Microcystin-LR regulates circadian clock and antioxidant gene expression in cultured rat cardiomyocytes

Background

Microcystins are waterborne environmental toxins that induce oxidative stress and cause injuries in the heart. On the other hand, many physiological processes, including antioxidant defense, are under precise control by the mammalian circadian clock.

Results

In the present study, we evaluated the effect of microcystin-LR (MC-LR) on the rhythmic expression patterns of circadian and antioxidant genes in rat cardiomyocytes using the serum shock technique. We found that a non-toxic dose (10 μm) of MC-LR decreased the amplitudes of rhythmic patterns of clock genes, while it increased the expression levels of antioxidant genes.

Conclusions

Our results indicate an influence of MC-LR on the circadian clock system and clock-controlled antioxidant genes, which will shed some light on the explanation of heart toxicity induced by MC-LR from the viewpoint of chronobiology.

     

Genetic sex determination of mice by simplex PCR

Background

Investigating fetal development in mice necessitates the determination of fetal sex. However, whilst the sex of adult and juvenile mice can be readily distinguished from anogenital distance, the sex of fetal and neonatal mice cannot be identified visually. Instead, genetic sex must be determined by PCR amplification of X chromosome genes with divergent Y chromosome gametologs. Existing simplex PCR methods are confounded by small size differences between amplicons, amplification of unexpected products, and biased amplification of the shorter amplicon.

Results

Primers were designed flanking an 84 bp deletion of the X-linked Rbm31x gene relative to its Y-linked gametolog Rbm31y. A single product was amplified from XX samples, with two products amplified from XY samples. Amplicons were resolved by gel electrophoresis for 20 min, with unbiased amplification of both products observed in XY samples.

Conclusion

This method achieves rapid and unequivocal genetic sex determination of mice in low volume PCR reactions, reducing reagent usage and simultaneously eliminating shortcomings of previous methods.

     

Sex-related factors influence expression of mood-related genes in the basolateral amygdala differentially depending on age and stress exposure

Background

Women are twice as likely to be diagnosed with major depressive disorder (MDD) compared to men, but the molecular mechanisms underlying this sex difference are unclear. Previous studies in the human postmortem brain suggest dysfunction in basolateral amygdala (BLA) inhibitory gamma-aminobutyric acid (GABA) signaling and brain-derived neurotrophic factor (BDNF) function, specifically in females with MDD.

Methods

We investigated the effects of sex chromosome complement, developmental gonadal sex, and circulating testosterone on expression of 3 GABA-related and 2 BDNF-related genes in the BLA using three cohorts of four core genotypes (FCG) mice. Cohort 1 included gonadally intact pre-pubertal FCG mice; results were analyzed using two-way ANOVA (sex chromosome complement-by-gonadal sex). We examined the same genes under adult non-stressed (cohort 2) and chronically stressed conditions (cohort 3). The results for cohorts 2 and 3 were analyzed by three-way ANOVA (sex chromosome complement-by-gonadal sex-by-hormone). The use of heatmaps and Spearman correlation of BLA gene expression and anxiety-like behavior provides a global interpretation of gene expression patterns.

Results

In weanlings, we found an effect of sex chromosome complement, with lower expression of GABA/BDNF-related genes in XY mice. Most of these effects did not persist into adulthood, although a number of interesting interactions between organizational and activational effects of hormones emerged. In our adult cohorts, we found that testosterone had different effects depending on stress conditions and/or gonadal sex. Notably, in our chronically stressed adults, we found that the BLA pattern of gene expression for the GABA-related gene, somatostatin (Sst), matched the anxiety-like behavior pattern (i.e., lower Sst and higher anxiety-like behavior in XY mice, while testosterone increased Sst and decreased anxiety-like behavior). Additionally, increased Sst gene expression was correlated with decreased anxiety-like behavior.

Conclusions

Sex chromosome complement is an important factor modulating expression of mood-related genes during pre-pubertal development. The observed sex differences under chronically stressed conditions suggest that different molecular profiles may characterize male and female MDD. Our findings here for Sst are especially interesting, and suggest an underlying XY vulnerability that is typically compensated for by circulating testosterone in “normal” males. Without testosterone, women may have lower SST expression in the amygdala, resulting in increased MDD vulnerability.

