12-506 Sigma-AldrichGoat Anti-Mouse IgG (H+L) Antibody, FITC conjugate

Growing evidence suggests a key role for RNA binding proteins (RBPs) in genome stability programs. Additionally, recent developments in RNA sequencing technologies, as well as mass-spectrometry techniques, have greatly expanded our knowledge on protein-RNA interactions. We here use full transcriptome sequencing and label-free LC/MS/MS to identify global changes in protein-RNA interactions in response to etoposide-induced genotoxic stress. We show that RBPs have distinct binding patterns in response to genotoxic stress and that inactivation of the RBP regulator module, p38/MK2, can affect the entire spectrum of protein-RNA interactions that take place in response to stress. In addition to validating the role of known RBPs like Srsf1, Srsf2, Elavl1 in the genotoxic stress response, we add a new collection of RBPs to the DNA damage response. We identify Khsrp as a highly regulated RBP in response to genotoxic stress and further validate its role as a driver of the G(1/)S transition through the suppression of Cdkn1a(P21) transcripts. Finally, we identify KHSRP as an indicator of overall survival, as well as disease free survival in glioblastoma multiforme.

Human metapneumovirus (hMPV) is a relatively recently identified paramyxovirus that causes acute upper and lower respiratory tract infection. Entry of hMPV is unusual among the paramyxoviruses, in that fusion is accomplished by the fusion (F) protein without the attachment glycoprotein (G protein). It has been suggested that hMPV F protein utilizes integrin αvβ1 as a cellular receptor. Consistent with this, the F proteins of all known hMPV strains possess an integrin-binding motif ((329)RGD(331)). The role of this motif in viral entry, infectivity, and pathogenesis is poorly understood. Here, we show that α5β1 and αv integrins are essential for cell-cell fusion and hMPV infection. Mutational analysis found that residues R329 and G330 in the (329)RGD(331) motif are essential for cell-cell fusion, whereas mutations at D331 did not significantly impact fusion activity. Furthermore, fusion-defective RGD mutations were either lethal to the virus or resulted in recombinant hMPVs that had defects in viral replication in cell culture. In cotton rats, recombinant hMPV with the R329K mutation in the F protein (rhMPV-R329K) and rhMPV-D331A exhibited significant defects in viral replication in nasal turbinates and lungs. Importantly, inoculation of cotton rats with these mutants triggered a high level of neutralizing antibodies and protected against hMPV challenge. Taken together, our data indicate that (i) α5β1 and αv integrins are essential for cell-cell fusion and viral replication, (ii) the first two residues in the RGD motif are essential for fusion activity, and (iii) inhibition of the interaction of the integrin-RGD motif may serve as a new target to rationally attenuate hMPV for the development of live attenuated vaccines.Human metapneumovirus (hMPV) is one of the major causative agents of acute respiratory disease in humans. Currently, there is no vaccine or antiviral drug for hMPV. hMPV enters host cells via a unique mechanism, in that viral fusion (F) protein mediates both attachment and fusion activity. Recently, it was suggested that hMPV F protein utilizes integrins as receptors for entry via a poorly understood mechanism. Here, we show that α5β1 and αv integrins are essential for hMPV infectivity and F protein-mediated cell-cell fusion and that the integrin-binding motif in the F protein plays a crucial role in these functions. Our results also identify the integrin-binding motif to be a new, attenuating target for the development of a live vaccine for hMPV. These findings not only will facilitate the development of antiviral drugs targeting viral entry steps but also will lead to the development new live attenuated vaccine candidates for hMPV.

