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Chronic lymphocytic leukemia (CLL) is a heterogeneous disease, whose presentation and clinical course are highly variable. Identification of novel prognostic factors may contribute to improving the CLL classification and providing indications for treatment options. The zinc finger protein ZNF224 plays a key role in cell transformation, through the control of apoptotic and survival pathways. In this study, we evaluated the potential application of ZNF224 as a novel marker of CLL progression and therapy responsiveness. To this aim, we analyzed ZNF224 expression levels in B lymphocytes from CLL patients at different stages of the disease and in patients showing different treatment outcomes. The expression of ZNF224 was significantly increased in disease progression and dramatically decreased in patients in complete remission after chemotherapy. Gene expression correlation analysis performed on datasets of CLL patients revealed that ZNF224 expression was well correlated with that of some prognostic and predictive markers. Moreover, bioinformatic analysis coupled ZNF224 to NF-κB pathway, and experimental data demonstrated that RNA interference of ZNF224 reduced the activity of the NF-κB survival pathway in CLL cells. Consistently with a pro-survival role, ZNF224 knockdown raised spontaneous and drug-induced apoptosis and inhibited the proliferation of peripheral blood mononuclear cells from CLL patients. Our findings provide evidence for the involvement of ZNF224 in the survival of CLL cells via NF-κB pathway modulation, and also suggest ZNF224 as a prognostic and predictive molecular marker of CLL disease.

"\"Il recettore degli androgeni controlla la morfogenesi, la gametogenesi nonché la crescita e lo sviluppo della ghiandola prostatica. Esso è implicato nella progressione del tumore prostatico. Tali evidenze suggeriscono che il recettore degli androgeni controlla la migrazione cellulare. I meccanismi molecolari con cui avviene tale controllo sono ancora oggi dibattuti.. Il manoscritto dimostra che il classico recettore degli androgeni espresso in cellule mesenchimali, normali o trasformate, interagisce nel compartimento extranucleare con la filmina A e che l'aggiunta di androgeni stabilizza tale legame ed induce l'assemblaggio di un complesso ternario formato da AR\\\/filamina A ed integrina beta 1. Questo complesso da un lato attiva Rac, modificando così' la velocità di migrazione delle cellule, dall'altro induce l'attivazione della chinasi FAK, modulando in tal modo l'adesione cellulare. Il manoscritto descrive per la prima volta il meccanismo molecolare con gli androgeni promuovono la migrazione e l'invalidità di cellule bersaglio attraverso l'attivazione di vie rapide, non genomiche.\"" "\"Androgen receptor (AR) controls male morphogenesis, gametogenesis and prostate growth as well as development of prostate cancer. These findings support a role for AR in cell migration and invasiveness. However, the molecular mechanism involved in AR-mediated cell migration still remains elusive.. . Methodology\\\/Principal Findings. Mouse embryo NIH3T3 fibroblasts and highly metastatic human fibrosarcoma HT1080 cells harbor low levels of transcriptionally incompetent AR. We now report that, through extra nuclear action, AR triggers migration of both cell types upon stimulation with physiological concentrations of the androgen R1881. We analyzed the initial events leading to androgen-induced cell migration and observed that challenging NIH3T3 cells with 10 nM R1881 rapidly induces interaction of AR with filamin A (FlnA) at cytoskeleton. AR\\\/FlnA complex recruits integrin beta 1, thus activating its dependent cascade. Silencing of AR, FlnA and integrin beta 1 shows that this ternary complex controls focal adhesion kinase (FAK), paxillin and Rac, thereby driving cell migration. FAK-null fibroblasts migrate poorly and Rac inhibition by EHT impairs motility of androgen-treated NIH3T3 cells. Interestingly, FAK and Rac activation by androgens are independent of each other. Findings in human fibrosarcoma HT1080 cells strengthen the role of Rac in androgen signaling. The Rac inhibitor significantly impairs androgen-induced migration in these cells. A mutant AR, deleted of the sequence interacting with FlnA, fails to mediate FAK activation and paxillin tyrosine phosphorylation in androgen-stimulated cells, further reinforcing the role of AR\\\/FlnA interaction in androgen-mediated motility.. . Conclusions\\\/Significance. The present report, for the first time, indicates that the extra nuclear AR\\\/FlnA\\\/integrin beta 1 complex is the key by which androgen activates signaling leading to cell migration. Assembly of this ternary complex may control organ development and prostate cancer metastasis.\""

