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The effect of schizophrenia (SCZ) genetic risk on gene expression in brain remains elusive. A popular approach to this problem has been the application of gene co-expression network algorithms (e.g., WGCNA). To improve reliability with this method it is critical to remove unwanted sources of variance while also preserving biological signals of interest. In this WCGNA study of RNA-Seq data from postmortem prefrontal cortex (78 neurotypical donors, EUR ancestry), we tested the effects of SCZ genetic risk on co-expression networks. Specifically, we implemented a novel design in which gene expression was adjusted by linear regression models to preserve or remove variance explained by biological signal of interest (GWAS genomic scores for SCZ risk—(GS-SCZ), and genomic scores- GS of height (GS-Ht) as a negative control), while removing variance explained by covariates of non-interest. We calculated co-expression networks from adjusted expression (GS-SCZ and GS-Ht preserved or removed), and consensus between them (representative of a "background" network free of genomic scores effects). We then tested the overlap between GS-SCZ preserved modules and background networks reasoning that modules with reduced overlap would be most affected by GS-SCZ biology. Additionally, we tested these modules for convergence of SCZ risk (i.e., enrichment in PGC3 SCZ GWAS priority genes, enrichment in SCZ risk heritability and relevant biological ontologies. Our results highlight key aspects of GS-SCZ effects on brain co-expression networks, specifically: 1) preserving/removing SCZ genetic risk alters the co-expression modules; 2) biological pathways enriched in modules affected by GS-SCZ implicate processes of transcription, translation and metabolism that converge to influence synaptic transmission; 3) priority PGC3 SCZ GWAS genes and SCZ risk heritability are enriched in modules associated with GS-SCZ effects. Overall, our results indicate that gene co-expression networks that selectively integrate information about genetic risk can reveal novel combinations of biological pathways involved in schizophrenia. Author summary: Genetic risk for schizophrenia (SCZ) is linked to brain connectivity at multiple levels, from molecular interactions to coherent cognitive processing. Biological mechanisms by which SCZ genetic risk affects brain connectivity are generally unknown. In this study we sought to uncover some of these mechanisms by focusing on gene co-expression networks calculated from RNA-Seq data extracted from postmortem DLPFC. We created co-expression networks from gene expression adjusted to account for, or to remove the likely effects of genomic (polygenic) scores of SCZ risk. We minimized potential confounding effects of illness state or treatment on gene expression by using only brains from donors without a history of mental disorder. We used as a control, co-expression networks from gene expression adjusted to account for effects of genomic scores of height- a normative trait. Our results show subtle and widespread effects of SCZ genomic scores on DLPFC co-expression networks. Gene sets from co-expression modules adjusted for SCZ genetic risk harbor biological significance represented by neuronal functions, but also by interacting pathways of general cellular processes like transcription, translation and metabolism. Our findings highlight possible avenues for identifying regulatory targets of gene expression in schizophrenia. [ABSTRACT FROM AUTHOR]

Background. Myasthenia gravis (MG) is a rare autoimmune disease with clinical symptoms of fluctuating muscle weakness. Due to the side effects of current therapies, there is an urgent need for a new medication for MG treatment. Nux vomica is a traditional Chinese medicine used in various diseases. However, the mechanism of action of Nux vomica against MG remains unclear. Methods. Network pharmacology was used to explore the underlying mechanisms of Nux vomica in MG treatment, which was validated using molecular docking and in vivo experiments in mice. Results. Twelve bioactive compounds and 72 targets in Nux vomica were screened. Seventy-nine myasthenia-related targets were obtained from the GENECARD and DisGeNET databases. PPI networks of Nux vomica- and myasthenia-related targets were constructed using Bisogenet, and these two networks were subsequently merged into an intersection to establish a core-target PPI network that consisted of 204 nodes and 4,668 edges. KEGG enrichment analysis indicated that 132 pathways were enriched in 204 core targets. In addition, we obtained 50 docking pairs via molecular docking. In vivo experiments revealed that Nux vomica can improve the symptoms of MG. Conclusion. Nux vomica is involved in the pathogenesis of MG through the "multicomponent-target-pathway" mechanism. [ABSTRACT FROM AUTHOR]

Abstract Superoxide dismutase is a key enzyme that scavenges superoxide anion and plays vital roles in plant antioxidant system. This study identified six SOD genes from the deciduous conifer Larix kaempferi , which is widely distributed across the cooler regions of the northern hemisphere. These six SOD genes were classified into three types: Cu/Zn-SOD (LkSOD1, 2, 3 and 4), Fe-SOD (LkSOD5) and Mn-SODs (LkSOD6). Three Cu/Zn-SOD proteins (LkSOD1, 3 and 4) were cytosolic-localized, while the other three proteins (LkSOD2, 5 and 6) were chloroplast-localized. Larix SOD proteins displayed catalytic activities toward superoxide anion, and retained >55% of its maximum enzymatic activity between 10 °C and 40 °C. Over expressions of three Larix SOD genes (LkSOD2 , 4 and 6) in Arabidopsis thaliana , respectively, showed increased germination rates and root lengths during salt stress. LkSOD5 gene could rescue pale green and dwarf phenotype of Arabidopsis atfsd2-2 mutant. Taken together, this study provided comprehensive insight into the gymnosperm SOD gene family. Highlights • Six SOD genes were identified from Larix kaempferi. • Larix SOD proteins displayed different subcellular location. • LkSOD5 gene could rescue the pale green and dwarf phenotype of atfsd2-2. • Over expressing of three Larix SOD genes in Arabidopsis could increase salt tolerance. [ABSTRACT FROM AUTHOR]

Objectives. Percutaneous coronary intervention (PCI) is the mainstay treatment for coronary artery disease but complications such as in-stent restenosis and thrombosis remain problematic. Radiofrequency balloon angioplasty (RBA) can improve lumen dimension, fusing intimal tears, and artery dissection but is associated with higher restenosis rate. Methods. After establishing an atherosclerosis model based on endothelial abrasion and high cholesterol diet, forty-five rabbits were randomly divided into three groups: RBA (n=20), percutaneous transluminal angioplasty (PTA) (n=20), and control groups (n=5). The RBA and PTA groups were subdivided according to harvested time posttreatment, respectively (1 hour, 7 days, 14 days, and 28 days). Aorta segments were then isolated for hematoxylin and eosin staining, Masson trichrome staining, immunohistochemistry, and Western blot for TLR-4, NF-κB, MCP-1, and VCAM-1expression. Results. At 28 days, intimal area was significantly lower in the RBA group compared to the PTA and control groups, whilst luminal and medial area were comparable in the RBA and PTA group but higher and lower than the control group, respectively. Expression of TLR-4, NF-κB, MCP-1, and VCAM-1 showed no significant difference between RBA and PTA groups. Conclusions. RBA can depress the intimal hyperplasia and promote dilatation of the artery to greater extents than PTA at 28 days. However, this did not involve TLR-4 signaling pathway, which likely plays a negligible role in mediating restenosis. Reduction of intimal hyperplasia may be due to injury of ablation to the tunica media and inhibition of VSMC proliferation and migration. [ABSTRACT FROM AUTHOR]

