Huajing Teng

MagicViewer: integrated solution for next-generation sequencing data visualization and genetic variation detection and annotation

New sequencing technologies, such as Roche 454, ABI SOLiD and Illumina, have been increasingly developed at an astounding pace with the advantages of high throughput, reduced time and cost. To satisfy the impending need for deciphering the large-scale data generated from next-generation sequencing, an integrated software MagicViewer is developed to easily visualize short read mapping, identify and annotate genetic variation based on the reference genome. MagicViewer provides a user-friendly environment in which large-scale short reads can be displayed in a zoomable interface under user-defined color scheme through an operating system-independent manner. Meanwhile, it also holds a versatile computational pipeline for genetic variation detection, filtration, annotation and visualization, providing details of search option, functional classification, subset selection, sequence association and primer design. In conclusion, MagicViewer is a sophisticated assembly visualization and genetic variation annotation tool for next-generation sequencing data, which can be widely used in a variety of sequencing-based researches, including genome re-sequencing and transcriptome studies. MagicViewer is freely available at http://bioinformatics.zj.cn/magicviewer/.

Deficiency of antinociception and excessive grooming induced by acute immobilization stress in Per1 mutant mice.

Acute stressors induce changes in numerous behavioral parameters through activation of the hypothalamic-pituitary-adrenal (HPA) axis. Several important hormones in paraventricular nucleus of the hypothalamus (PVN) play the roles in these stress-induced reactions. Corticotropin-releasing hormone (CRH), arginine-vasopressin (AVP) and corticosterone are considered as molecular markers for stress-induced grooming behavior. Oxytocin in PVN is an essential modulator for stress-induced antinociception. The clock gene, Per1, has been identified as an effecter response to the acute stresses, but its function in neuroendocrine stress systems remains unclear. In the present study we observed the alterations in grooming and nociceptive behaviors induced by acute immobilization stress in Per1 mutant mice and other genotypes (wild types and Per2 mutant). The results displayed that stress elicited a more robust effect on grooming behavior in Per1 mutant mice than in other genotypes. Subsequently, the obvious stress-induced antinociception was observed in the wild-type and Per2 mutant mice, however, in Per1 mutant, this antinociceptive effects were partially-reversed (mechanical sensitivity), or over-reversed to hyperalgesia (thermal sensitivity). The real-time qPCR results showed that in PVN, there were stress-induced up-regulations of Crh, Avp and c-fos in all of genotypes; moreover, the expression change of Crh in Per1 mutant mice was much larger than in others. Another hormonal gene, Oxt, was up-regulated induced by stress in wild-type and Per2 mutant but not in Per1 mutant. In addition, the stress significantly elevated the serum corticosterone levels without genotype-dependent differences, and accordingly the glucocorticoid receptor gene, Nr3c1, expressed with a similar pattern in PVN of all strains. Taken together, the present study indicated that in acute stress treated Per1 mutant mice, there are abnormal hormonal responses in PVN, correlating with the aberrant performance of stress-induced behaviors. Therefore, our findings suggest a novel functional role of Per1 in neuroendocrine stress system, which further participates in analgesic regulation.

Regulation of Peripheral Clock to Oscillation of Substance P Contributes to Circadian Inflammatory Pain

Background: The daily fluctuations of many physiologic and behavioral parameters are differentially influenced by either central or peripheral clocks in mammals. Since substance P (SP) oscillates in some brain tissues and plays an indispensable role in modulating inflammatory pain at the spinal level, we speculated that SP mediates circadian nociception transmission at the spinal level. Methods: In the present study behavioral observation, real-time polymerase chain reaction, luciferase assay, chromatin immunoprecipitation, and immunohistochemistry stain methods were used to investigate the role of SP in the spinal circadian nociception transmission and its regulation mechanism. Results: Our results showed that under transcriptional regulation of BMAL1:CLOCK heterodimers, SPs coding gene Tac1 expression oscillates in dorsal root ganglion (n = 36), but not in the spinal dorsal horn. Further, the expression of SP cycled in the spinal dorsal horn, and this rhythmicity was potentially determined by circadian expression of Tac1 in dorsal root ganglion. Furthermore, the variation of SP expression induced by formalin was fluctuated in a similar rhythm to behavioral nociceptive response induced by formalin (n = 48); and the nociceptive behavioral circadian rhythm could be abolished through blockade of the SP–Neurokinin 1 receptor pathway (n = 70). Lastly, the variations of spinal SP expression and behavioral nociceptive response were in step, and both were changed by the deletion mutation of clock gene. Conclusions: We conclude that spinal SP probably plays a pivotal role in modulating circadian inflammatory pain and suggest that peripheral circadian-regulated signaling is potentially an essential pathway for circadian nociceptive transmission.