     

Sex differences in kidney gene expression during the life cycle of F344 rats

Background

The kidney functions in key physiological processes to filter blood and regulate blood pressure via key molecular transporters and ion channels. Sex-specific differences have been observed in renal disease incidence and progression, as well as acute kidney injury in response to certain drugs. Although advances have been made in characterizing the molecular components involved in various kidney functions, the molecular mechanisms responsible for sex differences are not well understood. We hypothesized that the basal expression levels of genes involved in various kidney functions throughout the life cycle will influence sex-specific susceptibilities to adverse renal events.

Methods

Whole genome microarray gene expression analysis was performed on kidney samples collected from untreated male and female Fischer 344 (F344) rats at eight age groups between 2 and 104 weeks of age.

Results

A combined filtering approach using statistical (ANOVA or pairwise t test, FDR 0.05) and fold-change criteria (>1.5 relative fold change) was used to identify 7,447 unique differentially expressed genes (DEGs). Principal component analysis (PCA) of the 7,447 DEGs revealed sex-related differences in mRNA expression at early (2 weeks), middle (8, 15, and 21 weeks), and late (104 weeks) ages in the rat life cycle. Functional analysis (Ingenuity Pathway Analysis) of these sex-different genes indicated over-representation of specific pathways and networks including renal tubule injury, drug metabolism, and immune cell and inflammatory responses. The mRNAs that code for the qualified urinary protein kidney biomarkers KIM-1, Clu, Tff3, and Lcn2 were also observed to show sex differences.

Conclusions

These data represent one of the most comprehensive in-life time course studies to be published, assessing sex differences in global gene expression in the F344 rat kidney. PCA and Venn analyses reveal specific periods of sexually dimorphic gene expression which are associated with functional categories (xenobiotic metabolism and immune cell and inflammatory responses) of key relevance to acute kidney injury and chronic kidney disease, which may underlie sex-specific susceptibility. Analysis of the basal gene expression patterns of renal genes throughout the life cycle of the rat will improve the use of current and future renal biomarkers and inform our assessments of kidney injury and disease.

     

Knockdown of Rab21 inhibits proliferation and induces apoptosis in human glioma cells

Background

Gliomas are commonly malignant tumors that arise in the human central nervous system and have a low overall five-year survival rate. Previous studies reported that several members of Rab GTPase family are involved in the development of glioma, and abnormal expression of Rab small GTPases is known to cause aberrant tumor cell behavior. In this study, we characterized the roles of Rab21 (Rab GTPase 21), a member of Rab GTPase family, in glioma cells.

Methods

The study involved downregulation of Rab21 in two glioma cell lines (T98G and U87) through transfection with specific-siRNA. Experiments using the MTT assay, cell cycle analysis, apoptosis assay, real-time PCR and western blot were performed to establish the expression levels of related genes.

Results

The results show that downregulation of Rab21 can significantly inhibit cell growth and remarkably induce cell apoptosis in T98G and U87 cell lines. Silencing Rab21 resulted in significantly increased expression of apoptosis-related proteins (caspase7, Bim and Bax) in glioma cells.

Conclusions

We inferred that Rab21 silencing can induce apoptosis and inhibit proliferation in human glioma cells, indicating that Rab21 might act as an oncogene and serve as a novel target for glioma therapy.

     

Sex-specific differences in hepatic steatosis in obese spontaneously hypertensive (SHROB) rats

Background

Patients with metabolic syndrome, who are characterized by co-existence of insulin resistance, hypertension, hyperlipidemia, and obesity, are also prone to develop non-alcoholic fatty liver disease (NAFLD). Although the prevalence and severity of NAFLD is significantly greater in men than women, the mechanisms by which gender modulates the pathogenesis of hepatic steatosis are poorly defined. The obese spontaneously hypertensive (SHROB) rats represent an attractive model of metabolic syndrome without overt type 2 diabetes. Although pathological manifestation caused by the absence of a functional leptin receptor has been extensively studied in SHROB rats, it is unknown whether these animals elicited sex-specific differences in the development of hepatic steatosis.

Methods

We compared hepatic pathology in male and female SHROB rats. Additionally, we examined key biochemical and molecular parameters of signaling pathways linked with hyperinsulinemia and hyperlipidemia. Finally, using methods of quantitative polymerase chain reaction (qPCR) and western blot analysis, we quantified expression of 45 genes related to lipid biosynthesis and metabolism in the livers of male and female SHROB rats.

