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Background: Alcohol‐associated hepatic fibrosis is a widespread liver disease with no effective treatment. Recent studies have indicated that interleukin‐22 (IL‐22) can ameliorate alcohol‐associated liver disease. However, the mechanism underlying the role of IL‐22 in alcohol‐associated hepatic fibrosis remains unclear. Therefore, we investigated the effect of IL‐22 in a mouse model of alcohol‐associated hepatic fibrosis and its underlying mechanisms. Methods: Alcohol‐associated hepatic fibrosis was induced by feeding male C57BL/6J mice with a Lieber‐DeCarli liquid diet containing 4% ethyl alcohol for 8 weeks and injecting them with 5% tetrachloromethane (CCl4) intraperitoneally for the last 4 weeks. During the last 4 weeks, IL‐22 was also administered. We investigated the role of IL‐22 in autophagy and the PI3K/AKT/mTOR signaling pathway using a 3‐methyladenine intraperitoneal injection in the mice treated with IL‐22. The effects of IL‐22 on alcohol‐associated hepatic fibrosis, autophagy‐related gene expression, and PI3K/AKT/mTOR activity were assessed using histopathology, biochemical analysis, transmission electron microscopy, quantitative real‐time PCR, immunohistochemistry, and western blotting. Results: Mice treated with ethanol and CCl4 displayed distinct liver injuries, including hepatocyte necrosis, inflammatory cell infiltration, and hepatic fibrosis, which were substantially attenuated by IL‐22 treatment. In addition, we found that IL‐22 regulated the expression of autophagy‐related genes and inhibited the PI3K/AKT/mTOR pathway, as evidenced by the reduction in p‐PI3K, p‐AKT, and p‐mTOR expression after IL‐22 treatment. Conclusions: IL‐22 exerts a marked protective effect against alcohol‐associated hepatic fibrosis. Its effect may be partly related to the alteration of autophagy‐related gene expression and inhibition of the PI3K/AKT/mTOR pathway in the liver. [ABSTRACT FROM AUTHOR]

Electroacupuncture (EA) is a popular therapeutic therapy for premature ovarian insufficiency (POI). However, little has been known about the underlying processes of EA therapy. To investigate the benefit of EA and reveal the mechanism, thirty SD female rats were allocated into the control, model, sham, EA, and GnRHa groups at random. Vaginal smears were used to monitor the rats' estrous cycle. Serum liver and renal function (ALT, AST, BUN, and Cr), sex hormone (FSH, E2, and AMH), oxidative stress markers (SOD, GSH, and MDA), and inflammatory cytokine (IL6, IL1β, and TNFα) levels were measured by enzyme-linked immunosorbent assay (ELISA). Their ovary morphology was observed by hematoxylin-eosin staining. Transmission electron microscope was used to remark the ultrastructure of the granulocytes. Protein and gene expressions of Keap1/Nrf2/HO-1 pathway were detected by western blot and RT-PCR. Compared with the model group, in the EA group, the levels of serum sex hormones recovered to normal levels. Moreover, it reduced oxidative stress in rats, as demonstrated by increased SOD and GSH levels and decreased MDA levels. Meanwhile, Keap1 mRNA and protein expression dropped considerably in the EA group, while the mRNA and protein expressions of Nrf2 and HO-1 increased. We found that preventive EA might rescue rats with CTX-induced ovarian dysfunction. The anti-inflammatory and antioxidative stress properties of EA, which elevated the Keap1/Nrf2/HO-1 signaling pathway, might be the underlying mechanism. Furthermore, as compared to GnRHa, electroacupuncture did not raise the burden of the liver (ALT and AST) or the kidney (BUN and Cr). Electroacupuncture has a meaningful impact and a sufficient level of safety, making it useful for therapeutic setting in POI. [ABSTRACT FROM AUTHOR]

Transmembrane 9 superfamily proteins (TM9SFs) define a highly conserved protein family, each member of which is characterized by a variable extracellular domain and presumably nine transmembrane domains. Although previous studies have delineated the potential cytological roles of TM9SFs like autophagy and secretory pathway, their functions during development are largely unknown. To establish the basis for dissecting the functions of TM9SFs in vivo , we employed the open-source database, structure prediction, immunofluorescence and Western blot to describe the gene and protein expression patterns of TM9SFs in human and mouse. While TM9SFs are ubiquitously and homogeneously expressed in all tissues in human with RNA sequencing and proteomics analysis, we found that all mice Tm9sf proteins are preferentially expressed in lung except Tm9sf1 which is enriched in brain although they all distributed in various tissues we examined. In addition, we further explored their expression patterns in the mice central nervous system (CNS) and its extension tissue retina. Interestingly, we could show that Tm9sf1is developmentally up-regulated in brain. In addition, we also detected all Tm9sf proteins are located in neurons and microglia instead of astrocytes. Importantly, Tm9sf3 is localized in the nuclei which is distinct from the other members that are dominantly targeted to the plasma membrane/cytoplasm as expected. Finally, we also found that Tm9sf family members are broadly expressed in the layers of INL, OPL, and GCL of retina and likely targeted to the plasma membrane of retinal cells. Thus, our data provided a comprehensive overview of TM9SFs expression patterns, illustrating their ubiquitous roles in different organs, implying the possible roles of Tm9sf2/3/4 in lung functions and Tm9sf1 in neurodevelopment, and highlighting a unique cell biological functions of TM9SF3 in neuronal and microglia. [ABSTRACT FROM AUTHOR]

• Spermidine (Spd) play a positive role in plant responses to saline-alkaline stress. • Exogenous Spd stimulates NO synthesis under saline-alkaline stress. • Exogenous Spd enhances the tolerance of tomato plants to saline-alkaline stress depend on NO. Spermidine (Spd) and nitric oxide (NO) are closely intertwined in their synthesis and play a significant role in plant responses to saline-alkaline stress. However, the specific interactions between Spd and NO in alleviating the toxicity of saline-alkaline stress in plants remain poorly understood. In this study, we aimed to elucidate the involvement of NO in the regulation of saline-alkaline stress tolerance by spermidine (Spd). To achieve this, we employed exogenous Spd treatment, NO synthesis inhibitors, and NO scavengers. We found that exogenous Spd stimulates NO synthesis in the leaves and roots of tomato seedlings by upregulating the nitrate reductase (NR) activities and the expression of its coding gene SlNR1. Importantly, when NO synthesis is inhibited or NO is scavenged, the regulatory effects of Spd on proline content, antioxidant enzyme system, ionic homeostasis, and the expression of genes involved in Na+/ K +homeostasis in tomato seedlings are compromised under saline-alkaline stress. These observations suggest that NO plays a pivotal role in the defensive mechanisms induced by Spd against saline-alkaline stress in tomato seedlings. Consequently, Spd enhances the tolerance of tomato plants to saline-alkaline stress by regulating the nitrate reductase (NR) signaling pathway and mediating NO synthesis. [ABSTRACT FROM AUTHOR]

