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Secretory azoospermia is a severe form of male infertility caused by unknown factors. DAX-1 is predominantly expressed in mammalian reproductive tissues and plays an important role in spermatogenesis because Dax-1 knockout male mice show spermatogenesis defects. To examine whether DAX-1 is involved in the pathogenesis of secretory azoospermia in humans, we sequenced all of the exons of DAX-1 in 776 patients diagnosed with secretory azoospermia and 709 proven fertile men. A number of coding mutations unique to the patient group, including two synonymous mutations and six missense mutations, were identified. Of the missense mutations, our functional assay demonstrated that the V385L mutation caused the reduced functioning of DAX-1. This novel mutation (p. V385L) of DAX-1 is the first to be identified in association with secretory azoospermia, thereby highlighting the important role of DAX-1 in spermatogenesis. [ABSTRACT FROM AUTHOR]

Human CMTM3 has been proposed as a putative tumor suppressor gene. The loss of CMTM3 has been found in several carcinomas. However, the regulation of CMTM3 expression and its function in tumor progression remain largely unknown. Here, we investigated the regulation of CMTM3 expression, function and molecular mechanism in human testicular cancer cells. CMTM3 was frequently downregulated or silenced in testicular cancer cell lines and tumor tissues but highly expressed in normal testis tissues. The re-expression of CMTM3 significantly suppressed the colony formation, proliferation, and migration capacity of testicular cancer cells by inducing a G2 cell cycle arrest and apoptosis. Moreover, the re-expression of CMTM3 activated the transcription of p53, induced p53 accumulation, up-regulated the expression of p21, and increased the cleavage of caspase 9, 8, 3, and PARP. The downregulation of CMTM3 in clinical tumor tissues was associated with the methylation of a single CpG site located within the Sp1/Sp3-responsive region of the core promoter. These results indicate that CMTM3 can function as tumor suppressor through the induction of a G2 cell cycle arrest and apoptosis. CMTM3 is thus involved in testicular cancer pathogenesis, and it is frequently at least partially silenced by the methylation of a single, specific CpG site in tumor tissues. [ABSTRACT FROM AUTHOR]

Purpose: The aims of this study were to evaluate the clinical significance and potential prognostic value of pregnancy up-regulated non-ubiquitous calmodulin kinase (PNCK) in clear cell renal cell carcinoma (ccRCC) patients. Materials and Methods: The expression of PNCK mRNA was determined in 24 paired samples of ccRCCs and adjacent normal tissues using real-time RT-PCR. The expression of PNCK was determined in 248 samples of ccRCCs and 92 paired samples of adjacent normal tissues by immunohistochemical analysis. Statistical analysis was performed to define the relationship between PNCK expression and the clinical features of ccRCC. Results: The mRNA level of PNCK was significantly higher in tumorous tissues than in the adjacent non-tumorous tissues (p<0.001). An immunohistochemical analysis of 92 paired tissue specimens showed that PNCK expression was higher in tumorous tissues than in the adjacent non-tumorous tissues (p<0.001). Moreover, there was a significant correlation between the PNCK expression and various clinicopathological parameters such as Fuhrman grade (p = 0.011), tumor size (p<0.001), T stage (p<0.001) and N stage (p = 0.015). Patients with higher PNCK expression had shorter overall survival time than those with lower PNCK expression (p<0.001). Multivariate analysis indicated that PNCK expression was an independent predictor for poor survival of ccRCC patients. Conclusions: To our knowledge, this is the first study that determines the relationship between PNCK and prognosis in ccRCC. We found that increased PNCK expression is associated with poor prognosis in ccRCC. PNCK may represent a novel prognostic marker for ccRCC. [ABSTRACT FROM AUTHOR]

Background: Due to recent leaps forward in DNA synthesis and sequencing technology, DNA manipulation has been extended to the level of whole-genome synthesis. Bacteriophages occupy a special niche in the micro-organic ecosystem and have potential as a tool for therapeutic agent. The purpose of this study was to carry out chemical synthesis of the bacteriophage G4 and the study of its infectivity. Methodology/Principal Findings: Full-sized genomes of bacteriophage G4 molecules were completed from short overlapping synthetic oligonucleotides by direct assembly polymerase chain reaction and ligase chain reaction followed by fusion polymerase chain reaction with flanking primers. Three novel restriction endonuclease sites were introduced to distinguish the synthetic G4 from the wild type. G4 particles were recovered after electroporation into Escherichia coli and were efficient enough to infect another strain. The phage was validated by electron microscope. Specific polymerase chain reaction assay and restriction analyses of the plaques verified the accuracy of the chemical synthetic genomes. Conclusions: Our results showed that the bacteriophage G4 obtained is synthetic rather than a wild type. Our study demonstrated that a phage can be synthesized and manipulated genetically according to the sequences, and can be efficient enough to infect the Escherichia coli, showing the potential use of synthetic biology in medical application. [ABSTRACT FROM AUTHOR]

