Transcriptional control mechanisms in the wall of the urinary bladder. Myocardin family coactivators and the transcriptomic impact of denervation.

Urological problems can be caused by diseases that affect the bladder wall and its innervation. Contraction and relaxation of smooth muscle cells (SMCs) is essential for maintaining detrusor function and for emptying the bladder. It is well established that a family of regulatory proteins called Myocardin Related Transcription Factors (MRTFs) controls the property of SMCs. SMCs have a number of unique ultrastructural features, including caveolae in the membrane and “dense bodies” in the cytoplasm. An overarching aim of the work presented here was to investigate the regulatory mechanism of the key proteins in caveolae and dense bodies. The generation of caveolae depends on caveolins and cavins. We found that most caveolae proteins are regulated by MRTFs via proximal promoter sequences. MRTFs thus likely represent major drivers of formation of caveolae in SMCs. We also found that Cavin3 (Prkcdbp) is preferentially expressed in SMCs compared to other cells. Knockout of Cavin3 reduced the number of caveolae in the SMC membrane, but not elsewhere, and at the same time reduced Caveolin-1, Caveolin-3 and Cavin1. This suggests that Cavin3 contributes to the generation of caveolae in SMCs. Dense bodies in smooth muscle may be regarded as equivalents of Z-discs in striated muscle. The protein Nexilin (NEXN) was previously identified as a Z-disc-localized protein that controls mechanical stability. We found that Nexilin (NEXN) is highly expressed in SMCs and regulated by MRTF- and YAP/TAZ-coactivators. Nexilin was moreover found to be a dense body-associated protein in SMCs that promotes actin polymerization, differentiation and motility. Bladder wall remodelling in pathological situations is accompanied by reduced innervation. A second aim of this work was to define changes in gene expression after denervation. We show that numerous mRNAs and miRNAs are differentially expressed in the denervated bladder. Pathway analysis indicates that many of the differentially expressed genes are related to proliferation (60%). This is no surprise because the bladder weight increases 5-fold following denervation. Cthrc1 is upregulated at both the mRNA and protein levels, correlating with bladder weight, and overexpression and knockdown of this protein influences SMC proliferation in vitro. Neural plasticity is influenced by neurotrophic factors released from the target organs. We addressed if the bladder may influence its own nerve supply by such a mechanism. We demonstrate that neurotrophic factors, including NGF, BDNF, and NT-3, are synthesized in the bladder wall, and promote outgrowth of neurites from the pelvic ganglia in vitro, presumably via Trk-receptors. In conclusion, the studies summarized here provide understanding of the transcriptional control of key proteins and ultrastructural features of SMCs. They also unveil molecular mechanisms of bladder remodelling following denervation. Manipulation of some of the genes identified here represents promising strategies for recovering bladder function in disease

Neurite outgrowth in cultured mouse pelvic ganglia - Effects of neurotrophins and bladder tissue

Neurotrophic factors regulate survival and growth of neurons. The urinary bladder is innervated via both sympathetic and parasympathetic neurons located in the major pelvic ganglion. The aim of the present study was to characterize the effects of the neurotrophins nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3) on the sprouting rate of sympathetic and parasympathetic neurites from the female mouse ganglion. The pelvic ganglion was dissected out and attached to a petri dish and cultured in vitro. All three factors (BDNF, NT-3 and NGF) stimulated neurite outgrowth of both sympathetic and parasympathetic neurites although BDNF and NT-3 had a higher stimulatory effect on parasympathetic ganglion cells. The neurotrophin receptors TrkA, TrkB and TrkC were all expressed in neurons of the ganglia. Co-culture of ganglia with urinary bladder tissue, but not diaphragm tissue, increased the sprouting rate of neurites. Active forms of BDNF and NT-3 were detected in urinary bladder tissue using western blotting whereas tissue from the diaphragm expressed NGF. Neurite outgrowth from the pelvic ganglion was inhibited by a TrkB receptor antagonist. We therefore suggest that the urinary bladder releases trophic factors, including BDNF and NT-3, which regulate neurite outgrowth via activation of neuronal Trk-receptors. These findings could influence future strategies for developing pharmaceuticals to improve re-innervation due to bladder pathologies

Microstructure and Impact Toughness of Local-Dry Keyhole Tungsten Inert Gas Welded Joints

In this paper, the microstructure and impact toughness of a S32101 duplex stainless steel underwater local-dry keyhole tungsten inert gas welded joint were studied. The impact toughness value of the underwater weld metal reached 78% of the onshore weld metal, which is in accordance with the underwater welding standards. The proportion of austenite in the underwater weld metal was 0.9% lower than that of the onshore weld metal. The proportion of the Σ3 coincidence site lattice boundaries and random phase boundaries in the underwater weld metal, which significantly influence the impact toughness of the weld metal, were smaller than that of the onshore weld metal