Recently, accumulating evidence has demonstrated that non-coding RNAs (ncRNAs) play a vital role in oncogenicity. Nevertheless, the regulatory mechanisms and functions remain poorly understood, especially for lncRNAs and circRNAs. In this study, we simultaneously detected, for the first time, the expression profiles of the whole transcriptome, including miRNA, circRNA and lncRNA + mRNA, in five pairs of laryngeal squamous cell carcinoma (LSCC) and matched non-carcinoma tissues by microarrays. Five miRNAs, four circRNAs, three lncRNAs and five mRNAs that were dysregulated were selected to confirm the verification of the microarray data by quantitative real-time PCR (qRT-PCR) in 20 pairs of LSCC samples. We constructed LSCC-related competing endogenous RNA (ceRNA) networks of lncRNAs and circRNAs (circRNA or lncRNA-miRNA-mRNA) respectively. Functional annotation revealed the lncRNA-mediated ceRNA network were enriched for genes involved in the tumor-associated pathways. Hsa_circ_0033988 with the highest degree in the circRNA-mediated ceRNA network was associated with fatty acid degradation, which was responsible for the depletion of fat in tumor-associated cachexia. Finally, to clarify the ncRNA co-regulation mechanism, we constructed a circRNA-lncRNA co-regulated network by integrating the above two networks and identified 9 modules for further study. A subnetwork of module 2 with the most dysregulated microRNAs was extracted to establish the ncRNA-involved TGF-β-associated pathway. In conclusion, our findings provide a high-throughput microarray data of the coding and non-coding RNAs and establish the foundation for further functional research on the ceRNA regulatory mechanism of non-coding RNAs in LSCC. [ABSTRACT FROM AUTHOR]

Objective We sought to inspect the role of AMD3100, which acts as an antagonist of stromal cell–derived factor-1/CXC chemokine receptor 4 on the formation of neointima in rabbit saccular aneurysm after flow diverter (FD) treatment. Methods Twenty saccular aneurysm models were established by using porcine pancreatic elastase. Three weeks later, a Tubridge FD was implanted into the saccular aneurysm. All treated models were immediately divided into 2 groups: the AMD3100 group was subcutaneously injected with AMD3100 (5 mg/kg per day), while the control group received saline. Morphology and thickness of the neointima were investigated 2 and 4 weeks after FD treatment, using hard tissue section and masson trichrome staining. Scanning electron microscope was used to observe endothelial-like cells, and fluorescence quantitative polymerase chain reaction was used to determine mRNA expression of neointima biomarkers, such as kinase insert domain receptor, VE-cadherin, CD34, and Tie2. Results Two and 4 weeks after FD treatment, the AMD3100 group had more endothelial-like cells than the control group in the neointima. Masson trichrome staining showed that the neointima in the AMD3100 group was more intact and thicker than that in the control group. Furthermore, increased mRNA levels of kinase insert domain receptor, VE-cadherin, and Tie2 in the neointima were found in the AMD3100 group compared with the control group. Conclusions Interval use of AMD3100 promotes the formation of neointima in rabbit saccular aneurysm and facilitates the endothelialization of the neointima after FD treatment. [ABSTRACT FROM AUTHOR]

Portulaca oleracea L.is an annual plant widely distributed from the temperate to the tropical zones. POL-P3b, a polysaccharide fraction purified fromPortulaca oleracea L., is able to enhance immunity and inhibit tumor formation. Induction of antitumor immunity by dendritic-tumor fusion cells can be modulated by their activation status. Mature dendritic cells are significantly better than immature dendritic cells at cytotoxic T-lymphocyte induction. In this study, we analyzed the effects of POL-P3b on the maturation and function of murine bone-marrow-derived dendritic cells (DCs) and relevant mechanisms. The phenotypic maturation of DCs was confirmed by flow cytometry. We found that POL-P3b upregulated the expression of CD80, CD86, CD83, and major histocompatibility complex class II molecules on DCs, stimulated production of more interleukin (IL)-12, tumor necrosis factor-α, and less IL-10. Also, DCs pulsed POL-P3b and freeze-thaw antigen increased DCs-driven T cells' proliferation and promoted U14 cells' apoptosis. Furthermore, the expression of TLR-4 was significantly increased on DCs treated by POL-P3b. These results suggested that POL-P3b may induce DCs maturation through TLR-4. Taken together, our results may have important implications for the molecular mechanisms of immunopotentiation of POL-P3b, and provide direct evidence to suggest that POL-P3b should be considered as a potent adjuvant nutrient supplement for DC-based vaccines. [ABSTRACT FROM PUBLISHER]

Background Chromodomain helicase DNA binding protein 5 (CHD5) has recently been identified as a potent tumour suppressor by acting as a master regulator of a tumour-suppressive network. Its inactivation resulted from aberrant methylation in the promoter occurs in several types of human malignancy and is associated with malignant tumour behaviour. In human hepatocellular carcinoma (HCC), CHD5 gene expression, methylation status and tumour-suppressive function have not been elucidated. Aims In this study, we focused on the epigenetic modification and tumour-suppressive mechanism of CHD5 gene in HCC. Methods CHD5 expression in nine HCC cell lines and 30 pairs of HCC specimens and adjacent non-cancerous tissues were analysed by quantitative reverse transcription PCR and Western blotting. Methylation-specific sequencing and methylation-specific PCR were performed to examine DNA methylation status of the CHD5 promoter in HCC cell lines and samples. The effect of CHD5 restoration on proliferation, colony formation, senescence, apoptosis and tumourigenicity were examined. Results CHD5 expression was sinificantly down-regulated in HCC cell lines and tissues examined, and the −841 to −470 region of CHD5 promoter was hypermethylated in these samples. Treatment with DNA methyltransferase inhibitor 5-aza-2-deoxycytidine resulted in a striking regional demethylation of the −841 to −470 region of CHD5 promoter and an increase in CHD5 expression. The restoration of CHD5 expression inhibited tumour cell proliferation, colony formation and tumourigenicity and caused cellular senescence. Conclusions Our findings demonstrate that CHD5 is a potential tumour suppressor gene epigenetically silenced in HCC. [ABSTRACT FROM AUTHOR]

Chromodomain helicase DNA binding protein 5 (CHD5) was previously proposed to function as a potent tumor suppressor by acting as a master regulator of a tumor-suppressive network. CHD5 is down-regulated in several cancers, including leukemia and is responsible for tumor generation and progression. However, the mechanism of CHD5 down-regulation in leukemia is largely unknown. In this study, quantitative reverse-transcriptase polymerase chain reaction and western blotting analyses revealed that CHD5 was down-regulated in human leukemia cell lines and samples. Luciferase reporter assays showed that most of the baseline regulatory activity was localized from 500 to 200 bp upstream of the transcription start site. Bisulfite DNA sequencing of the identified regulatory element revealed that the CHD5 promoter was hypermethylated in human leukemia cells and samples. Thus, CHD5 expression was inversely correlated with promoter DNA methylation in these samples. Treatment with DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) activates CHD5 expression in human leukemia cell lines. In vitro luciferase reporter assays demonstrated that methylation of the CHD5 promoter repressed its promoter activity. Furthermore, a chromatin immunoprecipitation assay combined with qualitative PCR identified activating protein 2 (AP2) as a potential transcription factor involved in CHD5 expression and indicated that treatment with DAC increases the recruitment of AP2 to the CHD5 promoter. In vitro transcription-factor activity studies showed that AP2 over-expression was able to activate CHD5 promoter activity. Our findings indicate that repression of CHD5 gene expression in human leukemia is mediated in part by DNA methylation of its promoter. [ABSTRACT FROM AUTHOR]