In this paper, the glucose tolerance tests (GTT) were performed to evaluate the effects of three dietary carbohydrate levels on growth, plasma glucose, hormones (insulin and IGF‐I), glucose homeostasis and GLUT4 gene expression of Tilapia nilotica. Juvenile tilapia (av. wt.10 g) were randomly distributed into three groups with three replicates and fed for 8 weeks with three dietary carbohydrate levels: 40% (CH), 30% (CM) and 20% (CL). After 8 weeks of feeding trial, fish were injected with glucose (30 mg/100 g body weight) for GTT. The growth performance results showed the survival rate in the CH group was significantly lower than the other two groups (p < 0.05). The GTT results showed that the plasma glucose level positively correlates with the diets' carbohydrate level initially, peaked at 1 hr and declined thereafter. The plasma IGF‐1 levels were positively correlated with diets' carbohydrate levels; first the levels increase and then decrease after injection in all the three groups. There was a direct relationship between the carbohydrate levels and the activities of liver pyruvate kinase (PK) and glucokinase (GK) enzymes. Muscle GLUT4 expression in groups changed slightly in 0–3 hr and then increased sharply during 6–12 hr. Results of this study indicated that the moderate dietary carbohydrate level may improve the glucose tolerance of T. nilotica. The carbohydrate levels can affect plasma glucose, IGF‐1 and GLUT4 expression in T. nilotica. In addition, the IGF‐1 may play a more important role on the glucose metabolism than insulin. The delayed gene transcription of GLUT4 may be one of the reasons for glucose intolerance in T. nilotica. [ABSTRACT FROM AUTHOR]

Background. This study aims to identify the key genes and miRNAs in slow transit constipation (STC). Methods. MRNA and miRNA expression profiling were obtained. Differentially expressed genes (DEGs) and miRNAs were identified followed by the regulatory network construction. Functional annotation analysis and protein-protein interaction (PPI) network were conducted. The electronic validation was performed. Results. Hsa-miR-2116-3p, hsa-miR-3622a-5p, hsa-miR-424-5p, and hsa-miR-1273-3p covered most DEGs. HLA-DRB1, HLA-DRB5, C3, and ICAM were significantly involved in staphylococcus aureus infection. The PPI network generated several hub proteins including ZBTB16, FBN1, CCNF, and CDK1. Electronic validation of HLA-DRB1, PTGDR, MKI67, BIRC5, CCNF, and CDK1 was consistent with the RNA-sequencing analysis. Conclusion. Our study might be helpful in understanding the pathology of STC at the molecular level. [ABSTRACT FROM AUTHOR]

This study was aimed at exploring the effects of P2X receptor on BV2 microglia cell injury induced by glycoprotein gp120 (gp120) and its underlying mechanisms. We used the MTS method to study the influence of different gp120 concentrations on BV2 microglia cells, and to test the degree of cell injury in each gp120 treatment group; quantitative real-time PCR (qPCR) and Western blot were used to detect the P2X mRNA and receptor protein expressions. Immunocytochemistry and Western blot were used to detect the P2X receptor expression and P65 NF-κB, respectively. We also measured the content of TNFα, IL-1β, nitric oxide (NO) and reactive oxygen species (ROS). We found that the cell survival rate generally decreased as gp120 concentration increased, and the cell survival rate of the gp120 + Brilliant Blue G (BBG) group was higher than that of the gp120 group. Western blot and qPCR results showed that the expressions of P2X receptor protein and mRNA were positively dose-dependent with gp120 concentration; the results of immunocytochemistry and Western blot showed that the expressions of P2X receptor and P65 NF-κB in the gp120 group increased significantly compared to those of the control (Ctrl) group, but those in the gp120+BBG group decreased. Taken together, these results confirmed that the P2X receptor is involved in gp120-induced BV2 microglial cell injury and that the underlying mechanism may be associated with the over-activation of microglia caused by P2X receptor up-regulation, which leads to abundant release of inflammatory factors which exert toxic effects on the cells. [ABSTRACT FROM AUTHOR]

Background: Congenital pulmonary airway malformation (CPAM) is the most frequent pulmonary developmental malformation and the pathophysiology remains poorly understood. This study aimed to identify the characteristic gene expression patterns and the marker genes essential to CPAM. Methods: Tissues from the cystic area displaying CPAM and the area of normal appearance were obtained during surgery. Bulk RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq) were performed for integrating analysis. Iterative weighted gene correlation network analysis (iWGCNA) was used to identify specifically expressed genes to CPAM. Results: In total, 2074 genes were significantly differentially expressed between the CPAM and control areas. Of these differentially expressed genes (DEGs), 1675 genes were up-regulated and 399 genes were down-regulated. Gene ontology analysis revealed these DEGs were specifically enriched in ciliated epithelium and involved in immune response. We also identified several CPAM-related modules by iWGCNA, among them, P15_I4_M3 module was the most influential module for distinguishing CPAMs from controls. By combining the analysis of the expression dataset from RNA-seq and scRNA-seq, SPOCK2, STX11, and ZNF331 were highlighted in CPAM. Conclusions: Through our analysis of expression datasets from both scRNA-seq and bulk RNA-seq of tissues obtained from patients with CPAM, we identified the characteristic gene expression patterns associated with the condition. Our findings suggest that SPOCK2 could be a potential biomarker gene for the diagnosis and therapeutic target in the development of CPAM, whereas STX11 and ZNF331 might serve as prognostic markers for this condition. Further investigations with larger samples and function studies are necessary to confirm the involvement of these genes in CPAM. [ABSTRACT FROM AUTHOR]

Numerous clinical conditions have been linked to ectopic mineralization (EM). This process of pathological biomineralization is complex and not fully elucidated, but thought to be started within matrix vesicles (MVs). We hypothesized that high mobility group box 1 (HMGB1), a cytokine associated with biomineralizing process under physiological and pathological conditions, induces EM via promoting MVs secretion from macrophages. In this study, we found that HMGB1 significantly promoted secretion of MVs from macrophages and subsequently led to mineral deposition in elevated Ca/Pi medium in vitro. Transmission electron microscopy of calcifying MVs showed formation of hydroxyapatite crystals in the vesicle interior. Subcutaneous injection into mice with MVs derived from HMGB1-treated cells showed a greater potential to initiate regional mineralization. Mechanistic experiments revealed that HMGB1 activated neutral sphingomyelinase2 (nSMase2) that involved the receptor for advanced glycation end products (RAGE) and p38 MAPK (upstream of nSMase2). Inhibition of nSMase2 with GW4869 or p38 MAPK with SB-239063 prevented MVs secretion and mineral deposition. Collectively, HMGB1 induces MVs secretion from macrophages at least in part, via the RAGE/p38 MAPK/nSMase2 signaling pathway. Our findings thus reveal a novel mechanism by which HMGB1 induces ectopic mineralization. [ABSTRACT FROM AUTHOR]

Sirtuin 3 (SIRT3) is a major deacetylase in the mitochondria and sensitive to diverse nutrient signals. However, how dietary fatty acids regulate SIRT3 expression remains poorly understood. Here, sirt3 gene from large yellow croaker was characterized and can be activated by the oxidized nicotinamide adenine dinucleotide (NAD+) precursor, nicotinamide mononucleotide (NMN). Moreover, the expression of SIRT3 is regulated differently by dietary fatty acids. In vivo , soybean oil increased the mRNA expression of sirt3 in head kidney. In vitro , stearic acid (SA) decreased the expression of SIRT3 in macrophages, while DHA and EPA increased the mRNA expression of sirt3. Meanwhile, all long-chain polyunsaturated fatty acids (LC-PUFAs) enhanced the protein levels of SIRT3, which was consistent with the trend of fatty acids affecting NAD+ levels. Furthermore, inhibition of NAD+ de novo synthesis blocked DHA-induced increases in SIRT3 expression, and NMN supplement reversed SA-induced decreases in SIRT3 expression. In conclusion, these findings suggest that dietary fatty acids may regulate SIRT3 expression by affecting intracellular NAD+ synthesis. Regarding the important role of SIRT3 in regulating mitochondrial function, appropriate activation of SIRT3 may be an important way to alleviate metabolic disorders in aquaculture. • SIRT3 in large yellow croaker was a conserved sirtuin family member and could be activated by NMN. • The effects of fatty acids on SIRT3 regulation is affected by carbon chain saturation rather than carbon chain length. • Dietary fatty acids affected the expression of SIRT3 by regulating intracellular NAD+ synthesis. [ABSTRACT FROM AUTHOR]

