Systematic characterization of Gossypium GLN family genes reveals a potential function of GhGLN1.1a regulates nitrogen use efficiency in cotton.

The enzyme glutamine synthetase (GLN) is mainly responsible for the assimilation and reassimilation of nitrogen (N) in higher plants. Although the GLN gene has been identified in various plants, there is little information about the GLN family in cotton (Gossypium spp.). To elucidate the roles of GLN genes in cotton, we systematically investigated and characterized the GLN gene family across four cotton species (G. raimondii, G. arboreum, G. hirsutum, and G. barbadense). Our analysis encompassed analysis of members, gene structure, cis-element, intragenomic duplication, and exploration of collinear relationships. Gene duplication analysis indicated that segmental duplication was the primary driving force for the expansion of the GhGLN gene family. Transcriptomic and quantitative real-time reverse-transcription PCR (qRT-PCR) analyses indicated that the GhGLN1.1a gene is responsive to N induction treatment and several abiotic stresses. The results of virus-induced gene silencing revealed that the accumulation and N use efficiency (NUE) of cotton were affected by the inactivation of GhGLN1.1a. This study comprehensively analyzed the GhGLN genes in Gossypium spp., and provides a new perspective on the functional roles of GhGLN1.1a in regulating NUE in cotton.

Optimization of Enzymatic and Chemical Decellularization of Native Porcine Heart Valves for the Generation of Decellularized Xenografts.

One of the most important medical interventions for individuals with heart valvular disease is heart valve replacement, which is not without substantial challenges, particularly for pediatric patients. Due to their biological properties and biocompatibility, natural tissue-originated scaffolds derived from human or animal sources are one type of scaffold that is widely used in tissue engineering. However, they are known for their high potential for immunogenicity. Being free of cells and genetic material, decellularized xenografts, consequently, have low immunogenicity and, thus, are expected to be tolerated by the recipient's immune system. The scaffold ultrastructure and ECM composition can be affected by cell removal agents. Therefore, applying an appropriate method that preserves intact the structure of the ECM plays a critical role in the final result. So far, there has not been an effective decellularization technique that preserves the integrity of the heart valve's ultrastructure while securing the least amount of genetic material left. This study demonstrates a new protocol with untraceable cells and residual DNA, thereby maximally reducing any chance of immunogenicity. The mechanical and biochemical properties of the ECM resemble those of native heart valves. Results from this study strongly indicate that different critical factors, such as ionic detergent omission, the substitution of Triton X-100 with Tergitol, and using a lower concentration of trypsin and a higher concentration of DNase and RNase, play a significant role in maintaining intact the ultrastructure and function of the ECM.

Producing and Testing Prototype Tissue-Engineered 3D Tri-Leaflet Valved Stents on Biodegradable Poly-ε-Caprolactone Scaffolds.

Transcatheter pulmonary valve replacement is a minimally-invasive alternative treatment for right ventricular outflow tract dysfunction and has been rapidly evolving over the past years. Heart valve prostheses currently available still have major limitations. Therefore, one of the significant challenges for the future is the roll out of transcatheter tissue engineered pulmonary valve replacement to more patients. In the present study, biodegradable poly-ε-caprolactone (PCL) nanofiber scaffolds in the form of a 3D leaflet matrix were successfully seeded with human endothelial colony-forming cells (ECFCs), human induced pluripotent stem cell-derived MSCs (hMSCs), and porcine MSCs (pMSCs) for three weeks for the generation of 3D tissue-engineered tri-leaflet valved stent grafts. The cell adhesion, proliferation, and distribution of these 3D heart leaflets was analyzed using fluorescence microscopy and scanning electron microscopy (SEM). All cell lineages were able to increase the overgrown leaflet area within the three-week timeframe. While hMSCs showed a consistent growth rate over the course of three weeks, ECFSs showed almost no increase between days 7 and 14 until a growth spurt appeared between days 14 and 21. More than 90% of heart valve leaflets were covered with cells after the full three-week culturing cycle in nearly all leaflet areas, regardless of which cell type was used. This study shows that seeded biodegradable PCL nanofiber scaffolds incorporated in nitinol or biodegradable stents will offer a new therapeutic option in the future.

