MicroRNAs Regulate Pituitary Development,and MicroRNA 26b Specifically Targets Lymphoid Enhancer Factor 1 (Lef-1), Which Modulates Pituitary Transcription Factor 1 (Pit-1) Expression

To understand the role of microRNAs (miRNAs) in pituitary development, a group of pituitary-specific miRNAs were identified, and Dicer1 was then conditionally knocked out using the Pitx2-Cre mouse, resulting in the loss of mature miRNAs in the anterior pituitary. The Pitx2-Cre/Dicer1 mutant mice demonstrate growth retardation, and the pituitaries are hypoplastic with an abnormal branching of the anterior lobe, revealing a role for microRNAs in pituitary development. Growth hormone, prolactin, and thyroid-stimulating hormone β-subunit expression were decreased in the Dicer1 mutant mouse, whereas proopiomelanocortin and luteinizing hormone β-subunit expression were normal in the mutant pituitary. Further analyses revealed decreased Pit-1 and increased Lef-1 expression in the mutant mouse pituitary, consistent with the repression of the Pit-1 promoter by Lef-1. Lef-1 directly targets and represses the Pit-1 promoter. miRNA-26b (miR-26b) was identified as targeting Lef-1 expression, and miR-26b represses Lef-1 in pituitary and non-pituitary cell lines. Furthermore, miR-26b up-regulates Pit-1 and growth hormone expression by attenuating Lef-1 expression in GH3 cells. This study demonstrates that microRNAs are critical for anterior pituitary development and that miR-26b regulates Pit-1 expression by inhibiting Lef-1 expression and may promote Pit-1 lineage differentiation during pituitary development.     

Formamide as a denaturant for bisulfite conversion of genomic DNA: Bisulfite sequencing of the GSTPi and RARβ2 genes of 43 formalin-fixed paraffin-embedded prostate cancer specimens

Analysis of methylated DNA, which refers to 5-methycytosine (5mC) versus cytosine (C) at specific loci in genomic DNA (gDNA), has received increased attention in epigenomics, particularly in the area of cancer biomarkers. Many different methods for analysis of methylated DNA rely on initial reaction of gDNA with concentrated acidic sodium bisulfite to quantitatively convert C to uracil (U) via sulfonation of denatured, single-stranded gDNA under conditions where 5mC is resistant to analogous sulfonation leading to thymine (T). These methods typically employ polymerase chain reaction (PCR) amplification after bisulfite conversion, thereby leading to readily detectable amounts of amplicons where T and C are measured as surrogates for C and 5mC in the original unconverted gDNA. However, incomplete bisulfite conversion of C in gDNA has been reported to be a common source of error in analysis of methylated DNA. Incomplete conversion can be revealed during the course of bisulfite sequencing, which is the generally accepted “gold standard” for analysis of methylated DNA. Previous bisulfite sequencing investigations of conventional predenaturation of gDNA with NaOH followed by the use of bisulfite containing added urea to maintain denaturation and thus mitigate incomplete conversion of C have been reported to give conflicting results. The current study describes a new approach where conventional predenaturation of gDNA with NaOH is instead achieved with formamide and maintains denaturation during subsequent sample handling and sulfonation. This formamide-based method was applied to 46 formalin-fixed/paraffin-embedded (FFPE) biopsy tissue specimens from well-characterized patients with primary prostate cancer. These specimens were representative of difficult-to-analyze samples due to the chemically compromised nature of the gDNA, which was recovered by modifying the protocol for a commercially available total RNA/DNA extraction kit (RecoverALL). An additional novel aspect of this study was analysis of CpG-rich promoter regions of two prostate cancer-related genes: glutathione S-transferase pi (GSTPi) and retinoic acid receptor beta2 (RARβ2). High-quality bisulfite sequencing results were obtained for both genes in 43 of 46 (93%) specimens. Detection of methylated GSTPi and RARβ2 genes was significantly associated with primary prostate cancer as compared with the benign prostate (Fisher’s exact test, P < 0.001). The sensitivity and specificity of detection of methylated GSTPi and RARβ2 genes were 86% and 100% and 91% and 100%, respectively. Moreover, the presence of either methylated gene was detected in primary prostate cancer with sensitivity and specificity of 100% and 100%, respectively. The results demonstrated a high degree of reliability of formamide-based denaturation and bisulfite conversion that should extend, generally, to FFPE and other types of samples intended for any analytical method predicated on bisulfite conversion. This pilot study also demonstrated the efficacy of determining methylation of these two genes with high sensitivity and specificity in FFPE biopsy tissue specimens. Moreover, the results showed a highly significant association of methylated GSTPi and RARβ2 genes with primary prostate cancer. Finally, this improved procedure for determining these two methylated genes may allow the detection of prostate cancer cells in core biopsy specimens with insufficient numbers of cells and poor morphology.     