Summary The unfolded protein response (UPR) is a stress response program that reprograms cellular translation and gene expression in response to proteotoxic stress in the endoplasmic reticulum (ER). One of the primary means by which the UPR alleviates this stress is by reducing protein flux into the ER via a general suppression of protein synthesis and ER-specific mRNA degradation. We report here an additional UPR-induced mechanism for the reduction of protein flux into the ER, where mRNAs that encode signal sequences are released from the ER to the cytosol. By removing mRNAs from the site of translocation, this mechanism may serve as a potent means to transiently reduce ER protein folding load and restore proteostasis. These findings identify the dynamic subcellular localization of mRNAs and translation as a selective and rapid regulatory feature of the cellular response to protein folding stress. [ABSTRACT FROM AUTHOR]

Purpose: To study the molecular mechanisms of colorectal cancer liver metastasis. Methods: Cecal wall implantation was performed in nude mice to subclone a highly liver metastatic human colorectal cancer clone (SW1116-M) from SW1116. In vivo and in vitro assays were adopted to confirm the proliferation and metastasis potential. The human tumor metastasis PCR microarrays were used to analyze the differential gene expressions. The results were confirmed further by real-time quantitative PCR. Results: SW1116-M and SW1116-S5, two human colon cancer cell clones with different metastatic potential, were subcloned from SW1116. In SW1116-M, in vitro invasion, migration and in vivo metastatic potential were higher, and in vitro proliferation rate was lower than SW1116-S5. In tumor metastasis PCR microarray, 24 genes related to cell invading, adhesion, cellular growth and differentiation were found with a twofold difference between SW1116-S5 and SW1116-M. Sixteen of these, including E-cadherins, MTSS1, TRAIL and TRPM1, were up-regulated; eight genes including cathepsin L, EphB2, HGF, MET, MCAM and RORβ were down-regulated. Conclusions: We have established a highly liver metastatic clone. The subsequent metastasis PCR microarray analysis identified a procedure of cellular differentiation and mesenchymal to epithelial transition (MET) in liver metastasis. The colonization to from macrometastasis is not a switch from cell cycle arrest but a result of cell differentiation and MET. [ABSTRACT FROM AUTHOR]

Background: The single nucleotide polymorphism (SNP) rs1344706 in ZNF804A (2q32.1) has been associated with schizophrenia in a genome-wide association study (GWAS). A recent candidate gene study, which replicated the positive association with rs1344706, identified another positive SNP (rs7597593) in ZNF804A associated with schizophrenia. Methods: We performed an association study of rs7597593 in four GWAS cohorts of European ancestry. Postmortem human brain expression data of normal Caucasian individuals (n = 89) was also analyzed for examining the effect of rs7597593 on ZNF804A messenger RNA expression, using logistic regression and linear regression. Results: We found that rs7597593 was significantly associated with schizophrenia in the combined GWAS datasets (n = 5023, odds ratio [OR]combined = 1.15, p = .0011). Analysis of stratification by sex showed that the association was driven by the female subjects (OR = 1.29, p = .0002) and was not significant in male subjects (OR = 1.08, p = .148) in the combined sample of four cohorts. A sex by genotype interaction was near significant in both the Genetic Association Information Network sample (p = .0532) and the combined sample of four cohorts (p combined = .0531). Gene expression analysis showed no main effects but a significant female-specific association (p female = .047, p male = .335) and sex by genotype interaction (p = .0166) for rs7597593. Conclusions: Our data suggest a clinical and molecular modulation by sex of the association of ZNF804A SNP rs7597593 and risk of schizophrenia. [Copyright &y& Elsevier]

Abstract: Chronic hypoxia usually evokes sustained release of endogenous neurohormones, leading to β2-adrenergic receptor (β2AR) desensitization and downregulation of expression, which impacts cellular contractility. We investigated whether exogenous β2AR could compensate for the functional deficiency of β2AR in rabbit cardiomyocytes under chronic hypoxia, and whether this led to improved contractility and cellular survival. A surgical experimental model of cyanotic heart disease was established in rabbits. Adv.hβ2AR was transfected into cardiomyocytes isolated from animals subjected to 6-week systemic hypoxia. The levels of cellular contractile function, protein expression of hβ2AR, p-Akt, p-Erk, and caspase-3, and cellular survival pre- and post-Adv.hβ2AR delivery were determined. In the cyanotic cells, decreased shortening and lengthening of TPC and R50 were evident. Cellular diastolic functioning showed greater deterioration compared to the systolic function (P <0.05). In cyanotic cells, the positive inotropic response to isoproterenol was decreased (P <0.01), low levels of cellular survival were found, protein levels of β2AR, p-Akt, and p-Erk were downregulated, and protein levels of caspase-3 were upregulated. After Adv.hβ2AR delivery, enhanced contractile function was achieved (P <0.01), TPC and R50 levels recovered up to 99% and 81.7% of the normal control levels, respectively (P <0.05), and cellular survival improved (P <0.01). Our results demonstrate that overexpression of the β2AR gene in cardiomyocytes exposed to chronic hypoxia provides significant catecholamine-dependent inotropic support and cellular protection. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor alpha (TNF-α) is a proinflammatory cytokine produced by activated macrophages and lymphocytes and involved in many inflammatory diseases. Preventing the production or action of TNF-α is a potent therapeutic strategy for these inflammatory diseases. Since there is a lack of rapid and effective assay for examining the expression TNF-α in macrophages, we attempt to establish a reporter system to assess TNF-α gene expression through measuring luciferase activity. In this study, mouse macrophage cell line RAW 264.7 was stably transfected with a luciferase reporter pGL3-TNFPro-UTR, which contains TNF-α promoter and 3′-untranslated region (3′-UTR). The TNF-α-luciferase reporter cell line is used for assessing the expression of TNF-α gene induced by LPS in the presence or absence of chemicals that inhibit the biosynthesis of TNF-α such as dexamethasone and emodin, and also for measuring change of expression of TNF-α gene under downregulation of the expression of steroid receptor coactivator-3, a modulator for TNF-α. The luciferase activity correlated well with the ELISA results for TNF-α production, therefore, the TNF-α-luciferase reporter cell line is a sensitive, effective tool for studying the expression of TNF-α gene. [ABSTRACT FROM AUTHOR]