LncRNA MSTO2P promotes colorectal cancer progression through epigenetically silencing CDKN1A mediated by EZH2.

BACKGROUND: Pseudogene-derived long non-coding RNAs (lncRNAs) have been reported to act as key regulatory factors of cancers. However, the study focused on pseudogene misato family member 2 (MSTO2P) in the occurrence and development of colorectal cancer (CRC) remains unclear. METHODS: CCK-8, colony formation, and transwell assays clarified HT-29 and SW480 cell proliferation and invasion. Furthermore, flow cytometry was carried out to detect cell cycle and cell apoptosis. Subcellular localization assay indicated the location of MSTO2P in HT-29 cells. RIP and CHIP assays clarified the relationship of MSTO2P with target protein and gene in HT-29 cells. RESULTS: MSTO2P expression was upregulated in CRC tissues and cells. Functional experiments revealed that inhibition of MSTO2P suppressed HT-29 and SW480 cell proliferation and invasion, and promoted cell cycle arrest and cell apoptosis. Besides, MSTO2P epigenetically down-regulated cyclin-dependent kinase inhibitor 1A (CDKN1A) via binding to the enhancer of zeste homolog 2 (EZH2) in the nucleus. At last, rescue experiments proved the anti-tumor effect of inhibition of MSTO2P was partially recovered due to the knockdown of CDKN1A in HT-29 cells. CONCLUSION: LncRNA MSTO2P promoted colorectal cancer progression through epigenetically silencing CDKN1A mediated by EZH2.

OCT3/4 enhances tumor immune response by upregulating the TET1-dependent NRF2/MDM2 axis in bladder cancer.

This study aimed to investigate the function of OCT3/4 on tumor immune escape in bladder cancer. Initially, the expression of OCT3/4, TET1, NRF2 and MDM2 was quantified in tumor tissues and cells, followed by gain- or loss-of-function studies to define their roles in cell migration, invasion and apoptosis and tumorigenicity in nude mice. Bladder cancer presented with abundant expression levels of OCT3/4, TET1, NRF2 and MDM2. We found that OCT3/4 promoted TET1 expression via binding to its promoter and that TET1 recruited MLL protein to NRF2 promoter and upregulated its expression, while NRF2 enhanced MDM2 expression. Upregulated MDM2 accelerated tumor immune escape in bladder cancer in mice. OCT3/4 knockdown suppressed the cell migration and invasion while inducing apoptosis, and consequently prevented tumor growth and immune escape in mice. Collectively, OCT3/4 may promote the progression of tumor immune escape in bladder cancer through acting as a promoter of the TET1/NRF2/MDM2 axis.

Identification and Expression Analysis of the NPF Genes in Cotton.

The NPF (NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER FAMILY) transports various substrates, including nitrogen (N), which is essential for plant growth and development. Although many NPF homologs have been identified in various plants, limited studies on these proteins have been reported in cotton. This study identified 75, 71, and 150 NPF genes in Gossypium arboreum, G. raimondii, and G. hirsutum, respectively, via genome-wide analyses. The phylogenetic tree indicated that cotton NPF genes are subdivided into eight subgroups, closely clustered with Arabidopsis orthologues. The chromosomal location, gene structure, motif compositions, and cis-elements have been displayed. Moreover, the collinearity analysis showed that whole-genome duplication event has played an important role in the expansion and diversification of the NPF gene family in cotton. According to the transcriptome and qRT-PCR analyses, several GhNPFs were induced by the nitrogen deficiency treatment. Additional functional experiments revealed that virus-induced silencing (VIGS) of the GhNPF6.14 gene affects the growth and N absorption and accumulation in cotton. Thus, this study lays the foundation for further functional characterization of NPF genes in cotton.