EXPRESSION PROFILING REVEALS A POSITIVE REGULATION BY MPER2 ON CIRCADIAN RHYTHM OF CYTOTOXICITY RECEPTORS: LY49C AND NKG2D

The mammalian circadian gene, mPer2, an indispensable component of the mammalian circadian clock, not only modulates endogenous circadian rhythms but also plays a crucial role in regulating innate immune function. Previously, we showed that mPer2 plays a crucial role in regulating cytotoxic response. To investigate the molecular mechanism for mPer2-controlled cytotoxic response, in the present study we conducted mRNA expression for 11 genes participating in cytotoxicity regulation in wild-type (WT) and mPer2 knockout (mPer2  −; ; /  −; ; ) mice bone marrow, that is, Dap-10, Ly49C, Ly49I, Rac1, Mapk1, Map2k1, Nkg2d, Shp-1, Pak1, Pik3ca, and Vav1. The mRNA levels of Ly49C (p < 0.001), Ly49I (p = 0.039), and Nkg2d (p = 0.038) were significantly downregulated in mPer2  −; ; /  −; ;  mice. Time-dependence of expression profiling was then conducted for four core clock genes (Per1, Bmal1, Clock, Rev-erbα), and six out of these 11 cytotoxic regulation genes (Ly49C, Ly49I, Mapk1, Nkg2d, Shp-1, Pik3ca) in WT and mPer2  −; ; /  −; ;  entrained in light/dark (LD) or dark/dark (DD) cycles. Consistently, circadian oscillations were observed for Per1, Rev-erbα, Ly49C, and Nkg2d in WT mice under LD and DD cycles. However, these rhythmic expressions were either disrupted or dampened in mPer2  −; ; /  −; ;  mice. Comparison of gene expression between WT and mPer2  −; ; /  −; ;  mice showed that mPer2 knockout had systematically downregulated the mRNA expression of two cytotoxicity regulators, Ly49C and Nkg2d. FACS analysis further confirmed that the circadian expression of these genes was not due to the daily difference in cell numbers of NK, NKT, or T cells in bone marrow. Taken together, our results reveal that mPer2 is a critical clock component in modulating circadian rhythms in bone marrow. Furthermore, it implies that Ly49C and Nkg2d are two clock-controlled genes that may play an important role in mediating mPer2-controlled cytotoxic response. (Author correspondence: zsunusa@yahoo.com)

Evolutionary Mode and Functional Divergence of Vertebrate NMDA Receptor Subunit 2 Genes

Background Ionotropic glutamate receptors in the central nervous system play a major role in numerous brain functions including learning and memory in many vertebrate species. NR2 subunits have been regarded as rate-limiting molecules in controlling the optimal N-methyl-D-aspartate (NMDA) receptors coincidence-detection property and subsequent learning and memory function across multi-species. However, its evolutionary mode among vertebrate species remains unclear. Results With extensive analysis of phylogeny, exon structure, protein domain, paralogon and synteny, we demonstrated that two-round genome duplication generated quartet GRIN2 genes and the third-round fish-specific genome duplication generated extra copies of fish GRIN2 genes. In addition, in-depth investigation has enabled the identification of three novel genes, GRIN2C_Gg, GRIN2D-1_Ol and GRIN2D-2_Tr in the chicken, medaka and fugu genome, respectively. Furthermore, we showed functional divergence of NR2 genes mostly occurred at the first-round duplication, amino acid residues located at the N-terminal Lig_chan domain were responsible for type I functional divergence between these GRIN2 subfamilies and purifying selection has been the prominent natural pressure operating on these diversified GRIN2 genes. Conclusion and Significance These findings provide intriguing subjects for testing the 2R and 3R hypothesis and we expect it could provide new insights into the underlying evolution mechanisms of cognition in vertebrate.

Comparative RNA-seq analysis reveals potential mechanisms mediating the conversion to androgen independence in an LNCaP progression cell model

The androgen-independent phenotype is an important symptom of refractory prostate cancer. However, the molecular mechanisms underlying this phenotypic conversion remain unclear. Using RNA-seq analysis of androgen-dependent prostate cancer cells (LNCaP) vs. androgen-independent cancer cells (LNCaP-AI-F), we identified 788 differentially expressed genes, 315 alternative splicing events, and eight novel LNCaP-AI-F-specific fusion genes. The fusion genes EIF2AK1-ATR and GLYR1-SLC9A8 were predicted to be damaging and oncogenic. We also observed dramatic changes in androgen receptor (AR)-mediated pathway molecules, including prostate-specific antigen (PSA, a major biomarker of prostate cancer) and AR variants, as well as neuroendocrine-like (NE-like) and tumor stem cell-like characteristics, during androgen-independent phenotype progression. Our findings provide new insights into the regulatory complexities of refractory prostate cancers.

VarCards: an integrated genetic and clinical database for coding variants in the human genome.

