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Microsporidia are obligate intracellular parasites that infect a wide variety of host organisms, including humans. The sporoplasm is the initial stage of microsporidian infection and proliferation, but its morphological and molecular characteristics are poorly understood. In this study, the sporoplasm of Nosema bombycis was successfully isolated and characterized after the induction of spore germination in vitro The sporoplasm was spherical, 3.64 ± 0.41 μm in diameter, had the typical two nuclei, and was nonrefractive. Scanning and transmission electron microscopy analyses revealed that the sporoplasm was surrounded by a single membrane, and the cytoplasm was usually filled with relatively homogeneous granules, possibly ribosomes, and contained a vesicular structure comprising a concentric ring and coiled tubules. Propidium iodide staining revealed that the sporoplasm membrane showed stronger membrane permeability than did the cell plasma membrane. Transmission electron microscopy (TEM) revealed that the sporoplasm can gain entry to the host cell by phagocytosis. Transcriptome analysis of mature spores and sporoplasms showed that 541 significantly differentially expressed genes were screened (adjusted P value [ P adj ] < 0.05), of which 302 genes were upregulated and 239 genes were downregulated in the sporoplasm. The majority of the genes involved in trehalose synthesis metabolism, glycolysis, and the pentose phosphate pathway were downregulated, whereas 10 transporter genes were upregulated, suggesting that the sporoplasm may inhibit its own carbon metabolic activity and obtain the substances required for proliferation through transporter proteins. This study represents the first comprehensive and in-depth investigation of the sporoplasm at the morphological and molecular levels and provides novel insights into the biology of microsporidia and their infection mechanism. IMPORTANCE Once awoken from dormancy, the cellular matter of microsporidia is delivered directly into the host cell cytoplasm through the polar tube. This means that the microsporidia are difficult to study biologically in their active state without a contaminating signal from the host cell. Sporoplasm is a cell type of microsporidia in vitro , but relatively little attention has been paid to the sporoplasm in the past 150 years due to a lack of an effective separation method. Nosema bombycis , the first reported microsporidium, is a type of obligate intracellular parasite that infects silkworms and can be induced to germinate in alkaline solution in vitro We successfully separated the N. bombycis sporoplasm in vitro , and the morphological and structural characteristics were investigated. These results provide important insight into the biology and pathogenesis of microsporidia and potentially provide a possible strategy for genetic manipulation of microsporidia targeting the sporoplasm., (Copyright © 2020 He et al.)

Background: The complete and accurate human reference genome is important for functional genomics researches. Therefore, the incomplete reference genome and individual specific sequences have significant effects on various studies., Results: we used two RNA-Seq datasets from human brain tissues and 10 mixed cell lines to investigate the completeness of human reference genome. First, we demonstrated that in previously identified ~5 Mb Asian and ~5 Mb African novel sequences that are absent from the human reference genome of NCBI build 36, ~211 kb and ~201 kb of them could be transcribed, respectively. Our results suggest that many of those transcribed regions are not specific to Asian and African, but also present in Caucasian. Then, we found that the expressions of 104 RefSeq genes that are unalignable to NCBI build 37 in brain and cell lines are higher than 0.1 RPKM. 55 of them are conserved across human, chimpanzee and macaque, suggesting that there are still a significant number of functional human genes absent from the human reference genome. Moreover, we identified hundreds of novel transcript contigs that cannot be aligned to NCBI build 37, RefSeq genes and EST sequences. Some of those novel transcript contigs are also conserved among human, chimpanzee and macaque. By positioning those contigs onto the human genome, we identified several large deletions in the reference genome. Several conserved novel transcript contigs were further validated by RT-PCR., Conclusion: Our findings demonstrate that a significant number of genes are still absent from the incomplete human reference genome, highlighting the importance of further refining the human reference genome and curating those missing genes. Our study also shows the importance of de novo transcriptome assembly. The comparative approach between reference genome and other related human genomes based on the transcriptome provides an alternative way to refine the human reference genome.

Background: The exploration of the structural topology and the organizing principles of genome-based large-scale metabolic networks is essential for studying possible relations between structure and functionality of metabolic networks. Topological analysis of graph models has often been applied to study the structural characteristics of complex metabolic networks., Results: In this work, metabolic networks of 75 organisms were investigated from a topological point of view. Network decomposition of three microbes (Escherichia coli, Aeropyrum pernix and Saccharomyces cerevisiae) shows that almost all of the sub-networks exhibit a highly modularized bow-tie topological pattern similar to that of the global metabolic networks. Moreover, these small bow-ties are hierarchically nested into larger ones and collectively integrated into a large metabolic network, and important features of this modularity are not observed in the random shuffled network. In addition, such a bow-tie pattern appears to be present in certain chemically isolated functional modules and spatially separated modules including carbohydrate metabolism, cytosol and mitochondrion respectively., Conclusion: The highly modularized bow-tie pattern is present at different levels and scales, and in different chemical and spatial modules of metabolic networks, which is likely the result of the evolutionary process rather than a random accident. Identification and analysis of such a pattern is helpful for understanding the design principles and facilitate the modelling of metabolic networks.

Purpose: The incidence of prostate cancer is frequent, occurring in almost one-third of men older than 45 years. Only a fraction of the cases reach the stages displaying clinical significance. Despite the advances in our understanding of prostate carcinogenesis and disease progression, our knowledge of this disease is still fragmented. Identification of the genes and patterns of gene expression will provide a more cohesive picture of prostate cancer biology., Patients and Methods: In this study, we performed a comprehensive gene expression analysis on 152 human samples including prostate cancer tissues, prostate tissues adjacent to tumor, and organ donor prostate tissues, obtained from men of various ages, using the Affymetrix (Santa Clara, CA) U95a, U95b, and U95c chip sets (37,777 genes and expression sequence tags)., Results: Our results confirm an alteration of gene expression in prostate cancer when comparing with nontumor adjacent prostate tissues. However, our study also indicates that the gene expression pattern in tissues adjacent to cancer is so substantially altered that it resembles a cancer field effect., Conclusion: We also found that gene expression patterns can be used to predict the aggressiveness of prostate cancer using a novel model.

