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Multilocular adipocytes are a hallmark of thermogenic adipose tissue(1,2), but the factors that enforce this cellular phenotype are largely unknown. Here, we show that an adipocyte-selective product of the Clstn3 locus (CLSTN3β) present in only placental mammals facilitates the efficient use of stored triglyceride by limiting lipid droplet (LD) expansion. CLSTN3β is an integral endoplasmic reticulum (ER) membrane protein that localizes to ER–LD contact sites through a conserved hairpin-like domain. Mice lacking CLSTN3β have abnormal LD morphology and altered substrate use in brown adipose tissue, and are more susceptible to cold-induced hypothermia despite having no defect in adrenergic signalling. Conversely, forced expression of CLSTN3β is sufficient to enforce a multilocular LD phenotype in cultured cells and adipose tissue. CLSTN3β associates with cell death-inducing DFFA-like effector proteins and impairs their ability to transfer lipid between LDs, thereby restricting LD fusion and expansion. Functionally, increased LD surface area in CLSTN3β-expressing adipocytes promotes engagement of the lipolytic machinery and facilitates fatty acid oxidation. In human fat, CLSTN3B is a selective marker of multilocular adipocytes. These findings define a molecular mechanism that regulates LD form and function to facilitate lipid utilization in thermogenic adipocytes.

Fluorescence In Situ Hybridization (FISH) is a widely used tool for quantifying gene expression and determining the location of RNA molecules in cells. Here, we present an improved method for FISH probe production that yields high purity probes with a wide range of fluorophores using standard laboratory equipment at low cost. The method modifies an earlier protocol that uses terminal deoxynucleotidyl transferase to add fluorescently labeled nucleotides to synthetic deoxyoligonucleotides. In our protocol, Amino-11-ddUTP is joined to an oligonucleotide pool prior to its conjugation to a fluorescent dye, thereby generating pools of probes ready for a variety of modifications. This order of reaction steps allows for high labeling efficiencies regardless of the GC content or terminal base of the oligonucleotides. The Degree Of Labeling (DOL) for spectrally distinct fluorophores (Quasar, ATTO and Alexa dyes) was mostly greater than 90%, comparable to commercial probes. The ease and low cost of production allowed generation of probe-sets targeting a wide variety of RNA molecules. Using these probes, FISH assays in C2C12 cells showed the expected subcellular localization of mRNAs and pre-mRNAs forPolr2a(RNA polymerase II subunit 2a) andGapdh, and of the long noncoding RNAsMalat1andNeat1. Developing FISH probe sets for several transcripts containing retained introns, we found that retained introns in theGabbr1andNoc2ltranscripts are present in subnuclear foci separate from their sites of synthesis and partially coincident with nuclear speckles. This labeling protocol should have many applications in RNA biology.

Background: Inflammation has been proposed to play potential roles in the development and progression of chronic kidney disease (CKD). We evaluated the relationship of neutrophil-to-lymphocyte ratio (NLR), a systemic inflammation marker, with CKD in normal-weight and overweight/obese adults. Methods: This cross-sectional study included 2846 apparently healthy adults who underwent a health examination between August 2000 and April 2002. Normal-weight was defined as a body mass index (BMI, kg/m2) of 18.5–24, while overweight/obesity was defined as a BMI of ≥24. CKD was defined as an estimated glomerular filtration rate (eGFR) of

Steps of mRNA maturation are important gene regulatory events that occur in distinct cellular locations. However, transcriptomic analyses often lose information on the subcellular distribution of processed and unprocessed transcripts. We generated extensive RNA-seq data sets to track mRNA maturation across subcellular locations in mouse embryonic stem cells, neuronal progenitor cells, and postmitotic neurons. We find disparate patterns of RNA enrichment between the cytoplasmic, nucleoplasmic, and chromatin fractions, with some genes maintaining more polyadenylated RNA in chromatin than in the cytoplasm. We bioinformatically defined four regulatory groups for intron retention, including complete cotranscriptional splicing, complete intron retention in the cytoplasmic RNA, and two intron groups present in nuclear and chromatin transcripts but fully excised in cytoplasm. We found that introns switch their regulatory group between cell types, including neuronally excised introns repressed by polypyrimidine track binding protein 1 (PTBP1). Transcripts for the neuronal gamma-aminobutyric acid (GABA) B receptor, 1 (Gabbr1) are highly expressed in mESCs but are absent from the cytoplasm. Instead, incompletely spliced Gabbr1 RNA remains sequestered on chromatin, where it is bound by PTBP1, similar to certain long noncoding RNAs. Upon neuronal differentiation, Gabbr1 RNA becomes fully processed and exported for translation. Thus, splicing repression and chromatin anchoring of RNA combine to allow posttranscriptional regulation of Gabbr1 over development. For this and other genes, polyadenylated RNA abundance does not indicate functional gene expression. Our data sets provide a rich resource for analyzing many other aspects of mRNA maturation in subcellular locations and across development.

SUMMARYTo globally assess the distribution and processing of gene transcripts across subcellular compartments, we developed extensive RNA-seq datasets of both polyA+ and total RNA from chromatin, nucleoplasm and cytoplasm of mouse ESC, neuronal progenitors, and neurons. We identified protein-coding genes whose polyadenylated transcripts were more abundant in chromatin than cytoplasm. We defined introns exhibiting cotranscriptional splicing, complete intron retention in cytoplasmic RNA, and many introns retained in nucleoplasmic and chromatin RNA but absent from cytoplasmic RNA, including new introns controlled during neuronal development. In particular, we found that polyadenylated Gabbr1 transcripts are expressed in mESC but remain sequestered on chromatin until neuronal differentiation when they are processed and released to the cytoplasm. This developmental regulation of splicing and chromatin association demonstrates that the abundance of polyadenylated RNA is not always an indicator of functional gene expression. Our datasets provide a rich resource for analyzing many other aspects of mRNA maturation.