Summary: Macrobrachium nipponense, as one of the large‐yield farmed shrimp, is facing germplasm degradation. Genetic improvement through hybridization is one of the effective methods to solve this problem. In this study, using a three‐line hybrid strategy, two‐hybrid F1 populations were obtained using three local populations of M. nipponense as parents for crossbreeding. Five populations were then cultured for 3 months. Growth rate performance was measured by the hepatosomatic index, weight gain, body length growth rate and special growth rate. Biochemical components were also assessed. The results showed that the survival rate and growth performance of the hybrid progeny were better than those of the parents. The levels of triglycerides, total cholesterol, glycogen and lactic acid of the hybrid population were higher than those of the parents. This was consistent with variation in the activity of four digestive enzymes. Compared with the results of the fatty acid and amino acid analysis, it was found that the contents of polyunsaturated fatty acids such as eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid and eight essential amino acids in the hybrid populations were significantly higher than those of their parents, and the contents of flavor amino acids were higher. The expression level of molting genes related to the growth of the parent populations was lower than that of the hybrids. These results show that crossbreeding is effective for the genetic improvement of M. nipponense germplasm. Hybrids showed advantages in growth and nutrition and multigenerational breeding will be required to form a stable germplasm. [ABSTRACT FROM AUTHOR]

SUMMARY: Switch defective/sucrose non‐fermentable (SWI/SNF) chromatin remodeling complexes are evolutionarily conserved, multi‐subunit machinery that play vital roles in the regulation of gene expression by controlling nucleosome positioning and occupancy. However, little is known about the subunit composition of SPLAYED (SYD)‐containing SWI/SNF complexes in plants. Here, we show that the Arabidopsis thaliana Leaf and Flower Related (LFR) is a subunit of SYD‐containing SWI/SNF complexes. LFR interacts directly with multiple SWI/SNF subunits, including the catalytic ATPase subunit SYD, in vitro and in vivo. Phenotypic analyses of lfr‐2 mutant flowers revealed that LFR is important for proper filament and pistil development, resembling the function of SYD. Transcriptome profiling revealed that LFR and SYD shared a subset of co‐regulated genes. We further demonstrate that the LFR and SYD interdependently activate the transcription of AGAMOUS (AG), a C‐class floral organ identity gene, by regulating the occupation of nucleosome, chromatin loop, histone modification, and Pol II enrichment on the AG locus. Furthermore, the chromosome conformation capture (3C) assay revealed that the gene loop at AG locus is negatively correlated with the AG expression level, and LFR‐SYD was functional to demolish the AG chromatin loop to promote its transcription. Collectively, these results provide insight into the molecular mechanism of the Arabidopsis SYD‐SWI/SNF complex in the control of higher chromatin conformation of the floral identity gene essential to plant reproductive organ development. Significance Statement: This study provides insight into the molecular mechanism of the Arabidopsis SYD‐SWI/SNF complex in the control of higher chromatin conformation of the floral identity gene essential to plant reproductive organ development. [ABSTRACT FROM AUTHOR]

Cytoplasmic polyadenylation element binding protein 4 (CPEB4) could be an important regulator in variety of cancers. However, the biological function and the underlying molecular mechanism of CPEB4 in non‐small cell lung cancer (NSCLC) remains unknown. In this study, we investigated the metastasis role of CPEB4 in NSCLC cells, we knocked down CPEB4 using siRNA. Transwell migration assay and cell invasion assay on Matrigel were presented, and cell migration was also determined by scratch‐healing assay. ROS generation were determined by fluorescence probe DCFH2‐DA. The protein expression was assessed by ELISA and Western blot analysis. LY294002 were used to inhibit PI3 K/Akt signaling. The data showed that knockdown of CPEB4 inhibited the migration and invasion of NSCLC. Moreover, silencing of CPEB4 reduced Snail and MMP‐3 expression in vitro. We also indicated that CPEB4 knockdown increased the ROS expression. Furthermore, we found that silencing of CPEB4 decreased pAkt expression. Taken all together, our data demonstrated that silencing of CPEB4 induces ROS generation, thus suppressing the Akt expression, which finally prevents NSCLC cells invasion and migration. Therefore, CPEB4 may regard as a target to inhibit NSCLC invasion and metastasis through Akt pathway. [ABSTRACT FROM AUTHOR]

Necrosis caused by soybean mosaic virus (SMV) has not been specifically distinguished from susceptible symptoms. The molecular mechanism for the occurrence of necrosis is largely overlooked in soybean genetic research. Field evaluation reveals that SMV disease seriously influences soybean production as indicated by decreasing 22.4% ~ 77.0% and 8.8% ~ 17.0% of yield and quality production, respectively. To expand molecular mechanism behind necrotic reactions, transcriptomic data obtained from the asymptomatic, mosaic, and necrotic pools were assessed. Compared between asymptomatic and mosaic plants, 1689 and 1752 up- and down-regulated differentially expressed genes (DEGs) were specifically found in necrotic plants. Interestingly, the top five enriched pathways with up-regulated DEGs were highly related to the process of the stress response, whereas the top three enriched pathways with down-regulated DEGs were highly related to the process of photosynthesis, demonstrating that defense systems are extensively activated, while the photosynthesis systems were severely destroyed. Further, results of the phylogenetic tree based on gene expression pattern and an amino acid sequence and validation experiments discovered three PR1 genes, Glyma.15G062400, Glyma.15G062500, and Glyma.15G062700, which were especially expressed in necrotic leaves. Meanwhile, exogenous salicylic acid (SA) but not methyl jasmonate (MeJA) could induce the three PR1 gene expressions on healthy leaves. Contrastingly, exogenous SA obviously decreased the expression level of Glyma.15G062400, Glyma.15G062500, and concentration of SMV, but increased Glyma.15G062700 expression in necrotic leaves. These results showed that GmPR1 is associated with the development of SMV-induced necrotic symptoms in soybean. Glyma.15G062400, Glyma.15G062500, and Glyma.15G062700 is up-regulated in necrotic leaves at the transcriptional levels, which will greatly facilitate a better understanding of the mechanism behind necrosis caused by SMV disease. [ABSTRACT FROM AUTHOR]

The polysaccharides found in Lentinula edodes have a variety of medicinal properties, such as anti-tumor and anti-viral effects, but their content in L. edodes sporophores is very low. In this study, Fe2+ was added to the liquid fermentation medium of L. edodes to analyze its effects on mycelial growth, polysaccharide and enzyme production, gene expression, and the activities of enzymes involved in polysaccharide biosynthesis, and in vitro antioxidation of polysaccharides. The results showed that when 200 mg/L of Fe2+ was added, with 7 days of shaking at 150 rpm and 3 days of static culture, the biomass reached its highest value (0.28 mg/50 mL) 50 days after the addition of Fe2+. Besides, Fe2+ addition also enhanced intracellular polysaccharide (IPS) and exopolysaccharide (EPS) productions, the levels of which were 2.98- and 1.79-fold higher than the control. The activities of the enzymes involved in polysaccharides biosynthesis, including phosphoglucomutase (PGM), phosphoglucose isomerase (PGI), and UDPG-pyrophosphorylase (UGP) were also increased under Fe2+ addition. Maximum PGI activity reached 1525.20 U/mg 30 days after Fe2+ addition, whereas PGM and UGP activities reached 3607.05 U/mg and 3823.27 U/mg 60 days after Fe2+ addition, respectively. The Pearson correlation coefficient showed a strong correlation (p < 0.01) between IPS production and PGM and UGP activities. The corresponding coding genes of the three enzymes were also upregulated. When evaluating the in vitro antioxidant activities of polysaccharides, EPS from all Fe2+-treated cultures exhibited significantly better capacity (p < 0.05) for scavenging -OH radicals. The results of the two-way ANOVA indicated that the abilities of polysaccharides to scavenge O2− radicals were significantly (p < 0.01) affected by Fe2+ concentration and incubation time. These results indicated that the addition of iron provided a good way to achieve desirable biomass, polysaccharide production, and the in vitro antioxidation of polysaccharides from L. edodes. [ABSTRACT FROM AUTHOR]