Background: MicroRNAs (miRNAs) function as endogenous regulators of biological behaviors of human cancers. Several natural non-coding RNAs are reported to inhibit miRNAs by base-pairing interactions. These phenomena raise questions about the ability of artificial device to regulate miRNAs. The purpose of this study is to create synthetic devices that target a single miRNA or a miRNA cluster and to ascertain their therapeutic effects on the phenotypes of bladder cancer cells., Methodology/principal Findings: Tandem bulged miRNA binding sites were inserted into the 3' untranslated region (UTR) of the SV-40 promoter-driven Renilla luciferase gene to construct two "miRNA-mowers" for suppression of miR-183-96-182 cluster or miR-210. A third device with tandem repeat sequences not complementary to any known miRNA was generated as an untargeted-control. In functional analyses, bladder cancer T24 and UM-UC-3 cells were transfected with each of the three devices, followed by assays for detection of their impacts. Luciferase assays indicated that the activities of the luciferase reporters in the miRNA-mowers were decreased to 30-50% of the untargeted-control. Using Real-Time qPCR, the expression levels of the target miRNAs were shown to be reduced 2-3-fold by the corresponding miRNA-mower. Cell growth, apoptosis, and migration were tested by MTT assay, flow cytometry assay, and in vitro scratch assay, respectively. Cell growth inhibition, increased apoptosis, and decreased motility were observed in miRNA-mowers-transfected bladder cancer cells., Conclusions/significance: Not only a single target miRNA but also the whole members of a target miRNA cluster can be blocked using this modular design strategy. Anti-cancer effects are induced by the synthetic miRNA-mowers in the bladder cancer cell lines. miR-183/96/182 cluster and miR-210 are shown to play oncogenic roles in bladder cancer. A potentially useful synthetic biology platform for miRNA loss-of-function study and cancer treatment has been established in this work.

Background: Unprecedented progresses in high-throughput DNA sequencing and de novo gene synthesis technologies have allowed us to create living organisms in the absence of natural template., Methodology/principal Findings: The sequence of wild-type S13 phage genome was downloaded from GenBank. Two synonymous mutations were introduced into wt-S13 genome to generate m1-S13 genome. Another mutant, m2-S13 genome, was obtained by engineering two nonsynonymous mutations in the capsid protein coding region of wt-S13 genome. A chimeric phage genome was designed by replacing the F capsid protein open reading frame (ORF) from phage S13 with the F capsid protein ORF from phage G4. The whole genomes of all four phages were assembled from a series of chemically synthesized short overlapping oligonucleotides. The linear synthesized genomes were circularized and electroporated into E.coli C, the standard laboratory host of S13 phage. All four phages were recovered and plaques were visualized. The results of sequencing showed the accuracy of these synthetic genomes. The synthetic phages were capable of lysing their bacterial host and tolerating general environmental conditions. While no phenotypic differences among the variant strains were observed when grown in LB medium with CaCl(2), the S13/G4 chimera was found to be much more sensitive to the absence of calcium and to have a lower adsorption rate under calcium free condition., Conclusions/significance: The bacteriophage S13 and its variants can be chemically synthesized. The major capsid gene of phage G4 is functional in the phage S13 life cycle. These results support an evolutional hypothesis which has been proposed that a homologous recombination event involving gene F of quite divergent ancestral lineages should be included in the history of the microvirid family.

Background: MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression. They are aberrantly expressed in many types of cancers. In this study, we determined the genome-wide miRNA profiles in bladder urothelial carcinoma by deep sequencing., Methodology/principal Findings: We detected 656 differentially expressed known human miRNAs and miRNA antisense sequences (miRNA*s) in nine bladder urothelial carcinoma patients by deep sequencing. Many miRNAs and miRNA*s were significantly upregulated or downregulated in bladder urothelial carcinoma compared to matched histologically normal urothelium. hsa-miR-96 was the most significantly upregulated miRNA and hsa-miR-490-5p was the most significantly downregulated one. Upregulated miRNAs were more common than downregulated ones. The hsa-miR-183, hsa-miR-200b ∼ 429, hsa-miR-200c ∼ 141 and hsa-miR-17 ∼ 92 clusters were significantly upregulated. The hsa-miR-143 ∼ 145 cluster was significantly downregulated. hsa-miR-182, hsa-miR-183, hsa-miR-200a, hsa-miR-143 and hsa-miR-195 were evaluated by Real-Time qPCR in a total of fifty-one bladder urothelial carcinoma patients. They were aberrantly expressed in bladder urothelial carcinoma compared to matched histologically normal urothelium (p < 0.001 for each miRNA)., Conclusions/significance: To date, this is the first study to determine genome-wide miRNA expression patterns in human bladder urothelial carcinoma by deep sequencing. We found that a collection of miRNAs were aberrantly expressed in bladder urothelial carcinoma compared to matched histologically normal urothelium, suggesting that they might play roles as oncogenes or tumor suppressors in the development and/or progression of this cancer. Our data provide novel insights into cancer biology.