During spermatogenesis, extensive restructuring takes place at the cell-cell interface since developing germ cells migrate progressively from the basal to the adluminal compartment of the seminiferous epithelium. Since germ cells per se are not motile cells, their movement relies almost exclusively on the Sertoli cell. Nonetheless, extensive exchanges in signaling take place between these cells in the seminiferous epithelium. c-Yes, a nonreceptor protein tyrosine kinase belonging to the Src family kinases (SFKs) and a crucial signaling protein, was recently shown to be upregulated at the Sertoli cell-cell interface at the blood-testis barrier (BTB) at stages VIII-IX of the seminiferous epithelial cycle of spermatogenesis. It was also highly expressed at the Sertoli cell-spermatid interface known as apical ectoplasmic specialization (apical ES) at stage V to early stage VIII of the epithelial cycle during spermiogenesis. Herein, it was shown that the knockdown of c-Yes by RNAi in vitro and in vivo affected both Sertoli cell adhesion at the BTB and spermatid adhesion at the apical ES, causing a disruption of the Sertoli cell tight junction-permeability barrier function, germ cell loss from the seminiferous epithelium, and also a loss of spermatid polarity. These effects were shown to be mediated by changes in distribution and/or localization of adhesion proteins at the BTB (e.g., occludin, N-cadherin) and at the apical ES (e.g., nectin-3) and possibly the result of changes in the underlying actin filaments at the BTB and the apical ES. These findings implicate that c-Yes is a likely target of male contraceptive research.

Current approaches for detecting circulating tumour cells (CTCs) in blood are dependent on CTC enrichment and are based either on surface epithelial markers on CTCs or on cell size differences. The objectives of this study were to develop and characterise an ultrasensitive multiplex fluorescent RNA in situ hybridisation (ISH)-based CTC detection system called CTCscope. This method detects a multitude of tumour-specific markers at single-cell level in blood.Healthy blood samples spiked with tumour cell lines were used as a model system for the development and initial characterisation of CTCscope. To demonstrate the feasibility of CTC detection in patient blood, duplicate blood samples were drawn from 45 metastatic breast cancer patients for analysis by CTCscope and the CellSearch system. The association of CTCs with the tumour marker CA15-3 and progression-free survival (PFS) were assessed.CTCscope detected CTC transcripts of eight epithelial markers and three epithelial-mesenchymal-transition (EMT) markers for increased sensitivity. CTCscope was used to detect CTCs with minimal enrichment, and did not detect apoptotic or dead cells. In patient blood samples, CTCs detected by CellSearch, but not CTCscope, were positively correlated with CA15-3 levels. Circulating tumour cells detected by either CTCscope or CellSearch predicted PFS (CTCscope, HR (hazard ratio) 2.26, 95% CI 1.18-4.35, P=0.014; CellSearch, HR 2.50, 95% CI 1.27-4.90, P=0.008).CTCscope offers unique advantages over existing CTC detection approaches. By enumerating and characterising only viable CTCs, CTCscope provides additional prognostic and predictive information in therapy monitoring.

To investigate the effect of tacrolimus on chemokine production by corneal myofibroblasts compared with that of cyclosporine or dexamethasone.

Since leptin levels are independently correlated with risk of coronary heart disease, we have identified signaling pathways important in mediating leptin production and lipogenesis in human preadipocytes. We used inhibitors of p70(S6) kinase, p42/44 mitogen-activated protein kinase (MAPK), p38 MAPK, and phosphatidylinositol 3-kinase (PI3K). Human preadipocytes were induced to differentiate in insulin, dexamethasone, triiodothyronine, and 3-isobutyl-1-methylxanthine in the presence or absence of inhibitors and the peroxisome proliferator-activated receptor (PPAR)-gamma activator rosiglitazone. Differentiation was assessed by measuring leptin secretion, lipid content, and lipogenic activity. Rosiglitazone increased cell protein by 15%, the lipid content of the cell layer was doubled, and the lipogenic activity increased sevenfold but did not stimulate leptin secretion. None of the inhibitors significantly inhibited protein content over 20 days, but lipid content and lipogenic activity were inhibited by p70(S6) kinase and p38 MAPK inhibition but not by p42/44 MAPK or PI3K inhibition. All of the inhibitors significantly decreased leptin secretion, and these inhibitory effects were increased by coincubation with rosiglitazone. We conclude that PI3K and p42/44 MAPK pathways are not critical to the differentiation program leading to lipid accumulation, but stimulation of leptin secretion is dependent on these as well as the p70(S6) kinase and p38 MAPK signaling pathways.

The immunosuppressant drug rapamycin, has been reported to inhibit 3T3-L1 adipocyte differentiation by interfering with critical postconfluent mitoses that are required early on for successful differentiation of this cell line (clonal expansion phase). In contrast to the murine 3T3-L1 preadipocyte cell line, human preadipocytes in primary culture do not undergo clonal expansion during differentiation. We investigated whether rapamycin could inhibit human adipocyte differentiation.