Abstract A growing number of genomic tools and databases were developed to facilitate the interpretation of genomic variants, particularly in coding regions. However, these tools are separately available in different online websites or databases, making it challenging for general clinicians, geneticists and biologists to obtain the first-hand information regarding some particular variants and genes of interest. Starting with coding regions and splice sties, we artificially generated all possible single nucleotide variants (n = 110 154 363) and cataloged all reported insertion and deletions (n = 1 223 370). We then annotated these variants with respect to functional consequences from more than 60 genomic data sources to develop a database, named VarCards (http://varcards.biols.ac.cn/), by which users can conveniently search, browse and annotate the variant- and gene-level implications of given variants, including the following information: (i) functional effects; (ii) functional consequences through different in silico algorithms; (iii) allele frequencies in different populations; (iv) disease- and phenotype-related knowledge; (v) general meaningful gene-level information; and (vi) drug–gene interactions. As a case study, we successfully employed VarCards in interpretation of de novo mutations in autism spectrum disorders. In conclusion, VarCards provides an intuitive interface of necessary information for researchers to prioritize candidate variations and genes.

Demethylation of c-MYB binding site mediates upregulation of Bdnf IV in cocaine-conditioned place preference

Abnormal BDNF signaling contributes to the structural and behavioral plasticity induced by drugs of abuse. However, the mechanisms regulating expression of Bdnf in drug addiction remain elusive. In the present study, using the conditioned place preference (CPP) model, we showed that expression of Bdnf IV is upregulated in the nucleus accumbens (NAc) of conditioned animals while Bdnf I is upregulated in cocaine-treated mice irrespective of conditioning. The methylation level of a putative c-MYB binding site in the promoter region of Bdnf IV was significantly decreased in the NAc under cocaine CPP conditioning but remained unchanged without conditioning, concurrently with increased binding of c-MYB to this site. Exon IV promoter/luciferase reporter assays revealed that transactivation of Bdnf by c-MYB was blocked by methylation of this c-MYB binding site. Administration of methionine, a precursor of SAM, inhibited cocaine CPP, reversed demethylation of c-MYB binding site and induction of Bdnf IV expression by cocaine CPP. Our results imply that Bdnf IV demethylation at c-MYB binding site is involved in cocaine-triggered seeking behavior, whereas Bdnf I responds to the immediate pharmacological effects of cocaine.

Epigenetic Activation of ASCT2 in the Hippocampus Contributes to Depression-Like Behavior by Regulating D-Serine in Mice

The roles of D-serine in depression are raised concerned recently as an intrinsic co-agonist for the NMDA receptor. However, the mechanisms underlying its regulation are not fully elucidated. ASCT2 is a Na+-dependent D-serine transporter. We found that decreased D-serine and increased hippocampal ASCT2 levels correlated with chronic social defeat stress (CSDS) in mice. Lentivirus-mediated shRNA-mediated knockdown of ASCT2 and the administration of exogenous D-serine in the hippocampus alleviated CSDS-induced social avoidance and immobility. In vivo and in vitro experiments revealed that upregulation of ASCT2 expression in CSDS was regulated through histone hyper-acetylation, not DNA methylation in its promoter region. Immunohistochemistry demonstrated the co-localization of ASCT2 and D-serine. Uptake of D-serine by ASCT2 was demonstrated by in vivo and in vitro experiments. Our results indicate that CSDS induces ASCT2 expression through epigenetic activation and decreases hippocampal D-serine levels, leading to social avoidance, and immobility. Thus, targeting D-serine transport represents an attractive new strategy for treating depression.

Genetic landscape of papillary thyroid carcinoma in the Chinese population

Improvement in the clinical outcome of human cancers requires characterization of the genetic alterations underlying their pathogenesis. Large‐scale genomic and transcriptomic characterization of papillary thyroid carcinomas (PTCs) in Western populations has revealed multiple oncogenic drivers which are essential for understanding pathogenic mechanisms of this disease, while, so far, the genetic landscape in Chinese patients with PTC remains uncharacterized. Here, we conducted a large‐scale genetic analysis of PTCs from patients in China to determine the mutational landscape of this cancer. By performing targeted DNA amplicon and targeted RNA deep‐sequencing, we elucidated the landscape of somatic genetic alterations in 355 Chinese patients with PTC. A total of 88.7% of PTCs were found to harbor at least one candidate oncogenic driver genetic alteration. Among them, around 72.4% of the cases carried BRAF mutations; 2.8% of cases harbored RAS mutations; and 13.8% of cases were characterized with in‐frame gene fusions, including seven newly identified kinase gene fusions. TERT promoter mutations were likely to occur in a sub‐clonal manner in our PTC cohort. The prevalence of somatic genetic alterations in PTC was significantly different between our Chinese cohort and TCGA datasets for American patients. Additionally, combined analyses of genetic alterations and clinicopathologic features demonstrated that kinase gene fusion was associated with younger age at diagnosis, larger tumor size, and lymph node metastasis in PTC. With the analyses of DNA rearrangement sites of RET gene fusions in PTC, signatures of chromosome translocations related to RET fusion events were also depicted. Collectively, our results provide fundamental insight into the pathogenesis of PTC in the Chinese population. Copyright