Leiomyosarcoma of the somatic soft tissue is a rare malignant mesenchymal neoplasm that metastasizes to other organs in a subset of cases. Much remains to be learned about the mechanisms underlying the development of aggressive behavior of this tumor. It has been difficult to predict the clinical behavior of leiomyosarcomas using the morphology-based grading system, even though tumor size and histological grade have correlated with biologic behavior in some studies. In this study we analyzed the gene expression patterns of 35 samples of mesenchymal origin, including 11 cases of leiomyosarcomas of different histological grades arising in soft tissue and the retroperitoneum, using the Affymetrix U133a chips, which contain more than 22,000 genes and expression sequence tags (ESTs). We identified a set of genes whose expression was commonly altered in all leiomyosarcoma samples. In addition, we identified specific gene expression patterns in several subsets of the tumor. We used these alterations of gene expression to subclassify the leiomyosarcomas into 3 groups. Interestingly, the grouping of these samples correlated well with tumor differentiation and clinical aggressiveness. The analysis identified 92 genes that distinguish low-grade, well-differentiated leiomyosarcomas from less well-differentiated, high-grade, and metastatic leiomyosarcoma. Thesse alterations of gene expression appear to be correlated with the clinical behavior and histological grade of the tumor. The striking differences in terms of gene expression pattern among leiomyosarcomas of different differentiation status and clinical aggressiveness imply that several genetic abnormalities are responsible for the genesis and progression of this tumor.

Opsins are a class of transmembrane proteins encoded by opsin genes, and they play a variety of functional roles. Short wavelength-sensitive opsin 2 (sws2), one of the five classes of visual opsin genes, mainly senses blue light. Previous research has indicated that sws2 is essential for melanocyte formation in fish; however, its specific role in skin color differentiation remains to be elucidated. Here, we identified the sws2 gene in a prized reef-dwelling fish, Plectropomus leopardus. The full-length P. leopardus sws2 gene encodes a protein consisting of 351 amino acids, and exhibits substantial homology with other fish species. The expression of the sws2 gene was widespread across P. leopardus tissues, with high expression in eye and skin tissues. Through immunohistochemistry and in situ hybridization analyses, we discovered that the sws2 gene was primarily localized in the rod and cone cells of the retina, and epidermal cells of the skin. Furthermore, dsRNA interference was used for sws2 gene knockdown in living P. leopardus to elucidate its function in skin color differentiation. Black-color-related genes, melanin contents, and tyrosinase activity in the skin significantly decreased after sws2 knockdown (p < 0.05), but red-color-related genes and carotenoid and lutein contents significantly increased (p < 0.05). Retinoic acid injection produced the opposite results. Our results suggested that the sws2 gene influences P. leopardus skin color regulation by affecting vitamin synthesis and melanin-related gene expression levels. This study establishes a foundation for elucidating the molecular mechanisms by which sws2 regulates melanocyte formation in fish skin. [ABSTRACT FROM AUTHOR]

Homeobox A13 (HOXA13) has been verified as an oncogen in some malignancies. However, its role in nasopharyngeal carcinoma (NPC) is still unclear. This study aims to explore the role of HOXA13 in NPC and its underlying mechanism. The mRNA expression of HOXA13 in NPC was obtained from the GSE53819 and GSE64634 datasets in the Gene Expression Omnibus (GEO) database. MTT, colony formation and transwell assays and xenograft tumour models were used to investigate the effects of HOXA13 on NPC HNE1 cells in vitro and in vivo. The expression of HOXA13, epithelial-mesenchymal transition-transcription factor (EMT-TF) Snail and matrix metalloproteinase 2 (MMP-2) was detected by immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. The results showed that HOXA13 was upregulated in NPC. Silencing HOXA13 suppressed the proliferation, migration, and invasion of HNE1 cells, which inhibited tumour growth, while overexpression of HOXA13 induced the opposite effects. In addition, the expression of Snail and MMP-2 at the transcriptional and protein levels was associated with the expression of HOXA13. In summary, our results suggest that HOXA13 plays a role as a cancer-promoting gene in NPC. The underlying mechanism may be related to the upregulation of Snail and MMP-2. [ABSTRACT FROM AUTHOR]

Extensive evidence has explored the involvement of microRNAs (miRNAs) in osteosarcoma (OS). Limitedly, the concrete function of microRNA-18b-5p (miR-18b-5p) in OS remains unexplored and largely elusive. Here, we validated that miR-18b-5p significantly elevated in OS via analyzing the data from GEO database. The results showed that miR-18b-5p was overexpressed in human OS tissues and cell lines. The clinical evidence suggested that high level of miR-18b-5p was negatively correlated with the poor prognosis of OS. Meanwhile, miR-18b-5p upregulation facilitated the proliferation and metastasis of OS cells in vitro and in vivo. The mechanism exploration demonstrated that miR-18b-5p acted as a potential inhibitor of PHF2, a tumor suppressor gene, at post-transcriptional level. Moreover, hypoxia induced gene expression of miR-18b-5p was clarified to be transcriptionally mediated by HIF-1α. The clinicopathological analysis in samples of OS patients further supported that miR-18b-5p had a positive correlation with HIF-1α expression, and negative correlation with PHF2. Collectively, the present study uncovered a new molecular mechanism of OS tumorigenesis and development and miR-18b-5p might be a prognostic biomarker and potential therapeutic target for OS treatment. [ABSTRACT FROM AUTHOR]

To explore the feeding regulatory mechanism of the leptin gene and its receptor, the full‐length cDNA of Tbleptin‐B and TbleptinR were cloned from Trachinotus blochii, and their expression patterns in different dietary strategies were analysed. The preprandial expressions of Tbleptin‐A, Tbleptin‐B and TbleptinR in liver showed a downward trend as a function of time. However, 1 h after postprandial, Tbleptin‐A increased significantly, Tbleptin‐B decreased significantly, TbleptinR did not change, and their postprandial expressions were not significantly different with the control group at 3 h. This result indicated that Tbleptin‐A and Tbleptin‐B were involved in the postprandial regulation of liver within 3 h. Similarly, the expressions of Tbleptin‐A, Tbleptin‐B and TbleptinR in liver were not different from the control group after refeeding (d11), which showed that the expressions of them in liver would return to normal level after long‐term hungry golden pomfret getting enough food. In hypothalamus, the postprandial expressions of Tbleptin‐B and TbleptinR were significantly increased at 1 h, while Tbleptin‐A was inhibited. Similarly, after refeeding, the expression of Tbleptin‐A was significantly lower than that of the control group, and Tbleptin‐B was significantly higher than that of the control group. In summary, Tbleptin‐A, Tbleptin‐B and TbleptinR play different roles in coping with different dietary strategies in liver and hypothalamus. Tbleptin‐A is mainly involved in dietary regulation through liver, while Tbleptin‐B and TbleptinR are mainly active in the hypothalamus. These results provide valuable information to reveal the T. blochii feeding regulatory mechanism. [ABSTRACT FROM AUTHOR]