Experimental Modeling of the Bifurcation Set Equation of the Chip-Splitting Catastrophe in Symmetrical Straight Double-Edged Cutting

The chip-splitting catastrophe (CSC) generated by symmetrical cutting with a straight double-edged tool will lead to a significant reduction in cutting force. This has enormous potential for energy-saving machining and for the design of energy efficient cutting tools. The premise of the utilization is to establish a mathematical model that can predict the critical conditions of CSC. However, no related literature has studied the prediction model of CSC. Therefore, this paper proposes an experimental method based on catastrophe theory to establish a model of CSC bifurcation set equations that can predict critical conditions. A total of 355 groups of experiments are conducted to observe the critical conditions of CSC in symmetrical straight double-edged cutting, and 22 groups of experimental data of the critical conditions were acquired. The modeling process is converted into the optimal solution of the function coefficient value when the mapping function from a set of actual control parameters to theoretical control parameters (u, v, w) is a linear function. The bifurcation set equation of CSC is established, which can predict CSC in the symmetrical cutting of a straight double-edged turning tool with any combination of edge angle and rake angle. With verification, it is found that the occurrence of CSC has obvious regularity, and the occurrence of CSC will lead to a maximum reduction of 64.68% in the specific cutting force. The predicted values of the critical cutting thickness for the CSC of the established equation are in good agreement with the experimental results (the average absolute error is 5.34%). This study lays the foundation for the energy-saving optimization of tool geometry and process parameters through the reasonable utilization of CSC

Cavin-3 (PRKCDBP) deficiency reduces the density of caveolae in smooth muscle

Cavins belong to a family of proteins that contribute to the formation of caveolae, which are membrane organelles with functional roles in muscle and fat. Here, we investigate the effect of cavin-3 ablation on vascular and urinary bladder structure and function. Arteries and urinary bladders from mice lacking cavin-3 (knockout: KO) and from controls (wild type: WT) were examined. Our studies revealed that the loss of cavin-3 resulted in ∼40% reduction of the caveolae protein cavin-1 in vascular and bladder smooth muscle. Electron microscopy demonstrated that the loss of cavin-3 was accompanied by a reduction of caveolae abundance by 40-45% in smooth muscle, whereas the density of caveolae in endothelial cells was unchanged. Vascular contraction in response to an α1-adrenergic agonist was normal but nitric-oxide-dependent relaxation was enhanced, in parallel with an increased relaxation on direct activation of soluble guanylyl cyclase (sGC). This was associated with an elevated expression of sGC, although blood pressure was similar in WT and KO mice. Contraction of the urinary bladder was not affected by the loss of cavin-3. The proteomic response to outlet obstruction, including STAT3 phosphorylation, the induction of synthetic markers and the repression of contractile markers were identical in WT and KO mice, the only exception being a curtailed induction of the Golgi protein GM130. Loss of cavin-3 thus reduces the number of caveolae in smooth muscle and partly destabilizes cavin-1 but the functional consequences are modest and include an elevated vascular sensitivity to nitric oxide and slightly disturbed Golgi homeostasis in situations of severe cellular stress

Effects of He-D Interaction on Irradiation-Induced Swelling in Fe9Cr Alloys

The atomic-scale defects such as (deuterium, helium)-vacancy clusters in nuclear energy materials are one of the causes for the deterioration of the macroscopic properties of materials. Unfortunately, they cannot be observed by transmission electron microscopy (TEM) before they grow to the nanometer scale. Positron annihilation spectroscopy (PAS) has been proven to be sensitive to open-volume defects, and could characterize the evolution of the size and concentration of the vacancy-like nanoclusters. We have investigated the effects of He-D interaction on the formation of nanoscale cavities in Fe9Cr alloys by PAS and TEM. The results show that small-sized bubbles are formed in the specimen irradiated with 5 × 1016 He+/cm2, and the subsequent implanted D-ions contribute to the growth of these helium bubbles. The most likely reason is that helium bubbles previously formed in the sample captured deuterium injected later, causing bubbles to grow. In the lower dose He-irradiated samples, a large number of small dislocations and vacancies are generated and form helium-vacancy clusters with the helium atoms

Prolonged Inhibitory Effects of Repeated Tibial Nerve Stimulation on the Micturition Reflex in Decorticated Rats