C-type lectin-like receptors (CLRs) are important pathogen pattern recognition molecules that recognize carbohydrate structures. However, the functions of these receptors in fish keep less known. In this study, we characterized a novel CLR from a teleost fish, Plecoglossus altivelis (ayu), tentatively named PaCD209L. The cDNA of PaCD209L is 1464 nucleotides (nts) in length, encoding a polypeptide of 281 amino acid residues with a calculated molecular weight of 31.5 kDa. Multiple alignment of the deduced amino acid sequences of PaCD209L and other related fish CLRs revealed that the PaCD209L sequence had typical characteristics of fish CLRs, but without Ca 2+ -binding sites. Sequence comparison and phylogenetic tree analysis showed that PaCD209L shared the highest amino acid identity (44%) with rainbow trout ( Oncorhynchus mykiss ) CD209 aE PaCD209L transcripts were detected in all of the tissues examined, mainly expressed in the brain and heart. Upon Vibrio anguillarum infection, PaCD209L transcripts were upregulated in all tested tissues and in monocytes/macrophages (MO/MΦ). We prepared recombinant PaCD209L (rPaCD209L) by prokaryotic expression and raised antiserum against PaCD209L. Western blot analysis revealed that native PaCD209L was glycosylated, and its protein expression significantly increased in ayu MO/MΦ upon V. anguillarum infection. In addition, rPaCD209L was able to bind Gram-positive and Gram-negative bacteria in the absence of Ca 2+ . After PaCD209L was blocked by anti-PaCD209L IgG, the phagocytosis and bacterial killing activity of MO/MΦ significantly decreased. These results suggest that PaCD209L plays an important role in the regulation of MO/MΦ functions in ayu. [ABSTRACT FROM AUTHOR]

Forming tight interaction with both Purkinje and granule cells (GCs), Bergmann glia (BG) are essential for cerebellar morphogenesis and neuronal homeostasis. However, how BG act in this process is unclear without comprehensive transcriptome landscape of BG. Here, high temporal‐resolution investigation of transcriptomes with FACS‐sorted BG revealed the dynamic expression of genes within given functions and pathways enabled BG to assist neural migration and construct neuron‐glia network. It is found that the peak time of GCs migration (P7‐10) strikingly coincides with the downregulation of extracellular matrix (ECM) related genes, and the disruption of which by Setdb1 ablation at P7‐10 in BG leads to significant migration defect of GCs emphasizing the criticality of Nfix‐Setdb1 mediated H3K9me3 repressive complex for the precise regulation of GCs migration in vivo. Thus, BG's transcriptomic landscapes offer an insight into the mechanism by which BG are in depth integrated in cerebellar neural network. [ABSTRACT FROM AUTHOR]

Lentinan is a Lentinus edodes secondary metabolite that can regulate human immune function, but yields are low. Here, the effects of Ca2+ and Na+ on L. edodes lentinan content were investigated. Metal ion concentrations and induction times were optimized according to mycelial biomass, and intracellular polysaccharide (IPS), extracellular polysaccharide (EPS), and total polysaccharide (TPS) content. The activities and gene expression of phospho-glucose isomerase (PGI), phosphoglucomutase (PGM), and UDP-glcpyrophosphorylase (UGP) were also measured. Ca2+ and Na+ concentration and induction time affected biomass, IPS, and EPS concentrations. Na+ increased EPS, IPS and TPS, while Ca2+ increased biomass, IPS, and TPS. During fermentation, mycelial biomass varied greatly under Ca2+ induction, while IPS, EPS and TPS varied greatly under Na+ induction. PGM and UGP activities increased in the presence of Na+, while PGI increased with Ca2+. Compared to control samples, pgi and pgm expression under Na+ was greater at days 45 and 60, respectively, while under Ca2+, ugp expression was greater at day 45. IPS content correlated significantly with enzyme activity, while EPS correlated with PGM activity. Our data contributes to better understanding how Na+ and Ca2+ affect mycelial growth and secondary metabolite production, and of polysaccharide biosynthesis mechanisms of L. edodes. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide (LPS) is essential for successful nodulation during the symbiosis of rhizobia and legumes. However, the detailed mechanism of the LPS in this process has not yet been clearly elucidated. In this study, the effects of common bean seed exudates on the growth, lipopolysaccharide production, and lipopolysaccharide transport genes expression (lpt) of Rhizobium anhuiense were investigated. Rhizobium anhuiense exposed to exudates showed changes in LPS electrophoretic profiles and content, whereby the LPS band was wider and the LPS content was higher in R. anhuiense treated with seed exudates. Exudates enhanced cell growth of R. anhuiense in a concentration-dependent manner; R. anhuiense exposed to higher doses of the exudate showed faster growth. Seven lpt genes of R. anhuiense were amplified and sequenced. Sequences of six lpt genes, except for lptE, were the same as those found in previously analyzed R. anhuiense strains, while lptE shared low sequence similarity with other strains. Exposure to the exudates strongly stimulated the expression of all lpt genes. Approximately 6.7- (lptG) to 301-fold (lptE) increases in the transcriptional levels were observed after only 15 min of exposure to exudates. These results indicate that seed exudates affect the LPS by making the cell wall structure more conducive to symbiotic nodulation. [ABSTRACT FROM AUTHOR]

Two isoforms of Glutathione S-Transferase (GST) genes, belonging to mu (Dp-GSTm1 and Dp-GSTm2) and sigma (Dp-GSTs1 and Dp-GSTs2) classes, were cloned and characterised in the freshwater Daphnia pulex. No signal peptide was found in any of the four GST proteins, indicating that they were cytosolic GST. A highly conserved glutathione (GSH) binding site (G-site) occurred in the N-terminal sequence, and a substrate binding site (H-site), interacting non-specifically with the second hydrophobic substrate, was present in the C-terminal. A Tyr residue, for the stabilization of GSH, was found to be conserved in the analysed sequences. The secondary and tertiary structures indicated that these genes possess the typical cytosolic GST structure, including a conserved N-terminal domain with a βαβαββα motif. The μ loop (NVGPAPDYDR and NFIGAEWDR in Dp-GSTm1 and Dp-GSTm2, respectively) was identified between the βαβ (β1α1β2) and αββα motifs (α2β3β4α3) in the N-terminal domain. The expressions of Dp-GSTs1 , Dp-GSTs2 , and Dp-GSTm1 were higher in other age groups compared to the newly-born neonates (1 d); however, the expression of Dp-GSTm2 first increased and then decreased with age. Gene expression was significantly reduced by high concentration (1 and 2 mg/L) of 75 nm polystyrene nanoplastic. However, nanoplastic exposure at the predicted environmental concentration (1 μg/L) had a low effect. Exposure of mothers to nanoplastic (1 μg/L) elevated the Dp-GSTs2 level in their neonates. These results improve our understanding on the response of different types of Daphnid GST to environmental contaminants, especially nanoplastic. • Four GST genes of D. pulex exhibited all the features typical to the GST family. • Glutathione S-Transferase played a positive role against nanoplastic exposure. • Exposure of mothers to nanoplastic elevated the Dp-GSTs2 level in their neonates. [ABSTRACT FROM AUTHOR]