Significance Alternative pre-mRNA splicing is a regulated process that greatly diversifies gene products by changing the exons incorporated into mRNA. This process is dysregulated in cancers. Here, we studied exon usage in aggressive prostate cancers and linked exon incorporation decisions to cancer driver genes. Through computational and experimental studies, we found that a strong cancer driver gene, Myc, was linked to exon changes in genes that themselves regulate alternative splicing. These exons often encoded premature stop codons that would decrease gene expression, suggestive of a Myc-driven autoregulatory loop to help control levels of splicing regulatory proteins., We sought to define the landscape of alternative pre-mRNA splicing in prostate cancers and the relationship of exon choice to known cancer driver alterations. To do so, we compiled a metadataset composed of 876 RNA-sequencing (RNA-Seq) samples from five publicly available sources representing a range of prostate phenotypes from normal tissue to drug-resistant metastases. We subjected these samples to exon-level analysis with rMATS-turbo, purpose-built software designed for large-scale analyses of splicing, and identified 13,149 high-confidence cassette exon events with variable incorporation across samples. We then developed a computational framework, pathway enrichment-guided activity study of alternative splicing (PEGASAS), to correlate transcriptional signatures of 50 different cancer driver pathways with these alternative splicing events. We discovered that Myc signaling was correlated with incorporation of a set of 1,039 cassette exons enriched in genes encoding RNA binding proteins. Using a human prostate epithelial transformation assay, we confirmed the Myc regulation of 147 of these exons, many of which introduced frameshifts or encoded premature stop codons. Our results connect changes in alternative pre-mRNA splicing to oncogenic alterations common in prostate and many other cancers. We also establish a role for Myc in regulating RNA splicing by controlling the incorporation of nonsense-mediated decay-determinant exons in genes encoding RNA binding proteins.

MicroRNA (miRNA)-124 is expressed in neurons, where it represses genes inhibitory for neuronal differentiation, including the RNA binding protein PTBP1. PTBP1 maintains nonneuronal splicing patterns of mRNAs that switch to neuronal isoforms upon neuronal differentiation. We find that primary (pri)-miR-124-1 is expressed in mouse embryonic stem cells where mature miR-124 is absent. PTBP1 binds to this precursor RNA upstream of the miRNA stem-loop to inhibit mature miR-124 expression in vivo and DROSHA cleavage of pri-miR-124-1 in vitro. This function for PTBP1 in repressing miR-124 biogenesis defines an additional regulatory loop in the already intricate interplay between these two molecules. Applying mathematical modeling to examine the dynamics of this regulation, we find that the pool of pri-miR-124 whose maturation is blocked by PTBP1 creates a robust and self-reinforcing transition in gene expression as PTBP1 is depleted during early neuronal differentiation. While interlocking regulatory loops are often found between miRNAs and transcriptional regulators, our results indicate that miRNA targeting of posttranscriptional regulators also reinforces developmental decisions. Notably, induction of neuronal differentiation observed upon PTBP1 knockdown likely results from direct derepression of miR-124, in addition to indirect effects previously described.

Rbfox proteins control alternative splicing and posttranscriptional regulation in mammalian brain and are implicated in neurological disease. These proteins recognize the RNA sequence (U)GCAUG, but their structures and diverse roles imply a variety of protein-protein interactions. We find that nuclear Rbfox proteins are bound within a large assembly of splicing regulators (LASR), a multimeric complex containing the proteins hnRNP M, hnRNP H, hnRNP C, Matrin3, NF110/NFAR-2, NF45, and DDX5, all approximately equimolar to Rbfox. We show that splicing repression mediated by hnRNP M is stimulated by Rbfox. Virtually all the intron-bound Rbfox is associated with LASR, and hnRNP M motifs are enriched adjacent toRbfox crosslinking sites invivo. These findings demonstrate that Rbfox proteins bind RNA with a defined set of cofactors and affect a broader set ofexons than previously recognized. The function of this multimeric LASR complex has implications for deciphering the regulatory codes controlling splicing networks.

A solvent-free lift-off method has been introduced to fabricate the aluminum nano-hole array with diameter down to 80 nm as the base electrode for a vertical organic transistor. The imprinted vertical organic transistor exhibited base leakage current density as low as 5 × 10 −5 mA/cm 2 and high ON/OFF current ratio as high as 10 5 .

Summary Human genetic studies have identified the neuronal RNA binding protein, Rbfox1, as a candidate gene for autism spectrum disorders. While Rbfox1 functions as a splicing regulator in the nucleus, it is also alternatively spliced to produce cytoplasmic isoforms. To investigate the function of cytoplasmic Rbfox1, we knocked down Rbfox proteins in mouse neurons and rescued with cytoplasmic or nuclear Rbfox1. Transcriptome profiling showed that nuclear Rbfox1 rescued splicing changes, whereas cytoplasmic Rbfox1 rescued changes in mRNA levels. iCLIP-seq of subcellular fractions revealed that Rbfox1 bound predominantly to introns in nascent RNA, while cytoplasmic Rbox1 bound to 3ʹ UTRs. Cytoplasmic Rbfox1 binding increased target mRNA stability and translation, and Rbfox1 and miRNA binding sites overlapped significantly. Cytoplasmic Rbfox1 target mRNAs were enriched in genes involved in cortical development and autism. Our results uncover a new Rbfox1 regulatory network and highlight the importance of cytoplasmic RNA metabolism to cortical development and disease. Video Abstract

