Fluorescent turn-on probes for wash-free mRNA imaging via covalent site-specific enzymatic labeling.

Investigating the many roles RNA plays in cellular regulation and function has increased demand for tools to explore RNA tracking and localization within cells. Our recently reported RNA-TAG (transglycosylation at guanine) approach uses an RNA-modifying enzyme, tRNA-guanine transglycosylase (TGT), to accomplish covalent labeling of an RNA of interest with fluorescent tracking agents in a highly selective and efficient manner. Unfortunately, labeling by this method currently suffers from a high nonspecific fluorescent background and is currently unsuitable for imaging RNA within complex cellular environments. Herein we report the design and synthesis of novel fluorogenic thiazole orange probes that significantly lower nonspecific binding and background fluorescence and, as a result, provide up to a 100-fold fluorescence intensity increase after labeling. Using these fluorogenic labeling agents, we were able to image mRNA expressed in Chinese Hamster Ovary cells in a wash-free manner

Criteria of efficiency for conformal prediction

We study optimal conformity measures for various criteria of efficiency of classification in an idealised setting. This leads to an important class of criteria of efficiency that we call probabilistic; it turns out that the most standard criteria of efficiency used in literature on conformal prediction are not probabilistic unless the problem of classification is binary. We consider both unconditional and label-conditional conformal prediction.Comment: 31 page

Clinical significance of serum lipids in idiopathic pulmonary alveolar proteinosis

<p>Abstract</p> <p>Background</p> <p>It is well known that pulmonary alveolar proteinosis(PAP) is characterised by accumulation of surfactant lipids and proteins within airspaces. However, few previous data describe the serum lipid levels associated with PAP.</p> <p>Materials and methods</p> <p>We retrospectively reviewed 25 patients with idiopathic PAP(iPAP). The serum lipid levels of patients with idiopathic PAP were compared with those of the healthy volunteers. In patients and healthy subjects, the LDL-C/HDL-C ratios were 2.94 ± 1.21 and 1.60 ± 0.70, respectively (<it>p </it>< 0.001), HDL-C were 1.11 ± 0.27 and 1.71 ± 0.71 respectively (<it>p </it>< 0.001). The values of LDL-C correlated significantly with those of PaO2 and P_A-aO2 (r = -0.685, <it>p </it>= 0.003, and r = 0.688, <it>p </it>= 0.003, respectively). The values of LDL-C/HDL-C ratios also correlated with PaO2 levels and PA-aO2 levels (r = -0.698, p = 0.003, and r = 0.653, p = 0.006, respectively). 11 and 13 patients experienced respectively a decline in TC and LDL-C levels following whole lung lavage(WLL), the median decline was 0.71 mmol/L(<it>p </it>< 0.009) and 0.47 mmol/L(<it>p </it>< 0.003), respectively.</p> <p>Conclusions</p> <p>the serum lipid levels, especially the levels of LDL-C and LDL-C/HDL-C, may reflect the severity of the disease in PAP patients, and predict the therapeutic effect of WLL.</p

Attenuation of fibrosis in vitro and in vivo with SPARC siRNA

INTRODUCTION: SPARC is a matricellular protein, which, along with other extracellular matrix components including collagens, is commonly over-expressed in fibrotic diseases. The purpose of this study was to examine whether inhibition of SPARC can regulate collagen expression in vitro and in vivo, and subsequently attenuate fibrotic stimulation by bleomycin in mouse skin and lungs. METHODS: In in vitro studies, skin fibroblasts obtained from a Tgfbr1 knock-in mouse (TBR1CA; Cre-ER) were transfected with SPARC siRNA. Gene and protein expressions of the Col1a2 and the Ctgf were examined by real-time RT-PCR and Western blotting, respectively. In in vivo studies, C57BL/6 mice were induced for skin and lung fibrosis by bleomycin and followed by SPARC siRNA treatment through subcutaneous injection and intratracheal instillation, respectively. The pathological changes of skin and lungs were assessed by hematoxylin and eosin and Masson\u27s trichrome stains. The expression changes of collagen in the tissues were assessed by real-time RT-PCR and non-crosslinked fibrillar collagen content assays. RESULTS: SPARC siRNA significantly reduced gene and protein expression of collagen type 1 in fibroblasts obtained from the TBR1CA; Cre-ER mouse that was induced for constitutively active TGF-beta receptor I. Skin and lung fibrosis induced by bleomycin was markedly reduced by treatment with SPARC siRNA. The anti-fibrotic effect of SPARC siRNA in vivo was accompanied by an inhibition of Ctgf expression in these same tissues. CONCLUSIONS: Specific inhibition of SPARC effectively reduced fibrotic changes in vitro and in vivo. SPARC inhibition may represent a potential therapeutic approach to fibrotic diseases

WNT-1 Signaling Inhibits Apoptosis by Activating β-Catenin/T Cell Factor–Mediated Transcription

Wnt signaling plays a critical role in development and oncogenesis. Although significant progress has been made in understanding the downstream signaling cascade of Wnt signaling, little is known regarding Wnt signaling modification of the cell death machinery. Given that numerous oncogenes transform cells by providing cell survival function, we hypothesized that Wnt signaling may inhibit apoptosis. Here, we report that cells expressing Wnt-1 were resistant to cancer therapy–mediated apoptosis. Wnt-1 signaling inhibited the cytochrome c release and the subsequent caspase-9 activation induced by chemotherapeutic drugs, including both vincristine and vinblastine. Furthermore, we found that Wnt-1–mediated cell survival was dependent on the activation of β-catenin/T cell factor (Tcf) transcription. Inhibition of β-catenin/Tcf transcription by expression of the dominant-negative mutant of Tcf-4 blocked Wnt-1–mediated cell survival and rendered cells sensitive to apoptotic stimuli. These results provide the first demonstration that Wnt-1 inhibits cancer therapy–mediated apoptosis and suggests that Wnt-1 may exhibit its oncogenic potential through a mechanism of anti-apoptosis

Control of inducible chemoresistance: Enhanced anti-tumor therapy through increased apoptosis by inhibition of NF-κB

Programmed cell death (apoptosis) seems to be the principal mechanism whereby anti-oncogenic therapies such as chemotherapy and radiation effect their responses. Resistance to apoptosis, therefore, is probably a principal mechanism whereby tumors are able to overcome these cancer therapies. The transcription factor NF-κB is activated by chemotherapy and by irradiation in some cancer cell lines. Furthermore, inhibition of NF-κB in vitro leads to enhanced apoptosis in response to a variety of different stimuli. We show here that inhibition of NF-κB through the adenoviral delivery of a modified form of IκBα, the inhibitor of NF-κB, sensitizes chemoresistant tumors to the apoptotic potential of TNFκ and of the chemotherapeutic compound CPT-11, resulting in tumor regression. These results demonstrate that the activation of NF-κB in response to chemotherapy is a principal mechanism of inducible tumor chemoresistance, and establish the inhibition of NF-κB as a new approach to adjuvant therapy in cancer treatment