Tissue Engineered Transcatheter Pulmonary Valved Stent Implantation: Current State and Future Prospect.

Patients with the complex congenital heart disease (CHD) are usually associated with right ventricular outflow tract dysfunction and typically require multiple surgical interventions during their lives to relieve the right ventricular outflow tract abnormality. Transcatheter pulmonary valve replacement was used as a non-surgical, less invasive alternative treatment for right ventricular outflow tract dysfunction and has been rapidly developing over the past years. Despite the current favorable results of transcatheter pulmonary valve replacement, many patients eligible for pulmonary valve replacement are still not candidates for transcatheter pulmonary valve replacement. Therefore, one of the significant future challenges is to expand transcatheter pulmonary valve replacement to a broader patient population. This review describes the limitations and problems of existing techniques and focuses on decellularized tissue engineering for pulmonary valve stenting.

Predictors of Coronary Plaque Erosion in Current and Non-Current Smokers With ST-Segment Elevation Myocardial Infarction　- An Optical Coherence Tomography Study.

BACKGROUND: Smoking is an important risk factor of plaque erosion. This study aimed to investigate the predictors of plaque erosion in current and non-current smokers presenting with ST-segment elevation myocardial infarction (STEMI).Methods and Results:A total of 1,320 STEMI patients with culprit plaque rupture or plaque erosion detected by pre-intervention optical coherence tomography were divided into a current smoking group (n=715) and non-current smoking group (n=605). Plaque erosion accounted for 30.8% (220/715) of culprit lesions in the current smokers and 21.2% (128/605) in the non-current smokers. Multivariable analysis showed age <50 years, single-vessel disease and the absence of dyslipidemia were independently associated with plaque erosion rather than plaque rupture, regardless of smoking status. In current smokers, diabetes mellitus (odds ratio [OR]: 0.29; 95% confidence interval [CI]: 0.10-0.83; P=0.021) was negatively associated with plaque erosion as compared with plaque rupture. In non-current smokers, minimal lumen area (MLA, OR: 1.37; 95% CI: 1.16-1.62; P<0.001) and nearby bifurcation (OR: 3.20; 95% CI: 1.98-5.16; P<0.001) were positively related to plaque erosion, but not plaque rupture. CONCLUSIONS: In patients with STEMI, the presence of diabetes mellitus significantly increased the risk of rupture-based STEMI but may not have reduced the risk of plaque erosion-based STEMI in current smokers. Nearby bifurcation and larger MLA were associated with plaque erosion in non-current smokers.

Transcriptome Analysis Reveals Differences in Key Genes and Pathways Regulating Carbon and Nitrogen Metabolism in Cotton Genotypes under N Starvation and Resupply.

Nitrogen (N) is the most important limiting factor for cotton production worldwide. Genotype-dependent ability to cope with N shortage has been only partially explored in cotton, and in this context, the comparison of molecular responses of cotton genotypes with different nitrogen use efficiency (NUE) is of particular interest to dissect the key molecular mechanisms underlying NUE. In this study, we employed Illumina RNA-Sequencing to determine the genotypic difference in transcriptome profile using two cotton genotypes differing in NUE (CCRI-69, N-efficient, and XLZ-30, N-inefficient) under N starvation and resupply treatments. The results showed that a large genetic variation existed in differentially expressed genes (DEGs) related to amino acid, carbon, and nitrogen metabolism between CCRI-69 and XLZ-30. Further analysis of metabolic changes in cotton genotypes under N resupply showed that nitrogen metabolism and aromatic amino acid metabolism pathways were mainly enriched in CCRI-69 by regulating carbon metabolism pathways such as starch and sucrose metabolism, glycolysis/gluconeogenesis, and pentose phosphate pathway. Additionally, we performed an expression network analysis of genes related to amino acid, carbon, and nitrogen metabolism. In total, 75 and 33 genes were identified as hub genes in shoots and roots of cotton genotypes, respectively. In summary, the identified hub genes may provide new insights into coordinating carbon and nitrogen metabolism and improving NUE in cotton.