MicroRNA-124 is expressed in neurons, where it represses genes inhibitory for neuronal differentiation, including the RNA binding protein PTBP1. PTBP1 maintains non-neuronal splicing patterns of mRNAs that switch to neuronal isoforms upon neuronal differentiation. We find that pri-miR-124-1 is expressed in mouse embryonic stem cells (mESCs) where mature miR-124 is absent. PTBP1 binds to this precursor RNA upstream of the miRNA stem-loop to inhibit mature miR-124 expression in vivo, and DROSHA cleavage of pri-miR-124-1 in vitro. This new function for PTBP1 in repressing miR-124 biogenesis adds an additional regulatory loop to the already intricate interplay between these two molecules. Applying mathematical modeling to examine the dynamics of this regulation, we find that the pool of pri-miR-124 whose maturation is blocked by PTBP1 creates a robust and self-reinforcing transition in gene expression as PTBP1 is depleted during early neuronal differentiation. While interlocking regulatory loops are often modeled between miRNAs and transcriptional regulators, our results indicate that miRNA targeting of posttranscriptional regulators also reinforces developmental decisions. Notably, induction of neuronal differentiation observed upon PTBP1 knockdown likely results from direct de-repression of miR-124, in addition to indirect effects previously described.

High-resolution image processing is crucial for emerging applications in virtual reality. The trade-off between robustness/accuracy and speed pose a challenge to high-resolution image stitching. This paper accelerates the state-of-the-art image stitching algorithm by using the CUDA toolkit. We also use the mesh coordinates as an initial guess for solving warping function for an additional increase in speed. Experimental results show that our implementation achieves comparable quality to the state-of-the-art work, while our implementation is almost 2x faster on high-resolution images (3264×2448).

Proteins of the Rbfox family act with a complex ofproteins called the Large Assembly of Splicing Regulators (LASR). We find that Rbfox interacts with LASR via its C-terminal domain (CTD), and thisdomain is essential for its splicing activity. In addition to LASR recruitment, a low-complexity (LC) sequence within the CTD contains repeated tyrosines that mediate higher-order assembly of Rbfox/LASR and are required for splicing activation by Rbfox. This sequence spontaneously aggregates in solution to form fibrous structures and hydrogels, suggesting an assembly similar to the insoluble cellular inclusions formed by FUS and other proteins in neurologic disease. Unlike the pathological aggregates, we find that assembly of the Rbfox CTD plays an essential role in its normal splicing function. Rather than simple recruitment of individual regulators to a target exon, alternative splicing choices also depend on the higher-order assembly of these regulators within the nucleus.

Dysfunction of the neuronal RNA binding protein RBFOX1 has been linked to epilepsy and autism spectrum disorders. Rbfox1 loss in mice leads to neuronal hyper-excitability and seizures, but the physiological basis for this is unknown. We identify the vSNARE protein Vamp1 as a major Rbfox1 target. Vamp1 is strongly downregulated in Rbfox1 Nes-cKO mice due to loss of 3' UTR binding by RBFOX1. Cytoplasmic Rbfox1 stimulates Vamp1 expression in part by blocking microRNA-9. We find that Vamp1 is specifically expressed in inhibitory neurons, and that both Vamp1 knockdown and Rbfox1 loss lead to decreased inhibitory synaptic transmission and E/I imbalance. Re-expression of Vamp1 selectively within interneurons rescues the electrophysiological changes in the Rbfox1 cKO, indicating that Vamp1 loss is a major contributor to the Rbfox1 Nes-cKO phenotype. The regulation of interneuron-specific Vamp1 by Rbfox1 provides a paradigm for broadly expressed RNA-binding proteins performing specialized functions in defined neuronal subtypes.

Supplemental Digital Content is available in the text, The aim of this study was to investigate cancer risk in patients with a history of urolithiasis and to determine whether intervention for calculi attenuated the risk of subsequent urinary tract cancer (UTC). Using data from the National Health Insurance Research Database in Taiwan, we performed a nationwide cohort study enrolling participants (n = 42,732) aged > 30 years who were diagnosed with urinary tract calculi between 2000 and 2009. Age- and gender-matched insured individuals (n = 213,660) found in the health service records over the same period were recruited as the control group. The Cox proportional hazards model and competing risks regression model were used to examine the relationship between urolithiasis and UTC, as well as whether early intervention for urolithiasis decreased the subsequent cancer risk relative to late intervention. Participants with a previous diagnosis of urolithiasis (n = 695) had a 1.82-fold (95% CI: 1.66–1.99, P

We used the oxygen and glucose deprivation (OGD) method in cultured astrocytes as an in vitro ischemic model. We investigated whether activation of group-II metabotropic glutamate receptors (mGluR2/3) can reverse OGD-induced impairment in astrocytic glutamate/aspartate transporter (GLAST) expression and elucidated the signaling pathways involving the GLAST expression. Cultured astrocytes exposed to OGD for 6 h resulted in significant reductions in the GLAST expression and extracellular glutamate clearance. These reductions were effectively restored by mGluR2/3 activation with mGluR2/3 agonists, LY379268 or DCG-IV, after the 6 h OGD insult. These mGluR2/3-mediated restorative effects were inhibited by selective mGluR2/3 antagonists LY341459 or EGLU. The mGluR2/3 activation also induced activations of signaling pathways including extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K) and nuclear transcription factor-κB (NFκB). These activations were prevented by blocking mGluR2/3 with LY341459, an mGluR2/3 antagonist. Furthermore, blocking ERK, PI3K and NFκB signaling pathways with U0126, LY294002 and pyrrolidine dithiocarbamate, respectively, significantly inhibited the mGluR2/3-mediated restorative effects. These results suggest that application of mGluR2/3 agonists after OGD insult can effectively reverse the OGD-reduced expression of GLAST proteins and restore clearance of extracellular glutamate by serially activating ERK/PI3K/NFκB signaling pathways in cultured astrocytes.