Results

We show that all SHROB rats developed hepatic steatosis that was accompanied by enhanced expression of SREBP1, SREBP2, ACC1, and FASN proteins. The livers of male rats also elicited higher induction of Pparg, Ppara, Slc2a4, Atox1, Skp1, Angptl3, and Pnpla3 mRNAs. In contrast, the livers of female SHROB rats elicited constitutively higher levels of phosphorylated JNK and AMPK and enhanced expression of Cd36.

Conclusion

Based on these data, we conclude that the severity of hepatic steatosis in male and female SHROB rats was mainly driven by increased de novo lipogenesis. Moreover, male and female SHROB rats also elicited differential severity of hepatic steatosis that was coupled with sex-specific differences in fatty acid transport and esterification.

     

Effects of sex chromosome dosage on corpus callosum morphology in supernumerary sex chromosome aneuploidies

Background

Supernumerary sex chromosome aneuploidies (sSCA) are characterized by the presence of one or more additional sex chromosomes in an individual’s karyotype; they affect around 1 in 400 individuals. Although there is high variability, each sSCA subtype has a characteristic set of cognitive and physical phenotypes. Here, we investigated the differences in the morphometry of the human corpus callosum (CC) between sex-matched controls 46,XY (N =99), 46,XX (N =93), and six unique sSCA karyotypes: 47,XYY (N =29), 47,XXY (N =58), 48,XXYY (N =20), 47,XXX (N =30), 48,XXXY (N =5), and 49,XXXXY (N =6).

Methods

We investigated CC morphometry using local and global area, local curvature of the CC boundary, and between-landmark distance analysis (BLDA). We hypothesized that CC morphometry would vary differentially along a proposed spectrum of Y:X chromosome ratio with supernumerary Y karyotypes having the largest CC areas and supernumerary X karyotypes having significantly smaller CC areas. To investigate this, we defined an sSCA spectrum based on a descending Y:X karyotype ratio: 47,XYY, 46,XY, 48,XXYY, 47,XXY, 48,XXXY, 49,XXXXY, 46,XX, 47,XXX. We similarly explored the effects of both X and Y chromosome numbers within sex. Results of shape-based metrics were analyzed using permutation tests consisting of 5,000 iterations.

Results

Several subregional areas, local curvature, and BLDs differed between groups.Moderate associations were found between area and curvature in relation to the spectrum and X and Y chromosome counts. BLD was strongly associated with X chromosome count in both male and female groups.

Conclusions

Our results suggest that X- and Y-linked genes have differential effects on CC morphometry. To our knowledge, this is the first study to compare CC morphometry across these extremely rare groups.

     

Microarray data integration for genome-wide analysis of human tissue-selective gene expression

Background

Microarray gene expression data are accumulating in public databases. The expression profiles contain valuable information for understanding human gene expression patterns. However, the effective use of public microarray data requires integrating the expression profiles from heterogeneous sources.

Results

In this study, we have compiled a compendium of microarray expression profiles of various human tissue samples. The microarray raw data generated in different research laboratories have been obtained and combined into a single dataset after data normalization and transformation. To demonstrate the usefulness of the integrated microarray data for studying human gene expression patterns, we have analyzed the dataset to identify potential tissue-selective genes. A new method has been proposed for genome-wide identification of tissue-selective gene targets using both microarray intensity values and detection calls. The candidate genes for brain, liver and testis-selective expression have been examined, and the results suggest that our approach can select some interesting gene targets for further experimental studies.

Conclusion

A computational approach has been developed in this study for combining microarray expression profiles from heterogeneous sources. The integrated microarray data can be used to investigate tissue-selective expression patterns of human genes.

     

Case report of whole genome sequencing in the XY female: identification of a novel <Emphasis Type="Italic">SRY</Emphasis> mutation and revision of a misdiagnosis of androgen insensitivity syndrome

Background

The 46,XY female is characterised by a male karyotype and female phenotype arising due to any interruption in the sexual development pathways in utero. The cause is usually genetic and various genes are implicated.