Abstract: Chromodomain helicase DNA binding protein 5 (CHD5) is a potent tumor suppressor that serves as a master regulator of a tumor-suppressive network. Examination of the role played by CHD5 in a wide range of human cancers is warranted. In this study, we focused on the epigenetic modification and tumor-suppressive role of CHD5 in lung cancer. We measured CHD5 mRNA and protein expression in lung cancer cells, lung cancer tissues, and their corresponding noncancerous lung tissues using real-time PCR and Western blot analysis. We then determined the methylation status of the CHD5 promoter in these samples using methylation-specific sequencing and analyzed CHD5 re-expression in lung cancer cells treated with or without 5-aza-2-deoxycytidine, an inhibitor of DNA methylation. Next, the lung cancer cell clones stably expressing EGFP-CHD5 protein or EGFP protein, respectively, were obtained and the effects of restored CHD5 expression on cell proliferation, colony formation, and tumorigenicity were assessed. CHD5 expression ranged from low to absent in the lung cancer cell lines and tissues examined; the CHD5 promoter was hyperethylated in these samples. Treatment with 5-aza-dC resulted in a localized decrease in methylation density and an increase in CHD5 expression. Clonogenicity and tumor growth were abrogated in A549 and H1299 cells upon restoration of CHD5 expression. A significant reduction in clonogenicity was observed; an average of 47.83±4.6% reduction for A549-EGFP-CHD5 was observed compared to A549-EGFP, and an average of 56.39±5.3% reduction for H1299-EGFP-CHD5 was observed compared to H1299-EGFP. A549-EGFP exhibited an average tumor size of 452.3±36.5mm3, whereas A549-EGFP-CHD5 exhibited an average tumor size of only 57.7±18.5mm3. Thus, our findings indicate that CHD5 is a potential tumor suppressor gene that is inactivated via an epigenetic mechanism in lung cancer. [Copyright &y& Elsevier]

Abstract: Recent evidence indicates that alterations to epigenetic DNA methylation patterns contribute to many autoimmune diseases. Biliary atresia (BA) is a virus-induced autoimmune disease characterized by impaired T cells, which may be due to aberrant DNA methylation. CD11a, a subunit of the β2-integrin LFA-1 (CD11a/CD18) with costimulatory functions, is overexpressed due to hypomethylation of its promoter regulatory elements in CD4+ T cells from patients with many autoimmune diseases. However, it is unknown whether aberrant expression and methylation of CD11a occur in T cells from infants with BA. We aimed to compare the CD11a expression level and the methylation status of the CD11a promoter region in CD4+ T cells from BA infants and healthy controls (HC). We used real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) to examine CD11a mRNA levels in CD4+ T cells from BA and HC infants. Bisulfite sequencing was used to determine the methylation status of the CD11a promoter and flanking regions in CD4+ T cells from BA and HC infants, and in CD4+ T cells with DNA methylation inhibitors. We found that CD11a expression is significantly decreased in BA CD4+ T cells (P =0.007). This was associated with hypermethylation of the CD11a promoter region in CD4+ T cells from infants with BA. Treatment with a DNA methylation inhibitor decreased CD11a promoter methylation and increased CD11a mRNA. Therefore, DNA hypermethylation at the CD11a locus contributes to the lowered expression of CD11a in BA CD4+ T cells. [Copyright &y& Elsevier]

Although transmission of the gene expression program from mother to daughter cells has been suggested to be mediated by gene bookmarking, the precise mechanism by which bookmarking mediates post-mitotic transcriptional re-activation has been unclear. Here, we used a real-time gene expression system to quantitatively demonstrate that transcriptional activation of the same genetic locus occurs with a significantly more rapid kinetics in post-mitotic cells versus interphase cells. RNA polymerase II large subunit (Pol II) and bromodomain protein 4 (BRD4) were recruited to the locus in a different sequential order on interphase initiation versus post-mitotic re-activation resulting from the recognition by BRD4 of increased levels of histone H4 Lys 5 acetylation (H4K5ac) on the previously activated locus. BRD4 accelerated the dynamics of messenger RNA synthesis by de-compacting chromatin and hence facilitating transcriptional re-activation. Using a real-time quantitative approach, we identified differences in the kinetics of transcriptional activation between interphase and post-mitotic cells that are mediated by a chromatin-based epigenetic mechanism. [ABSTRACT FROM AUTHOR]

Diosmetin (DGVL) extracted from the traditional Chinese herb L. has been found to have anticancer activity. In this study, the effects of DGVL on the thymus of U14-bearing mice were investigated. Using flow cytometry, peripheral blood lymphocytes were characterized based on the expression of surface markers for T helper cells (CD4+) and T suppressor cells (CD8+). Serum levels of tumor necrosis factor α (TNF-α), interleukin-2 (IL-2), IL-10, and transforming growth factor β1 (TGF-β1) and a cell proliferation assay were determined with an enzyme-linked immunosorbent assay. The expression of Fas and Fas ligand (FasL) on the thymus was determined by Western blotting. Our results showed that DGVL inhibited tumor growth and significantly increased the thymus weight compared with the control. Also, DGVL elevated serum levels of IL-2 and significantly reduced levels of TNF-α, TGF-β1, and IL-10 in a dose-dependent manner. Histological study and terminal dUTP nick end labeling staining results showed that DGVL protected thymus tissue against the onslaught of tumor growth by inhibiting thymus lymphocyte apoptosis. The cell proliferation assay revealed that DGVL might promote more thymus lymphocytes towards proliferation. Furthermore, the ratio of CD4+/CD8+ T lymphocytes was significantly increased from 0.69 to 2.29 by treatment with DGVL. Immunoblotting analyses revealed that the expression of Fas and FasL on the thymus was lower in mice in the DGVL treatment group than in the control mice. In conclusion, DGVL can inhibit tumor growth and protect tumor-induced apoptosis of the thymus, and the mechanism is closely associated with reduced cell death in the thymus and a Fas-FasL-dependent pathway. [ABSTRACT FROM AUTHOR]

The eukaryotic nucleus is a highly compartmentalized and dynamic environment. Chromosome territories are arranged nonrandomly within the nucleus and numerous studies have indicated that a gene''s position in the nucleus can impact its transcriptional activity. Here, we focus on recent advances in our understanding of the influence of specific nuclear neighborhoods on gene expression or repression. Nuclear neighborhoods associated with transcriptional repression include the inner nuclear membrane/nuclear lamina and perinucleolar chromatin, whereas neighborhoods surrounding the nuclear pore complex, PML nuclear bodies, and nuclear speckles seem to be transcriptionally permissive. While nuclear position appears to play an important role in gene expression, it is likely to be only one piece of a flexible puzzle that incorporates numerous parameters. We are still at a very early, yet exciting stage in our journey toward deciphering the mechanism(s) that govern(s) the permissiveness of gene expression/repression within different nuclear neighborhoods. [Copyright &y& Elsevier]