Objectives The effect of agmatine on bladder contractility and the diabetes-induced alteration of this action were studied in the rat. Methods Bladder strips were isolated from 9-week-old streptozotocin (STZ)-diabetic rats and control Wistar rats. Strips were hung in an organ bath for measurement of isometric tension and pre-contracted with either 1 µmol/L acetylcholine (ACh) or 50 mmol/L KCl. Dose-dependent relaxation of the bladder strips was studied by cumulative administration of agmatine 1–100 µmol/L into the organ bath. Effects of specific imidazoline receptor (IR) antagonists on the agmatine-induced relaxation were studied. Western blotting analysis was used to measure bladder IR, sulphonylurea receptor (SUR) and inwardly rectifying K+ channel subunit 6.2 (Kir 6.2) protein levels. Results Agmatine reduced ACh and KCl pre-contracted bladder strip tension in a dose-dependent fashion. Relaxation was significantly increased in STZ-diabetic rats. The relaxation was inhibited by BU224, a selective I2 IR antagonist; but not by efaroxan (I1 IR antagonist) or KU14R (I3 IR antagonist). Moreover, the agmatine-induced relaxation was attenuated by glibenclamide (inhibitor of KATP channel) and H-89 (inhibitor of protein kinase A), but enhanced by 3-isobutyl-1-methylxanthine (IBMX, inhibitor of cyclic AMP phosphodiesterase). Western blotting showed increased expression of bladder IR but not SUR or Kir 6.2 in the STZ-diabetic rat. Conclusion Agmatine causes rat bladder relaxation by activation of the I2 IR, which opens KATP channels through the cyclic AMP/protein kinase A pathway. Agmatine-induced bladder relaxation in STZ-diabetic rats is increased due to a higher expression of IR.

Objectives: The effect of agmatine on prostate contractility as well as the roles of imidazoline receptors and potassium channels in this action were studied using isolated Wistar rat prostate tissue. Methods: Rat prostate strips were pre-contracted with 1 µmol/L phenylephrine or 50 mmol/L KCl. The relaxation response to agmatine (1–100 µmol/L) was measured. The effects of imidazoline receptor blockers: efaroxan, BU224, KU14R; ATP-sensitive K+ channels (KATP) channel inhibitor: glibenclamide; cyclic AMP (cAMP) phosphodiesterase inhibitor: IBMX; or protein kinase A (PKA) inhibitor: H-89 on the agmatine-induced relaxation were studied. Results: Agmatine produced relaxation in prostate strips pre-contracted with phenylephrine or KCl in a dose-dependent manner. This relaxation was significantly reduced by BU224, a selective I2 imidazoline receptor (IR) blocker, but not by I1 or I3 IR blockers (efaroxan, KU14R respectively). Moreover, the agmatine-induced relaxation was attenuated by glibenclamide and H-89, but enhanced by IBMX. Conclusion: The results suggest that agmatine causes rat prostate relaxation by activation of the I2 IR, which opens KATP channels through cAMP/PKA pathway.

Neuronal death induced by I6 displayed apoptotic characteristics but the precise mechanism has not been fully elucidated. In the present studies, I6 at 24 h after intraperitoneal administration significantly decreased the density of surviving neurons and increased caspase-3 activity in frontal cortex, suggesting that peripherally administered I6 may cross BBB to induce CNS toxicity. In rat embryonic primary cortical cells, I6-induced reduction of mitochondrial viability and neuronal apoptosis was inhibited by vitamin E. In addition, I6-induced reactive oxygen species (ROS) caused the disruption of mitochondria membrane potential (MMP), the release of cytochrome c, the activation of caspase-9 and caspase-3, and cleavage of poly(ADP-ribose) polymerase (PARP), resulting in activation of mitochondrial-mediated intrinsic death pathway. Pre-treatment with antioxidant vitamin E or N-acetylcysteine (NAC) completely abolished the I6-induced generation of ROS, loss of MMP, release of cytochrome c, activation of caspase-9 and caspase-3, and cleavage of PARP. Carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), a mitochondrial uncoupler, significantly reduced I6-induced neuronal death as well as caspase-3 activation and PARP cleavage. These results suggest that I6 induces neuronal death by promoting intracellular ROS production to cause a loss of MMP that result in release of cytochrome c and activation of mitochondria-mediated intrinsic death pathway.

