Genetic Elimination of Connective Tissue Growth Factor in the Forebrain Affects Subplate Neurons in the Cortex and Oligodendrocytes in the Underlying White Matter

Connective tissue growth factor (CTGF) is a secreted extracellular matrix-associated protein, which play a role in regulating various cellular functions. Although the expression of CTGF has been reported in the cortical subplate, its function is still not clear. Thus, to explore the significance of CTGF in the brain, we created a forebrain-specific Ctgf knockout (FbCtgf KO) mouse model. By crossing Ctgffl/fl mice with Emx1-Cre transgenic mice, in which the expression of Cre is prenatally initiated, the full length Ctgf is removed in the forebrain structures. In young adult (2–3 months old) FbCtgf KO mice, subplate markers such as Nurr1 and Cplx3 are still expressed in the cortical layer VIb; however, the density of the subplate neurons is increased. Interestingly, in these mutants, we found a reduced structural complexity in the subplate neurons. The distribution patterns of neurons and glial cells, examined by immunohistochemistry, are comparable between genotypes in the somatosensory cortex. However, increased densities of mature oligodendrocytes, but not immature ones, were noticed in the external capsule underneath the cortical layer VIb in young adult FbCtgf KO mice. The features of myelinated axons in the external capsule were then examined using electron microscopy. Unexpectedly, the thickness of the myelin sheath was reduced in middle-aged (>12 months old), but not young adult FbCtgf KO mice. Our results suggest a secretory function of the subplate neurons, through the release of CTGF, which regulates the density and dendritic branching of subplate neurons as well as the maturation and function of nearby oligodendrocytes in the white matter

Bcl-x Pre-mRNA splicing regulates brain injury after neonatal hypoxia-ischemia

The bcl-x gene appears to play a critical role in regulating apoptosis in the developing and mature central nervous system (CNS) and following CNS injury. Two isoforms of Bcl-x are produced as a result of alternative pre-mRNA splicing: Bcl-x(L) (the long form) is anti-apoptotic, while Bcl-x(S) (short form) is pro-apoptotic. Despite the antagonistic activities of these two isoforms, little is known about how regulation of alternative splicing of bcl-x may mediate neural cell apoptosis. Here, we report that apoptotic stimuli (staurosporine or C2-ceramide) reciprocally altered Bcl-x splicing in neural cells, decreasing Bcl-x(L) while increasing Bcl-x(S). Specific knockdown of Bcl-x(S) attenuated apoptosis. In order to further define regulatory elements that influenced Bcl-x splicing, a Bcl-x minigene was constructed. Deletional analysis revealed several consensus sequences within intron 2 that altered splicing. We found that the splicing factor, CUG-binding-protein-1 (CUGBP1), bound to a consensus sequence close to the Bcl-x(L) 5′ splice site, altering the Bcl-x(L)/Bcl-x(S) ratio and influencing cell death. In vivo, neonatal hypoxia-ischemia reciprocally altered Bcl-x pre-mRNA splicing, similar to the in vitro studies. Manipulation of the splice isoforms using viral gene transfer of Bcl-x(S) shRNA into the hippocampus of rats prior to neonatal hypoxia-ischemia decreased vulnerability to injury. Moreover, alterations in nuclear CUGBP1 preceded Bcl-x splicing changes. These results suggest that alternative pre-mRNA splicing may be an important regulatory mechanism for cell death after acute neurological injury, and may potentially provide novel targets for intervention

The design of a prospective, randomized, open-labeled study to compare the efficacy of lercanidipine with amlodipine on renal function in hypertensive patients aged at least 55 years (LEADER study)