Case presentation

Herein we describe a 46,XY woman who was first diagnosed with androgen insensitivity syndrome (testicular feminisation) at 18 years; however, this was later questioned due to the presence of intact Müllerian structures. The clinical phenotype suggested several susceptibility genes including SRY, DHH, NR5A1, NR0B1, AR, AMH, and AMHR2. To study candidate genes simultaneously, we performed whole genome sequencing. This revealed a novel and likely pathogenic missense variant (p.Arg130Pro, c.389G>C) in SRY, one of the major genes implicated in complete gonadal dysgenesis, hence securing this condition over androgen insensitivity syndrome as the cause of the patient’s disorder of sexual development.

Conclusion

This case highlights the emerging clinical utility of whole genome sequencing as a tool in differentiating disorders of sexual development.

     

Efficient activation of gene expression using a heat-shock inducible Gal4/Vp16-UAS system in medaka

Background

Genetic interference by DNA, mRNA or morpholino injection is a widely used approach to study gene function in developmental biology. However, the lack of temporal control over the activity of interfering molecules often hampers investigation of gene function required during later stages of embryogenesis. To elucidate the roles of genes during embryogenesis a precise temporal control of transgene expression levels in the developing organism is on demand.

Results

We have generated a transgenic Gal4/Vp16 activator line that is heat-shock inducible, thereby providing a tool to drive the expression of specific effector genes via Gal4/Vp16. Merging the Gal4/Vp16-UAS system with the I-SceI meganuclease and the Sleeping Beauty transposon system allows inducible gene expression in an entirely uniform manner without the need to generate transgenic effector lines. Combination of this system with fluorescent protein reporters furthermore facilitates the direct visualization of transgene expressing cells in live embryos.

Conclusion

The combinatorial properties of this expression system provide a powerful tool for the analysis of gene function during embryonic and larval development in fish by ectopic expression of gene products.

     

Clusterin confers gmcitabine resistance in pancreatic cancer

Objective

To measure clusterin expression in pancreatic cancer tissues and cell lines and to evaluate whether clusterin confers resistance to gmcitabine in pancreatic cancer cells.

Methods

Immunohistochemistry for clusterin was performed on 50 primary pancreatic cancer tissues and 25 matched backgrounds, and clusterin expression in 5 pancreatic cancer cell lines was quantified by Western blot and PT-PCR. The correlation between clusterin expression level and gmcitabine IC50 in pancreatic cancer cell lines was evaluated. The effect of an antisense oligonucleotide (ASO) against clusterin(OGX-011) on gmcitabine resistance was evaluated by MTT assays. Xenograft model was used to demonstrate tumor growth.

Results

Pancreatic cancer tissues expressed significantly higher levels of clusterin than did normal pancreatic tissues (P < 0.01). Clusterin expression levels were correlated with gmcitabine resistance in pancreatic cancer cell lines, and OGX-011 significantly decreased BxPc-3 cells resistance to gmcitabine (P < 0.01). In vivo systemic administration of AS clusterin and gmcitabine significantly decreased the s.c. BxPC-3 tumor volume compared with mismatch control ODN plus gmcitabine.

Conclusion

Our finding that clusterin expression was significantly higher in pancreatic cancer than in normal pancreatic tissues suggests that clusterin may confer gmcitabine resistance in pancreatic cancer cells.

     

Differentiation of human adipose-derived stem cells into cardiomyocyte-like cells in fibrin scaffold by a histone deacetylase inhibitor

Background

Human adipose-derived stem cells (hADSCs) are capable of differentiating into many cells such as cardiac cells. Different types of inducers are used for cardiac cell differentiation, but this question still remains to be investigated, which one is the best. The aim of this paper was to investigate the effect of combination of fibrin scaffold and trichostatin A (TSA), for differentiation of hADSCs into cardiomyocyte-like cells.

Methods

After approval of characteristics of hADSCs and fibrin scaffold, hADSCs were cultured in fibrin scaffold with 10 µM TSA for 72 h and kept in standard conditions for 4 weeks. QRT-PCR and immunostaining assay were performed for evaluating the expression pattern of special cardiac genes and proteins.

Results

In particular, our study showed that fibrin scaffold alongside TSA enhanced expression of the selected genes and proteins.

Conclusions

We concluded that the TSA alone or with fibrin scaffold can lead to the generation of cardiac like cells in a short period of time.