The RNA-binding proteins PTBP1 and PTBP2 control programs of alternative splicing during neuronal development. PTBP2 was found to maintain embryonic splicing patterns of many synaptic and cytoskeletal proteins during differentiation of neuronal progenitor cells (NPCs) into early neurons. However, the role of the earlier PTBP1 program in embryonic stem cells (ESCs) and NPCs was not clear. We show that PTBP1 controls a program of neuronal gene expression that includes the transcription factor Pbx1. We identify exons specifically regulated by PTBP1 and not PTBP2 as mouse ESCs differentiate into NPCs. We find that PTBP1 represses Pbx1 exon 7 and the expression of the neuronal Pbx1a isoform in ESCs. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of neuronal genes. Thus, PTBP1 controls the activity of Pbx1 to suppress its neuronal transcriptional program prior to induction of NPC development. DOI: http://dx.doi.org/10.7554/eLife.09268.001, eLife digest The neurons that transmit information around the nervous system develop in several stages. Embryonic stem cells specialize to form neuronal progenitor cells, which then develop into neurons. These cell types have different characteristics, in part because they make different proteins or different versions of the same proteins. To make a protein, the DNA sequence of a gene is used to build a molecule of ribonucleic acid (RNA) that acts as a template for the protein. However, not all of this sequence codes for the protein. The non-coding regions must be removed from the RNA, and the remaining “exons” joined together to form the final “mRNA” template. Not all of the exons are necessarily included in the final mRNA molecule. By joining together different combinations of exons, several different versions of a protein can be produced from a single gene. This process is known as alternative splicing. One way that alternative splicing is controlled is through proteins that bind to RNA and determine which exons are included or excluded from the final mRNA molecule. PTBP1 is an RNA-binding protein that controls alternative splicing in embryonic stem cells and neuronal progenitor cells. Embryonic stem cells have the ability to develop into all the cells of the body. In contrast, neuronal progenitor cells are restricted in their development and only give rise to specialized cells of the nervous system. The role of PTBP1 in these properties was not clear. Linares et al. have now used a range of techniques to study the RNA molecules produced in these two cell types and how these RNAs change when PTBP1 is removed. This identified many RNAs whose splicing is regulated by PTBP1, including mRNAs of the gene that produces a protein called Pbx1, which is an important regulator of neuronal development. Further investigation revealed that PTBP1 prevents a particular exon being included in the mRNA template for Pbx1. This creates an embryonic stem cell form of Pbx1 that does not affect neuronal genes. Removal of PTBP1 allows splicing of the Pbx1 exon and produces a version of Pbx1 that is found in neuronal progenitor cells and which turns on neuronal genes. Thus, through its action on Pbx1, one role of PTBP1 is to enable stem cells to maintain their non-neuronal properties and prevent their premature development into neuronal progenitor cells. The gene for Pbx1 is only one of many genes controlled by PTBP1 at the level of splicing. One challenge for the future will be to understand how these genes work together in a common program that determines the properties of stem cells. Another question regards how the different Pbx1 proteins in stem cells and in neuronal progenitors can exert different effects in the cells where they are made. DOI: http://dx.doi.org/10.7554/eLife.09268.002