Background: DNA methylation aberration and microRNA (miRNA) deregulation have been observed in many types of cancers. A systematic study of methylome and transcriptome in bladder urothelial carcinoma has never been reported., Methodology/principal Findings: The DNA methylation was profiled by modified methylation-specific digital karyotyping (MMSDK) and the expression of mRNAs and miRNAs was analyzed by digital gene expression (DGE) sequencing in tumors and matched normal adjacent tissues obtained from 9 bladder urothelial carcinoma patients. We found that a set of significantly enriched pathways disrupted in bladder urothelial carcinoma primarily related to "neurogenesis" and "cell differentiation" by integrated analysis of -omics data. Furthermore, we identified an intriguing collection of cancer-related genes that were deregulated at the levels of DNA methylation and mRNA expression, and we validated several of these genes (HIC1, SLIT2, RASAL1, and KRT17) by Bisulfite Sequencing PCR and Reverse Transcription qPCR in a panel of 33 bladder cancer samples., Conclusions/significance: We characterized the profiles between methylome and transcriptome in bladder urothelial carcinoma, identified a set of significantly enriched key pathways, and screened four aberrantly methylated and expressed genes. Conclusively, our findings shed light on a new avenue for basic bladder cancer research.

Background: Genome-wide gene expression profile using deep sequencing technologies can drive the discovery of cancer biomarkers and therapeutic targets. Such efforts are often limited to profiling the expression signature of either mRNA or microRNA (miRNA) in a single type of cancer., Methodology: Here we provided an integrated analysis of the genome-wide mRNA and miRNA expression profiles of three different genitourinary cancers: carcinomas of the bladder, kidney and testis., Principal Findings: Our results highlight the general or cancer-specific roles of several genes and miRNAs that may serve as candidate oncogenes or suppressors of tumor development. Further comparative analyses at the systems level revealed that significant aberrations of the cell adhesion process, p53 signaling, calcium signaling, the ECM-receptor and cell cycle pathways, the DNA repair and replication processes and the immune and inflammatory response processes were the common hallmarks of human cancers. Gene sets showing testicular cancer-specific deregulation patterns were mainly implicated in processes related to male reproductive function, and general disruptions of multiple metabolic pathways and processes related to cell migration were the characteristic molecular events for renal and bladder cancer, respectively. Furthermore, we also demonstrated that tumors with the same histological origins and genes with similar functions tended to group together in a clustering analysis. By assessing the correlation between the expression of each miRNA and its targets, we determined that deregulation of 'key' miRNAs may result in the global aberration of one or more pathways or processes as a whole., Conclusions: This systematic analysis deciphered the molecular phenotypes of three genitourinary cancers and investigated their variations at the miRNA level simultaneously. Our results provided a valuable source for future studies and highlighted some promising genes, miRNAs, pathways and processes that may be useful for diagnostic or therapeutic applications.

Background: With the advent of second-generation sequencing, the expression of gene transcripts can be digitally measured with high accuracy. The purpose of this study was to systematically profile the expression of both mRNA and miRNA genes in clear cell renal cell carcinoma (ccRCC) using massively parallel sequencing technology., Methodology: The expression of mRNAs and miRNAs were analyzed in tumor tissues and matched normal adjacent tissues obtained from 10 ccRCC patients without distant metastases. In a prevalence screen, some of the most interesting results were validated in a large cohort of ccRCC patients., Principal Findings: A total of 404 miRNAs and 9,799 mRNAs were detected to be differentially expressed in the 10 ccRCC patients. We also identified 56 novel miRNA candidates in at least two samples. In addition to confirming that canonical cancer genes and miRNAs (including VEGFA, DUSP9 and ERBB4; miR-210, miR-184 and miR-206) play pivotal roles in ccRCC development, promising novel candidates (such as PNCK and miR-122) without previous annotation in ccRCC carcinogenesis were also discovered in this study. Pathways controlling cell fates (e.g., cell cycle and apoptosis pathways) and cell communication (e.g., focal adhesion and ECM-receptor interaction) were found to be significantly more likely to be disrupted in ccRCC. Additionally, the results of the prevalence screen revealed that the expression of a miRNA gene cluster located on Xq27.3 was consistently downregulated in at least 76.7% of ∼50 ccRCC patients., Conclusions: Our study provided a two-dimensional map of the mRNA and miRNA expression profiles of ccRCC using deep sequencing technology. Our results indicate that the phenotypic status of ccRCC is characterized by a loss of normal renal function, downregulation of metabolic genes, and upregulation of many signal transduction genes in key pathways. Furthermore, it can be concluded that downregulation of miRNA genes clustered on Xq27.3 is associated with ccRCC.