Application of the Perme Score to assess mobility in patients with COVID-19 in inpatient units.

Objective: To evaluate the ability of the Perme Score to detect changes in the level of mobility of patients with COVID-19 outside the intensive care unit. Method: A retrospective cohort study was conducted in inpatient units of a private hospital. Patients older than 18, diagnosed with COVID-19, who were discharged from the intensive care unit and remained in the inpatient units were included. The variables collected included demographic characterization data, length of hospital stay, respiratory support, Perme Score values at admission to the inpatient unit and at hospital discharge and the mobilization phases performed during physical therapy. Result: A total of 69 patients were included, 80% male and with a mean age of 61.9 years (SD=12.5 years). The comparison of the Perme Score between the times of admission to the inpatient unit and at hospital discharge shows significant variation, with a mean increase of 7.3 points (95%CI:5.7-8.8; p<0.001), with estimated mean values of Perme Score at admission of 17.5 (15.8; 19.3) and hospital discharge of 24.8 (23.3; 26.3). There was no association between Perme Score values and length of hospital stay (measure of effect and 95%CI 0.929 (0.861; 1.002; p=0.058)). Conclusion: The Perme Score proved effective for assessing mobility in patients diagnosed with COVID-19 with prolonged hospitalization outside the intensive care setting. In addition, we demonstrated by the value of the Perme Score that the level of mobility increases significantly from the time of admission to inpatient units until hospital discharge. There was no association between the Perme Score value and length of hospital stay.

Analysis of mouse faecal dysbiosis, during the development of cachexia, induced by transplantation with Lewis lung carcinoma cells.

Cachexia (CC) is a complex wasting syndrome that significantly affects life quality and life expectancy among cancer patients. Original studies, in which CC was induced in mouse models through inoculation with BaF and C26 tumour cells, demonstrated that CC development correlates with bacterial gut dysbiosis in these animals. In both cases, a common microbial signature was observed, based on the expansion of Enterobacteriaceae in the gut of CC animals. However, these two types of tumours induce unique microbial profiles, suggesting that different CC induction mechanisms significantly impact the outcome of gut dysbiosis. The present study sought to expand the scope of such analyses by characterizing the CC-associated dysbiosis that develops when mice are inoculated with Lewis lung carcinoma (LLC) cells, which constitutes one of the most widely employed mechanisms for CC induction. Interestingly, Enterobacteriaceae expansion is also observed in LLC-induced CC. However, the dysbiosis identified herein displays a more complex pattern, involving representatives from seven different bacterial phyla, which were consistently identified across successive levels of taxonomic hierarchy. These results are supported by a predictive analysis of gene content, which identified a series of functional/structural changes that potentially occur in the gut bacterial population of these animals, providing a complementary and alternative approach to microbiome analyses based solely on taxonomic classification.

Preserved activity of soluble guanylate cyclase (sGC) in iliac artery from middle-aged rats: Role of sGC modulators.

Vascular aging leads to structural and functional changes. Iliac arteries (IA) provide blood flow to lower urinary tract and pelvic ischemia has been reported as an important factor for bladder remodeling and overactivity. Dysfunction of the nitric oxide (NO)-cyclic guanosine monophosphate pathway (cGMP) is one factor involved in the development of lower urinary tract (LUT) disorders. Therefore, we hypothesized that ageing-associated LUT disorders is a consequence of lower cGMP productions due to an oxidation of soluble guanylate cylase (sGC) that results in local ischemia. In the present study IA from middle-aged and young rats were isolated and the levels of NO, reactive oxygen species (ROS), the gene expression of the enzymes involved in the NO-pathway and concentration-response curves to the soluble guanylate (sGC) stimulator (BAY 41-2272), sGC activator (BAY 58-2667), tadalafil, acetylcholine (ACh) and sodium nitroprusside (SNP) were determined. In IA from middle-aged rats the gene expression for endothelial nitric oxide synthase and the ROS were lower and higher, respectively than the young group. The relaxations induced by ACh and SNP were significantly lower in IA from middle-aged rats. In IA from middle-aged rats the mRNA expression of PDE5 was 55% higher, accompanied by lower relaxation induced by tadalafil. On the other hand, the gene expression for sGCα1 were similar in IA from both groups. Both BAY 41-2272 and BAY 58-2667 produced concentration-dependent relaxations in IA from both groups, however, the latter was 9-times more potent than BAY 41-2272 and produced similar relaxations in IA in both middle-aged and young groups. Yet, the sGC oxidant, ODQ increased the relaxation and the cGMP levels induced by BAY 58-2667. On the other hand, in tissues stimulated with SNP, tadalafil and BAY-2272, the intracellular levels of cGMP were lower in IA from middle-aged than young rats. In conclusion, our results clearly showed that the relaxations induced by the endothelium-dependent and -independent agents, by the PDE5 inhibitor and by sGC stimulator were impaired in IA from aged rats, while that induced by sGC activator was preserved. It suggests that sGC activator may be advantageous in treating ischemia-related functional changes in the lower urinary tract organs in situations where the NO levels are reduced.