s / Urological Science 26 (2015) 301e310 303 * Abbreviations: LN, lymph node; Lt, left; Rt, right; OPN, open partial nephrectomy; ORN, open radical nephrectomy; lapa., laparoscopic ; NED, no evidence of disease; AML, angiomyolipoma; RSG, radical subtotal gastrectomy Conclusion: Among these 9 cases, 2 cases (22.2%) had distant metastases and ominent outcome. 3 cases with symptoms (flank pain, palpable mass, body weight loss) were all diagnosed as malignant epithelioid AML, indicating symptomatic renal tumor may need to be managed more cautiously as potential malignancies. NDP03: EXPLORE THE CHARACTERISTICS OF BLADDER FUNCTION OF THE PATIENTS WITH PELVIC ORGAN MALIGNANCY Chian-Shiung Lin , Chih-Cheng Lu , Chia-Ho Lin , Eric W. Fan , Tse-Chou Cheng . Divisions of Urology, Department of Surgery, Chi Mei Medical Center, Liouying, Tainan City, Taiwan; Min-Hwei Junior College of Health Care Management, Tainan City, Taiwan Purpose: The primary function of urinary bladder is for urine storage and voiding. Pelvic organ malignancy may deteriorate the originally normal bladder function via either neurogenic or myogenic way. The aim of this study is to compare urodynamic bladder dysfunction among the patients with different pelvic organ malignancies. Materials and Methods: From January 2010 to June 2015, there were 640 patients eligible for urodynamic analysis. Patients were stratified into 3 groups based on cancer origins (prostate, bladder, and colorectum). The data including gender, age, clinical tumor staging, lower urinary tract symptoms or acute urinary retention, and the parameter values of urodynamic studies were compared among groups. SPSS 17.0.1 for Windows and Microsoft Office Excel 2007 were used for all statistical analysis. Results: Pelvic organ malignancies included prostatic (40.9%), bladder (33.5%), and colorectal (25.6%) origin. Age ranged from 39 to 95 years (mean 72.93± 9.08 years). In uroflowmetry (525 cases), the mean± standard deviation of maximal urine flow rate was significant lower in prostatic group (11.49± 5.53 ml/sec). In cystometrogram (115 cases), the first desire was more sensitive in bladder group (85.52± 49.78 ml); the cystometric capacity was obvious decreased in prostatic group (161.50± 94.29 ml); the maximum voiding pressure and compliance were significant lower in colorectal group (64.58± 50.09 cmH2O). Conclusions: Patients having pelvic organ malignancies may suffer from urination dysfunction. Urinary flow rate obvious decreased in prostate group, more hypersensitive bladder was found in bladder group and bladder dysfunction was poor in colorectal group. The physicians are encouraged to be aware of these urinary complications in patient with pelvic organ oncology. NDP04: THE PATTERN OF CALCIFICATION CAN PREDICT THE TYPE OF MALIGNANCY IN UPPER URINARY TRACT Li-Hua Huang , Chuan-Shu Chen , Yen-Chuan Ou . Divisions of Urology, Department of Surgery, Tungs' Taichung MetroHarbor Hospital, Taichung, Taiwan; Divisions of Urology, Department of Surgery, Taichung Veterans General Hospital, Taichung, Taiwan Purpose: In upper urinary tract (UUT), squamous cell carcinoma(SCC) and urothelial carcinoma (UC) are twodifferent disease in its etiology, incidence and prognosis. I contrast to UC, the development SCC is believed to bemore related to chronic inflammation of urothelial epithelium secondary to renal calculi. In this study, we investigated the relationship between calcification patternandsquamousneoplasminupperurinarytract. Materials and Methods: The study was approved by institutional review board. From 2000 to 2007, there are 373 patients with localized upper urinary tract malignancy receiving radical nephroureterectomy with bladder cuff excision (RNU) at Taichung Veterans General Hospital. Only patients having UC, UC with squamous differentiation (SqD) and SCC were enrolled. These patients' clinical and pathologic data were retrospectively reviewed. The calcification pattern in tumors was analyzed by computer tomography(CT). Among these, 62 patients' preoperative CT films were lost and they were excluded from the study. Finally, there were 232 pure UC, 24 UC with SqD and 9 pure SCC(2.7%) patients. The differences of calcification pattern were analyzed by Fisher's exact test and chisquare test (Categorical variables). Continuous variables were asscssed by MannWhitney-U (two categories) and Kruskal-Wallis tests (three categories) Results: Computer tomography shows the rates of calcification change are 7.8% (18/232), 25.0% (6/24) and 55.6% (5/9) in UC, SqD and SCC, respectively. In SCC, the calcification tends to be bigger, and more numerous. In addition, all SCC patients had multifocal distribution of calcification while SqD and UC patients often had single calcification. Conclusions: We can predict the type of tumor in upper urinary tract according to the calcification pattern at CT. When CT revealed multiple, dispersive and bigger calcification in the tumor, squamous cell carcinoma should be considered and the surgical field should be more extended. NDP05: VARIATIONS OF CLINICAL PRACTICE GUIDELINES BETWEEN CHINA AND TAIWAN IN ROBOT-ASSISTED LAPAROSCOPIC RADICAL PROSTATECTOMY Chih-Cheng Lu, Wen-Chou Fan. Division of Urology, Department of Surgery, Chi Mei Medical Center, Liouying, Tainan, Taiwan Purpose: To explore the role of robot-assisted Laparoscopic radical prostatectomy (RALP) from the documented clinical practice guidelines (CPGs) between China and Taiwan. Materials andMethods: The printed and onlinematerials in guidelines for PCa from China and Taiwan were analyzed. We focused on the RALP treatment for PCa. Results: The online guidelines for PCa by Chinese Urological Association (CUA)were available in 2011(since 2007 as the first version). Taiwanese first version was available by Taiwan Cooperation Oncology Group (TCOG) PCa practice guidelines in 1999, the second edition in 2003, and the third edition in 2010. Normal range of prostate specific antigen (PSA) is defined from Chinese people datawith age specific consideration in CUA, but not inTCOG. PCa Staging by AJCC 2002 is noted in CUA but by AJCC 2010 (the seventh edition) in TCOG. In treatment, RALP takes the advantage of less blood loss and blood transfusion rate compared with traditional approach in both CPGs. Some significant improvement in postoperative urinary control compared with traditional approach in TCOG but no conclusions in CUA. Better outcome in sexual function from RALP in TCOG but not in CUA. High installment cost is mentioned in both CPGs. No Definite evidence-based level of evidence or grade of recommendation is mentioned in both CPGs. Conclusion: There are differences in staging system, merits of RALP treatment between China and Taiwan in CPGs. Also this limited study could do some help for the revision of the CPG in Taiwan. Asian urologists and oncologists are suggested to realize the differences at managing prostate cancer patients. NDP06: INFLAMMATORY MYOFIBROBLASTIC TUMOR OF URINARY BLADDER IN A PATIENT WITH HUMAN IMMUNODEFICIENCY VIRUS e A RARE CASE REPORT AND LITERATUR REVIEW Wei-Jen Chen , Chih-Chieh Lin , Alex T.L. Lin , Kuang-Kuo Chen . Department of Urology, Taipei Veterans General Hospital, Taiwan; Department of Urology, School of Medicine, National Yang-Ming University, Taiwan; 3 Shu-Tien Urological Science Research Center, Taiwan Purpose: Inflammatorymyofibroblastic tumor (IMT) is a rare tumor with a generally indolent, but sometimes aggressive behavior. It had been described inmajor organs. The first case of IMTof the bladder was reported in 1980. Until recent 15 years, IMT have gained a distinct entity with established characteristic features in pathological diagnosis. Reported literatures are limited in case report and case series. A systemic review article published in 2014 presented a total of 182 patients with IMT in urinary bladder. However, there is no related literature in patient with human immunodeficiency virus (HIV). Here, we report a HIV patient with IMT of the urinary bladder and discuss its clinical presentation, diagnosis and management. Materials and Methods: A 45 year-old HIV infected man, came to our outpatient department due to painless gross hematuria with repeated

Minocycline has been shown to alleviate several neurological disorders. Unexpectedly, we found that minocycline had opposite effects on glioma cells: minocycline induced nonapoptotic cell death in glioma cells. The glioma cell death was associated with the presence of autophagic vacuoles in the cytoplasm. Minocycline induced autophagy was confirmed by acridine orange, monodansylcadaverine (MDC) stainings of vesicle formation and the conversion of microtubule-associated proteins light chain 3 (LC3-I) to LC3-II. Pretreatment with autophagy inhibitor 3-methyladenine (3-MA) suppressed the induction of acidic vesicular organelles and the accumulation of LC3-II to the autophagosome membrane in glioma cells treated with minocycline. Despite the pretreatment of 3-MA, minocycline induced cell death which could result from the activation of caspase-3. Minocycline effectively inhibited tumor growth and induced autophagy in the xenograft tumor model of C6 glioma cells. These results suggest that minocycline may kill glioma cells by inducing autophagic cell death. When autophagy was inhibited, minocycline still induced cell death through the activation of caspase-3. Thus, minocycline is a promising agent in the treatment of malignant gliomas.

