MicroRNAs are short single-stranded RNAs that are associated with gene regulation at the transcriptional and translational level. Changes in their expression were found in a variety of human cancers. Only few data are available on microRNAs in clear cell renal cell carcinoma (ccRCC). We performed genome-wide expression profiling of microRNAs using microarray analysis and quantification of specific microRNAs by TaqMan real-time RT-PCR. Matched malignant and non-malignant tissue samples from two independent sets of 12 and 72 ccRCC were profiled. The microarray-based experiments identified 13 over-expressed and 20 down-regulated microRNAs in malignant samples. Expression in ccRCC tissue samples compared with matched non-malignant samples measured by RT-PCR was increased on average by 2.7- to 23-fold for the hsa-miR-16, −452*, −224, −155 and −210, but decreased by 4.8- to 138-fold for hsa-miR-200b, −363, −429, −200c, −514 and −141. No significant associations between these differentially expressed microRNAs and the clinico-pathological factors tumour stage, tumour grade and survival rate were found. Nevertheless, malignant and non-malignant tissue could clearly be differentiated by their microRNA profile. A combination of miR-141 and miR-155 resulted in a 97% overall correct classification of samples. The presented differential microRNA pattern provides a solid basis for further validation, including functional studies.

Chlamydia trachomatis is an obligate intracellular pathogenic bacterium that has been refractory to genetic manipulations. Although the genomes of several strains have been sequenced, very little information is available on the gene structure of these bacteria. We used deep sequencing to define the transcriptome of purified elementary bodies (EB) and reticulate bodies (RB) of C. trachomatis L2b, respectively. Using an RNA-seq approach, we have mapped 363 transcriptional start sites (TSS) of annotated genes. Semi-quantitative analysis of mapped cDNA reads revealed differences in the RNA levels of 84 genes isolated from EB and RB, respectively. We have identified and in part confirmed 42 genome- and 1 plasmid-derived novel non-coding RNAs. The genome encoded non-coding RNA, ctrR0332 was one of the most abundantly and differentially expressed RNA in EB and RB, implying an important role in the developmental cycle of C. trachomatis. The detailed map of TSS in a thus far unprecedented resolution as a complement to the genome sequence will help to understand the organization, control and function of genes of this important pathogen.

The WHO has recently classified Neisseria gonorrhoeae as a super-bacterium due to the rapid spread of antibiotic resistant derivatives and an overall dramatic increase in infection incidences. Genome sequencing has identified potential genes, however, little is known about the transcriptional organization and the presence of non-coding RNAs in gonococci. We performed RNA sequencing to define the transcriptome and the transcriptional start sites of all gonococcal genes and operons. Numerous new transcripts including 253 potentially non-coding RNAs transcribed from intergenic regions or antisense to coding genes were identified. Strikingly, strong antisense transcription was detected for the phase-variable opa genes coding for a family of adhesins and invasins in pathogenic Neisseria, that may have regulatory functions. Based on the defined transcriptional start sites, promoter motifs were identified. We further generated and sequenced a high density Tn5 transposon library to predict a core of 827 gonococcal essential genes, 133 of which have no known function. Our combined RNA-Seq and Tn-Seq approach establishes a detailed map of gonococcal genes and defines the first core set of essential gonococcal genes.

The v‐abl oncogene of Abelson murine leukemia virus (A‐MuLV) induces two opposite phenotypes in NIH3T3 cells. In the majority of cells, v‐abl causes a growth arrest at the G1 phase of the cell cycle; while in a minority of cells, v‐abl abrogates the requirement for growth factors. Using temperature sensitive mutants, it can be demonstrated that v‐Abl tyrosine kinase is required for growth inhibition or stimulation. The two phenotypes are not caused by mutations or differences in the expression of v‐Abl, but are dependent on the cell context. Two stable subclones of NIH3T3 cells have been isolated that exhibit similar morphology and growth characteristics. However, upon infection with A‐MuLV, the ‘positive’ cells become serum‐ and anchorage‐independent, whereas the ‘negative’ cells become arrested in G1. The positive phenotype is dominant, shown by cell fusion, and treatment with 5‐azacytidine converts the negative cells to the positive phenotype. Activation of v‐Abl tyrosine kinase induces the serum‐responsive genes in the positive but not in the negative cells. Transactivation of the c‐fos promoter by v‐Abl in transient assays is also restricted to the positive cells. These results show that v‐Abl tyrosine kinase is not an obligatory activator of growth, but requires a permissive cellular context to manifest its mitogenic function.