Nitrogen preference and genetic variation of cotton genotypes for nitrogen use efficiency.

BACKGROUND: Although nitrogen (N) availability is a major determinant of cotton production, little is known about the importance of plants' preference for ammonium versus nitrate for better growth and nitrogen use efficiency (NUE). We aimed to assess the growth, physiology, and NUE of contrasting N-efficient cotton genotypes (Z-1017, N-efficient and GD-89, N-inefficient) supplied with low and high concentrations of ammonium- and nitrate-N. RESULTS: The results revealed that ammonium fed plants showed poor root growth, lower dry biomass, N content, leaf chlorophyll and gas exchange than those under nitrate irrespective of the concentration. However, the highest N uptake and utilization efficiency were obtained with nitrate fed plants, which also resulted in the highest dry biomass, N content, leaf chlorophyll and gas exchange as well as root growth. The results further confirmed that N-efficient (Z-1017) genotype performed better under both N sources, showing more flexibility to contrasting N condition by increasing growth and NUE in either source of N. Moreover, multivariate analysis showed a strong relationship of root morphological traits with N utilization efficiency, suggesting the physiological importance of roots over shoots in response to low nitrate concentration. CONCLUSION: Thus, it was confirmed that nitrate-N is superior to ammonium-N and the use of nitrate and N-efficient genotype is the best option for optimum cotton growth and NUE. Further, field evaluation is required to confirm the hypothesis that nitrate is a preferred N source for better cotton production and NUE. © 2020 Society of Chemical Industry.

Anticancer sulfonamide hybrids that inhibit bladder cancer cells growth and migration as tubulin polymerisation inhibitors.

Novel sulfonamide-dithiocarbamate hybrids were designed and synthesised via the molecular hybridisation strategy. Among them, compound 13d displayed a potent activity with IC50 values of 0.9, 0.7, 1.9 and 2.6 µM against UM-UC-3, RT-112, RT4 and T24. Compound 13d inhibited the migration and regulated the migration-related markers (E-cadherin, N-cadherin, Vimentin, Snail and Slung) against RT-112 cells in a concentration dependent manner. By the tubulin polymerisation assay in vitro and immunostaining assay, compound 13d was identified as a novel tubulin polymerisation inhibitor. Intragastric administration of compound 13d could inhibit the growth of RT-112 cells in vivo in a xenograft mouse model with the low toxicity, indicating that it may be a leading candidate with antitumor properties to treat bladder cancer.

Connexin43 Is Required for the Effective Activation of Spleen Cells and Immunoglobulin Production.

Gap junctions (Gjs), formed by specific protein termed connexins (Cxs), regulate many important cellular processes in cellular immunity. However, little is known about their effects on humoral immunity. Here we tested whether and how Gj protein connexin43 (Cx43) affected antibody production in spleen cells. Detection of IgG in mouse tissues and serum revealed that wild-type (Cx43+/+) mouse had a significantly higher level of IgG than Cx43 heterozygous (Cx43+/-) mouse. Consistently, spleen cells from Cx43+/+ mouse produced more IgG under both basal and lipopolysaccharide (LPS)-stimulated conditions. Further analysis showed that LPS induced a more dramatic activation of ERK and cell proliferation in Cx43+/+ spleen cells, which was associated with a higher pro-oxidative state, as indicated by the increased NADPH oxidase 2 (NOX2), TXNIP, p38 activation and protein carbonylation. In support of a role of the oxidative state in the control of lymphocyte activation, exposure of spleen cells to exogenous superoxide induced Cx43 expression, p38 activation and IgG production. On the contrary, inhibition of NOX attenuated the effects of LPS. Collectively, our study characterized Cx43 as a novel molecule involved in the control of spleen cell activation and IgG production. Targeting Cx43 could be developed to treat certain antibody-related immune diseases.

