the first evidence that PSD-93 directly interacts with SynGAP and mediates its ubiquitination and degradation to aggravate ischemic brain damage. Tat-SynGAP (670-685aa) may be considered as a candidate for treatment of acute ischemic stroke. Show less

Long non-coding RNAs (lncRNAs) are a heterogeneous class of non-coding RNAs whose biological roles are still poorly understood. LncRNAs serve as gene expression regulators, frequently interacting with epigenetic factors to shape the outcomes of crucial biological processes, and playing roles in different pathologies including cancer. Over the last years, growing scientific evidence supports the key role of some lncRNAs in tumor development and proposes them as valuable biomarkers for the clinic. In this study, we aimed to characterize lncRNAs whose expression is altered in tumor samples from patients with lung adenocarcinoma (LUAD) compared to adjacent normal tissue samples. On an RT-qPCR survey of 90 cancer-related lncRNAs, we found one lncRNA, DLG2-AS1, which was consistently downregulated in 70 LUAD patients. To gain insight into its biological function, DLG2-AS1 was cloned and successfully re-expressed in LUAD cancer cell lines. We determined that DLG2-AS1 is not a cis-regulatory element of its overlapping gene DLG2, as their transcription levels were not correlated, nor did DLG2-AS1 restoration modify the expression of DLG2 protein. Furthermore, after generating a receiver operating curve (ROC) and calculating the area under curve (AUC), we found that DLG2-AS1 expression showed high sensitivity and specificity (AUC = 0.726) for the classification of LUAD and normal samples, determining its value as a potential lung cancer biomarker. Show less

High-risk neuroblastomas typically display an undifferentiated or poorly differentiated morphology. It is therefore vital to understand molecular mechanisms that block the differentiation process. We identify an important role for oncogenic ALK-ERK1/2-SP1 signaling in the maintenance of undifferentiated neural crest-derived progenitors through the repression of DLG2, a candidate tumor suppressor gene in neuroblastoma. DLG2 is expressed in the murine "bridge signature" that represents the transcriptional transition state when neural crest cells or Schwann cell precursors differentiate to chromaffin cells of the adrenal gland. We show that the restoration of DLG2 expression spontaneously drives neuroblastoma cell differentiation, highlighting the importance of DLG2 in this process. These findings are supported by genetic analyses of high-risk 11q deletion neuroblastomas, which identified genetic lesions in the DLG2 gene. Our data also suggest that further exploration of other bridge genes may help elucidate the mechanisms underlying the differentiation of NC-derived progenitors and their contribution to neuroblastomas. Show less

Interactions among genomic loci have often been overlooked in genome-wide association studies, revealing the combinatorial effects of variants on phenotype or disease manifestation. Unexplained genetic variance, interactions among causal genes of small effects, and biological pathways could be identified using a network biology approach. The main objective of this study was to determine the genome-wide epistatic variants affecting feed efficiency traits [feed conversion ratio (FCR) and residual feed intake (RFI)] based on weighted interaction SNP hub (WISH-R) method. Herein, we detected highly interconnected epistatic SNP modules, pathways, and potential biomarkers for the FCR and RFI in Duroc and Landrace purebreds considering the whole population, and separately for low and high feed efficient groups. Highly interacting SNP modules in Duroc (1,247 SNPs) and Landrace (1,215 SNPs) across the population and for low feed efficient (Duroc-80 SNPs, Landrace-146 SNPs) and high feed efficient group (Duroc-198 SNPs, Landrace-232 SNPs) for FCR and RFI were identified. Gene and pathway analyses identified Show less