Sam68 (Src-associated in mitosis, 68 kDa) is a KH domain RNA binding protein implicated in a variety of cellular processes, including alternative pre-mRNA splicing, but its functions are not well understood. Using RNA interference knockdown of Sam68 expression and splicing-sensitive microarrays, we identified a set of alternative exons whose splicing depends on Sam68. Detailed analysis of one newly identified target exon in epsilon sarcoglycan (Sgce) showed that both RNA elements distributed across the adjacent introns and the RNA binding activity of Sam68 are necessary to repress the Sgce exon. Sam68 protein is upregulated upon neuronal differentiation of P19 cells, and many Sam68 RNA targets change in expression and splicing during this process. When Sam68 is knocked down by short hairpin RNAs, many Sam68-dependent splicing changes do not occur and P19 cells fail to differentiate. We also found that the differentiation of primary neuronal progenitor cells from embryonic mouse neocortex is suppressed by Sam68 depletion and promoted by Sam68 overexpression. Thus, Sam68 controls neurogenesis through its effects on a specific set of RNA targets.

A miniature independent dual-band hybrid dielectric resonator antenna (DRA) fed by a coplanar waveguide (CPW) inductive slot is proposed. In this configuration, the CPW inductive slot performs the functions of an effective radiator and the feeding structure of the DR. By optimizing the structure parameters, the hybrid structure resonates at two different frequencies independently. One is from the DRA with the broadside patterns and the other from the inductive slot with the dipole-like patterns. In order to determine the performance of varying design parameters on bandwidth and resonance frequency, parametric study is carried out using simulation software HFSS and experimental results. The measured and simulated results show excellent agreement.

Although previous studies have indicated that the neuroprotective effect of N-acetylcysteine (NAC) required activation of the Ras-extracellular-signal-regulated kinase (ERK) pathway, the detailed mechanisms and signal cascades leading to activation ERK are not clear. In the present study, we investigated the effect of NAC on A beta(25-35)-induced neuronal death. Pretreatment of neurons with NAC 1 hr before application of A beta prevented A beta-mediated cell death. NAC increased cyclin-dependent kinase 5 (Cdk5) phosphorylation, an effect that was blocked by Cdk5 inhibitor. The neuroprotective effect of NAC was significantly attenuated by Cdk5 inhibitors or in neurons transfected with Cdk5 or p35 small interfering RNA (siRNA). Conversely, pretreatment of neurons with the calpain inhibitors calpeptin or MDL28170 enhanced the neuroprotective effect of NAC. A beta(25-35) caused a significant decrease in the level of p35, with a concomitant increase in p25, which was completely prevented by NAC. This effect of NAC was blocked by the Cdk5 inhibitors roscovitine and butyrolactone. In addition, NAC increased Cdk5/p35 kinase activity but reduced Cdk5 kinase activity. A beta(25-35) treatment decreased phosphorylated levels of ERK, which could be reversed by NAC. The effect of NAC was completely blocked by Cdk5 inhibitors. NAC reversed the A beta(25-35)-induced decrease in the expression of Bcl-2, which could be blocked by the MAPK kinase (MEK) inhibitor or Cdk5 inhibitors. These results suggest that NAC-mediated neuroprotection against A beta toxicity is likely mediated by the p35/Cdk5-ERKs-Bcl-2 signal pathway.

Alternative splicing has emerged as a promising therapeutic target in a number of human disorders. However, the discovery of compounds that target the splicing reaction has been hindered by the lack of suitable high-throughput screening assays. Conversely, the effects of known drugs on the splicing reaction are mostly unclear and not routinely assessed. We have developed a two-color fluorescent reporter for cellular assays of exon inclusion that can accommodate nearly any cassette exon and minimizes interfering effects from changes in transcription and translation. We used microtubule-associated protein tau (MAPT) exon 10, whose missplicing causes frontotemporal dementia, to test the reporter in screening libraries of known bioactive compounds. These screens yielded several compounds that alter the splicing of the exon, both in the reporter and in the endogenous MAPT mRNA. One compound, digoxin, has long been used in the treatment of heart failure, but was not known to modulate splicing. The positive compounds target different signal transduction pathways, and microarray analysis shows that each compound affects the splicing of a different set of exons in addition to MAPT exon 10. Our results identify currently prescribed cardiotonic steroids as modulators of alternative splicing and demonstrate the feasibility of screening for drugs that alter exon inclusion.

Many metazoan gene transcripts exhibit neuron-specific splicing patterns, but the developmental control of these splicing events is poorly understood. We show that the splicing of a large group of exons is reprogrammed during neuronal development by a switch in expression between two highly similar polypyrimidine tract-binding proteins, PTB and nPTB (neural PTB). PTB is a well-studied regulator of alternative splicing, but nPTB is a closely related paralog whose functional relationship to PTB is unknown. In the brain, nPTB protein is specifically expressed in post-mitotic neurons, whereas PTB is restricted to neuronal precursor cells (NPC), glia, and other nonneuronal cells. Interestingly, nPTB mRNA transcripts are found in NPCs and other nonneuronal cells, but in these cells nPTB protein expression is repressed. This repression is due in part to PTB-induced alternative splicing of nPTB mRNA, leading to nonsense-mediated decay (NMD). However, we find that even properly spliced mRNA fails to express nPTB protein when PTB is present, indicating contributions from additional post-transcriptional mechanisms. The PTB-controlled repression of nPTB results in a mutually exclusive pattern of expression in the brain, where the loss of PTB in maturing neurons allows the synthesis of nPTB in these cells. To examine the consequences of this switch, we used splicing-sensitive microarrays to identify different sets of exons regulated by PTB, nPTB, or both proteins. During neuronal differentiation, the splicing of these exon sets is altered as predicted from the observed changes in PTB and nPTB expression. These data show that the post-transcriptional switch from PTB to nPTB controls a widespread alternative splicing program during neuronal development.