Introduction: Whether and how mesoderm posterior 1 (MESP1) plays a role in the proliferation of gastric cancer cells remain unclear. Methods: The expression of MESP1 was compared in 48 human gastric cancer tissues and adjacent normal tissues. Knockdown of MESP1 was performed to investigate the role of MESP1 in the proliferation and apoptosis of BGC-823 and MGC-803 gastric cancer cells. Knockdown of alternative reading frame (ARF) was performed to study the role of ARF in the inhibitory effect of MESP1 knockdown on cell proliferation in gastric cancer cells. Mouse subcutaneous xenograft tumor model bearing BGC-823 cells was used to investigate the role of MESP1 in the growth of gastric tumor in vivo. The effect of seven active ingredients from T. terrestris on MESP1 expression was tested. The anti-cancer effect of diosgenin was confirmed in gastric cancer cells. MESP1 dependence of the anti-cancer effect of diosgenin was confirmed by MESP1 knockdown. Results: MESP1 was highly expressed in human gastric cancer tissues (p < 0.05). MESP1 knockdown induced apoptosis and up-regulated the expression of ARF in gastric cancer cells (p < 0.05). Knockdown of ARF attenuated the anti-cancer effect of MESP1 knockdown (p < 0.05). In addition, MESP1 knockdown also suppressed tumor growth in vivo (p < 0.05). Diosgenin inhibits both mRNA and protein expression of MESP1 (p < 0.05). MESP1 knockdown attenuated the anti-cancer effect of diosgenin (p < 0.05). Conclusions: MESP1 promotes the proliferation of gastric cancer cells via inhibiting ARF expression. Diosgenin exerts anti-cancer effect through inhibiting MESP1 expression in gastric cancer cells. [ABSTRACT FROM AUTHOR]

Acetylcholinesterase (AChE, EC3.1.1.7) is a key enzyme in neuronal signal transduction that hydrolyzes the neurotransmitter acetylcholine (ACh). The potential roles of AChEs involved in the toxicological and physiological impacts of insecticides on the destructive pest Spodoptera frugiperda, however, are still exclusive. In the present study, two acetylcholinesterase genes, ace1 and ace2, were characterized from S. frugiperda transcriptome and genome. Spatial−temporal expression analysis indicated that both Sfruace1 and Sfruace2 had an enriched expression in the heads among all larval tissues, and in 3rd instar larvae among all developmental stages. Notably, the expression levels of Sfruace1 were much higher than Sfruace2 in all tested tissues and developmental stages. The RNA interference (RNAi) with specific designed small interfering RNA (siRNA) significantly reduced the expression of Sfruace1 to 30%, and Sfruace2 to 39%. The knockdown of Sfruace1 expression resulted in mortality of 37.6%, which is significantly higher than 17.06% in the siSfruace2-treated group. Furthermore, the RNAi of Sfruace1 and Sfruace2 expressions reduced the AChE enzymatic activity to 32.81% and 65.77%, respectively, compared with those of the untreated group, 24 h after injection of 70 ng siRNA per insect. The survivors after the siRNA treatments showed an apparent motor retardation to the artificial diet, and an apparent growth inhibition in the parental generation (F0) larvae. There was no significant inhibition effect on the growth of F0 and the first filial generation (F1) pupae and the fecundity of female adult. Insecticide bioassay showed that the siSfruace1-treated larvae were more susceptible to acephate than the siSfruace2-treated and untreated larvae. Our study suggests that Sfruace1 plays more important roles than Sfruace2 in larval survivorship and susceptibility to acephate, and both ace genes have differential roles in regulating larval growth, motor ability and insecticidal sensitivity in S. frugiperda. [ABSTRACT FROM AUTHOR]

Simple Summary: Porcine rotavirus (PoRV) is a major pathogen causing dehydrating diarrhea and fatality in newborn piglets, resulting in substantial economic losses in the swine industry worldwide. Therefore, the development of a safe and effective medication for treating PoRV infection is imperative. This study provides the first report on the anti-PoRV activity of Portulaca oleracea L. (POL). Our findings demonstrate that POL-P has antiviral activity against PoRV infection in vitro, and thus might be developed into a novel antiviral agent to control PoRV in pig farms. Diarrhea is one of the most common causes of death in young piglets. Porcine rotavirus (PoRV) belongs to the genus Rotavirus within the family Reoviridae, and is considered to be the primary pathogen causing diarrhea in piglets. Portulaca oleracea L. (POL) has been reported to alleviate diarrhea and viral infections. However, the antiviral effect of Portulaca oleracea L. polysaccharide (POL-P), an active component of POL, on PoRV infection remains unclear. This study demonstrated that the safe concentration range of POL-P in IPEC-J2 cells is 0–400 μg/mL. POL-P (400 μg/mL) effectively inhibits PoRV infection in IPEC-J2 cells, reducing the expression of rotavirus VP6 protein, mRNA and virus titer. Furthermore, on the basis of viral life cycle analysis, we showed that POL-P can decrease the expression of PoRV VP6 protein, mRNA, and virus titer during the internalization and replication stages of PoRV. POL-P exerts antiviral effects by increasing IFN-α expression and decreasing the expression levels of TNF-α, IL-6, and IL-10 inflammatory factors. Overall, our study found that POL-P is a promising candidate for anti-PoRV drugs. [ABSTRACT FROM AUTHOR]

Trisomy 21 is the most common chromosomal disorder and underlies Down syndrome. Epigenetics, such as DNA methylation and post-translational histone modifications, plays a vital role in Down syndrome. However, the functions of epigenetics-related long noncoding RNAs (lncRNAs), found to have an impact on neural diseases such as Alzheimer’s disease, remain unknown in Down syndrome. In this study, we analyzed the RNA sequencing data from Down syndrome-induced pluripotent stem cells (iPSCs) and normal iPSCs. A large number of lncRNAs were identified differentially expressed in Down syndrome-iPSCs. Notably, stronger perturbation was shown in the expression of lncRNAs compared to protein coding genes (Kolmogorov–Smirnov test, P < 0.05), suggesting that lncRNAs play more important roles in Down syndrome. Through gene set enrichment analysis and bi-clustering, we also found that most of the differential expressed lncRNAs were closely associated with mitochondrial functions (e.g. mitochondrion organization, P = 3.21 × 10 −17 ; mitochondrial ATP synthesis coupled electron transport, P = 1.73 × 10 −19 and mitochondrial membrane organization, P = 4.04 × 10 −8 ). PCR-array and qRT-PCR results revealed that almost all genes related to mitochondria were down-regulated in Down syndrome-iPSCs, implying that mitochondria were dysfunctional in Down syndrome (e.g. ATP5B , Fold Change = −8.2317; COX6A1 , Fold Change = −12.7788 and SLC25A17 , Fold Change = −22.1296). All in all, our study indicated that a stronger perturbation of lncRNAs expression may lead to the dysfunction of mitochondria in Down syndrome. [ABSTRACT FROM AUTHOR]

