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The immunomodulatory drug (IMiD) thalidomide and its structural analogs lenalidomide and pomalidomide are highly effective in treating clinical indications. Thalidomide binds to cereblon (CRBN), a substrate receptor of the cullin-4 really interesting new gene (RING) E3 ligase complex. Here, we examine the effect of thalidomide and its analogs on CRBN ubiquitination and its functions in human cell lines. We find that the ubiquitin modification of CRBN includes K48-linked polyubiquitin chains and that thalidomide blocks the formation of CRBN-ubiquitin conjugates. Furthermore, we show that ubiquitinated CRBN is targeted for proteasomal degradation. Treatment of human myeloma cell lines such as MM1.S, OPM2, and U266 with thalidomide (100 μM) and its structural analog lenalidomide (10 μM) results in stabilization of CRBN and elevation of CRBN protein levels. This in turn leads to the reduced level of CRBN target proteins and enhances the sensitivity of human multiple myeloma cells to IMiDs. Our results reveal a novel mechanism by which thalidomide and its analogs modulate the CRBN function in cells. Through inhibition of CRBN ubiquitination, thalidomide and its analogs allow CRBN to accumulate, leading to the increased cullin-4 RING E3 ligase-mediated degradation of target proteins.—Liu, Y., Huang, X., He, X., Zhou, Y., Jiang, X., Chen-Kiang, S., Jaffrey, S. R., Xu, G. A novel effect of thalidomide and its analogs: suppression of cereblon ubiquitination enhances ubiquitin ligase function.

We reported a novel fiber sensor based on two cascaded single mode fiber corners, which has a high sensitivity for refractive index (RI) sensing and a low temperature crosstalk. RI sensing was experimentally demonstrated with an average sensitivity of 235 nm/RI unit in a RI range from 1.333 to 1.418, which is relatively high among the previously reported similar fiber sensors (e.g., it is about 10 times higher than that of the normal two or three-cascaded-taper based Mach–Zehnder interferometer). The proposed RI sensor was fabricated by fiber fusion splicer, where the fiber corner was fabricated by splicing the two overlapping single mode fibers, which ensures that the proposed RI sensor is compact and strong. © 2014 Wiley Periodicals, Inc. Microwave Opt Technol Lett 56:2642–2645, 2014

We propose and demonstrate a novel all-fiber temperature sensor that is a single-end fiber modal interferometer (FMI) based on a segment of a few mode fiber (FMF) and a segment of a single-mode fiber (SMF).The FMF spliced to a lead-in SMF is used to expend the optical beam in the fiber and the sensing SMF with a well-cleaved end is spliced to the FMF. Owing to the core mismatch between the FMF and the sensing SMF, part of the guided light propagates in the cladding of the sensing SMF and part of the guided light propagates in the core of the sensing SMF, which finally forms the FMI when the reflective light comes back to the FMF. Temperature sensing is achieved by measuring the wavelength shift of the FMI. A temperature sensor with an average sensitivity of 0.1 nm/°C in a temperature range from 33 to 833°C is presented. © 2013 Wiley Periodicals, Inc. Microwave Opt Technol Lett 55:1700–1703, 2013; View this article online at wileyonlinelibrary.com. DOI 10.1002/mop.27657

Aims We investigated the mechanisms of diabetic bladder dysfunction (BD) through analysis of the roles of L- and T-type voltage-gated calcium channels (VGCCs), with the ultimate goal of identifying potential drug targets for diabetic BD. Methods Bladder function of db/db (type 2 diabetes) and wild type (Wt) mice was evaluated by behavioral tests and in vivo cystometry. Contractile responses of bladder strips to carbachol were measured with or without pre-treatment with nifedipine (a L-type VGCC blocker) or mibefradil (a T-type VGCC blocker). Furthermore, the effects of mibefradil and nifedipine on the proliferation of human bladder smooth muscle cells (BSMCs) were studied. Results db/db mice had significantly increased voiding frequency, bladder weight, bladder compliance and capacity, and heightened contractile response to carbachol, compared to Wt mice. Nifedipine, but not mibefradil, dramatically suppressed bladder tissue contraction in Wt mice. Whereas nifedipine nearly completely inhibited bladder contraction in db/db mice, mibefradil “normalized” the heightened bladder contractility of db/db mice to the level of Wt mice. In culture, mibefradil, but not nifedipine, inhibited the proliferation of human BSMCs. Conclusion Our results indicate that while L-type VGCCs play a major role in the contraction of both diabetic and non-diabetic bladders, T-type VGCCs are involved in the contraction of diabetic bladders and mediate BSMC proliferation. This study provides support for further investigations on the effect of blockade of T-type VGCC or combined blockade of both types of VGCCs in the treatment of diabetic BD. Neurourol. Urodynam. 33:147–152, 2014. © 2013 Wiley Periodicals, Inc.

