Integrated genomics analysis of gene and miRNA expression profiles in clear cell renal carcinoma cell lines

Clear cell renal cell carcinoma (ccRCC) is the most common and malignant tumor in the adult kidney, representing 75-80% of renal primary malignancies. Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene, by mutation, deletion and promoter methylation, occurs in most sporadic ccRCCs and in all inherited cases. Recent evidences showed that microRNAs (miRNAs) are often dysregulated in many tumors, including ccRCC. We used Caki-1, Caki-2 and A498 cell lines as in vitro model of ccRCC pathology, and HK-2 (normal proximal tubular epithelial cell line) as reference sample. We characterized the VHL status by direct sequencing and the HIF status by western blot. Affymetrix microarray technology was applied to assess miRNA (onto GeneChip\uae miRNA Array) and gene expression profiles (onto GeneChip\uae Human Gene 1.0 ST Array). Analysis of common differentially expressed miRNAs (DEMs) in RCC cell lines outlined specific miRNAs to be involved in ccRCC and in cancer (e.g. miR-145, miR-29a, miR-342-3p, miR-205, miR-183, miR-197, miR-132, miR-146a and miR-34a). Functional enrichment analysis of common differentially expressed genes (DEGs) highlighted some genes involved in leucocyte transendothelial migration, focal adhesion and p53 signalling pathways (e.g. ATM, FER, CDC27 and GRB10). Additionally, we conducted an integrated analysis to combine gene and miRNA expression profiles and to reconstruct miRNA-gene post-transcriptional regulatory networks involved in RCC pathology. We also compared our expression data with RCC datasets publicly available from NCBI GEO microarray repository. We selected potentially interesting miRNAs and target genes for further validation (by qPCR). This integrated analysis approach may help to unravel the molecular complexity characterizing ccRCC biology, and it will facilitate the elucidation of regulatory circuits important for tumorigenesis and the biological processes under relevant post-transcriptional regulation in ccRCC

Grapevine rootstocks shape underground bacterial microbiome and networking but not potential functionality

BackgroundThe plant compartments of Vitis vinifera, including the rhizosphere, rhizoplane, root endosphere, phyllosphere and carposphere, provide unique niches that drive specific bacterial microbiome associations. The majority of phyllosphere endophytes originate from the soil and migrate up to the aerial compartments through the root endosphere. Thus, the soil and root endosphere partially define the aerial endosphere in the leaves and berries, contributing to the terroir of the fruit. However, V. vinifera cultivars are invariably grafted onto the rootstocks of other Vitis species and hybrids. It has been hypothesized that the plant species determines the microbiome of the root endosphere and, as a consequence, the aerial endosphere. In this work, we test the first part of this hypothesis. We investigate whether different rootstocks influence the bacteria selected from the surrounding soil, affecting the bacterial diversity and potential functionality of the rhizosphere and root endosphere.MethodsBacterial microbiomes from both the root tissues and the rhizosphere of Barbera cultivars, both ungrafted and grafted on four different rootstocks, cultivated in the same soil from the same vineyard, were characterized by 16S rRNA high-throughput sequencing. To assess the influence of the root genotype on the bacterial communities’ recruitment in the root system, (i) the phylogenetic diversity coupled with the predicted functional profiles and (ii) the co-occurrence bacterial networks were determined. Cultivation-dependent approaches were used to reveal the plant-growth promoting (PGP) potential associated with the grafted and ungrafted root systems.ResultsRichness, diversity and bacterial community networking in the root compartments were significantly influenced by the rootstocks. Complementary to a shared bacterial microbiome, different subsets of soil bacteria, including those endowed with PGP traits, were selected by the root system compartments of different rootstocks. The interaction between the root compartments and the rootstock exerted a unique selective pressure that enhanced niche differentiation, but rootstock-specific bacterial communities were still recruited with conserved PGP traits.ConclusionWhile the rootstock significantly influences the taxonomy, structure and network properties of the bacterial community in grapevine roots, a homeostatic effect on the distribution of the predicted and potential functional PGP traits was found

Ultrarapid Detection of blaKPC1/2-12 from Perirectal and Nasal Swabs by Use of Real-Time PCR

The novel real-time PCR assay developed as described here was able to detect bla(KPC1/2-12) (bla(KPC-1/2) to bla(KPC-12)) from easily available clinical specimens in less than 2 h. The genotypic assay was highly sensitive (100%) and specific (98%). In some cases, it was able to detect bla(KPC) 48 h before positive detection by standard phenotypic assay on patients who were monitored daily. The high sensitivity and rapidity of the molecular method make it the method of choice for KPC surveillance and, thus, containment purposes

Acyclovir resistance in herpes simplex virus type 1: biochemical and functional studies on the thymidine kinase of the highly resistant R100 strain

The biochemical and functional properties of the thymidine kinase (TK) of the herpes simplex virus type 1 mutant R100, that is highly resistant to 9-(2-hydroxyethoxymethyl)guanine (acyclovir), are reported in comparison with the properties of its parental strain, wt. The mutant induced the production of a TK activity that accounted for only 10% of the wt one. This feature was not apparently related to a defective expression of the TK gene but it was rather connected to some functional characteristics of R100 enzyme. Although affinities of this enzyme for ATP and thymidine were unchanged, apparent Vmax values for thymidine were much reduced. In addition, affinities for antiviral analogues acyclovir, 9-(1,3-dihydroxymethyl)guanine (DHPG), 5-(2-bromovinyl)2'-deoxyuridine (BVdU), and 5-iodo-2'deoxycytidine (IdCyd) were drastically diminished (between 50-fold and more than 100-fold). This mutation therefore seems to affect the active site of the enzyme which is involved in the catalytic conversion of thymidine and in the binding of the analogues. The above features of HSV-1 R100 seem quite distinct from those of previously described HSV-1 resistant mutants

Acyclovir resistance in Herpes simplex virus type 1: functional studies on the thymidine kinase of the highly resistant R100 strain.

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The Sp1 transcription factor does not directly interact with the HIV-1 Tat protein

The role of Sp1 in regulating the trans-activating activity of the human immunodeficiency virus type 1 (HIV-1) Tat protein has not yet been clearly defined. In fact, studies on the physical and functional interaction between Sp1 and Tat have yielded contradictory results. Here we investigated whether a physical interaction between Sp1 and Tat indeed occurs, exploiting both biochemical and genetic techniques that allow detection of direct protein-protein interactions. Studies performed with the yeast two-hybrid system indicate that Sp1 does not directly interact with the HIV-1 Tat protein. Control experiments demonstrated that both proteins are functionally expressed in the yeast cells. In vitro binding assays further confirmed that Sp1 does not physically bind Tat. These data suggest that in vivo Tat and Sp1 most likely take part of a multicomponent complex and thus encourage the search of the molecule(s) which mediate Tat-Sp1 interaction