The characterization of human gene expression is limited by short read lengths, high error rates and large input requirements. Here, we used a synthetic long read (SLR) sequencing approach, LoopSeq, to generate accurate sequencing reads that span full length transcripts using standard short read data. LoopSeq identified isoforms from control samples with 99.4% accuracy and a 0.01% per-base error rate, exceeding the accuracy reported for other long-read technologies. Applied to targeted transcriptome sequencing from colon cancers and their metastatic counterparts, LoopSeq revealed large scale isoform redistributions from benign colon mucosa to primary colon cancer and metastatic cancer and identified several previously unknown fusion isoforms. Strikingly, single nucleotide variants (SNVs) occurred dominantly in specific isoforms and some SNVs underwent isoform switching in cancer progression. The ability to use short reads to generate accurate long-read data as the raw unit of information holds promise as a widely accessible approach in transcriptome sequencing. Silvia Liu et al. present LoopSeq, a synthetic long-read sequencing method that generates accurate long-read transcriptome data from short Illumina reads. As an example of possible applications, they use LoopSeq to investigate differential isoform expression, isoform-specific single nucleotide variants, and potential fusion genes in multiple stages of colon cancer. Altogether, LoopSeq is a valuable method for analyzing complex transcriptomes and investigating gene expression at the isoform level. [ABSTRACT FROM AUTHOR]

Circulating tumor cells/microemboli (CTCs/CTMs) are malignant cells that depart from cancerous lesions and shed into the bloodstream. Analysis of CTCs can allow the investigation of tumor cell biomarker expression from a non-invasive liquid biopsy. To date, high-throughput technologies have become a powerful tool to provide a genome-wide view of transcriptomic changes associated with CTCs/CTMs. These data provided us much information to understand the tumor heterogeneity, and the underlying molecular mechanism of tumor metastases. Unfortunately, these data have been deposited into various repositories, and a uniform resource for the cancer metastasis is still unavailable. To this end, we integrated previously published transcriptome datasets of CTCs/CTMs and constructed a web-accessible database. The first release of ctcRbase contains 526 CTCs/CTM samples across seven cancer types. The expression of 14 631 mRNAs and 3642 long non-coding RNAs of CTCs/CTMs were included. Experimental validations from the published literature are also included. Since CTCs/CTMs are considered to be precursors of metastases, ctcRbase also collected the expression data of primary tumors and metastases, which allows user to discover a unique 'circulating tumor cell gene signature' that is distinct from primary tumor and metastases. An easy-to-use database was constructed to query and browse CTCs/CTMs genes. ctcRbase can be freely accessible at http://www.origin-gene.cn/database/ctcRbase/. [ABSTRACT FROM AUTHOR]

Plectropomus leopardus is a valuable marine fish whose skin color is strongly affected by the background color. However, the influence of the visual sense on the skin color variation of P. leopardus remains unknown. In the present study, transcriptome analysis was used to examine the visual response mechanism under different background colors. Paraffin sections of the eyes showed that the background color caused morphological changes in the pigment cells (PCs) and outer nuclear layer (ONL) and the darkening of the iris color. The transcriptome analysis results indicated that the gene expressions in the eyes of P. leopardus were significantly different for different background colors. We identified 4845, 3069, 5874, and 6309 differentially expressed genes (DEGs) in the pairwise comparisons of white vs. initial, blue vs. initial, red vs. initial, and black vs. initial groups, respectively. Some hub genes and key pathways regulating the adaptive mechanism of P. leopardus 's eyes to the background color were identified, i.e., the JAK-STAT, mTOR, and Ras signaling pathways, and the ndufb7 , slc6a13 , and novel.3553 gene. This adaptation was achieved through the synthesis of stress proteins and energy balance supply mediated by hub genes and key pathways. In addition, the phenylalanine metabolism, tyrosine metabolism, and actin cytoskeleton-related processes or pathways and genes were responsible for iris and skin color adaptation. In summary, we inferred that stress protein synthesis, phenylalanine metabolism, and energy homeostasis were critical stress pathways for P. leopardus to adapt its skin color to the environment. These new findings indicate that the P. leopardus skin color variation may have been caused by the environmental adaption of the eyes. The results provide new insights into the molecular mechanisms underlying the skin color adaptation of P. leopardus. [Display omitted] • Background color caused the morphology change of the pigment cells (PC) and outer nuclear layer (ONL). • The darkening trend of the P. leopardus iris color is consistent with that of skin color response to the background color. • The adaptive mechanism of P. leopardus's eyes to the background color regulated by JAK-STAT, mTOR, and Ras signaling pathways. • The skin color variation is most likely driven by environmental adaption of the eyes of P. leopardus. [ABSTRACT FROM AUTHOR]

Distant metastasis is the main cause of death in patients with clear cell renal carcinoma (ccRCC). The dysregulation of the tumor microenvironment is responsible for tumorigenesis and metastasis in ccRCC. The role of long non-coding RNAs in the tumor immune of ccRCC remains unclear. The present study screened differentially expressed protein-coding genes and non-coding genes between ccRCC and normal tissues based on three datasets. The commonly deregulated genes were used to identify distant metastasis-related long non-coding RNAs (lncRNAs) and prognostic lncRNAs. Pearson correlation analysis was used to identify immune-related lncRNAs. A competing endogenous RNA network was constructed and hub lncRNAs were identified. A total of 1650 coding genes, 821 lncRNAs and 62 miRNAs were commonly deregulated in the three datasets. A total of 408 lncRNAs associated with the overall survival of patients with ccRCC were identified. Among them, 82 lncRNAs were distant metastasis-related. Further analysis identified 52 lncRNAs associated with the immune pathway. Functional analyses concordantly demonstrated the role of the 52 lncRNAs in metastasis and tumor immunology. The ceRNA network analysis indicated lncRNA DSCR9 as the key lncRNA regulator. Univariate and multivariate analysis in two independent cohorts validated that DSCR9 could be an independent risk factor for the progression-free survival of patients with ccRCC. Further analyses indicated that DSCR9 might be associated with the immunotherapeutic response. reverse transcription-quantitative PCR demonstrated that the RNA expression level of DSCR9 was upregulated in ccRCC compared with normal kidney samples. The present study demonstrated the potential of LncRNA DSCR9 in assessing the prognosis and developing future immunotherapy for patients with metastatic ccRCC. [ABSTRACT FROM AUTHOR]