The effect of high superficial gas velocities in continuous and batch cultures of the strains Dunaliella tertiolecta, Chlamydomonas reinhardtii wild-type and cell wall-lacking mutant was studied in bubble columns. No cell damage was found for D. tertiolecta and C. reinhardtii (wild-type) up to superficial gas velocities of 0.076 and 0.085 m s(-1), respectively, suggesting that high superficial gas velocities alone cannot be responsible for cell death and, consequently, bubble bursting cannot be the sole cause for cell injury. A death rate of 0.46 +/- 0.08 h(-1) was found for C. reinhardtii (cell wall-lacking mutant) at a superficial gas velocity of 0.076 m s(-1), and increased to 1.01 +/- 0.29 h(-1) on increasing superficial gas velocity to 0.085 m s(-1). Shear sensitivity is thus strain-dependent and to some extent the cell wall plays a role in the protection against hydrodynamic shear. When studying the effect of bubble formation at the sparger in batch cultures of D. tertiolecta by varying the number of nozzles, a death rate of 0.047 +/- 0.016 h(-1) was obtained at high gas entrance velocities. D. tertiolecta was cultivated in a pilot-plant reactor under different superficial gas velocities of up to 0.026 m s(-1), with relatively low gas entrance velocities and no cell damage was observed. There is some indication that the main parameter causing cell death and damage was the gas entrance velocity at the sparger.

BackgroundGene function analysis of the obligate intracellular bacterium Chlamydia pneumoniae is hampered by the facts that this organism is inaccessible to genetic manipulations and not cultivable outside the host. The genomes of several strains have been sequenced; however, very little information is available on the gene structure and transcriptome of C. pneumoniae.ResultsUsing a differential RNA-sequencing approach with specific enrichment of primary transcripts, we defined the transcriptome of purified elementary bodies and reticulate bodies of C. pneumoniae strain CWL-029; 565 transcriptional start sites of annotated genes and novel transcripts were mapped. Analysis of adjacent genes for co-transcription revealed 246 polycistronic transcripts. In total, a distinct transcription start site or an affiliation to an operon could be assigned to 862 out of 1,074 annotated protein coding genes. Semi-quantitative analysis of mapped cDNA reads revealed significant differences for 288 genes in the RNA levels of genes isolated from elementary bodies and reticulate bodies. We have identified and in part confirmed 75 novel putative non-coding RNAs. The detailed map of transcription start sites at single nucleotide resolution allowed for the first time a comprehensive and saturating analysis of promoter consensus sequences in Chlamydia.ConclusionsThe precise transcriptional landscape as a complement to the genome sequence will provide new insights into the organization, control and function of genes. Novel non-coding RNAs and identified common promoter motifs will help to understand gene regulation of this important human pathogen.

Obligate intracellular

Most lung cancer deaths are related to metastases, which indicates the necessity of detecting and inhibiting tumor cell dissemination. Here, we aimed to identify miRNAs involved in metastasis of lung adenocarcinoma as prognostic biomarkers and therapeutic targets. To that end, lymph node metastasis-associated miRNAs were identified in The Cancer Genome Atlas lung adenocarcinoma patient cohort (sequencing data; = 449) and subsequently validated by qRT-PCR in an independent clinical cohort ( = 108). Overexpression of miRNAs located on chromosome 14q32 was associated with metastasis in lung adenocarcinoma patients. Importantly, Kaplan-Meier analysis and log-rank test revealed that higher expression levels of individual 14q32 miRNAs (mir-539, mir-323b, and mir-487a) associated with worse disease-free survival of never-smoker patients. Epigenetic analysis including DNA methylation microarray data and bisulfite sequencing validation demonstrated that the induction of 14q32 cluster correlated with genomic hypomethylation of the 14q32 locus. CRISPR activation technology, applied for the first time to functionally study the increase of clustered miRNA levels in a coordinated manner, showed that simultaneous overexpression of 14q32 miRNAs promoted tumor cell migratory and invasive properties. Analysis of individual miRNAs by mimic transfection further illustrated that miR-323b-3p, miR-487a-3p, and miR-539-5p significantly contributed to the invasive phenotype through the indirect regulation of different target genes. In conclusion, overexpression of 14q32 miRNAs, associated with the respective genomic hypomethylation, promotes metastasis and correlates with poor patient prognosis in lung adenocarcinoma. This study points to chromosome 14q32 miRNAs as promising targets to inhibit tumor cell dissemination and to predict patient prognosis in lung adenocarcinoma. .