MicroRNAs (miRNAs) play an important role in proper function and differentiation of mouse embryonic stem cells (ESCs). We performed a systematic comparison of miRNA expression in undifferentiated vs. differentiating ESCs. We report that 138 miRNAs are increased on the induction of differentiation. We compared the entire list of candidate mRNA targets of up-regulated miRNAs with that of mRNA downregulated in ESCs on induction of differentiation. Among the candidate targets emerging from this analysis, we found three genes, Smarca5, Jarid1b, and Sirt1, previously demonstrated to be involved in sustaining the undifferentiated phenotype in ESCs. On this basis, we first demonstrated that Smarca5 is a direct target of miR-100, Jarid1b of miR-137, and we also confirmed previously published data demonstrating that Sirt1 is a direct target of miR-34a in a different context. The suppression of these three miRNAs by anti-miRs caused the block of ESC differentiation induced by LIF withdrawal. On the other hand, the overexpression of the three miRNAs resulted in an altered expression of differentiation markers. These results demonstrate that miR-34a, miR-100, and miR-137 are required for proper differentiation of mouse ESCs, and that they function in part by targeting Sirt1, Smarca5, and Jarid1b mRNAs.—Tarantino, C., Paolella, G., Cozzuto, L., Minopoli, G., Pastore, L., Parisi, S., Russo, T. miRNA 34a, 100, and 137 modulate differentiation of mouse embryonic stem cells.

In the large intestine organic cation transporter type-2 (OCTN2) is recognized as a transporter of compounds such as carnitine and colony sporulation factor, promoting health of the colon intestinal epithelium. Recent reports suggest that OCTN2 expression in small intestine is under control of peroxisome proliferator-activated receptor-alpha (PPAR alpha). However, PPAR alpha contribution to colonic OCTN2 expression remains controversial. Here we examined the transcriptional regulation of colon OCTN2 gene by PPAR alpha. To exclude any additional modulation of other PPAR to OCTN2 expression, we used both in vivo and in vitro PPAR-null models and specific PPAR inhibitors. The PPAR gamma agonists thiazolidinediones increased both OCTN2 mRNA and protein expression in colonic epithelial cell lines independently by PPAR alpha expression. The induction was blocked only by PPAR gamma antagonists or by gamma ORF4, a PPAR gamma isoform with dominant negative activity, suggesting a PPAR gamma-dependent mechanism. A conserved noncanonical PPAR-responsive element was found by computational analysis in the first intron of human OCTN2 gene and validated by EMSA assay. Promoter-reporter assays further confirmed transcriptional functionality of the putative PPAR response element, whereas selective mutation caused complete loss of responsiveness to PPAR gamma activation. Finally, adenovirus-mediated overexpression of constitutively active PPAR gamma mutant increased colon OCTN2 expression in PPAR alpha(-/-) mice. Interestingly, animals overexpressing colon PPAR gamma showed a significant increase in plasma carnitine, thus demonstrating the functional contribution of large intestine to systemic carnitine homeostasis. This study reveals a PPAR gamma-dependent absorption machinery in colon that is likely involved in the health of colon epithelium, in the microbiota-host interactions and in the absorption of nutraceuticals and drugs.