DNA methylation is an essential epigenetic modification involved in regulating gene expression and maintaining epigenetic information across generations. However, how these marks are recognized and interpreted to activate or repress imprinted genes is not fully understood. Preliminary evidence describes the transcriptional repressor TRIM28 as a key regulator of imprinted gene expression during and after early genome-wide reprogramming. Aberrant expression of imprinted genes maybe one possible cause of incomplete epigenetic reprogramming and low efficiency in somatic cell nuclear transfer. Here, we perform a series of experiments to determine whether knockdown of Trim28 alters imprinted gene expression and DMR methylation in sheep embryonic fibroblast (SEF) cells. siRNA-mediated Trim28 silencing in SEF cells resulted in significantly decreased expression of Gtl2 to 30% and increased expression of Dlk 1 (~ 1.7-fold). Moreover, knocking down Trim28 induced DNA methylation at the IG-DMR and the Gtl2 promoter was disrupted. Here, we uncover an important role for Trim28 in the maintenance of DNA methylation at IG-DMR during replication-dependent dilution of methylated cytosine during cellular proliferation. Unlike Dlk1-Gtl2 however, knocking down Trim28 does not affect DMR methylation in the Igf2-H19 gene cluster, yet results in increased expression of Igf2 and H19 . Interestingly, Peg3 expression decreased by 60% in Trim28 knockdown cells. PEG3 as a transcriptional repressor to the H19-ICR that interacts with the co-repressor protein TRIM28 through KRAB-A. Trim28 therefore appears to control the Igf2 - H19 imprinted cluster indirectly via PEG3, which is distinct from its classical role in preserving DNA methylation during DNA replication. Our results therefore indicate that Trim28 regulates imprinted gene expression through at least two distinct mechanisms during cells proliferation. [ABSTRACT FROM AUTHOR]

This study was conducted to examine the effects of dietary valine on the physical and flavor characteristics, fatty acid (FA) profile, antioxidant status and Nrf2-dependent antioxidant enzyme gene expression in the muscle of young grass carp (Ctenopharyngodon idella) fed increasing levels of valine (4.3, 8.0, 10.6, 13.1, 16.9 and 19.1 g/kg) for 8 weeks. Compared with the control group, the group fed valine showed improved physical characteristics of fish fillets (increased relative shear force, hydroxyproline, protein and lipid levels and decreased cathepsin B and L activities, as well as cooking loss, were observed). Moreover, valine improved the flavor of young grass carp fillets by increasing the amino acid (AA) concentration in fish muscle (increased aspartic acid, threonine, glutamine, cystine, methionine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine and valine concentrations were observed). Additionally, optimal valine supplementation increased the potential health benefits to humans by decreasing the saturated FA (C15:0 and C16:0) concentration and increasing the unsaturated FA (monounsaturated FAs (MUFAs), such as C16:1, C18:1c+t and C20:1, and polyunsaturated FAs (PUFAs), such as C18:3n-3, C20:2 and C22:6) concentration. In addition, the reduced glutathione (GSH) content and the activities of Cu/Zn superoxide dismutase (SOD1), catalase (CAT) and Selenium-dependent glutathione peroxydase (Se-GPx) increased under valine supplementation (P < 0.05). Furthermore, the SOD1, CAT and Se-GPx mRNA levels increased with dietary valine levels, possibly due to the up-regulation of NF-E2-related factor 2 (Nrf2), target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1) and the down-regulation of Kelch-like-ECH-associated protein 1 (Keap1) in muscle (P < 0.05). In conclusion, valine improved the physical and flavor characteristics, FA profile, and antioxidant status and regulated the expression of the antioxidant enzyme genes Nrf2, Keap1, TOR and S6K1 in fish fillets. [ABSTRACT FROM AUTHOR]

Objective: Calretinin (CALB2), a calcium-binding protein, may be expressed in human testes, suggesting that CALB2 can play roles in regulating testicular functions. This study was designed to explore the different expression of CALB2 in the testes of rats at different development stags, and to discuss its role in regulating androgen production. Methods: The 3-4-week-old SD male rats as group A(the pre-sexual maturity, re=35), 16-week-old as group B(the sexual maturity, n=16), and over-12-month as group C(the older rats, n= 10)were used in the study. Sera testosterone concentration was detected by radioimmunoassay (RIA).Cellular location of CALB2 in testes was observed by immunohistochemistry. Expression levels of CALB2 mRNA and protein were quantificationally analyzed by qPCR and western blot. Results: CALB2 was mainly located in the cytoplasm of Leydig cells. Sera testosterone level was the lowest in the group A, and the highest in the group B (P<0.05). Compared to the group A and the group C, expressions of CALB2 were significantly increased(P< 0.05). Conclusions: Calretinin is located in the cytoplasm of Leydig cells, and differently expressed in testes of rats at different development stages, which suggested that calretinin regulated testosterone production. [ABSTRACT FROM AUTHOR]

Background: Toll-like receptors (TLRs) play a pivotal role in the defense against invading pathogens by detecting pathogen-associated molecular patterns (PAMPs). TLR4 recognizes lipopolysaccharides (LPS) in the cell walls of Gram-negative bacteria, resulting in the induction and secretion of proinflammatory cytokines such as TNF-α and IL-6. The WW domain containing E3 ubiquitin protein ligase 1 (WWP1) regulates a variety of cellular biological processes. Here, we investigated whether WWP1 acts as an E3 ubiquitin ligase in TLR-mediated inflammation. Methodology/Results: Knocking down WWP1 enhanced the TNF-α and IL-6 production induced by LPS, and over-expression of WWP1 inhibited the TNF-α and IL-6 production induced by LPS, but not by TNF-α. WWP1 also inhibited the IκB-α, NF-κB, and MAPK activation stimulated by LPS. Additionally, WWP1 could degrade TRAF6, but not IRAK1, in the proteasome pathway, and knocking down WWP1 reduced the LPS-induced K48-linked, but not K63-linked, polyubiquitination of endogenous TRAF6. Conclusions/Significance: We identified WWP1 as an important negative regulator of TLR4-mediated TNF-α and IL-6 production. We also showed that WWP1 functions as an E3 ligase when cells are stimulated with LPS by binding to TRAF6 and promoting K48-linked polyubiquitination. This results in the proteasomal degradation of TRAF6. [ABSTRACT FROM AUTHOR]