Transcriptomic profiling identifies novel transcripts, isomorphs, and noncoding RNAs in Paracoccidioides brasiliensis.

This paper describes a transcriptomic profiling of Paracoccidioides brasiliensis (Pb) performed with the aid of an RNA-seq-based approach, aimed at characterizing the general transcriptome in this human pathogenic fungus, responsible for paracoccidioidomycosis (PCM). Results confirm that ∼75% of the genes currently annotated in the P. brasiliensis genome are, in fact, transcribed in vivo and that ∼19% of them may display alternative isomorphs. Moreover, we identified 627 transcripts that do not match any gene currently mapped in the genome, represented by 114 coding transcripts (probably derived from previously unmapped protein-coding genes) and 513 noncoding RNAs (ncRNAs), including 203 long-noncoding RNAs (lncRNAs).

Anti-Brucella canis antibodies in dogs naturally infected with Leishmania infantum and associated histological alterations in the genital tract.

In canine visceral leishmaniasis, coinfections can aggravate the disease. Our aim was to investigate Brucella canis in dogs infected with Leishmania infantum. One hundred and six L. infantum-seropositive dogs were submitted to serology for B. canis, PCR for B. canis and L. infantum, and histopathological analysis of the genital tract. Anti-B. canis antibodies were detected in seven dogs whose clinical signs, L. infantum load and histological alterations were similar to those of seronegative animals. The circulation of anti-B. canis antibodies was low but demonstrates the exposure of dogs to this bacterium in a visceral leishmaniasis-endemic area.

The Quorum Sensing Auto-Inducer 2 (AI-2) Stimulates Nitrogen Fixation and Favors Ethanol Production over Biomass Accumulation in Zymomonas mobilis.

Autoinducer 2 (or AI-2) is one of the molecules used by bacteria to trigger the Quorum Sensing (QS) response, which activates expression of genes involved in a series of alternative mechanisms, when cells reach high population densities (including bioluminescence, motility, biofilm formation, stress resistance, and production of public goods, or pathogenicity factors, among others). Contrary to most autoinducers, AI-2 can induce QS responses in both Gram-negative and Gram-positive bacteria, and has been suggested to constitute a trans-specific system of bacterial communication, capable of affecting even bacteria that cannot produce this autoinducer. In this work, we demonstrate that the ethanologenic Gram-negative bacterium Zymomonas mobilis (a non-AI-2 producer) responds to exogenous AI-2 by modulating expression of genes involved in mechanisms typically associated with QS in other bacteria, such as motility, DNA repair, and nitrogen fixation. Interestingly, the metabolism of AI-2-induced Z. mobilis cells seems to favor ethanol production over biomass accumulation, probably as an adaptation to the high-energy demand of N2 fixation. This opens the possibility of employing AI-2 during the industrial production of second-generation ethanol, as a way to boost N2 fixation by these bacteria, which could reduce costs associated with the use of nitrogen-based fertilizers, without compromising ethanol production in industrial plants.

Characterization of noise emitted by a low-profile tractor and its influence on the health of rural workers.

Noise is one of the main physical agents in the rural work environment and it can be harmful to the health of workers exposed to it. Thus, research on the spatialization of noise emitted by agricultural machinery can help minimize harmful health effects. This study sets out to evaluate the spatial distribution of the noise emitted by an agricultural tractor with and without the activation of a rotary cutter. A wave model geostatistical analysis was used to identify healthy working zones according to current legislation. The experiment was carried out using a 75 hp tractor at 2000 rpm with the rotary cutter on the on and off modes. A digital sound level meter was used to register noise in a regular 2.0 x 2.0 m sample mesh within a 10-meter radius. The semivariogram was adjusted using the weighted least squares method, wave model and kriging interpolation to obtain the maps. The magnitude and spatial structure of the noise emitted by the tractor were identified. The results show that the equipment produced noise levels above the limits recommended by Brazilian regulatory standard NR 15. Thus, both the machine operator and the workers involved in the operation should use Personal Protective Equipment.