Abstract: Recent studies have demonstrated that apoptosis plays a pivotal role during skin wound healing and apoptosis-related factors in cell signaling regulate a variety of cellular function. In this study, the expressions of p38MAPK, and JNK, iNOS, eNOS were detected and the activations of caspase-6, -7, -8, -9, and calpain, another signaling pathway of apoptosis, were also investigated by immunohistochemical staining and Western blotting in mice. A time-dependent increase of each protein level was observed by immunohistochemistry and Western blot in mouse skin incision. p38MAPK level peaked at 12h and 3 d, calpain level peaked at 1 d and 5 d, iNOS level peaked at 1 d and 10 d, while the peak levels of eNOS, caspase-6, -7, -8, and -9 occurred at 3 d and p-JNK at 1 d post-injury. In the early phase of wound healing, infiltrating polymorphonulcear cells were labeled with all the factors except caspase-8. Thereafter, infiltrating mononuclear cells and proliferating spindle-shaped fibroblastic cells showed positive staining for p38MAPK, JNK, calpain, caspases and NOS. The activation of caspase-8, -9, -6, and -7 as detected by Western blot indicated that caspase apoptotic pathway may take effect in cellular elimination during skin wound healing. From the viewpoint of forensic pathology, the time-dependent expressions of the factors in apoptotic pathway during skin incised wound healing may be used as potential markers for wound age estimation. [Copyright &y& Elsevier]

Clara cells (CCs) are a morphologically and operationally heterogeneous population of Secretoglobin Scgb1a1-expressing secretory cells that are crucial for airway homeostasis and post-injury repair. Analysis of the extent and origin of CC diversity are limited by knowledge of genes expressed in these cells and their precursors. To identify novel putative markers of CCs and explore the origins of CC diversity, we characterized global changes in gene expression in embryonic lungs in which CCs do not form due to conditional disruption of Notch signaling (RbpjkCNULL). Microarray profiling, Real Time PCR (qRT-PCR), and RNA in situ hybridization (ISH) identified eleven genes downregulated in the E18.5 airways of Rbpjkcnull compared to controls, nearly half not previously known to mark CCs. ISH revealed that several genes had overlapping but distinct domains of expression of in the normal developing lung (E18.5). Notably, Reg3g, Chad, Gabrp and Lrrc26 were enriched in proximal airways, Hp in the distal airways and Upk3a in clusters of cells surrounding Neuroepithelial Bodies (NEBs). Seven of the eleven genes, including Reg3g, Hp, and Upk3a, were expressed in the adult lung in CCs in a pattern similar to that observed in the developing airways. qRT-PCR-based analysis of gene expression of CCs isolated from different airway regions of B1-EGFP reporter mice corroborated the spatial enrichment in gene expression observed by ISH. Our study identifies candidate markers for CC-precursors and CCs and supports the idea that the diversification of the CC phenotype occurs already during embryonic development. [ABSTRACT FROM AUTHOR]

Hypoxia-inducible factor 1α (HIF1α) is an important cellular survival protein under hypoxic conditions, regulating the cellular response to low oxygen tension via recruitment of a transcriptional co-activator, p300/CBP. p300/CBP induces expression of multiple genes involved in cell survival, proliferation, angiogenesis, and tumor development. Thus, a strategy to inhibit hypoxic responses in tumors may be to target the protein-protein interaction between HIF1α and p300/CBP. Here, we document, for the first time, that the aminocoumarin antibiotic, novobiocin, directly blocks the protein-protein interaction between the HIF1α C-terminal activation domain (CTAD) and the cysteine-histidine rich (CH1) region of p300/CBP. Also, novobiocin down-regulated HIF1α-controlled gene expression, specifically CA9, which is related to tumorigenesis. In a monolayer cell culture, novobiocin inhibited cell proliferation and colony formation in the MCF-7 human breast adenocarcinoma cell line and the A549 human lung cancer cell line. Rescue experiments revealed that the recombinant CTAD fragment of HIF1α partially reversed novobiocin’s inhibitory effects on cell proliferation and colony formation in MCF-7 cells. These findings suggest a novel mechanism of action for novobiocin which has the potential for innovative therapeutic use in tumor treatment. [ABSTRACT FROM AUTHOR]

Summary: Bim is a proapoptotic BH3-only Bcl-2 family member. In response to death stimuli, Bim dissociates from the dynein light chain 1 (DYNLL1/LC8), where it is inactive, and can then initiate Bax/Bak-mediated mitochondria-dependent apoptosis. We found that Bim depletion increases autophagosome synthesis in cells and in vivo, and this effect is inhibited by overexpression of cell death-deficient Bim. Bim inhibits autophagy by interacting with Beclin 1, an autophagy regulator, and this interaction is facilitated by LC8. Bim bridges the Beclin 1-LC8 interaction and thereby inhibits autophagy by mislocalizing Beclin 1 to the dynein motor complex. Starvation, an autophagic stimulus, induces Bim phosphorylation, which abrogates LC8 binding to Bim, leading to dissociation of Bim and Beclin 1. Our data suggest that Bim switches locations between apoptosis-inactive/autophagy-inhibitory and apoptosis-active/autophagy-permissive sites. [Copyright &y& Elsevier]

The expression of the N-methyl-D-aspartate receptor (NMDA-R) in astrocytes is controversial. The receptor is commonly considered neuron-specific. We showed that astrocytes in primary cultures differentially expressed mRNA of NMDA-R subunits, NR1, NR2A and NR2B, in development, ischemia and post-ischemia. One-week-old cultures expressed detectable NR1 mRNA, which fell significantly at 2 weeks and became barely detectable at 4 weeks. NR2A and NR2B mRNA were both significantly up-regulated from 1 to 2 weeks. In 4 weeks, 2 h of ischemia caused a significant up-regulation of NR1 and NR2B mRNA; while 6 h caused down-regulation of NR2A mRNA. Under 3 h of post-ischemia, only NR1 mRNA was increased. Ischemia induced the expression of major NMDA-R effecter, nitric oxide synthase 1, which was unaffected by AMPA-R antagonist CNQX, but dose-dependently inhibited by NMDA-R specific antagonist MK-801. These findings reflected that astrocyte could express inducible functional NMDA receptors without the presence of neurons. [ABSTRACT FROM AUTHOR]