Background: microRNAs (miRNAs) are a class of non-coding, single-stranded RNA molecules that regulate gene expression at the posttranscriptional level. Methyl-CpG-binding domain proteins (MBPs) are transcription repressors through binding to methylated gene promoters. Recent studies have shown that the effect of miRNAs on DNA methylation by targeting DNA methyltransferase (DNMTs) and/or MBPs plays an important role in various human cancers.Aims: This study focuses on the regulation of MBPs by miR-373 and its downstream effect in hilar cholangiocarcinoma.Methods: miR-373 was investigated by TaqMan miRNA Assay; mRNA and protein of MBD1, MBD2, and Mecp2 were determined by QuantiTect(®) Primer Assays and Western blotting, respectively; RASSF1A mRNA was measured by SYBR-Green real-time PCR; The targeting at MBD2-3'UTR by miR-373 was evaluated by dual-luciferase reporter gene assay.Results: miR-373 decreased and closely associated with poor cell differentiation, advanced clinical stage, and shorter survival in hilar cholangiocarcinoma; MBD2 exclusively over-expressed and reciprocally related to miR-373; precursor miR-373 inhibited the luciferase activity of MBD2-3'UTR construct; exogenous miR-373 suppressed the expression of MBD2 and enhanced RASSF1A mRNA in QBC(939) cells; anti-miR-373 inhibitor up-regulated the expression of MBD2 and reduced RASSF1A mRNA in HIBEpic cells.Conclusions: miR-373 is one negative regulator of MBD2. In hilar cholangiocarcinoma, down-expression of miR-373 leads to increase of MBD2, which in turn suppresses the methylation-mediated gene such as RASSF1A. [ABSTRACT FROM AUTHOR]

E-cadherin is a transmembrane glycoprotein which mediates epithelial cell-to-cell adhesion function as a tumor suppressor and frequently loss of expression in a wide spectrum of human cancer. However, recent studies demonstrated that E-cadherin was always over-expressed in inflammatory breast cancer (IBC) specimen and cell lines, which is a clinical extreme malignancy of breast cancer. It is hypothesized that the gain and not the loss of the E-cadherin axis contributes to the IBC unique phenotype. To test this assumption, we generated dominant negative mutant E-cadherin high-expression inflammatory breast cancer cells by introduced dominant negative mutant E-cadherin (H-2kd-E-cad) cDNA into human IBC SUM149 cells. Our studies demonstrated that the ability of invasion of SUM149 cells was significantly inhibited by H-2kd-E-cad via down-regulation of MMP-1 and MMP-9 expression. The underlying signal pathway of MAPK phosphorylated Erk 1/2(P44/42) in H-2kd-E-cad-transfected SUM149 cells was significantly down-regulated compared to parental and mock contrast. Our studies provided further functional evidence as the gain of E-cadherin expression dedicated to the IBC malignant phenotype and the blockage of MAPK/Erk activation maybe a promising therapeutic target. [ABSTRACT FROM AUTHOR]

Prostate cancer is one of the leading causes of cancer-related death in the USA. In the past decade, tremendous progress has been made in the identification and understanding of the genetic factors related to prostate cancer development. Unlike many other types of cancers, only a small fraction of prostate cancer cases are aggressive and life-threatening. The factors related to prostate cancer development and progression appear complex and diverse. This review summarises some of the important findings in the areas of genome and gene expression abnormalities in prostate cancer, and aims to provide a comprehensive view of new developments in these areas. [ABSTRACT FROM PUBLISHER]

Enteritis poses a significant threat to fish farming, characterized by symptoms of intestinal and hepatic inflammation, physiological dysfunction, and dysbiosis. Focused on the leopard coral grouper (Plectropomus leopardu s) with an enteritis outbreak on a South China Sea farm, our prior scrutiny did not find any abnormalities in feeding or conventional water quality factors, nor were any specific pathogen infections related to enteritis identified. This study further elucidates their intestinal flora alterations, host responses, and their interactions to uncover the underlying pathogenetic mechanisms and facilitate effective prevention and management strategies. Enteritis-affected fish exhibited substantial differences in intestinal flora compared to control fish (P = 0.001). Notably, norank_f_Alcaligenaceae , which has a negative impact on fish health, predominated in enteritis-affected fish (91.76 %), while the probiotic genus Lactococcus dominated in controls (93.90 %). Additionally, certain genera with pathogenesis potentials like Achromobacter , Sphingomonas , and Streptococcus were more abundant in diseased fish, whereas Enterococcus and Clostridium_sensu_stricto with probiotic potentials were enriched in control fish. At the transcriptomic level, strong inflammatory responses, accompanied by impaired metabolic functions, tissue damage, and iron death signaling activation were observed in the intestines and liver during enteritis. Furthermore, correlation analysis highlighted that potential pathogen groups were positively associated with inflammation and tissue damage genes while presenting negatively correlated with metabolic function-related genes. In conclusion, dysbiosis in the intestinal microbiome, particularly an aberrantly high abundance of Alcaligenaceae with pathogenic potential may be the main trigger for this enteritis outbreak. Alcaligenaceae alongside Achromobacter , Sphingomonas , and Streptococcus emerged as biomarkers for enteritis, whereas some species of Lactococcus , Clostridium_sensu_stricto , and Enterococcus showed promise as probiotics to alleviate enteritis symptoms. These findings enhance our understanding of enteritis pathogenesis, highlight intestinal microbiota shifts in leopard coral grouper, and propose biomarkers for monitoring, probiotic selection, and enteritis management. • Enteritis induced significant intestinal flora changes in Plectropomus leopardus. • Immune and metabolism genes significantly changed in enteritis-affected fish. • Correlations were identified between the top 20 abundant intestinal bacteria genera and fish gene expressions. [ABSTRACT FROM AUTHOR]

[Display omitted] • CFA intraplantar injection caused a significant decrease in the level of spinal IRF7. • IRF7 plays an important role in the regulation of inflammatory pain. • IRF7 regulates the activation of NF-κB pathways in the spinal cord. • IRF7 modulates the expression of inflammatory mediators in the spinal cord. It is known that nerve signals arising from sites of inflammation lead to persistent changes in the spinal cord and contribute to the amplification and persistence of pain. Nevertheless, the underlying mechanisms have not yet been completely elucidated. We identified differentially expressed genes in the lumbar (L4–L6) segment of the spinal cord from complete Freund's adjuvant (CFA) rats compared to control animals via high throughput sequencing. Based on differential gene expression analysis, we selected interferon regulatory factor 7 (IRF7) for follow-up experiments to explore its antinociceptive potential. An animal model of inflammatory pain was induced by intraplantar injection of CFA. We evaluated the effects of adeno-associated viral (AAV)-mediated overexpression of IRF7 in the spinal cord on pain-related behavior after CFA injection. Moreover, the activation of the nuclear factor-κB (NF-κB) and the expression of inflammatory cytokines were investigated to understand the underlying mechanisms related to the contribution of IRF7 to inflammatory pain. CFA intraplantar injection caused a significant decrease in the level of spinal IRF7, which is mainly expressed in the dorsal horn neurons and astrocytes. Moreover, IRF7 overexpression significantly attenuated pain-related behaviors, as well as the activity of NF-κB/p65 and the production of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the spinal cord of CFA rats. Our data indicated that spinal IRF7 plays an important role in the regulation of inflammatory pain. Thus, IRF7 overexpression at the spinal cord level might represent a potential target for the treatment of inflammatory pain. [ABSTRACT FROM AUTHOR]