Angiotensin II Suppresses Rev-erbα Expression in THP-1 Macrophages via the Ang II Type 1 Receptor/Liver X Receptor α Pathway.

BACKGROUND/AIMS: Angiotensin II (Ang II) regulates the expression of some core clock genes; excess Ang II leads to atherosclerosis advancement. Macrophage Rev-erbα mediates clockwork and inflammation, and plays a role in atherosclerotic lesion progression. However, the role of Ang II in regulating Rev-erbα expression in macrophages remains unclarified. METHODS: We induced THP-1 macrophages by phorbol 12-myristate 13-acetate and investigated the effect of Ang II on Rev-erbα expression via real-time polymerase chain reaction, western blotting and small interfering RNA (siRNA) techniques. The cytotoxicity of the Rev-erbα agonist SR9009 was analyzed using a (3-[4,5-dimethylthiazol-2-yl])-2,5- diphenyltetrazolium bromide assay. RESULTS: Ang II suppressed Rev-erbα mRNA and protein expression in THP-1 macrophages in a dose and time dependent manner. This effect was mediated via Ang II type 1 receptor (AT1R), and not Ang II type 2 receptor or peroxisome proliferator-activated receptor γ (PPARγ). Consistent with Rev-erbα expression regulated by Ang II, the liver X receptor α (LXRα) protein expression was downregulated in a time-dependent manner after Ang II treatment. The activation or silence of LXRα significantly increased or decreased Rev-erbα expression regulated by Ang II, respectively. This suggests that LXRα is involved in the effect of Ang II on Rev-erbα expression. MMP-9 mRNA expressions were significantly suppressed by SR9009 in THP-1 and RAW264.7 macrophages; moreover, SR9009-treatment significantly reduced Ang II-induced MMP-9 protein expressions in two types of macrophages. CONCLUSION: Ang II downregulates Rev-erbα expression in THP-1 macrophages via the AT1R/LXRα pathway.

Rab11 Functions as an Oncoprotein via Nuclear Factor kappa B (NF-κB) Signaling Pathway in Human Bladder Carcinoma.

BACKGROUND Elevated expression of Rab11 has been reported in different human cancers, including human bladder carcinoma. This study, we investigated the biological effects and mechanism of Rab11 overexpression in human bladder carcinoma for the first time. MATERIAL AND METHODS Rab11 expression in bladder cancer tissues was detected using immunohistochemistry and Western blot analysis. Then, Rab11 expression was inhibited in T24 cells and it was overexpressed in BIU-87 cells. The effects of Rab11 perturbations on cell growth rate and invasion were analyzed by CCK8, cell cycle assay, and matrix gel invasion assay. MMP-9, cyclin E, and cyclin D1 levels were studied using Western blot and qPCR. NF-κB activity was studied by luciferase assay. RESULTS High expression of Rab11 was detected in 41.5% (66/159) of tumor specimens. We found a significant correlation between high Rab11 expression and depth of tumor invasion (P=0.004). Rab11 overexpression was observed to promote the growth rate and invasiveness of cancer cells through upregulation of MMP9, cyclin E, and cyclin D1 levels. Rab11 overexpression further elevated NF-κB reporter activity and enhanced p-IκB expression. Use of BAY 11-7082, a noted NF-κB inhibitor, partially abolished overexpression of MMP9 and cyclin D1 by Rab11. CONCLUSIONS Our research proved that high Rab11 expression enhances cellular multiplication and invasiveness of bladder cancer, possibly by regulating the NF-κB signaling pathway.

Carbenoxolone inhibits TRPV4 channel-initiated oxidative urothelial injury and ameliorates cyclophosphamide-induced bladder dysfunction.