Background. Human Schlafen 5 (SLFN5) is reported to inhibit or promote the proliferation of several specific types of cancer cells by our lab and other researchers. We are curious about its implications in lung adenocarcinoma (LUAC), a malignant tumor with a high incidence rate and high mortality. Method. Lentiviral stable transfections of SLFN5-specific shRNA for knockdown and SLFN5 full-length coding sequence for overexpression were performed in LUAC cell for proliferation analysis in vitro and in vivo in nude mice. Clinical LUAC samples were collected for immunohistochemical analysis of SLFN5 protein levels. Results. We found that knockdown of endogenous SLFN5 upregulates cancer cell proliferation while inhibiting apoptosis. Besides, SLFN5 inhibition on proliferation was also observed in a nude mouse xenograft model. In contrast, overexpression of exogenous SLFN5 inhibited cell proliferation in vitro and in vivo and promoted apoptosis. As to the signaling pathway, we found phosphatase and tensin homolog on chromosome 10 (PTEN) was positively regulated by SLFN5, while its downstream signaling pathway AKT/mammalian target of rapamycin (mTOR) was inhibited. Moreover, compared with adjacent normal tissues, SLFN5 protein levels were markedly decreased in lung adenocarcinoma tissues. In conclusion, these suggest that human SLFN5 plays inhibitory roles in LUAC progression through the PTEN/PI3K/AKT/mTOR pathway, providing a potential target for developing drugs for lung cancer therapy in the future. [ABSTRACT FROM AUTHOR]

• Frequency-specific brain activities within the sensorimotor network (SMN), visual network (VN), and default mode network (DMN) that were associated with the varying degrees of symptom severity in DCM patients. • Frequency-specific brain activity within the SMN correlated with functional recovery in DCM patients. • A spatial correlation between the brain-wide expression of genes involved in neuronal migration and the brain functional activity pattern was discovered and associated with symptom severity in DCM patients. Neuroimaging techniques provide insights into the brain abnormalities secondary to degenerative cervical myelopathy (DCM) and their association with neurological deficits. However, the neural correlates underlying the discrepancy between symptom severity and the degree of spinal cord compression, as well as the transcriptional correlates of these cortical abnormalities, remain unknown in DCM patients. In this cross-sectional study, which collected resting-state functional MRI (rs-fMRI) images and the Japanese Orthopedic Association (JOA) score, enrolled 104 participants (54 patients and 50 healthy controls). The frequency-dependent amplitude of low-frequency fluctuation (ALFF) was obtained for all participants. We investigated the ALFF differences between mild-symptom DCM patients and severe-symptom DCM patients while carefully matching the degree of compression between these two groups via both univariate comparison and searchlight classification for three frequency bands (e.g., Slow-4, Slow-5, and Full-band). Additionally, we identified genes associated with symptom severity in DCM patients by linking the spatial patterns of gene expression of Allen Human Brain Atlas and brain functional differences between mild symptom and severe symptom groups. (1) We found that the frequency-specific brain activities within the sensorimotor network (SMN), visual network (VN), and default mode network (DMN) were associated with the varying degrees of functional impairment in DCM patients; (2) the frequency-specific brain activity within the SMN correlated with the functional recovery in patients with DCM; (3) a spatial correlation between the brain-wide expression of genes involved in neuronal migration and the brain functional activities associated with symptom severity was identified in DCM patients. In conclusion, our study bridges gaps between genes, cell classes, biological processes, and brain functional correlates of DCM. While our findings are correlational in nature, they suggest that the neural activities of sensorimotor cortices in DCM are associated with the severity of symptoms and might be associated with neuronal migration within the brain. [ABSTRACT FROM AUTHOR]

Plasma membrane proton pumps play a crucial role in maintaining ionic homeostasis in salt-resistant Populus euphratica under saline conditions. High levels of NaCl (200 mM) induced PeHA1 expression in P. euphratica roots and leaves. We isolated a 2022 bp promoter fragment upstream of the translational start of PeHA1 from P. euphratica. The promoter–reporter construct PeHA1-pro :: GUS was transferred to tobacco plants, demonstrating that β-glucuronidase activities increased in root, leaf, and stem tissues under salt stress. DNA affinity purification sequencing revealed that PeWRKY1 protein targeted the PeHA1 gene. We assessed the salt-induced transcriptional response of PeWRKY1 and its interaction with PeHA1 in P. euphratica. PeWRKY1 binding to the PeHA1 W-box in the promoter region was verified by a yeast one-hybrid assay, EMSA, luciferase reporter assay, and virus-induced gene silencing. Transgenic tobacco plants overexpressing PeWRKY1 had improved expression of NtHA4 , which has a cis -acting W-box in the regulatory region, and improved H+ pumping activity in both in vivo and in vitro assays. We conclude that salt stress up-regulated PeHA1 transcription due to the binding of PeWRKY1 to the W-box in the promoter region of PeHA1. Thus, we conclude that enhanced H+ pumping activity enabled salt-stressed plants to retain Na+ homeostasis. [ABSTRACT FROM AUTHOR]

CircRNAs are involved in a variety of human diseases, however, the expression profiles and the potential diagnostic value of circRNAs in human acute Stanford type A aortic dissection (AAAD) remains largely unknown. In this study, high-throughput RNA sequencing (RNA-Seq) was used to investigate the differentially expressed circRNAs, microRNAs (miRs) and mRNAs in human AAAD tissues (n = 10) compared with normal aortic tissues (n = 10). The results of RNA-Seq revealed that 506 circRNAs were significantly dysregulated (P<0.05, false discovery rate, FDR<0.05, fold change>2). The subsequent weighted gene correlation network analysis and the following co-expression network analysis revealed that tyrosine-protein kinase Fgr might play important roles in the occurrence and development of AAAD. According to the circRNA-miRNA-mRNA network, we found that the upstream regulatory molecule of Fgr is circMARK3. Finally, a receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of the serum circMARK3 as biomarkers for AAAD (cutoff value = 1.497, area under the curve = 0.9344, P < 0.0001, sensitivity = 90.0%, specificity = 86.7%). These results provided a preliminary landscape of circRNAs expression profiles and indicated that circMARK3 was a potential biomarker for AAAD diagnosis. [ABSTRACT FROM AUTHOR]

Objective. By observing the needle-knife of KOA rabbit morphology, knee joint cartilage p-FAK, p-PI3K, Aggrecan gene, and protein expression, to study the effect of needle-knife to promote cartilage cell synthesis metabolism mechanism. Method. 49 male New Zealand rabbits, randomly divided into normal group (Z), model group (M), model-inhibitors (MP), needle-knife group (D), needle-knife inhibitors group (DP), electroacupuncture group (E), and electroacupuncture inhibitors (EP). RT-PCR and Western Blot were used to test each animal cartilage p-FAK, p-PI3K, and Aggrecan gene and protein expression level. Results. Compared with N group, p-FAK and p-PI3K protein and mRNA expression of M group, D group, and E group increased (P < 0.05), while the protein and mRNA expression of Aggrecan reduced (P < 0.05). Compared with M group, p-FAK, p-PI3K, Aggrecan protein, and mRNA of E and D group increased (P < 0.05). Compared with E group, p-FAK, p-PI3K, Aggrecan protein, and mRNA expression of D group increased (P < 0.05); after adding inhibitors, p-FAK, p-PI3K, Aggrecan protein, and mRNA expression reduced (P < 0.05). Conclusion. Needle-knife therapy can promote the repairment of cartilage cells by activating FAK-PI3K signaling pathways, promoting the synthesis of cartilage cell metabolism. [ABSTRACT FROM AUTHOR]