The selection of relevant genes for breast cancer metastasis is critical for the treatment and prognosis of cancer patients. Although much effort has been devoted to the gene selection procedures by use of different statistical analysis methods or computational techniques, the interpretation of the variables in the resulting survival models has been limited so far. This article proposes a new Random Forest (RF)-based algorithm to identify important variables highly related with breast cancer metastasis, which is based on the important scores of two variable selection algorithms, including the mean decrease Gini (MDG) criteria of Random Forest and the GeneRank algorithm with protein-protein interaction (PPI) information. The new gene selection algorithm can be called PPIRF. The improved prediction accuracy fully illustrated the reliability and high interpretability of gene list selected by the PPIRF approach. [ABSTRACT FROM AUTHOR]

The immunity of insects plays a vital role in their survival. Our experiments found that lipopolysaccharide (LPS) and glucono-δ-lactone (GDL) could influence the virulence of Serratia marcescens Bizio (SM1) to Odontotermes formosanus (Shiraki) by affecting the immunity. Gram-negative binding proteins (GNBPs) are an important pattern recognition proteins that play a crucial role in the innate immune system. Therefore, two OfGNBP s were cloned in O. formosanus. The expression of OfGNBP s was significantly changed by LPS,SM1 and GDL, not prick. In addition, the immune-related gene expression, the phenoloxidase activity and antibacterial activity of donor termites and recipient termites were significantly induced by SM1. Furthermore, the knockdown of OfGNBP by RNA interference reduced not only individual immunity but also social immunity in O. formosanus , which increased the virulence of SM1 to O. formosanus. Importantly, ds OfGNBP alone also had good control effect on O. formosanus. In summary, we concluded that ds OfGNBP s are important termite immunosuppressants. [Display omitted] • Two OfGNBP s play an important role in the individual immunity of O. formosanus. • Two OfGNBP s play an important role in the social immunity of O. formosanus. • Two OfGNBP s were first confirmed to play an important role in resistanting SM1. [ABSTRACT FROM AUTHOR]

Citri Reticulata Pericarpium Viride (also known Qing-Pi or QP) is a plant in the Rutaceae family, QP is a traditional Qi-regulating medicine in Chinese medicine that is compatible with other Chinese medicine components and has extensive clinical practice in treating anxiety and depression. Reports on the pharmacological effects of QP have demonstrated its neuroprotective effects and antioxidant capacities. Numerous pharmacological benefits of QP are attributed to its antioxidant abilities. Anxiety disorders are a broadly defined category of mental illnesses. Oxidative stress and an imbalance in the antioxidant defense system are typical pathological features of these disorders. The aim of this study was to evaluate the effects of QP essential oil on anxiety using animal models and investigate the underlying neurobiological mechanisms. This study aimed to develop an animal model of anxiety using chronic restraint stress and investigate the effects of inhalation of Citri Reticulata Pericarpium Viride essential oil on anxiety-like behavior, olfactory function, and olfactory bulb neurogenesis in mice with anxiety. The results showed that long-term chronic restraint stimulation caused a decrease in olfactory function, significant anxiety-like behavior, and a notable reduction in the number of neurons in the olfactory bulb. However, inhalation of Citri Reticulata Pericarpium Viride essential oil reversed these effects, improving the olfactory function, neuro-stimulating effect, alleviating anxiety-like behavior, and regulating theta (4–12Hz) oscillation in the hippocampus DG area. These effects were associated with changes in the expression levels of glutamate receptor NMDAR and NeuN in olfactory bulb. The study revealed that mice with anxiety induced by chronic restraint stress exhibited significant olfactory dysfunction, providing strong evidence for the causal relationship between anxiety disorders and olfactory dysfunction. Moreover, QP essential oil has the potential to be developed as a therapeutic drug for anxiety disorders, in addition to its role as a complementary anxiolytic. [Display omitted] • Long-term chronic restraint stimulation leads to a decrease in olfactory function. • Inhalation of essential oils is a powerful pathway to improve olfactory function and have a neuro-stimulating effect. • Inhalation administration of QP essential oil can stimulate θ waves vibrate with alleviation of anxiety-like behavior. • The mechanism is related to regulation glutamate/NMDAR and NeuN changes in olfactory bulb. [ABSTRACT FROM AUTHOR]

Pompano fishes have been widely farmed worldwide. As a representative commercial marine species of the Carangidae family, the golden pompano (Trachinotus blochii) has gained significant popularity in China and worldwide. However, because of rapid growth and high-density aquaculture, the golden pompano has become seriously threatened by various diseases. Cell lines are the most cost-effective resource for in vitro studies and are widely used for physiological and pathological research owing to their accessibility and convenience. In this study, we established a novel immortal cell line, GPF (Golden pompano fin cells). GPF has been passaged over 69 generations for 10 months. The morphology, adhesion and extension processes of GPF were evaluated using light and electron microscopy. GPF cells were passaged every 3 days with L-15 containing 20 % fetal bovine serum (FBS) at 1:3. The optimum conditions for GPF growth were 28 °C and a 20 % FBS concentration. DNA sequencing of 18S rRNA and mitochondrial 16S rRNA confirmed that GPF was derived from the golden pompano. Chromosomal analysis revealed that the number pattern of GPF was 48 chromosomes. Transfection experiments demonstrated that GPF could be utilized to express foreign genes. Furthermore, heavy metals (Cd, Cu, and Fe) exhibited dose-dependent cytotoxicity against GPF. After polyinosinic-polycytidylic acid (poly I:C) treatment, transcription of the retinoic acid-inducible gene I-like receptor (RLR) pathway genes, including mda5 , mita , tbk1 , irf3 , and irf7 increased, inducing the expression of interferon (IFN) and anti-viral proteins in GPF cells. In addition, lipopolysaccharide (LPS) stimulation up-regulated the expression of inflammation-related factors, including myd88 , irak1 , nfκb , il1β , il6 , and cxcl10 expression. To the best of our knowledge, this is the first study on the immune response signaling pathways of the golden pompano using an established fin cell line. In this study, we describe a preliminary investigation of the GPF cell line immune response to poly I:C and LPS, and provide a more rapid and efficient experimental material for research on marine fish immunology. • GPF was established and became a stable fin cell line of Trachinotus blochii. • GPF can be used to assess the cytotoxicity of heavy metals. • GPF can be utilized to express foreign genes with 32 % transfection efficiency. • GPF's immune response to LPS and poly I: C challenge proved it can be used for in vitro immunology study. [ABSTRACT FROM AUTHOR]