Carbenoxolone (CBX) is a clinically prescribed drug for the treatment of digestive ulcer and inflammation. It is also a widely used pharmacological inhibitor of several channels in basic research. Given that the overactivity of several channels, including those inhibitable by CBX, underlies bladder dysfunction, we tested the potential therapeutic application and mechanism of CBX in the treatment of voiding dysfunction. In a mouse model of cystitis induced by cyclophosphamide (CYP), CBX administration prevented the CYP-elicited increase in bladder weight, oedema, haemorrhage, and urothelial injury. CBX also greatly improved micturition pattern, as manifested by the apparently decreased micturition frequency and increased micturition volume. Western blot results showed that CBX suppressed CYP-induced increase in protein carbonyls, COX-2, and iNOS. Further analysis using cultured urothelial cells revealed that acrolein, the major metabolite of CYP, caused protein oxidation, p38 activation, and urothelial injury. These effects of acrolein were reproduced by TRPV4 agonists and significantly prevented by antioxidant NAC, p38 inhibitor SB203580, TRPV4 antagonist RN-1734, and CBX. Further studies showed that CBX potently suppressed TRPV4 agonist-initiated calcium influx and subsequent cell injury. CBX attenuated CYP-induced cystitis in vivo and reduced acrolein-induced cell injury in vitro, through mechanisms involving inhibition of TRPV4 channels and attenuation of the channel-mediated oxidative stress. CBX might be a promising agent for the treatment of bladder dysfunction.

p53 protein-mediated up-regulation of MAP kinase phosphatase 3 (MKP-3) contributes to the establishment of the cellular senescent phenotype through dephosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2).

Growth arrest is one of the essential features of cellular senescence. At present, the precise mechanisms responsible for the establishment of the senescence-associated arrested phenotype are still incompletely understood. Given that ERK1/2 is one of the major kinases controlling cell growth and proliferation, we examined the possible implication of ERK1/2. Exposure of normal rat epithelial cells to etoposide caused cellular senescence, as manifested by enlarged cell size, a flattened cell body, reduced cell proliferation, enhanced ß-galactosidase activity, and elevated p53 and p21. Senescent cells displayed a blunted response to growth factor-induced cell proliferation, which was preceded by impaired ERK1/2 activation. Further analysis revealed that senescent cells expressed a significantly higher level of mitogen-activated protein phosphatase 3 (MKP-3, a cytosolic ERK1/2-targeted phosphatase), which was suppressed by blocking the transcriptional activity of the tumor suppressor p53 with pifithrin-α. Inhibition of MKP-3 activity with a specific inhibitor or siRNA enhanced basal ERK1/2 phosphorylation and promoted cell proliferation. Apart from its role in growth arrest, impairment of ERK1/2 also contributed to the resistance of senescent cells to oxidant-elicited cell injury. These results therefore indicate that p53-mediated up-regulation of MKP-3 contributes to the establishment of the senescent cellular phenotype through dephosphorylating ERK1/2. Impairment of ERK1/2 activation could be an important mechanism by which p53 controls cellular senescence.

Upregulation of connexin43 contributes to PX-12-induced oxidative cell death.

Thioredoxin (Trx) is a small redox protein that underlies aggressive tumor growth and resistance to chemotherapy. Inhibition of Trx with the chemical inhibitor PX-12 suppresses tumor growth and induces cell apoptosis. Currently, the mechanism underlying the therapeutic actions of PX-12 and the molecules influencing cell susceptibility to PX-12 are incompletely understood. Given that connexin43 (Cx43), a tumor suppressor, regulates tumor cell susceptibility to chemotherapy, we examined the possible involvement of Cx43 in PX-12-induced cell death. Exposure of cells to PX-12 led to a loss of cell viability, which was associated with the activation of oxidative sensitive c-Jun N-terminal kinase (JNK). Inhibition of JNK or supplement of cells with anti-oxidants prevented the cell-killing action of PX-12. The forced expression of Cx43 in normal and tumor cells increased cell sensitivity to PX-12-induced JNK activation and cell death. In contrast, the downregulation of Cx43 with siRNA or the suppression of gap junctions with chemical inhibitors attenuated JNK activation and enhanced cell resistance to PX-12. Further analysis revealed that PX-12 at low concentrations induced a JNK-dependent elevation in the Cx43 protein, which was also preventable by supplementing the cells with anti-oxidants. Our results thus indicate that Cx43 is a determinant in the regulation of cell susceptibility to PX-12 and that the upregulation of Cx43 may be an additional mechanism by which PX-12 exerts its anti-tumor actions.