AbstractBackgroundAlthough all classes of antihypertensive treatment can successfully reduce morbidity and mortality of cardiac pathology, prevention of target organ damages is of great importance beyond blood pressure lowering. Unlike most dihydropyridines, lercanidipine dilates both afferent and the efferent arterioles of nephrons, so it may provide renoprotective effects, which other CCBs may not have. The main purpose of this study is to compare the renoprotective effect of lercanidipine and amlodipine among hypertensive people aged 55years and older with newly diagnosed hypertension or those who were treatment-naïve for one month.MethodsThe study is a prospective, open-labelled, randomized, controlled trial to enrol 232 hypertensive patients aged ≥55 years. Subjects will be randomized into lercanidipine arm (10–20mg/day) and amlodipine arm (5–10mg/day) by 1:1 ratio. The dosage can be up-titrated to 20mg/day (lercanidipine group) and 10mg/day (amlodipine group), respectively, at week 4 or any following visit thereafter. Efficacy and safety data will be collected at week 4, 12 and 24 by evaluating the blood pressure lowering, estimated glomerular filtration rate, creatinine clearance, and urine albumin-creatinine ratio.ConclusionsThe reno-protective effects of new generation of CCBs such as lercanidipine administered to patients with hypertension are not investigated well. After all, this study will bring benefit to older patients who need drugs with both excellent anti-hypertensive and reno-protective efficacy. And the results will be provided for future treatment guideline of elder population in Taiwan

Abnormal splicing switch of DMD's penultimate exon compromises muscle fibre maintenance in myotonic dystrophy

International audienceMyotonic Dystrophy type 1 (DM1) is a dominant neuromuscular disease caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors resulting in alternative splicing misregulation and muscular dysfunction. Here we show that the abnormal splicing of DMD exon 78 found in dystrophic muscles of DM1 patients is due to the functional loss of MBNL1 and leads to the re-expression of an embryonic dystrophin in place of the adult isoform. Forced expression of embryonic dystrophin in zebrafish using an exon-skipping approach severely impairs the mobility and muscle architecture. Moreover, reproducing Dmd exon 78 missplicing switch in mice induces muscle fibre remodelling and ultrastructural abnormalities including ringed fibres, sarcoplasmic masses or Z-band disorganization, which are characteristic features of dystrophic DM1 skeletal muscles. Thus, we propose that splicing misregulation of DMD exon 78 compromises muscle fibre maintenance and contributes to the progressive dystrophic process in DM

Non-nosocomial healthcare-associated infective endocarditis in Taiwan: an underrecognized disease with poor outcome

<p>Abstract</p> <p>Background</p> <p>Non-nosocomial healthcare-associated infective endocarditis (NNHCA-IE) is a new category of IE of increasing importance. This study described the clinical and microbiological characteristics and outcome of NNHCA-IE in Taiwan.</p> <p>Methods</p> <p>A retrospective study was conducted of all patients with IE admitted to the Kaohsiung Veterans General Hospital in Kaohsiung, Taiwan over a five-year period from July 2004 to July 2009. The clinical and microbiological features of NNHCA-IE were compared to those of community-acquired and nosocomial IE. Predictors for in-hospital death were determined.</p> <p>Results</p> <p>Two-hundred episodes of confirmed IE occurred during the study period. These included 148 (74%) community-acquired, 30 (15%) non-nosocomial healthcare-associated, and 22 (11%) nosocomial healthcare-associated IE. <it>Staphylococcus aureus </it>was the most frequent pathogen. Patients with NNHCA-IE compared to community-acquired IE, were older (median age, 67 vs. 44, years, <it>p </it>< 0.001), had more MRSA (43.3% vs. 9.5%, <it>p </it>< 0.001), more comorbidity conditions (median Charlson comorbidity index [interquartile range], 4[2-6] vs. 0[0-1], <it>p </it>< 0.001), a higher in-hospital mortality (50.0% vs. 17.6%, <it>p </it>< 0.001) and were less frequently recognized by clinicians on admission (16.7% vs. 47.7%, <it>p </it>= 0.002). The overall in-hospital mortality rate for all patients with IE was 25%. Shock was the strongest risk factor for in-hospital death (odds ratio 7.8, 95% confidence interval 2.4-25.2, <it>p </it>< 0.001).</p> <p>Conclusions</p> <p>NNHCA-IE is underrecognized and carries a high mortality rate. Early recognition is crucial to provide optimal management and improve outcome.</p