Genetic factors significantly affect alcohol consumption and vulnerability to withdrawal. Furthermore, some genetic models showing predisposition to severe withdrawal are also predisposed to low ethanol (EtOH) consumption and vice versa, even when tested independently in naïve animals. Beginning with a C57BL/6J × DBA/2J F2 intercross founder population, animals were simultaneously selectively bred for both high alcohol consumption and low acute withdrawal (SOT line), or vice versa (NOT line). Using randomly chosen fourth selected generation (S4) mice (N = 18-22/sex/line), RNA-Seq was employed to assess genome-wide gene expression in ventral striatum. The MegaMUGA array was used to detect genome-wide genotypic differences. Differential gene expression and the weighted gene co-expression network analysis were implemented as described elsewhere (Genes Brain Behav 16, 2017, 462). The new selection of the SOT and NOT lines was similar to that reported previously (Alcohol Clin Exp Res 38, 2014, 2915). One thousand eight hundred and sixteen transcripts were detected as differentially expressed between the lines. For genes more highly expressed in the SOT line, there was enrichment in genes associated with cell adhesion, synapse organization, and postsynaptic membrane. The genes with a cell adhesion annotation included 23 protocadherins, Mpdz and Dlg2. Genes with a postsynaptic membrane annotation included Gabrb3, Gphn, Grid1, Grin2b, Grin2c, and Grm3. The genes more highly expressed in the NOT line were enriched in a network module (red) with annotations associated with mitochondrial function. Several of these genes were module hub nodes, and these included Nedd8, Guk1, Elof1, Ndufa8, and Atp6v1f. Marked effects of selection on gene expression were detected. The NOT line was characterized by higher expression of hub nodes associated with mitochondrial function. Genes more highly expressed in the SOT aligned with previous findings, for example, Colville and colleagues (Genes Brain Behav 16, 2017, 462) that both high EtOH preference and consumption are associated with effects on cell adhesion and glutamate synaptic plasticity. Show less

Low back pain (LBP) is a common disabling condition. Lumbar disc degeneration (LDD) may be a contributing factor for LBP. Modic change (MC), a distinct phenotype of LDD, is presented as a pathological bone marrow signal change adjacent to vertebral endplate on MRI. It is strongly associated with LBP and has heritability around 30%. Our objective was to identify genetic loci associated with MC using a genome-wide meta-analysis. Presence of MC was evaluated in lumbar MRI in the Northern Finland Birth Cohort 1966 (n=1182) and TwinsUK (n=647). Genome-wide association analyses were carried out using linear regression model. Inverse-variance weighting approach was used in the meta-analysis. A locus associated with MC (p<5e-8) was found on chromosome 9 with the lead SNP rs1934268 in an intron of the Show less

Ovarian cancer (OC) is xenogeneic that is influenced by many generated factors related to epigenetic factors to accelerate tumor metastasis. This study was conducted with the objective of investigating the effect of microRNA-23a-3p (miR-23a) on the biological characteristics of OC stem cells by targeting discs large homolog 2 (DLG2). OC-related differentially expressed genes were screened by microarray-based gene expression analysis, after which a list of miRNAs that regulate the genes was predicted. In total, 50 patients diagnosed with OC were enrolled in this study. DLG2 positive protein expression was measured in OC tissues. The interaction between DLG2 and miR-23a was predicted and analyzed through luciferase activity measurement. With the intervention of miR-23a and/or DLG2 expression in OC stem cells, the expression of miR-23a, DLG2, Bax, Bcl-2, Oct-4, and Nanog was determined. Afterward, different cell experiments were conducted to examine the regulation effect of miR-23a in OC stem cells. Tumor formation in vivo was also evaluated in nude mice. DLG2 had low expression in OC. The results showed that there was a decrease in the expression of Bcl-2, Oct-4, and Nanog, while DLG2 and Bax were increased as a result of miR-23a depletion. In addition, when miR-23a was suppressed, cell viability, migration, invasion, cloning, and renewal abilities of OC stem cells were decreased, while apoptosis ability was enhanced. As a target gene of miR-23a, DLG2 downregulation reversed the suppressive function of miR-23a in the inhibition of OC development. Finally, in vivo experiment verified that miR-23a downregulation restrained the tumor growth in OC stem cells. In conclusion, our findings suggested that the inhibition of miR-23a results in the suppression of OC progression by releasing DLG2, which provides new understanding on the potential therapeutic effect of miR-23a inhibition in OC patients. Show less

A typical feature of the contextual fear memory is increased fear generalization with time. Though much attention has been given to the neural structures that underlie the long-term consolidation of a contextual fear memory, the molecular mechanisms regulating fear generalization remain unclear. We observed that retrieval of contextual fear in a novel context at a remote time point increased coupling of neuronal nitric oxide synthase (nNOS) with postsynaptic density-95 (PSD-95) and c-Fos expression in the anterior cingulate cortex (ACC). Disrupting nNOS-PSD-95 coupling in the ACC decreased the expression of Histone deacetylase 2 (HDAC Show less