Comprehensive Proteomics Analysis of Stressed Human Islets Identifies GDF15 as a Target for Type 1 Diabetes Intervention

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Combined Single-Cell Profiling of lncRNAs and Functional Screening Reveals that H19 Is Pivotal for Embryonic Hematopoietic Stem Cell Development

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Validation of differential GDAP1 DNA methylation in alcohol dependence and its potential function as a biomarker for disease severity and therapy outcome

Alcohol dependence is a severe disorder contributing substantially to the global burden of disease. Despite the detrimental consequences of chronic alcohol abuse and dependence, effective prevention strategies as well as treatment options are largely missing to date. Accumulating evidence suggests that gene-environment interactions, including epigenetic mechanisms, play a role in the etiology of alcohol dependence. A recent epigenome-wide study reported widespread alterations of DNA methylation patterns in alcohol dependent patients compared to control individuals. In the present study, we validate and replicate one of the top findings from this previous investigation in an independent cohort: the hypomethylation of GDAP1 in patients. To our knowledge, this is the first independent replication of an epigenome-wide finding in alcohol dependence. Furthermore, the AUDIT as well as the GSI score were negatively associated with GDAP1 methylation and we found a trend toward a negative association between GDAP1 methylation and the years of alcohol dependency, pointing toward a potential role of GDAP1 hypomethylation as biomarker for disease severity. In addition, we show that the hypomethylation of GDAP1 in patients reverses during a short-term alcohol treatment program, suggesting that GDAP1 DNA methylation could also serve as a potential biomarker for treatment outcome. Our data add to the growing body of knowledge on epigenetic effects in alcohol dependence and support GDAP1 as a novel candidate gene implicated in this disorder. As the role of GDAP1 in alcohol dependence is unknown, this novel candidate gene should be followed up in future studies.     

Molecular functions of the iron-regulated metastasis suppressor,NDRG1, and its potential as a molecular target for cancer therapy

N-myc down-regulated gene 1 (NDRG1) is a known metastasis suppressor in multiple cancers, being also involved in embryogenesis and development, cell growth and differentiation, lipid biosynthesis and myelination, stress responses and immunity. In addition to its primary role as a metastasis suppressor, NDRG1 can also influence other stages of carcinogenesis, namely angiogenesis and primary tumour growth. NDRG1 is regulated by multiple effectors in normal and neoplastic cells, including N-myc, histone acetylation, hypoxia, cellular iron levels and intracellular calcium. Further, studies have found that NDRG1 is up-regulated in neoplastic cells after treatment with novel iron chelators, which are a promising therapy for effective cancer management. Although the pathways by which NDRG1 exerts its functions in cancers have been documented, the relationship between the molecular structure of this protein and its functions remains unclear. In fact, recent studies suggest that, in certain cancers, NDRG1 is post-translationally modified, possibly by the activity of endogenous trypsins, leading to a subsequent alteration in its metastasis suppressor activity. This review describes the role of this important metastasis suppressor and discusses interesting unresolved issues regarding this protein.     