A comparison of ARMS and direct sequencing for EGFR mutation analysis and Tyrosine Kinase Inhibitors treatment prediction in body fluid samples of Non-Small-Cell Lung Cancer patients

<p>Abstract</p> <p>Background</p> <p>Epidermal growth factor receptor (<it>EGFR</it>) mutation is strongly associated with the therapeutic effect of tyrosine kinase inhibitors (TKIs) in patients with non-small-cell lung cancer (NSCLC). Nevertheless, tumor tissue that needed for mutation analysis is frequently unavailable. Body fluid was considered to be a feasible substitute for the analysis, but arising problems in clinical practice such as relatively lower mutation rate and poor clinical correlation are not yet fully resolved.</p> <p>Method</p> <p>In this study, 50 patients (32 pleural fluids and 18 plasmas) with TKIs therapy experience and with direct sequencing results were selected from 220 patients for further analysis. The <it>EGFR </it>mutation status was re-evaluated by Amplification Refractory Mutation System (ARMS), and the clinical outcomes of TKIs were analyzed retrospectively.</p> <p>Results</p> <p>As compared with direct sequencing, 16 positive and 23 negative patients were confirmed by ARMS, and the other 11 former negative patients (6 pleural fluids and 5 plasmas) were redefined as positive, with a fairly well clinical outcome (7 PR, 3 SD, and 1 PD). The objective response rate (ORR) of positive patients was significant, 81.3% (direct sequencing) and 72.7% (ARMS) for pleural fluids, and 80% (ARMS) for plasma. Notably, even reclassified by ARMS, the ORR for negative patients was still relatively high, 60% for pleural fluids and 46.2% for plasma.</p> <p>Conclusions</p> <p>When using body fluids for <it>EGFR </it>mutation analysis, positive result is consistently a good indicator for TKIs therapy, and the predictive effect was no less than that of tumor tissue, no matter what method was employed. However, even reclassified by ARMS, the correlation between negative results and clinical outcome of TKIs was still unsatisfied. The results indicated that false negative mutation still existed, which may be settled by using method with sensitivity to single DNA molecule or by optimizing the extraction procedure with RNA or CTC to ensure adequate amount of tumor-derived nucleic acid for the test.</p

Muscleblind-Like 1 Knockout Mice Reveal Novel Splicing Defects in the Myotonic Dystrophy Brain

Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by a CTG trinucleotide repeat expansion (CTGexp) in the DMPK gene. In skeletal muscle, nuclear sequestration of the alternative splicing factor muscleblind-like 1 (MBNL1) explains the majority of the alternative splicing defects observed in the HSALR transgenic mouse model which expresses a pathogenic range CTGexp. In the present study, we addressed the possibility that MBNL1 sequestration by CUGexp RNA also contributes to splicing defects in the mammalian brain. We examined RNA from the brains of homozygous Mbnl1ΔE3/ΔE3 knockout mice using splicing-sensitive microarrays. We used RT-PCR to validate a subset of alternative cassette exons identified by microarray analysis with brain tissues from Mbnl1ΔE3/ΔE3 knockout mice and post-mortem DM1 patients. Surprisingly, splicing-sensitive microarray analysis of Mbnl1ΔE3/ΔE3 brains yielded only 14 candidates for mis-spliced exons. While we confirmed that several of these splicing events are perturbed in both Mbnl1 knockout and DM1 brains, the extent of splicing mis-regulation in the mouse model was significantly less than observed in DM1. Additionally, several alternative exons, including Grin1 exon 4, App exon 7 and Mapt exons 3 and 9, which have previously been reported to be aberrantly spliced in human DM1 brain, were spliced normally in the Mbnl1 knockout brain. The sequestration of MBNL1 by CUGexp RNA results in some of the aberrant splicing events in the DM1 brain. However, we conclude that other factors, possibly other MBNL proteins, likely contribute to splicing mis-regulation in the DM1 brain