To explore the molecular mechanism of insulin on proliferation and differentiation of MC3T3-E1 cell under high glucose conditions. We first investigated the effect of different concentrations of insulin on the osteoblast cell proliferation and cell differentiation at various time points by MTT analysis, cell cycle analysis, and expression detection of differentiation genes. Then, we used 200 ng/mL of insulin to treat the osteoblast cell at different time points for identifying the common differentially expressed mRNAs among various time points by RNA sequencing. Thirdly, we performed the gene ontology (GO) and the Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis to explore the biological function of these common differentially expressed mRNAs. The results showed that insulin promoted the cell proliferation and differentiation of osteoblast cell. In RNA sequencing, a total of 31 common differentially expressed mRNAs were identified between different time points. Mt1, Tmem135, Avp, and Dlg2 were found to be associated with the new bone formation. In addition, three important signalling pathways, namely, lysosome, glutamatergic synapse, and chemokine signalling pathways, were found in the KEGG enrichment analysis. Our work demonstrated that insulin could promote the osteoblast cell proliferation and cell differentiation, which may play a key role in bone formation. SIGNIFICANCE OF THE STUDY: Our result showed that insulin could promote the proliferation and differentiation of osteoblast at both cellular and molecular levels, which may promote the new bone formation in the osteoblasts. Show less

Overall survival of patients with low-grade glioma (LGG) has shown no significant improvement over the past 30 years, with survival averaging approximately 7 years. This study aimed to identify novel promising biomarkers of LGG and reveal its potential molecular mechanisms by integrated bioinformatics analysis. The microarray datasets of GSE68848 and GSE4290 were selected from GEO database for integrated analysis. In total, 293 overlapping differentially expressed genes (DEGs) were detected using the limma package. One hundred and eighty-eight nodes with 603 interactions were obtained from the establishment of protein-protein interaction (PPI) network. Functional and signaling pathway enriched were significantly correlated with the synapse and calcium signaling pathway, respectively. Module analysis revealed eight hub genes with high connectivity, which included CHRM1, DLG2, GABRD, GRIN1, HTR2A, KCNJ3, KCNJ9, and NUSAP1, and they were markedly correlated with patients' prognosis. The mining of the Gene Expression Profiling Interactive Analysis database and qPCR further confirmed the abnormal expression of these key genes with their prognostic value in LGG. We eventually predicted the 20 most vital small molecule drugs, which potentially reverse the carcinogenic state of LGG, as per the CMap (connectivity map) database and these DEGs, and MS-275 (enrichment score = -0.939) was considered as the most promising small molecule to treat LGG. In conclusion, our study provided eight reliable novel molecular biomarkers for diagnosis, prognosis prediction, and treatment targets for LGG. These conclusions will contribute to a better comprehension of molecular mechanisms fundamental to LGG occurrence and progression, and providing new insights for future development of genomic individualized treatment in LGG. Show less

We performed a comprehensive assessment of rare inherited variation in autism spectrum disorder (ASD) by analyzing whole-genome sequences of 2,308 individuals from families with multiple affected children. We implicate 69 genes in ASD risk, including 24 passing genome-wide Bonferroni correction and 16 new ASD risk genes, most supported by rare inherited variants, a substantial extension of previous findings. Biological pathways enriched for genes harboring inherited variants represent cytoskeletal organization and ion transport, which are distinct from pathways implicated in previous studies. Nevertheless, the de novo and inherited genes contribute to a common protein-protein interaction network. We also identified structural variants (SVs) affecting non-coding regions, implicating recurrent deletions in the promoters of DLG2 and NR3C2. Loss of nr3c2 function in zebrafish disrupts sleep and social function, overlapping with human ASD-related phenotypes. These data support the utility of studying multiplex families in ASD and are available through the Hartwell Autism Research and Technology portal. Show less

MicroRNAs (miRNAs) have emerged as critical regulators of ischemic stroke, a condition that affects neuronal survival. However, the precise role of miRNAs in regulating neuronal injury during ischemic stroke remains largely unknown. In this study, we investigated the potential role of miR-152-3p in regulating oxygen-glucose-deprivation/reoxygenation (OGD/R)-induced neuronal injury in vitro. We found that OGD/R-exposed neurons expressed less miR-152-3p. Functional analysis revealed that miR-152-3p overexpression increased the viability and reduced the apoptosis and reactive oxygen species (ROS) production of OGD/R-exposed neurons. By contrast, miR-152-3p inhibition exacerbated OGD/R-induced injury. Notably, we identified postsynaptic density protein-93 (PSD-93), an important regulator of neuroprotection during ischemic stroke, as a miR-152-3p target gene. PSD-93 inhibition by small interfering RNA (siRNA) or miR-152-3p reinforced the activation of nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) antioxidant signaling in OGD/R-exposed neurons. However, PSD-93 overexpression or Nrf2 silencing partially reversed miR-152-3p-mediated neuroprotection in OGD/R-exposed neurons. Overall, these results demonstrated that miR-152-3p protected neurons from OGD/R-induced apoptosis and ROS production by reinforcing Nrf2/ARE antioxidant signaling through targeting and inhibiting PSD-93, findings that suggest miR-152-3p is a potential target for neuroprotection during ischemic stroke. Show less