Objective: This study aimed to determine whether a short-term repeated stimulation of tibial nerve afferents induces a prolonged modulation effect on the micturition reflex in a decorticated rat model. Material and Methods: Fifteen female Sprague-Dawley rats (250-350 g) were fully decorticated and paralyzed in the study. Tibial nerve stimulation (TNS) was delivered by inserting two pairs of needle electrodes close to the nerves at the level of the medial malleolus. Constant flow cystometries (0.07 mL/min) at approximately ten-minute intervals were performed, and the micturition threshold volume (MTV) was recorded and used as a dependent variable. After four to five stable recordings, the tibial nerves of both sides were stimulated continuously for five minutes at 10 Hz and at an intensity of three times the threshold for alpha-motor axons. Six same stimulations were applied repeatedly, with an interval of five minutes between each stimulation. Mean MTV was calculated on the basis of several cystometries in each half-hour period before, during, and after the six repeated TNS. Results: During the experiment, all the animals survived in good condition with relatively stable micturition reflexes, and a significant increase in MTV was detected after TNS. The strongest effect (mean = 178%) was observed during the first 30 minutes after six repeated stimulations. This obvious threshold increase remained for at least five hours. Conclusions: A prolonged poststimulation modulatory effect on the micturition reflex was induced by short-term repeated TNS in decorticated rats. This study provides a theoretical explanation for the clinical benefit of TNS in patients with overactive bladder and suggests decorticated rats as a promising model for further investigation of the neurophysiological mechanisms underlying the bladder inhibitory response induced by TNS.Funding Agencies|Medical Scienti fic Research Foundation of Guangdong Province, China; Natural Science Foundation of Guangdong Province, China; National Natural Science Foundation of China; Chinese Postdoctoral Science Foundation; [2013A806]; [B2020011]; [2016A030307033]; [81802551]; [2020M672593]</p

Formation of vacancy-type defects and hydride introduced by irradiation in pure titanium

The effect of hydrogen ion irradiation on the formation of hydride and vacancy-type defects in pure titanium was investigated by X-ray diffraction (XRD), Doppler broadening spectrum (DBS), and coincidence Doppler broadening spectrum (CDB) based on slow positron beam. The XRD results showed that γ-TiH was observed in single hydrogen ion-irradiated and hydrogen + deuterium (H + D) sequentially irradiated specimens but not in the single deuterium plasma-implanted sample. This finding indicates that the vacancy-type defects caused by H ion irradiation greatly promoted the formation of γ-TiH. The DBS results showed that for the single D plasma-implanted sample, the damage peak was closer to the surface region and lower compared to the other two samples containing γ-TiH, indicating that vacancy generated by lattice distortion didn't diffuse to the depth of the matrix, and the concentration of that was smaller, which was not conducive to the deuteride formation. For the H + D sequentially irradiated sample, the number of hydrogen-vacancy complexes (HmVn) further increased compared with the single H ion-irradiated sample. Thus, the effective volume of vacancy defects was reduced, which led to the decrease in the S parameter

Array profiling reveals contribution of Cthrc1 to growth of the denervated rat urinary bladder

Bladder denervation and bladder outlet obstruction are urological conditions that cause bladder growth. Transcriptomic surveys in outlet obstruction have identified differentially expressed genes, but similar studies following denervation have not been done. This was addressed using a rat model in which the pelvic ganglia were cryo-ablated followed by bladder microarray analyses. At 10 days following denervation, bladder weight had increased 5.6-fold, and 2,890 mRNAs and 135 micro-RNAs (miRNAs) were differentially expressed. Comparison with array data from obstructed bladders demonstrated overlap between the conditions, and 10% of mRNAs changed significantly and in the same direction. Many mRNAs, including collagen triple helix repeat containing 1 ( Cthrc1), Prc1, Plod2, and Dkk3, and miRNAs, such as miR-212 and miR-29, resided in the shared signature. Discordantly regulated transcripts in the two models were rare, making up for <0.07% of all changes, and the gene products in this category localized to the urothelium of normal bladders. These transcripts may potentially be used to diagnose sensory denervation. Western blotting demonstrated directionally consistent changes at the protein level, with increases of, e.g., Cthrc1, Prc1, Plod2, and Dkk3. We chose Cthrc1 for further studies and found that Cthrc1 was induced in the smooth muscle cell (SMC) layer following denervation. TGF-β1 stimulation and miR-30d-5p inhibition increased Cthrc1 in bladder SMCs, and knockdown and overexpression of Cthrc1 reduced and increased SMC proliferation. This work defines common and distinguishing features of bladder denervation and obstruction and suggests a role for Cthrc1 in bladder growth following denervation