The metabotropic glutamate receptors (mGluRs) are G-proteincoupled receptors whose activation modulates neural activity via their linkage to multiple second-messenger systems (Pin and Duvoisin 1995; Anwyl 1999). Eight mGluRs have been cloned and classified into three groups based on their sequence homologies, signal transduction mechanisms, and agonist pharmacology (Nakanishi 1992; Pin and Duvoisin 1995). Group I (mGluR1 and mGluR5) receptors stimulate phosphatidylinositol hydrolysis, whereas group II (mGluR2 and mGluR3) and group III (mGluR4, mGluR6, mGluR7, and mGluR8) receptors are negatively coupled to cAMP production. The group II mGluRs are implicated as negative feedback autoreceptors to modulate neurotransmission by presynaptic mechanisms (Pin and Duvoisin 1995). In situ hybridization and immunohistochemical studies of rat brain have revealed that the intense mGluR II-like immunoreactivity was seen predominantly in the cortical and limbic regions including the amygdala (Neki et al. 1996; Schaffhauser et al. 1998), suggesting that these receptors may modulate higher CNS functions such as emotion and cognition. Indeed, mGluR II agonists have been shown to inhibit the release of glutamate in vivo (Battaglia et al. 1997) and exhibit potent anxiolytic effects in rats (Helton et al. 1998). These compounds also ameliorated the symptoms of nicotine withdrawal (Helton et al. 1997) and lactate-induced panic responses (Shekhar and Keim 2000). Moreover, mice lacking mGluR II display impaired hippocampal mossy fiber long-term depression (LTD) (Yokoi et al. 1996). In external capsule (EC)-basolateral amygdala synapses, it has been shown that low-frequency stimulation (LFS) induced long-term potentiation (LTP), whereas brief high-frequency stimulation (HFS) evoked short-term potentiation that generally decayed within 10 min. When LFS was applied after recovery from the short-term potentiating effect of HFS, there was an initial transient enhancement followed by LTD. The switch in the direction of the enduring change in synaptic strength required activation of mGluR II (Li et al. 1998). LTP of synaptic responses could be reversed by LFS of afferent fibers, a phenomenon termed “depotentiation” (Barrionuevo et al. 1980; Staubli and Chun 1996). Previous studies indicated that LTP of synapses from auditory thalamus and cortex to the lateral amygdala (LA) underlay the encoding of fear memory (McKernan and Shinnick-Gallagher 1997; Rogan et al. 1997). Yet, it is not clear whether depotentiation contributes to behavioral extinction of fear memory. In the present study, we obtained evidence that activation of mGluR II induces depotentiation in rat amygdala slices. This chemical depotentiation is independent of NMDA receptor, L-type Ca++ channels, and calcineurin activity, but requires presynaptic activity and extracellular Ca++. By behavioral tests, we further demonstrate that administration of mGluR II agonists to the LA or basolateral amygdala (BLA) blocks the consolidation of fear memory measured with fear-potentiated startle.

Carbon profiles of PM1 and PM2.5 were measured simultaneously in the indoor and outdoor air of 10 homes in the Taipei urban area in both summer and winter seasons. It was indicated that the average indoor and outdoor concentrations were found to be 25.88 and 25.86 μg m −3 for PM1, as well as 37.60 and 37.26 μg m −3 for PM2.5, respectively. In addition, the average summer and winter levels were found to be 25.13 and 26.70 μg m −3 for PM1, as well as 36.45 and 38.51 μg m −3 for PM2.5, respectively. There were no significant concentration differences observed in indoor/outdoor (I/O) and summer/winter PM1/PM2.5 levels. Moreover, the median value of I/O ratios of PM1 and PM2.5 was observed to be 1.0 which might demonstrate that there was no presence of significant indoor PM sources in this subtropical region. In addition, it was found that PM1/PM2.5 mass ratios were 0.68. Regarding carbon profiles, it was observed that mass fractions of total carbon in PM1 and PM2.5 were 0.50 and 0.46. It was demonstrated that indoor/ambient combustion-related fine particle play an important role in urban residential environments.

Characteristics of PM 1 /PM 2.5 /PM 10 were evaluated in the ambient general (Chung-Shan) and traffic (Da-Tung) monitoring stations in the Taipei area. It was observed that average concentrations of PM 1 , PM 2.5 , and PM 10 were 14.0, 24.4, and 48.1 w g/m 3 in the Chung-Shan station, respectively. In the Da-Tung station, the mean levels of PM 1 , PM 2.5 , and PM 10 were 37.6, 42.8, and 61.5 w g/m 3 , respectively. In addition, it was found that PM 1 /PM 2.5 mass ratios could be as high as 0.90 in the traffic station. Regarding carbon profiles, it was observed that mass fractions of total carbon in PM 1 , PM 2.5 , and PM 10 were 0.58, 0.49, and 0.35 in the Chung-Shan station and 0.74, 0.66, and 0.53 in the Da-Tung station, respectively. It was clearly indicated that fine particles related to combustion sources play an important role in urban atmosphere. Further investigation is needed to evaluate chemical compositions and health effects of PM 1 .