Cardiac differentiation involves a cascade of coordinated gene expression that regulates cell proliferation and matrix protein formation in a defined temporo-spatial manner. The zinc-finger-containing transcription factor has been implicated as a critical regulator of multiple cardiac-expressed genes as well as a regulator of inducible gene expression in response to hypertrophic stimulation. Mitogen-activated protein kinase (MAPK) signal transduction pathways are among the most widespread mechanisms of eukaryotic cell regulation. The MAPKs function inside the nucleus and target transcription factors that are prebound to DNA. Many transcription factors are probably important MAPK targets. Here, we have cloned a new zinc-finger gene named ZNF411 using degenerate primers from an early embryo heart cDNA library, which mapped to 19p13.11. The ZNF411 gene consists of 2360 nucleotides and encodes a protein of 499 amino acids with an amino-terminal KRAB domain and eleven carboxy-terminal C2H2 zinc-finger units. Northern blot analysis indicates that a 2.4 kb transcript specific for ZNF411 is expressed in heart, skeletal muscle, and placenta at adult stage and is expressed in most of the examined embryonic tissues, especially at a higher level in skeletal muscle, heart, and pancreas. ZNF411 protein distributes evenly in nuclei when overexpressed in the cells. Reporter gene assays show that ZNF411 is a transcriptional repressor and overexpression of ZNF411 in the COS-7 cells inhibits the transcriptional activities of AP-1 and SRE. These results indicate that ZNF411 is a member of the zinc-finger transcription factor family and may be involved in the heart development, and it probably works as a negative regulator in MAPK signaling pathway. [Copyright &y& Elsevier]

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway is one of the main pleiotropic cascades used to transmit information from extracellular receptors to the nucleus, which results in DNA transcription and expression of genes involved in immunity, proliferation, differentiation, migration, apoptosis, and cell survival. Members of JAK family and STAT family have been extensively studied in different mammalian species because of their important roles in innate and adaptive immune responses. However, they have not been systematically studied among teleost fish species. In this study, five JAK family members and eight STAT family members were identified and characterized from channel catfish. Phylogenetic analysis was conducted to properly annotate these genes. Syntenic analysis was also conducted to establish orthology, and confirm the results from phylogenetic analysis. Compared to mammals, more members of the JAK and STAT family were identified in channel catfish genome. Expression of JAK and STAT family members was detected in healthy catfish tissues, but was induced in gill, liver, and intestine after bacterial challenge. Notably, the significant upregulation of STAT1b gene in catfish liver, gill and intestine after Edwardsiella ictaluri infection supported the notion that high STAT1 expression are involved in defense against pathogens. Collectively, the increased expression of JAK and STAT members in tested tissues suggested their crucial function in defending the host against pathogen invasion. [ABSTRACT FROM AUTHOR]

The ideal therapeutic drug for rheumatoid arthritis (RA) should not only inhibit inflammation, but also prevent articular joint damage and particularly inhibit osteoclastogenesis. Betulinic acid (BA) is a natural pentacyclic triterpene that has displayed moderate anti-inflammatory and anti-osteoclastogenesis activities in various experimental systems, suggesting that BA or its derivatives could have an inhibitory effect on RA. In this study, we screened BA derivatives and found a heterocyclic ring-fused BA derivative, SH479, which had greater inhibitory effect than BA on Th17 differentiation. Moreover, we investigated the immune regulatory activity and potential therapeutic effects of SH479 in an experimental model of rheumatoid arthritis, the collagen-induced arthritis (CIA) mouse model. SH479 significantly inhibited Th1 and Th17 polarization, antigen-specific T cell proliferation and splenic lymphocyte-induced osteoclastogenesis. Furthermore, it diminished arthritis scores as well as bone destruction and cartilage depletion in the CIA mouse model. The protective effect of SH479 was accompanied by decreased levels of pro-inflammatory cytokines IL-17 and IFN-γ, together with enhanced anti-inflammatory cytokine expression including IL-10 and IL-4, as well as elevated CD4 + Foxp3 + cell number. At the molecular level, our results indicated that SH479 alleviated CIA through regulation of CD4 + T cell subtypes by JAK-STAT pathways. In conclusion, this study demonstrates that SH479 has therapeutic potential for rheumatoid arthritis through an anti-inflammatory effect by shifting a pathogenic Th17/Th1 response to a Th2/Treg phenotype, and also through an additional articular bone protection effect. [ABSTRACT FROM AUTHOR]

In the present study, a new water-soluble polysaccharide (LJP-11) was obtained from Laminaria japonica by anion exchange DEAE-cellulose chromatography and Sephacryl S-500 chromatography. The average molecular weight of this polysaccharide was estimated to be about 2.89 × 10 6 Da by high performance liquid chromatography system. Gas chromatography showed that LJP-11 was composed of arabinose, mannose and glucose in a molar ratio of 1.0:1.16:6.33. LJP-11 contains a long backbone consisting of (1→4)-β- d -Glc p Ac, (1→4)-α- d -Glc p , (1→6)-β- d -Glc p and (1→3,6)-α- d -Man p . The 1-linked β- l -Ara f was linked to the C-6 of (1→3)-α- d -Man p and the sulfate group was attached to the C-4 of (1→6)-β- d -Glc p . Pharmacological tests displayed that LJP-11 can stimulate macrophages to release NO, IL-6, TNF-α and IL-10 as well as the up-regulation of their gene expressions, indicating LJP-11 has beneficial effects on immunostimulation. Moreover, LJP-11 exhibited positive effects on the translocation of NF-κB p65 from cytoplasm to nucleus and the phosphorylation of IκBα, ERK1/2, JNK1/2 and P38 in macrophages. These results suggested that the activation of MAPK and NF-κB signaling pathways is one of the mechanisms responsible for the immunostimulating activity of LJP-11. [ABSTRACT FROM AUTHOR]