Background Cell motility plays a central role in development, wound-healing and tumour invasion. Cultures of eucariotic cells are a complex system where most cells move according to 'random' patterns, but may also be induced to a more coordinate migration by means of specific stimuli, such as the presence of chemical attractants or the introduction of a mechanical stimulus. Various tools have been developed that work by keeping track of the paths followed by specific objects and by performing statistical analysis on the recorded path data. The available tools include desktop applications or macros running within a commercial package, which address specific aspects of the process. Results An online application, MotoCell, was developed to evaluate the motility of cell populations maintained in various experimental conditions. Statistical analysis of cell behaviour consists of the evaluation of descriptive parameters such as average speed and angle, directional persistence, path vector length, calculated for the whole population as well as for each cell and for each step of the migration; in this way the behaviour of a whole cell population may be assessed as a whole or as a sum of individual entities. The directional movement of objects may be studied by eliminating the modulo effect in circular statistics analysis, able to evaluate linear dispersion coefficient (R) and angular dispersion (S) values together with average angles. A case study is provided where the system is used to characterize motility of RasV12 transformed NIH3T3 fibroblasts. Conclusion Here we describe a comprehensive tool which takes care of all steps in cell motility analysis, including interactive cell tracking, path editing and statistical analysis of cell movement, all within a freely available online service. Although based on a standard web interface, the program is very fast and interactive and is immediately available to a large number of users, while exploiting the web approach in a very effective way. The ability to evaluate the behaviour of single cells allows to draw the attention on specific correlations, such as linearity of movement and deviation from the expected direction. In addition to population statistics, the analysis of single cells allows to group the cells into subpopulations, or even to evaluate the behaviour of each cell with respect to a variable reference, such as the direction of a wound or the position of the closest cell.

Background Protein kinases are a well defined family of proteins, characterized by the presence of a common kinase catalytic domain and playing a significant role in many important cellular processes, such as proliferation, maintenance of cell shape, apoptosys. In many members of the family, additional non-kinase domains contribute further specialization, resulting in subcellular localization, protein binding and regulation of activity, among others. About 500 genes encode members of the kinase family in the human genome, and although many of them represent well known genes, a larger number of genes code for proteins of more recent identification, or for unknown proteins identified as kinase only after computational studies. Results A systematic in silico study performed on the human genome, led to the identification of 5 genes, on chromosome 1, 11, 13, 15 and 16 respectively, and 1 pseudogene on chromosome X; some of these genes are reported as kinases from NCBI but are absent in other databases, such as KinBase. Comparative analysis of 483 gene regions and subsequent computational analysis, aimed at identifying unannotated exons, indicates that a large number of kinase may code for alternately spliced forms or be incorrectly annotated. An InterProScan automated analysis was perfomed to study domain distribution and combination in the various families. At the same time, other structural features were also added to the annotation process, including the putative presence of transmembrane alpha helices, and the cystein propensity to participate into a disulfide bridge. Conclusion The predicted human kinome was extended by identifiying both additional genes and potential splice variants, resulting in a varied panorama where functionality may be searched at the gene and protein level. Structural analysis of kinase proteins domains as defined in multiple sources together with transmembrane alpha helices and signal peptide prediction provides hints to function assignment. The results of the human kinome analysis are collected in the KinWeb database, available for browsing and searching over the internet, where all results from the comparative analysis and the gene structure annotation are made available, alongside the domain information. Kinases may be searched by domain combinations and the relative genes may be viewed in a graphic browser at various level of magnification up to gene organization on the full chromosome set.

S -Adenosylhomocysteine (SAH), a potent inhibitor of methyltransferases, and several thioethers structurally related to SAH, have been tested as potential inhibitors of tRNA (guanine-7)-methyltransferase from Salmonella typhimurium . The tested compounds are l -, d -, dl - S -adenosylhomocysteine, S -adenosylcysteine, methylthioadenosine, butylthioadenosine, thioethanoladenosine, isobutylthioadenosine, S -inosylhomocysteine, and methylthioinosine. Among them the highest inhibitory activity has been shown by SAH ( K i = 8 μ M), whereas butylthioadenosine, isobutylthioadenosine, and thioethanoladenosine are almost inactive as inhibitors. The other compounds inhibit the enzyme with K i values ranging between 400 and 800 μ m . From these data it is possible to evaluate the importance of the -NH 2 and -COOH groups of the substrate in the binding to the enzyme molecule, as well as other features such as the chirality at the α-carbon atom and the length of the hydrocarbon chain connecting the -NH 2 and -COOH groups to the aromatic ring of adenosine. The aminic group of the adenosine is also critical, because S -inosylhornocysteine and methylthioinosine are poorer inhibitors in comparison with SAH and methylthioadenosine.