Crystallography is a powerful tool to determine the atomic structures of proteins and RNAs. X-ray crystallography has been used to determine the structure of many splicing related proteins and RNAs, making major contributions to our understanding of the molecular mechanism and regulation of pre-mRNA splicing. Compared to other structural methods, crystallography has its own advantage in the high-resolution structural information it can provide and the unique biological questions it can answer. In addition, two new crystallographic methods – the serial femtosecond crystallography and 3D electron crystallography – were developed to overcome some of the limitations of traditional X-ray crystallography and broaden the range of biological problems that crystallography can solve. This review discusses the theoretical basis, instrument requirements, troubleshooting, and exciting potential of these crystallographic methods to further our understanding of pre-mRNA splicing, a critical event in gene expression of all eukaryotes. [ABSTRACT FROM AUTHOR]

It has become increasingly clear that proper cellular control of pluripotency and differentiation is related to the regulation of rRNA synthesis. To further our understanding of the role that the regulation of rRNA synthesis has in pluripotency we monitored rRNA synthesis during the directed differentiation of human embryonic stem cells (hESCs). We discovered that the rRNA synthesis rate is reduced ~50% within 6 hours of ACTIVIN A treatment. This precedes reductions in expression of specific stem cell markers and increases in expression of specific germ layer markers. The reduction in rRNA synthesis is concomitant with dissociation of the Pol I transcription factor, UBTF, from the rRNA gene promoter and precedes any increase to heterochromatin throughout the rRNA gene. To directly investigate the role of rRNA synthesis in pluripotency, hESCs were treated with the Pol I inhibitor, CX-5461. The direct reduction of rRNA synthesis by CX-5461 induces the expression of markers for all three germ layers, reduces the expression of pluripotency markers, and is overall similar to the ACTIVIN A induced changes. This work indicates that the dissociation of UBTF from the rRNA gene, and corresponding reduction in transcription, represent early regulatory events during the directed differentiation of pluripotent stem cells. [ABSTRACT FROM AUTHOR]

Muscle wound healing process is a typical inflammation-evoked event. The monoacylglycerol lipase (MAGL) inhibitor (4-nitrophenyl)4-[bis(1,3-benzodioxol -5-yl)-hydroxymethyl]piperidine-1-carboxylate (JZL184) has been previously reported to reduce inflammation in colitis and acute lung injury in mice, which provide a new strategy for primary care of skeletal muscle injury. We investigated the effect of JZL184 on inflammation in rat muscle contusion model, and found decreased neutrophil and macrophage infiltration and pro-inflammatory cytokine expression. With extension of post-traumatic interval, myofiber regeneration was significantly hindered with increased collagen types I and ІІІ mRNAfibroblast infiltration as well as promoted fibrosis. Furthermore, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-morpholin-4-ylpyrazole-3-carboxamide (AM281, a selective cannabinoid CB 1 receptor antagonist) and [6-iodo-2-methyl-1-(2-morpholin-4-ylethyl)indol-3-yl]-(4-methoxyphenyl)methanone (AM630, a selective cannabinoid CB 2 receptor antagonist) treatment alleviated the anti-inflammatory effect of JZL184. Our findings demonstrate that JZL184 is able to inhibit the inflammatory response and interfere with contused muscle healing, in which the anti-inflammatory action may be mediated through cannabinoid CB 1 and CB 2 receptors. [ABSTRACT FROM AUTHOR]

RT-qPCR is a commonly used method for evaluating gene expression; however, its accuracy and reliability are dependent upon the choice of appropriate reference gene(s), and there is limited information available on suitable reference gene(s) that can be used in mouse testis at different stages. In this study, using the RT-qPCR method, we investigated the expression variations of six reference genes representing different functional classes ( Actb, Gapdh, Ppia, Tbp, Rps29, Hprt1) in mice testis during embryonic and postnatal development. The expression stabilities of putative reference genes were evaluated using five algorithms: geNorm, NormFinder, Bestkeeper, the comparative delta C method and integrated tool RefFinder. Analysis of the results showed that Ppia, Gapdh and Actb were identified as the most stable genes and the geometric mean of Ppia, Gapdh and Actb constitutes an appropriate normalization factor for gene expression studies. The mRNA expression of AT1 as a test gene of interest varied depending upon which of the reference gene(s) was used as an internal control(s). This study suggested that Ppia, Gapdh and Actb are suitable reference genes among the six genes used for RT-qPCR normalization and provide crucial information for transcriptional analyses in future studies of gene expression in the developing mouse testis. [ABSTRACT FROM AUTHOR]

Abstract: Glioma stem-like cells have been demonstrated to have highly invasive activity, which is the major cause of glioma recurrence after therapy. Leptin plays a role in glioma invasion, however, whether and how leptin contributes to the biological properties of glioma stem-like cells, such as invasion, remains to be explored. In the current study, we aimed to explore the role of leptin during glioma stem-like cells invasion as well as the signaling pathway. We found that glioma stem-like cells exhibited high invasive potential, especially in the presence of leptin, Ob-R coexpressed with CD133 in glioma stem-like cells was showed to be responsible for leptin mediated invasion of glioma stem-like cells. Our results indicated that leptin served as a key intermediary linking the accumulation of excess adipokine to the invasion of glioma stem-like cells, which may be a novel therapeutic target for suppressing tumor invasion and recurrence. [Copyright &y& Elsevier]

Abstract: Secreted protein acidic and rich in cysteine (SPARC) is a secretory multifunctional matricellular glycoprotein. High circulating levels of SPARC have been reported to be associated with obesity and insulin resistance. The aim of the present study was to investigate whether SPARC induces insulin resistance and mitochondrial dysfunction in adipocytes. Our results showed that feeding high fat diet to rats for 12 weeks significantly increased SPARC expression in adipose tissues at both mRNA and protein levels. Moreover, SPARC overexpression in stably transfected 3T3-L1 cells induced insulin resistance and mitochondrial dysfunction, as evidenced by inhibition of insulin-stimulated glucose transport, lower ATP synthesis and mitochondrial membrane potential, reduced expression of glucose transporter 4 (GLUT4), and increased levels of reactive oxygen species (ROS) in mature adipocytes. Finally, overexpression of SPARC also modulated the expression levels of several inflammatory cytokines, which play important roles in insulin resistance, glucose and lipid metabolism during adipogenesis. In conclusion, our data suggest that SPARC is involved in obesity-induced adipose insulin resistance and may serve as a potential target in the treatment of obesity and obesity-related insulin resistance. [Copyright &y& Elsevier]