Animal growth and development is a complicated process and is regulated by multi-genes. Myostatin (Mstn) and myogenin (Myog) are a pair of negative and positive regulators respectively, which play an important role in the generation of muscle cells. In order to study the function of these two genes in muscle growth of Trachinotus blochii , full lengths of two mstn genes (mstn -1 and mstn -2) and myog gene were cloned using RACE. We first identified and characterized the complete cDNA sequences of mstn -1, mstn -2, and myog genes derived from T. blochii , an economically important mariculture species in China. Multiple sequence alignment of amino acids and phylogenetic analysis revealed that the Mstn and Myog were highly conserved to the other Perciformes. In addition, gene duplication of mstn in T. blochii was observed. mstn -1 mRNA was mainly expressed in the muscle and gonad, while mstn -2 and myog transcripts were detectable mainly in the brain and muscle, respectively. Moreover, the nutritional status and temperature influenced abundance levels in brain and muscle. Results suggested that mstn and myog genes play an important role in muscle growth of T. blochii , mstn may not be limited to control of muscle growth in fish and could also be involved in other biological functions. [Display omitted] • The m stn -1 and m stn -2 exhibit different tissue distribution patterns in T. blochii as reported for other fish species. • Temperature influenced abundance levels of mstn and myog transcripts in brain and muscle, showing opposite trends. • The functions of Mstn and Myog may not be limited to muscle growth in T. blochii as suggested for other fish species. [ABSTRACT FROM AUTHOR]

Abstract: Viroporins are a group of viral proteins that participate in viral replication cycles, including modification of membrane permeability and promotion of viral release. Although biological data have been accumulated on viroporion-like proteins of other viruses belonging to family Flaviviridae, the viroporin activity and membrane topology of p7 protein from classical swine fever virus (CSFV), a member of the genus Pestivirus of the family Flaviviridae, are largely unknown. In this study, sequence analysis of the primary structure of p7 polypeptide demonstrates that p7 contains two putative transmembrane regions connected by a short hydrophilic segment. Expression of p7 protein in Escherichia coli leads to the permeabilization of bacterial cells to small molecules. The p7 protein also enhances the permeability of mammalian cells, increasing the intracellular Ca2+ concentration and the permeability of cells to the translation inhibitor Hygromycin B. This protein is an integral membrane protein and can form homo-oligomers. It mainly localizes to the ER at the early stage of the expression and can be transferred to the plasma membrane at the late stage of the expression. Detergent permeabilization assays confirmed that the p7 protein is a 2-pass transmembrane protein and its N and C termini are exposed to the ER lumen. Deletion analysis showed that amino acid residues 41–63 may be essential for the viroporin activity of the protein. Our studies demonstrate that CSFV p7 possesses properties commonly associated with viroporins, which could be a potential target for the development of a therapeutic intervention for classic swine fever virus infection. [Copyright &y& Elsevier]

Abstract: Cudratricusxanthone G (CTXG), a natural bioactive cudratricusxanthone extracted from C. tricuspidata, has shown anti-cancer properties. However, the function and mechanism of CTXG in tumor invasion have not been elucidated to date. In this study, we investigated the inhibitory effect of CTXG on the proliferation, migration and invasion of SW620 cells. We found that MMP-2, a pivotal factor in tumor invasion, was suppressed in both expression and activation by CTXG in a dose-dependent manner. The suppression of MMP-2 expression by CTXG led to an inhibition of SW620 cells invasion and migration by inactivating Rac1 and Cdc42 but not RhoA GTPase. Furthermore, CTXG also inhibited the transcriptional activity of AP-1 (activator protein-1). In conclusion, our data demonstrate that CTXG exerted anti-invasion action in SW620 cells by targeting MMP-2 though regulating the activities of Rac1, Cdc42 and their downstream transcriptional factor AP-1. These results are the first to reveal the novel functions of CTXG in cancer cell invasion and its molecular basis for the anti-cancer action. [Copyright &y& Elsevier]

Abstract: The use of radioactive in situ hybridization (ISH) to quantitatively determine low-to-moderate abundant mRNA expression in formalin-fixed, paraffin-embedded archival post-mortem human brain tissue is often limited by non-specific-deposits, visible as speckles. In the present study, optimal hybridization conditions were achieved for quantifying the mRNA expression of histidine decarboxylase (HDC) by a number of alterations in a routine protocol, which included (1) during purification of the oligo-probes, glycogen was omitted as a carrier for precipitation, (2) after precipitation, the labeled probe contained within the pellet was first dissolved in water instead of in hybridization buffer (HBF), (3) during hybridization, the dithiothreitol (DTT) concentration was increased from 200 to 800mM in HBF, and (4) stringencies during hybridization and post-hybridization washes were increased by increasing the temperature. The effect of the adjustment was quantified on adjacent sections from 18 subjects (9 with Parkinson’s disease and 9 controls), by comparing the data from the standard and new protocol. The results showed that the improved protocol brought about significantly clearer background with higher signal-to-noise ratios (p =0.001). We propose that this protocol is also applicable for detection of other lower-abundant genes in human brain tissue and probably in other tissues as well. In the present study, this is not only illustrated for HDC ISH, but also for corticotrophin-releasing hormone mRNA expression in the hypothalamic paraventricular nucleus. [Copyright &y& Elsevier]

Zinc finger proteins play important roles in a variety of cellular functions, including cell growth, proliferation, apoptosis, and intracellular signal transduction, and the zinc finger-containing transcription factor has been implicated as a critical regulator of multiple cardiac-expressed genes as well as a regulator of inducible gene expression in response to hypertrophic stimulation. With the aim of identifying the genes involved in human heart development and diseases, we have isolated a novel LER-related zinc finger gene named ZNF394 from human heart cDNA library. ZNF394 gene has a predicted 561-amino acid open reading frame, encoding a 64 kDa zinc finger protein. The N-terminus of ZNF394 protein has a leucine-rich region (LER or SCAN domain), followed by a well-conserved krűppel-associated box domain. The C-terminus of the protein contains 7 C2H2 zinc finger motifs in tandem arrays with the highly conserved space region of the H/C-link. ZNF394 gene is mapped to chromosome 7q11.21. Northern blot analysis indicates that a 2.18 kb transcript specific for ZNF394 is specifically expressed in the heart, skeletal muscle, and brain in human adult tissues. ZNF394 protein is expressed in cell nucleus. Overexpression of ZNF394 in the cell inhibits the transcriptional activities of c-Jun and AP-1 reporters, suggesting that ZNF394 is a new transcriptional repressor in mitogen-activated protein kinase signaling pathways and may play an important role in cardiac development and/or cardiac function. [Copyright &y& Elsevier]