Misdiagnosis as steatohepatitis in a family with mild glycogen storage disease type 1a

The manifestations of glycogen storage disease type 1a (GSD 1a) are usually so prominent in childhood that it is readily diagnosed by pediatricians. However, a mild form of the disease may only become apparent during adolescence or adulthood. We observed a brother and sister with subtle manifestations of the disease, which was discovered after the brother's son was diagnosed with typical GSD 1a. The adult siblings never suffered from hypoglycemia, had normal fasting blood glucose and liver transaminases at the time of diagnosis, and were taller than average for Chinese. Their only notable disease manifestations were recurrent gouty arthritis associated with hyperuricemia and hyperlipidemia during adolescence. When diagnosed, the brother had multiple benign and malignant hepatic tumors, and died of fulminant metastatic hepatocellular carcinoma 6months after liver transplantation. p.M121V/p.R83H and p.M121V/p.M121V genotypic constellations of the G6PC gene were identified in this family. Both siblings were homozygous for the newly identified p.M121V mutation. The infant had compound heterozygous mutations, p.R83H and p.M121V. We recommend that mild GSD should be considered in the adolescents with unexplained hyperuricemia and hyperlipidemia, despite the presence of normal blood glucose levels. This report also reminds us that hepatocellular carcinoma could develop even in very mild GSD 1a patients.     

Chromium downregulates the expression of Acetyl CoA Carboxylase 1 gene in lipogenic tissues of domestic goats: a potential strategy for meat quality improvement

Acetyl CoA Carboxylase 1 (ACC1) is a biotin-dependent enzyme that catalyzes the carboxylation of Acetyl CoA to form Malonyl CoA, the key intermediate metabolite in fatty acid synthesis. In this study, the mRNA expression of the ACC1 gene was evaluated in four different tissues (liver, visceral fat, subcutaneous fat, and longissimus muscle) of the domestic goat (Capra hircus) kids feeding on four different levels of trivalent chromium (0, 0.5, 1, and 1.5 mg/day) as food supplementation. RT-qPCR technique was used for expression analyses and heat shock protein 90 gene (HSP-90) was considered as reference gene for data normalization. Our results revealed that 1.5 mg/day chromium significantly reduced the expression of the ACC1 gene in liver, visceral fat, and subcutaneous fat tissues, but not in longissimus muscles (P < 0.05). We measured some phenotypic traits of kid's carcasses to detect their probable correlations with chromium-mediated downregulation of ACC1 expression. Interestingly, changes in ACC1 expression were accompanied with decreased accumulation of fats in adipose tissues such that the subcutaneous fat thickness and heart fat percentage decreased in kids feeding on chromium. By contrast, chromium supplemented kids showed higher percentage of muscles despite the fact that their total body weight did not differ from that of non-supplemented kids. Our study suggests that trivalent chromium alters the direction of energy accumulation towards muscles rather than fats and provides insights into application of chromium supplementation as a useful strategy for improvement of meat quality in domestic animals.     

The complete mitochondrial genome of a turbinid vetigastropod from MiSeq Illumina sequencing of genomic DNA and steps towards a resolved gastropod phylogeny

A need to increase sampling of mitochondrial genomes for Vetigastropoda has been identified as an important step towards resolving relationships within the Gastropoda. We used shotgun sequencing of genomic DNA, using an Illumina MiSeq, to obtain the first mitochondrial genome for the vetigastropod family Turbinidae, doubling the number of genomes for the species-rich superfamily Trochoidea. This method avoids the necessity of finding suitable primers for long PCRs or primer-walking amplicons, resulting in a timely and cost-effective method for obtaining whole mitochondrial genomes from ethanol-preserved tissue samples. Bayesian analysis of amino acid variation for all available gastropod genomes including the new turbinid mtgenome produced a well resolved tree with high nodal support for most nodes. Major clades within Gastropoda were recovered with strong support, with the exception of Littorinimorpha, which was polyphyletic. We confirm here that mitogenomics is a useful tool for molluscan phylogenetics, especially when using powerful new models of amino acid evolution, but recognise that increased taxon sampling is still required to resolve existing differences between nuclear and mitochondrial gene trees.