Suramin inhibits antibody binding to cell surface antigens and disrupts complement-mediated mesangial cell lysis.

Suramin inhibits immune responses and protects cells against inflammatory cell injury. However, little is known about its mechanisms. Using an in vitro model of glomerular mesangial cell (MC) lysis induced by antibodies plus complement, we investigated the potential protective effects and mechanisms of suramin on immunologic cell injury. Exposure of rat MCs to anti-Thy-1 antibody plus complement or anti-MC rabbit serum caused complement-dependent cell lysis, which was blocked by suramin and its structural analogue NF023 and NF049, but not by PPADS, an antagonist of purinergic receptors. Addition of exogenous ATP also failed to affect MC lysis. Further analysis revealed that suramin interfered with antibody binding to cell membrane antigens and suppressed antibody-induced phosphorylation of several proteins, including p38. Inhibition of p38 with chemical inhibitor significantly attenuated cell injury. Collectively, our results indicate that suramin protects cells against antibody-initiated and complement-dependent cell injury through inhibition of antibody binding to cell surface antigens and suppression of p38 activation. Our study thus provides novel mechanistic insights into the actions of suramin and suggests that suramin might be used to treat certain immune diseases.

[Analysis of DIAPH3 gene mutation in a boy with autism spectrum disorder].

OBJECTIVE: To analyze the clinical manifestations and gene mutation of a 6 year old boy with autism spectrum disorders (ASD). METHODS: Peripheral blood of the boy and his parents were subjected to genetic testing. RESULTS: The patient was diagnosed with typical autism. Exome sequencing has identified mutations of four candidate genes, namely TUT1, DIAPH3, REELIN and SETD2, which were confirmed with Sanger sequencing. Analysis of family members confirmed that the missense mutations of DIAPH3 and SETD2 genes were of de novo origin. CONCLUSION: Missense mutations of DIAPH3 and SETD2 genes may have contributed to the risk of ASD. Disrupted neurogenesis associated with such mutations may have been the underlying mechanism for ASD.

A novel TXNIP-based mechanism for Cx43-mediated regulation of oxidative drug injury.

Gap junctions (GJs) play an important role in the regulation of cell response to many drugs. However, little is known about their mechanisms. Using an in vitro model of cytotoxicity induced by geneticin (G418), we explored the potential signalling mechanisms involved. Incubation of cells with G418 resulted in cell death, as indicated by the change in cell morphology, loss of cell viability and activation of caspase-3. Before the onset of cell injury, G418 induced reactive oxygen species (ROS) generation, activated oxidative sensitive kinase P38 and caused a shift of connexin 43 (Cx43) from non-phosphorylated form to hyperphosphorylated form. These changes were largely prevented by antioxidants, suggesting an implication of oxidative stress. Downregulation of Cx43 with inhibitors or siRNA suppressed the expression of thioredoxin-interacting protein (TXNIP), activated Akt and protected cells against the toxicity of G418. Further analysis revealed that inhibition of TXNIP with siRNA activated Akt and reproduced the protective effect of Cx43-inhibiting agents, whereas suppression of Akt sensitized cells to the toxicity of G418. Furthermore, interference of TXNIP/Akt also affected puromycin- and adriamycin-induced cell injury. Our study thus characterized TXNIP as a presently unrecognized molecule implicated in the regulatory actions of Cx43 on oxidative drug injury. Targeting Cx43/TXNIP/Akt signalling cascade might be a promising approach to modulate cell response to drugs.