LncRNA TUC338 has recently been characterized as an oncogene in several types of cancer. Our study aimed to characterize the functionality of TUC338 in prostate carcinoma. It was observed that TUC338 was upregulated in tumor tissues comparing to adjacent healthy tissues of prostate carcinoma patients. Plasma levels of TUC338 were also higher in prostate carcinoma patients than in healthy controls. A 5-year follow-up study showed that high plasma level of TUC338 was correlated with poor survival. miR-466 was downregulated in tumor tissues compared with adjacent healthy tissues of prostate carcinoma patients. TUC338 and miR-466 were inversely correlated in tumor tissues. miR-466 overexpression failed to affect TUC338 expression, while TUC338 overexpression led to downregulated miR-466 expression. TUC338 overexpression failed to significantly affect cancer cell proliferation, but promoted cancer cell migration and invasion. MiR-466 overexpression resulted in reduced rates of cancer cell migration and invasion, and also attenuated the effect of TUC338 overexpression. Therefore, TUC338 may serve as an oncogenic lncRNA in prostate carcinoma by downregulating miR-466. • Our study aimed to characterize the functionality of TUC338 in prostate carcinoma. • TUC338 may regulate miR-466 through unknown pathways. • TUC338 is upregulated and may serve as an oncogene in prostate carcinoma patients by downregulating miR-466. [ABSTRACT FROM AUTHOR]

The aim of the present study was to evaluate the protective effect and mechanism of Letinous edodes foot peptides on ethanol‑induced L02 cells. A cell model of ethanol‑induced damage was established in vitro to study the effects of the Letinous edodes foot peptides on human L02 hepatocytes. The expression and activity of superoxide dismutase (SOD), malondialdehyde (MDA), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH), following treatment were examined to determine the anti‑alcoholism and hepatoprotective functions of Letinous edodes foot peptides. Taking Letinous edodes foot peptides prior to ethanol exposure was more beneficial, which significantly increased SOD activity and the mRNA expression of ADH and ALDH suppressed by ethanol. In addition, the intracellular MDA content, and AST and ALT activity decreased in ethanol‑induced L02 cells pretreated with the peptides, when compared with the control. Furthermore, Letinous edodes foot peptides inhibited the ethanol‑induced activation of the proinflammatory cytokines, interleukin‑6 and tumor necrosis factor‑α, and promoted the metabolic regulation factors, AMP‑activated protein kinase‑α2 and peroxisome proliferator‑activated receptor‑α. [ABSTRACT FROM AUTHOR]

Understanding the cardiomyocyte hypertrophic response to stress is pivotal to elucidating its role in normal and abnormal cardiac physiology. While previous studies have characterized transcriptome remodeling in cardiac hypertrophy, these studies did not resolve cell type-specific changes and were limited to assessments of steady state mRNA abundance. In addition, very little is known regarding the dynamic changes that occur upon initiation of the hypertrophic process. Here, we assessed the immediate and sustained cardiomyocyte transcriptional and translational regulatory responses to acute exercise. We generated a mouse line that specifically expresses GFP-tagged L10a, a large ribosomal subunit protein, in the cardiomyocytes. Mice were subjected to forced swimming for varying times ranging from 30 min to 90 min, or rest for 3 h after a 90 min swim. Left ventricles were then excised and GFP-tagged ribosomes affinity-purified via GFP antibody capture. RNA-Seq was utilized to assess the transcriptome and a modified Ribo-Seq protocol was used to measure the translational status of the mRNA transcriptome. In immuno-staining, GFP co-localized with the Troponin positive cardiomyocytes, but not with either PDGFRα (fibroblasts) or CD31 (endothelial cells), indicating cardiomyocyte-specific expression of GFP-L10a. By comparing the mRNA abundance of known cell-type markers in the immunoprecipitated vs. unbound fractions, we established that cardiomyocyte genes were enriched while others were eliminated. For Ribo-Seq analyses, polyribosomes were subjected to ribonuclease digestion and the ribosome protected fragments were isolated, purified and used to prepare cDNA libraries for deep sequencing. In preliminary studies with qPCR, a number of known early responsive genes (EGR1, c-Fos, UPC3) was increased at different time of acute exercise. In ongoing studies, Next Gen Sequencing is being applied. This study is expected to reveal the dynamic transcriptional and translational regulation in cardiomyocytes in the physiological setting, in a cell-type specific manner and at single codon resolution, to provide insights into mechanisms of the cardiomyocyte transcriptional and translational regulatory response to a physiological stress. [ABSTRACT FROM AUTHOR]

Lentinula edodes (shiitake mushroom) is a common edible mushroom with a number of potential therapeutic and nutritional applications. It contains various medically important molecules, such as polysaccharides, terpenoids, sterols, and lipids, were contained in this mushroom. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool to analyze the mechanisms underlying the biosynthetic pathways of these substances. qRT-PCR is used for accurate analyses of transcript levels owing to its rapidity, sensitivity, and reliability. However, its accuracy and reliability for the quantification of transcripts rely on the expression stability of the reference genes used for data normalization. To ensure the reliability of gene expression analyses using qRT-PCR in L. edodes molecular biology research, it is necessary to systematically evaluate reference genes. In the current study, ten potential reference genes were selected from L. edodes genomic data and their expression levels were measured by qRT-PCR using various samples. The expression stability of each candidate gene was analyzed by three commonly used software packages: geNorm, NormFinder, and BestKeeper. Base on the results, Rpl4 was the most stable reference gene across all experimental conditions, and Atu was the most stable gene among strains. 18S was found to be the best reference gene for different development stages, and Rpl4 was the most stably expressed gene under various nutrient conditions. The present work will contribute to qRT-PCR studies in L. edodes. [ABSTRACT FROM AUTHOR]

Diabetic neuropathic pain (DNP), one of the early symptoms of diabetic neuropathy, relates to metabolic disorders induced by high blood glucose, neurotrophic vascular ischemia and hypoxia, and autoimmune factors. This study was aimed at exploring the effects of long noncoding RNA (lncRNA) BC168687 siRNA on DNP mediated by P2X7 receptor on SGCs in DRG of rats. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) of rats, the expression levels of P2X7 mRNA and protein in the DRG, and nitric oxide (NO) in the serum were, respectively, detected in our study. Our experimental results showed that the level of BC168687 mRNA in DNP group was markedly higher than that of control group; the MWT and TWL of DNP + BC168687 si group were significantly increased, and the expression levels of P2X7 in DRG and the concentrations of NO in serum of DNP + BC168687 si group were decreased compared to those of the DNP group. In conclusion, lncRNA BC168687 may participate in the pathogenesis of DNP mediated by P2X7 receptor, which will provide a novel way for the study of the pathogenesis of diabetes mellitus complicated with neuropathic pain and its prevention and treatment. [ABSTRACT FROM AUTHOR]

Myoglobin (Mb) is a monomeric hemoprotein which plays an important role in oxygen storage and transport in cardiac and skeletal muscle under hypoxia. The red tail toad-headed lizard Phrynocephalus erythrurus (Lacertilia: Agamidae), which inhabits at an elevation of 4500–5300 m on the Qinghai–Tibetan plateau, is known to be the highest living lizard in the world. To investigate the characters of myoglobin of this unique species, another low altitude lizard Phrynocephalus przewalskii (Lacertilia: Agamidae) was selected as a reference. The open reading frame (ORF) of myoglobin in two lizards was 465 bp which encodes a polypeptide of 154 amino acids with different theoretical molecular weight and isoelectric point. The amino acid substitutions of myoglobin between two species were found at Thr13Ile, Lys87Thr and His118Asn. Homology modeling results indicated that P. erythrurus myoglobin has a greater heme pocket, which may be more favorable to oxygen binding and unloading. On the other hand, the mRNA levels of myoglobin in both cardiac and skeletal muscle in P. erythrurus were significantly larger than those in low altitude P. przewalskii . At protein level, myoglobin concentration in skeletal muscle in P. erythrurus was notably increased, but no significant difference was observed in cardiac muscle. [ABSTRACT FROM AUTHOR]