Many single nucleotide polymorphisms (SNPs) have been reported to be associated with migraine susceptibility. However, evidences for their associations with migraine endophenotypes or subtypes are scarce. We aimed to investigate the associations of pre-identified migraine susceptibility loci in Taiwanese with migraine endophenotypes or subtypes, including chronic migraine and allodynia. The associations of six SNPs identified from our previous study, including TRPM8 rs10166942, LRP1 rs1172113, DLG2 rs655484, GFRA1 rs3781545, UPP2 rs7565931, and GPR39 rs10803531, and migraine endophenotypes, including chronic migraine and allodynia were tested. Significant associations in the discovery cohort were validated in the replication cohort. The adjusted odds ratios (aOR) were calculated after controlling for confounders. In total, 1904 patients (mean age 37.5 ± 12.2 years old, female ratio: 77.7%) including 1077 in the discovery cohort and 827 in the replication cohort were recruited. Of them, 584 (30.7%) had chronic migraine. Of the 6 investigated SNPs, TRPM8 rs10166942 T allele-carrying patients were more likely to have chronic migraine than non-T allele carriers in both discovery and replication cohorts and combined samples (33.7% vs. 25.8%, p = 0.004, aOR = 1.62). In addition, T allele carriers reported more allodynic symptoms than non-T allele carriers (3.5 ± 3.7 vs. 2.6 ± 2.8, p < 0.001). However, allodynia severity did not differ between episodic and chronic migraine patients. No further correlations between genetic variants and endophenotypes were noted for the other SNPs. TRPM8 may contribute to the pathogenesis of chronic migraine. However, our study did not support allodynia as a link between them. The underlying mechanisms deserve further investigations. Show less

Leveraging the conserved cancer genomes across mammals has the potential to transform driver gene discovery in orphan cancers. Here, we combine cross-species genomics with validation across human-dog-mouse systems to uncover a new bone tumor suppressor gene. Comparative genomics of spontaneous human and dog osteosarcomas (OS) expose Disks Large Homolog 2 (DLG2) as a tumor suppressor candidate. DLG2 copy number loss occurs in 42% of human and 56% of canine OS. Functional validation through pertinent human and canine OS DLG2-deficient cell lines identifies a regulatory role of DLG2 in cell division, migration and tumorigenesis. Moreover, osteoblast-specific deletion of Dlg2 in a clinically relevant genetically engineered mouse model leads to acceleration of OS development, establishing DLG2 as a critical determinant of OS. This widely applicable cross-species approach serves as a platform to expedite the search of cancer drivers in rare human malignancies, offering new targets for cancer therapy. Show less

The disc-large (DLG)-membrane-associated guanylate kinase (MAGUK) family of proteins forms a central signaling hub of the glutamate receptor complex. Among this family, some proteins regulate developmental maturation of glutamatergic synapses, a process vulnerable to aberrations, which may lead to neurodevelopmental disorders. As is typical for paralogs, the DLG-MAGUK proteins postsynaptic density (PSD)-95 and PSD-93 share similar functional domains and were previously thought to regulate glutamatergic synapses similarly. Here, we show that they play opposing roles in glutamatergic synapse maturation. Specifically, PSD-95 promoted, whereas PSD-93 inhibited maturation of immature α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptor (AMPAR)-silent synapses in mouse cortex during development. Furthermore, through experience-dependent regulation of its protein levels, PSD-93 directly inhibited PSD-95's promoting effect on silent synapse maturation in the visual cortex. The concerted function of these two paralogs governed the critical period of juvenile ocular dominance plasticity (jODP), and fine-tuned visual perception during development. In contrast to the silent synapse-based mechanism of adjusting visual perception, visual acuity improved by different mechanisms. Thus, by controlling the pace of silent synapse maturation, the opposing but properly balanced actions of PSD-93 and PSD-95 are essential for fine-tuning cortical networks for receptive field integration during developmental critical periods, and imply aberrations in either direction of this process as potential causes for neurodevelopmental disorders. Show less