Study Type – Aetiology (case control) Level of Evidence 3b What's known on the subject? and What does the study add? As one of the major components of the extracellular matrix, elastic fibres are believed to enhance tissue compliance. However, the role of elastic fibres in normal bladder function and dysfunction remains speculative. Although transgenic mice overexpressing elastin showed increased bladder compliance, the findings in patients with non-compliant bladders are inconsistent. Using transgenic elastin-deficient mouse models, this study provides the first direct evidence that sufficient elastin content is critical for healthy bladder function, and elastin is involved in the detrusor response to partial bladder outlet obstruction. OBJECTIVE • To examine functional and molecular changes of the bladders from elastin-haploinsufficient mice (Eln+/−) at baseline as well as in response to partial bladder outlet obstruction (pBOO). MATERIALS AND METHODS • Female Eln+/− and wild type (Wt) mice (3–4 months old) were studied. • The bladder elastin content was quantified by measuring desmosine. • Mice were divided into two groups to undergo surgery to create pBOO or to undergo sham surgery. Three days after surgery, bladder function was evaluated by in vivo cystometry, and the contractile response of bladder strips exposed to electrical field stimulation (EFS) and carbachol was examined by ex vivo myography. RESULTS • The Eln+/−-sham mice had a 33.6% decrease in bladder elastin compared with Wt-sham mice. • Cystometry showed significantly decreased bladder compliance and capacity in Eln+/−-sham vs Wt-sham mice; pBOO increased bladder compliance and capacity to a greater extent in Eln+/− mice compared with Wt mice. • Bladder strips from Eln+/−-sham mice showed a significantly heightened contractile response to both EFS and carbachol compared with Wt-sham mice. • A significantly increased contractile response to carbachol was detected in Wt-pBOO vs Wt-sham but not between Eln+/−-pBOO and Eln+/−-sham mice. CONCLUSION • The results that elastin-deficient mice had decreased bladder compliance and capacity and increased bladder contractility; and that Wt-pBOO mice showed an enhanced contractile response to carbachol, but Eln+/−-pBOO mice did not, suggest that elastin is critical for normal bladder function and is involved in bladder response to pBOO.

Although great progresses have been made in proteomics during the last decade, proteomics is still in its infancy. Extreme complexity of proteome sample and large dynamic range of protein abundance overwhelm the capability of all currently available analytical platforms. Sample pretreatment is a good approach to reduce the complexity of proteome sample and decrease the dynamic range. In this article, we present an overview of different technologies and methods for sample pretreatment in efficient proteome and peptidome analysis. Methods for isolation of rare amino acid-containing peptides, terminal peptides, PTM peptides and endogenous peptides are reviewed. In addition, two automated sample pretreatment technologies, i.e. automated sample injection and on-line digestion, are also covered.

Large-scale characterization of phosphoproteins requires highly specific methods for the purification of phosphopeptides because of the low abundance of phosphoproteins and substoichiometry of phosphorylation. A phosphopeptide enrichment method using ZrO2 nanoparticles is presented. The high specificity of this approach was demonstrated by the isolation of phosphopeptides from the digests of model phosphoproteins. The strong affinity of ZrO2 nanoparticles to phosphopeptides enables the specific enrichment of phosphopeptides from a complex peptide mixture in which the abundance of phosphopeptides is two orders of magnitude lower than that of nonphosphopeptides. Superior selectivity of ZrO2 nanoparticles for the enrichment of phosphorylated peptides than that of conventional immobilized metal affinity chromatography was observed. Femtomole phosphopeptides from digestion products could be enriched by ZrO2 nanoparticles and can be well detected by MALDI mass spectrometric analysis. ZrO2 nanoparticles were further applied to selectively isolate phosphopeptides from the tryptic digestion of mouse liver lysate for phosphoproteome analysis by nanoliter LC MS/MS (nano-LC-MS/MS) and MS/MS/MS. A total of 248 defining phosphorylation sites and 140 phosphorylated peptides were identified by manual validation using a series of rigid criteria.

Human plasma contains a complex matrix of proteolytically derived peptides (plasma peptidome) that may provide a correlate of biological events occurring in the entire organism. Analyzing these peptides from a small amount of serum/ plasma is difficult due to the complexity of the sample and the low levels of these peptides. Here, we describe a novel peptidome analysis approach using multiwalled carbon nanotubes (MWCNTs) as an alternative adsorbent to capture endogenous peptides from human plasma. Harvested peptides were analyzed by using liquid chromatography-mass spectrometry as a means of detecting and assessing the adsorbed molecules. The improved sensitivity and resolution obtained by using liquid chromatography-mass spectrometry allowed detection of 2521 peptide features (m/z 300-1800 range) in about 50 microL of plasma. 374 unique peptides were identified with high confidence by two-dimensional liquid chromatography system coupled to a nano-spray ionization linear ion trap-mass spectrometer. High recovery of BSA digest peptides enriched with MWCNTs, in both standard buffer and high abundance protein solution, was observed. Comparative studies showed that MWCNTs were superior to C18 and C8 for the capture of the smaller peptides. This approach could hold promise of routine plasma peptidome analysis.

The mechanisms involved in bleomycin-induced lung toxicity have not been fully understood to date. This work aimed to compare the proteome profiling of bleomycin-induced lung toxicity by using 2D-nano-LC-MS/MS and spectral counting. By comparing the spectral counts of identified proteins between control and bleomycin-treated groups, we noted that 102 proteins were upregulated and 28 proteins were downregulated in the bleomycin-treated group. Among these differently expressed proteins, five proteins were chosen for validation by Western blot analysis. The levels of these five proteins were consistent with proteomic results. These potential mediators can facilitate the translation of the underlying mechanisms of bleomycin-induced lung toxicity to molecular targets in the clinical arena.

Zymogen granule (ZG) constituents play important roles in pancreatic injury and disease. In previous studies, proteomic analyses with rat zymogen granules were separated by two-dimensional gel electrophoresis or one-dimensional SDS-PAGE, followed by in-gel tryptic digestion. In order to overcome the disadvantage of in-gel digestion and to carry out further in-depth proteomic analysis of the zymogen granules, in this study, by combining a filter-aided sample preparation method and fully automated 2D-LC-MS/MS technique, 800 ZG proteins were identified with at least two unique peptides for each protein, 75% of which have not been previously reported. The identified proteins revealed broad diversity in protein identity and function. This is the largest dataset of ZG proteome, and also the first dataset of the mouse ZG proteome, which may help elucidate on the molecular architecture of ZGs and their functions.