This study was designed to detect the expression, detergent resistance, subcellular localization, and channel and hemichannel functions of mutant Cx50 to understand the forming mechanism for inducing congenital cataract by a novel mutation p.S276F in connexin 50 (Cx50) reported previously by us. HeLa and human lens epithelial (HLE) cells were transfected with wild-type Cx50 and mutant Cx50 (S276F). We examined the functional characteristics of mutant Cx50 (S276F) in comparison with those of wild-type Cx50 using immunoblot, confocal fluorescence microscopy, dye transfer analysis and dye uptake assay. The mutant and wild-type Cx50 were expressed in equal levels and could efficiently localize to the plasma membrane without transportation and assembly problems. Scrape loading dye transfer was significantly evident in cells transfected with wild-type Cx50 compared to those in cells transfected with mutant Cx50 and cotransfected with wild-type and mutant Cx50. The dye uptake was found to be significantly lower in cells transfected with mutant Cx50 than in cells transfected with wild-type Cx50 and cells cotransfected with wild-type and mutant Cx50. The transfected HeLa and HLE cell lines showed similar performance in all the experiments. These results indicated that the mutant Cx50 (S276F) might inhibit the function of gap junction channel in a dominant negative manner, but inhibit the hemichannel function in a recessive negative manner. [ABSTRACT FROM AUTHOR]

Abstract: Background: Cell proliferation, renewal, and apoptosis factors are related to hemorrhagic shock (HS) survival. Objective: Hepatic gene expression before and 24 h after induced HS were compared. Methods: Male Sprague-Dawley rats aged 8–9 wk (n = 11) were subjected to blood loss, and HS was induced in 9 rats (blood loss <0.1 mL) by left lobular hepatectomy with fixed-volume blood loss (2.5 mL/100 g) for this self-controlled study. In 3 randomly selected rats surviving >24 h post-HS, hepatic tissue samples collected pre-HS (n = 3; group A) and 24 h post-HS (n = 3; group B) were used for microarray analysis (21,793 genes) of differentially expressed genes using pathway, gene ontology, and network analyses. Real-time reverse transcriptase polymerase chain reaction confirmed Aldh1a1, Aldh1a7, amine oxidase, copper containing 3, cytochrome P450 26A1, histidine decarboxylase 1, and epoxide hydrolase 2 expression using a beta-actin reference. Results: Four rats survived 24 h after HS. Microarray revealed 562 upregulated and 634 downregulated genes in group A compared with group B. Gene ontology analysis revealed differentially expressed genes involved in cholesterol metabolic processes, extracellular stimuli response, sterol metabolic processes, hormonal stimuli response, steroid metabolic processes, endogenous stimulus response, oxidation and reduction reactions, organic substance response, and fatty acid metabolic processes. Conclusions: HS pathogenesis involves numerous interrelated signaling pathways. Redox reaction and fatty acid metabolism pathway involvement in traumatic HS recovery, as well as other pathways, may provide novel targets for better understanding the pathology of HS and developing treatments to limit post-HS organ failure. [Copyright &y& Elsevier]

Abstract: This study was to investigate whether sulforaphane (SFN) can prevent diabetic cardiomyopathy. Type 1 diabetes was induced in FVB mice by multiple intraperitoneal injections with low-dose streptozotocin. Hyperglycemic and age-matched control mice were treated with or without SFN at 0.5mg/kg daily in five days of each week for 3months and then kept until 6months. At 3 and 6months of diabetes, blood pressure and cardiac function were assessed. Cardiac fibrosis, inflammation, and oxidative damage were assessed by Western blot, real-time qPCR, and histopathological examination. SFN significantly prevented diabetes-induced high blood pressure and cardiac dysfunction at both 3 and 6months, and also prevented diabetes-induced cardiac hypertrophy (increased the ratio of heart weight to tibia length and the expression of atrial natriuretic peptide mRNA and protein) and fibrosis (increased the accumulation of collagen and expression of connective tissue growth factor and tissue growth factor-β). SFN also almost completely prevented diabetes-induced cardiac oxidative damage (increased accumulation of 3-nitrotyrosine and 4-hydroxynonenal) and inflammation (increased tumor necrotic factor-α and plasminogen activator inhibitor 1 expression). SFN up-regulated NFE2-related factor 2 (Nrf2) expression and transcription activity that was reflected by increased Nrf2 nuclear accumulation and phosphorylation as well as the mRNA and protein expression of Nrf2 downstream antioxidants. Furthermore, in cultured H9c2 cardiac cells silencing Nrf2 gene with its siRNA abolished the SFN''s prevention of high glucose-induced fibrotic response. These results suggest that diabetes-induced cardiomyopathy can be prevented by SFN, which was associated with the up-regulated Nrf2 expression and transcription function. [Copyright &y& Elsevier]

Summary Filoviruses (Ebola and Marburg viruses) cause severe and often fatal haemorrhagic fever in humans and non-human primates. The US Centers for Disease Control identifies Ebola and Marburg viruses as 'category A' pathogens (defined as posing a risk to national security as bioterrorism agents), which has lead to a search for vaccines that could prevent the disease. Because the use of such vaccines would be in the service of public health, the cost of production is an important component of their development. The use of plant biotechnology is one possible way to cost-effectively produce subunit vaccines. In this work, a geminiviral replicon system was used to produce an Ebola immune complex (EIC) in Nicotiana benthamiana. Ebola glycoprotein (GP1) was fused at the C-terminus of the heavy chain of humanized 6D8 IgG monoclonal antibody, which specifically binds to a linear epitope on GP1. Co-expression of the GP1-heavy chain fusion and the 6D8 light chain using a geminiviral vector in leaves of N. benthamiana produced assembled immunoglobulin, which was purified by ammonium sulphate precipitation and protein G affinity chromatography. Immune complex formation was confirmed by assays to show that the recombinant protein bound the complement factor C1q. Size measurements of purified recombinant protein by dynamic light scattering and size-exclusion chromatography also indicated complex formation. Subcutaneous immunization of BALB/C mice with purified EIC resulted in anti-Ebola virus antibody production at levels comparable to those obtained with a GP1 virus-like particle. These results show excellent potential for a plant-expressed EIC as a human vaccine. [ABSTRACT FROM AUTHOR]

Abstract: Mutations in the parkin gene are the most common cause of autosomal recessive Parkinson’s disease (PD). As an E3-ubiquitin ligase, Parkin is associated with mitochondrial dynamics and mitophagy. Mortalin, a molecular chaperone, is located primarily in mitochondria, where it functions to maintain mitochondrial homeostasis and antagonize oxidative stress injury. A reduced expression level of mortalin has been observed in the affected brain regions of PD patients. Mortalin also interacts with a variety of PD-related proteins and plays an indispensible role in helping native protein refolding and importing proteins into the mitochondrial matrix. Thus, the main aims of the present study were to investigate mitochondrial dysfunction induced by knockdown of mortalin and to test whether Parkin overexpression could rescue this effect. We found that lentivirus-mediated knockdown of mortalin in HeLa cells resulted in a collapse of mitochondrial membrane potential, an abnormal accumulation of reactive oxygen species and apparent alterations in mitochondrial morphology under H2O2-induced stress conditions. Remarkably, Parkin overexpression rescued these mitochondrial abnormalities. In HeLa cells expressing Parkin, co-immunoprecipitation of endogenous mortalin and wild-type Parkin was detected when they were treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP). In conclusion, we indicate that the relatively decreased mortalin expression level and its impaired interaction with Parkin could affect its roles in mitochondrial function. [Copyright &y& Elsevier]