Abstract: The PRNP gene encodes a cellular protein named prion, whose misfolded form has been implicated in a number of neuropathic diseases in mammals such as the Bovine Spongiform Encephalopathy (BSE) in cattle. BSE has brought devastating impact on the world economy and human health. Recently, several groups have performed the gene targeting strategy to disrupt the PRNP gene in bovine fibroblast cells and produce BSE-resistant cattle by somatic cell nuclear transfer (SCNT). However, the enrichment efficiency of the gene targeting vector was low. Here, we constructed a novel promoterless gene targeting vector to sequentially disrupt the PRNP gene in bovine fibroblast cells and generate gene targeted cattle by SCNT. The enrichment efficiency of the novel vector was 100% and 60%, respectively. After nuclear transfer, no significant difference was found in the rate of cleavage and blastocyst formation between the knockout and wild type cloned embryos. One PRNP +/− calf was born with no obvious abnormal development by now. Fusion RT-PCR and real-time PCR showed one allele of the PRNP gene was functionally disrupted, and the mRNA expression reduced dramatically in the PRNP +/− cattle. The reconstituted PRNP −/− embryos showed double alleles disruption, and no difference in the rate of cleavage and blastocyst formation. [Copyright &y& Elsevier]

The phytohormone abscisic acid (ABA) plays a vital role in plant development and response to environmental challenges, but the complex networks of ABA signaling pathways are poorly understood. We previously reported that a chloroplast protein, the magnesium-protoporphyrin IX chelatase H subunit (CHLH/ABAR), functions as a receptor for ABA in Arabidopsis thaliana. Here, we report that ABAR spans the chloroplast envelope and that the cytosolic C terminus of ABAR interacts with a group of WRKY transcription factors (WRKY40, WRKY18, and WRKY60) that function as negative regulators of ABA signaling in seed germination and postgermination growth. WRKY40, a central negative regulator, inhibits expression of ABA-responsive genes, such as ABI5. In response to a high level of ABA signal that recruits WRKY40 from the nucleus to the cytosol and promotes ABAR–WRKY40 interaction, ABAR relieves the ABI5 gene of inhibition by repressing WRKY40 expression. These findings describe a unique ABA signaling pathway from the early signaling events to downstream gene expression. [ABSTRACT FROM AUTHOR]

Abstract: After cellular injury many endogenous toxins are released from injured cells and result in secondary injury. To elucidate mechanisms of such injury many of these toxins have been studied individually. However, the data obtained is only useful for reference and does not accurately represent the multifactorial situation under pathophysiological conditions. Primary astrocytic cultures were treated individually and simultaneously with two well-studied toxins, glutamate (Glu) and arachidonic acid (AA). Both are simultaneously released from neural cells during injury. Measurements of cellular protein content, intracellular water space, lactate dehydrogenase release, and malondialdehyde formation indicated that Glu and AA act through different mechanisms. Glu+AA applied together had a synergistic effect on the levels of Caspase-3 gene expression, and Bcl-2 and Hsp70 protein. Atomic force microscopy observed that Glu caused cell membrane roughness and nuclear swelling, while AA induced pores in the cell membrane and nuclear shrinkage. Glu+AA accelerated nuclear shrinkage and resulted in more serious cell damage. This study not only distinguishes the different responses of astrocytes to Glu and AA, but also provides a new view into the synergistic effect of these biochemicals; highlighting the need to be cautious in applying single factor experimental data to interpret complex physiological and pathological conditions in animals. Two or more factors may act not only on different targets but also on the same target synergistically. [Copyright &y& Elsevier]

Abstract: To analyze stage-specific gene expression profiles of pre-implantation embryos and evaluate potential viability, techniques were adapted to generate 3-end enriched cDNA libraries from individual embryos of cattle based on RT-PCR methodology. The reproducibility of constructing a cDNA library was tested by five independent PCR experiments with specific primers for the presence of several rare genes such as DNMT1 (DNA methylation transferase 1), DNMT2, DNMT3A, Oct-4/3 (octmer-binding transcription factor), IFN-ι, IGF-2r (insulin like growth factor 2 receptor), and the housekeeping genes, H2A and β-actin. Results indicated repeatability and that a proportion of expressed genes in the cDNA library from an individual embryo was not affected by limited PCR amplification. From the cDNA library, 134 clones were randomly selected for sequencing and showed that structure related elements accounted for 33.5% of transcripts and the energy- and metabolism-related genes were also an important component being 11.9% in the cDNA library. Approximately 14% of genes in the library were functionally unknown including greater than 5% of genes that were likely novel because there was no identity in Genbank. The frequency of structure-related genes such as β-actin and ribosomal proteins in the cDNA library corresponded to other reports and suggested that the cDNA library constructed by RT-PCR might be proportional to the mRNA populations. The cDNA libraries constructed from different stage embryos will provide a powerful tool to explore novel genes relevant to embryogenesis, determine the profiling of stage-specific gene expression, and evaluate the potential viability of embryos. [Copyright &y& Elsevier]

To investigate multitissue engraftment of human primitive hematopoietic cells and their differentiation in goats, human CD34+Lin- cord blood cells transduced with a GFP vector were transplanted into fetal goats at 45-55 days of gestation. GFP+ cells were detected in hematopoietic and nonhematopoietic organs including blood, bone marrow, spleen, liver, kidney, muscle, lung, and heart of the recipient goats (1.2-36% of all cells examined). We identified human β2 microglobulin-positive cells in multiple tissues. GFP+ cells sorted from the perfused liver of a transplant goat showed human insulin-like growth factor 1 gene sequences, indicating that the engrafted GFP+ cells were of human origin. A substantial fraction of cells engrafted in goat livers expressed the human hepatocyte-specific antigen, proliferating cell nuclear antigen, albumin, hepatocyte nuclear factor, and GFP. DNA content analysis showed no evidence for cellular fusion. Long-term engraftment of GFP+ cells could be detected in the blood of goats for up to 2 yr. Microarray analysis indicated that human genes from a variety of functional categories were expressed in chimeric livers and blood. The human/goat xenotransplant model provides a unique system to study the kinetics of hematopoietic stem cell engraftment, gene expression, and possible stem cell plasticity under noninjured conditions. [ABSTRACT FROM AUTHOR]

The locus control region (LCR) is the most important cis-element in the regulation of β-globin gene expression. DNaseI-hypersensitive site (HS) 2 and HS3 are two significant components of β-LCR. To examine the effect of HS2, HS3, and HS2–HS3 (combination of HS2 and HS3) on the spatial and temporal expression of the human β-globin gene, we have produced transgenic mice with constructs, in which the gene encoding enhanced green fluorescent protein (EGFP) is driven by β-globin promoter and under the control of HS2, HS3, and HS2–HS3, respectively. The results showed that HS2 and HS3 each had the same enhancement activity in regulation of β-globin gene expression in transgenic mice. When HS2 and HS3 were in combination (HS2–HS3), the two cis-elements showed a marked synergy in regulating β-globin gene spatial and temporal expression as well as its expression level in transgenic mice although the EGFP expression varied largely among different transgenic mouse litters. The results also showed that HS2 was able to confer β-globin gene expression in embryonic yolk sac, fetal liver, and adult bone marrow, which was not developmentally stage-specific, while HS3 could confer the same β-globin gene expression in the adult. Thus, HS3 was different from HS2, the former being more important for specific expression of β-globin gene in the developmental stages and the switch of γ→β-globin genes. Our results indicate that the mechanism of γ→β switch could be best explained by the “divided model.” [Copyright &y& Elsevier]