Abstract: Fab antibody display on filamentous phage is widely applied to de novo antibody discovery and engineering. Here we describe a phagemid system for the efficient display and affinity selection of Fabs through linkage to the minor coat protein pIX. Display was successful by fusion of either Fd or Lc through a short linker to the amino terminus of pIX and co-expression of the counter Lc or Fd as a secreted, soluble fragment. Assembly of functional Fab was confirmed by demonstration of antigen-specific binding using antibodies of known specificity. Phage displaying a Fab specific for RSV-F protein with Fd linked to pIX showed efficient, antigen-specific enrichment when mixed with phage displaying a different specificity. The functionality of this system for antibody engineering was evaluated in an optimization study. A RSV-F protein specific antibody with an affinity of about 2nM was randomized at 4 positions in light chain CDR1. Three rounds of selection with decreasing antigen concentration yielded Fabs with an affinity improvement up to 70-fold and showed a general correlation between enrichment frequency and affinity. We conclude that the pIX coat protein complements other display systems in filamentous phage as an efficient vehicle for low copy display and selection of Fab proteins. [ABSTRACT FROM AUTHOR]

Schizophrenia polygenic risk is plausibly manifested by complex transcriptional dysregulation in the brain, involving networks of co-expressed and functionally related genes. 1. Samples: DLPFC gene expression (RNA-Seq) abundance was quantified (RPKM) in postmortem human brains from the LIBD Postmortem Human Brain Repository (90 controls- CTRL, 76 schizophrenia- SCZ, Caucasians, age: 16–80, RIN≥7). Common Mind Consortium (CMC) RNA-Seq postmortem DLPFC (125 CTRL, 109 SCZ, Caucasians, age, RIN as above) was used for replication of LIBD co-expression networks. 2. Expression measures/ quality control: only genes with median RPKM≥0.1 (N=23132 genes; LIBD samples) were used. Expression data was normalized by log2 transformation and adjusted for RNA quality measures (RIN, PMI, total gene assignment, proportion of mitochondrial RNA) by empirical Bayesian models. 3. Data analysis: WGCNA was applied to the adjusted expression data to construct co-expression networks, separately for LIBD- CTRL and LIBD- SCZ. Modules of co-expressed genes were detected by dynamic tree cutting method and summarized as "module eigengenes" for further analyses. 4. Modules of LIBD (CTRL; SCZ) were tested for enrichment in PGC2 genes within the 108 loci associated with the schizophrenia risk in the latest GWAS. 5. Composite module preservation statistics- median rank and Zsummary was used for assessing the preservation of LIBD SCZ modules in LIBD CTRL network and for replication of LIBD co-expression modules in the CMC independent data sets. Gene enrichment analyses for testing modules' enrichment in Gene Ontology (GO) biological processes (BP) was performed with AmiGO/ PANTHER. 17 co-expression modules were identified in each LIBD group. Module preservation analysis showed a good preservation of co-expression modules between LIBD CTRL and SCZ networks, with all but one SCZ module showing evidence for strong preservation (Zsummary≥10). PGC2 genes were overrepresented in two CTRL modules (p=0.001973, p=0.028297) enriched for GO-BP such as translation, ATP synthesis, oxidative phosphorylation, homophilic cell adhesion via plasma membrane adhesion molecules, chemical synaptic transmission, axonogenesis, and in one SCZ module (p=0.000519) enriched for RNA processing, nervous system development and modulation of synaptic transmission. 58 PGC2 genes overlapped with the SCZ module (e.g., DRD2, FURIN, TSNARE1), whereas 48 + 17 PGC2 genes were overrepresented in the two CTRL modules (e.g., CACNA1C, CHRM4, CACNA1I, TCF20). Of note, only 9 overrepresented PGC2 genes were shared by CTRL and SCZ: AMBRA1, ANKRD63, HSPA9, LRP1, PRR12, PTPRF, RERE, TOM1L2, ZDHHC5. Replication of co-expression LIBD networks in CMC data set showed evidence of weak to strong preservation (Zsummary>2, respectively ≥10) for 16/ 17 modules for each LIBD group. Our results illustrate the complexity of SCZ risk distribution within the DLPFC co-expression networks in postmortem brains of affected and unaffected individuals. Importantly, not the same PGC2 genes are members of co-expression modules in CTRL and SCZ, nor do they aggregate in similar modules. These observations may be explained by differential transcriptional regulation due to pleiotropy, epistasis, epigenetic dysregulation, or even hidden RNA quality artifacts. Future studies are necessary to elucidate the participation of SCZ risk genes in brain co-expression networks. [ABSTRACT FROM AUTHOR]

• Effects of polystyrene nanoplastic were measured at the molecular and biochemical levels. • Nanoplastic induced the overproduction of ROS. • The ERK gene in Daphnia pulex was cloned. • Oxidative stress is a possible mechanism for the nanoplastic effect. Recently, research on the biological effects of nanoplastics has grown exponentially. However, studies on the effects of nanoplastics on freshwater organisms and the mechanisms of the biological effects of nanoplastics are limited. In this study, the content of reactive oxygen species (ROS), gene and protein expression in the MAPK-HIF-1/NFkB pathway, and antioxidant gene expressions and enzyme activities were measured in Daphnia pulex exposed to polystyrene nanoplastic. In addition, the full-length extracellular signal-regulated kinases (ERK) gene, which plays an important role in the MAPK pathway, was cloned in D. pulex , and the amino acid sequence, function domain, and phylogenetic tree were analyzed. The results show that nanoplastic caused the overproduction of ROS along with other dose-dependent effects. Low nanoplastic concentrations (0.1 and/or 0.5 mg/L) significantly increased the expressions of genes of the MAPK pathway (ERK ; p38 mitogen-activated protein kinases, p38 ; c-Jun amino-terminal kinases, JNK ; and protein kinase B, AKT), HIF-1 pathway (prolyl hydroxylasedomain, PHD ; vascular endothelial growth factor, VEGF; glucose transporter, GLUT ; pyruvate kinase M, PKM ; hypoxia-inducible factor 1, HIF1), and CuZn superoxide dismutase (SOD) along with the activity of glutathione- S -transferase. As the nanoplastic concentration increased, these indicators were significantly suppressed. The protein expression ratio of ERK, JNK, AKT, HIF1α, and NFkBp65 (nuclear transcription factor-kB p65) as well as the phosphorylation of ERK and NFkBp65 were increased in a dose-dependent manner. The activities of other antioxidant enzymes (catalase, total SOD, and CuZn SOD) were significantly decreased upon exposure to nanoplastic. Combined with our previous work, these results suggest that polystyrene nanoplastic causes the overproduction of ROS and activates the downstream pathway, resulting in inhibited growth, development, and reproduction. The present study fosters a better understanding of the biological effects of nanoplastics on zooplankton. [ABSTRACT FROM AUTHOR]