Reconstitution of autophagy ameliorates vascular function and arterial stiffening in spontaneously hypertensive rats.

Insufficient autophagy has been proposed as a mechanism of cellular aging, as this leads to the accumulation of dysfunctional macromolecules and organelles. Premature vascular aging occurs in hypertension. In fact, many factors that contribute to the deterioration of vascular function as we age are accelerated in clinical and experimental hypertension. Previously, we have reported decreased autophagy in arteries from spontaneously hypertensive rats (SHRs); however, the effects of restoring autophagic activity on blood pressure and vascular function are currently unknown. We hypothesized that reconstitution of arterial autophagy in SHRs would decrease blood pressure and improve endothelium-dependent relaxation. We treated 14- to 18-wk-old Wistar rats (n = 7 vehicle and n = 8 trehalose) and SHRs (n = 7/group) with autophagy activator trehalose (2% in drinking water) for 28 days. Blood pressure was measured by radiotelemetry, and vascular function and structure were measured in isolated mesenteric resistance arteries (MRAs) using wire and pressure myographs, respectively. Treatment with trehalose had no effect on blood pressure in SHRs; however, isolated MRAs presented enhanced relaxation to acetylcholine, in a cyclooxygenase- and reactive oxygen species-dependent manner. Similarly, trehalose treatment shifted the relaxation to the Rho kinase (ROCK) inhibitor Y-27632 to the right, indicating reduced ROCK activity. Finally, trehalose treatment decreased arterial stiffness as indicated by the slope of the stress-strain curve. Overall these data indicate that reconstitution of arterial autophagy in SHRs improves endothelial and vascular smooth muscle function, which could synergize to prevent stiffening. As a result, restoration of autophagic activity could be a novel therapeutic for premature vascular aging in hypertension.NEW & NOTEWORTHY This work supports the concept that diminished arterial autophagy contributes to premature vascular aging in hypertension and that therapeutic reconstitution of autophagic activity can ameliorate this phenotype. As vascular age is a new clinically used index for cardiovascular risk, understanding this mechanism may assist in the development of new drugs to prevent premature vascular aging in hypertension.

Dugong: a Docker image, based on Ubuntu Linux, focused on reproducibility and replicability for bioinformatics analyses.

Summary: This manuscript introduces and describes Dugong, a Docker image based on Ubuntu 16.04, which automates installation of more than 3500 bioinformatics tools (along with their respective libraries and dependencies), in alternative computational environments. The software operates through a user-friendly XFCE4 graphic interface that allows software management and installation by users not fully familiarized with the Linux command line and provides the Jupyter Notebook to assist in the delivery and exchange of consistent and reproducible protocols and results across laboratories, assisting in the development of open science projects. Availability and implementation: Source code and instructions for local installation are available at https://github.com/DugongBioinformatics, under the MIT open source license. Contact: Luiz.nunes@ufabc.edu.br.

Formyl peptide receptor-1 activation exerts a critical role for the dynamic plasticity of arteries via actin polymerization.

Several human diseases, include cancer and stroke are characterized by changes in immune system activation and vascular contractility. However, the mechanistic foundation of a vascular immuno-physiology network is still largely unknown. Formyl peptide receptor-1 (FPR-1), which plays a vital role in the function of the innate immune system, is widely expressed in arteries, but its role in vascular plasticity is unclear. We questioned why a receptor that is crucial for immune defense, and cell motility in leukocytes, would be expressed in vascular smooth muscle cells (VSMCs). We hypothesized that activation of FPR-1 in arteries is important for the temporal reorganization of actin filaments, and consequently, changes in vascular function, similar to what is observed in neutrophils. To address our hypothesis, we used FPR-1 knockout and VSMCs lacking FPR-1. We observed that FPR-1 activation induces actin polymerization in wild type VSMCs. Absence of FPR-1 in the vasculature significantly decreased vascular contraction and induced loss of myogenic tone to elevated intraluminal pressures via disruption of actin polymerization. Actin polymerization activator ameliorated these responses. In conclusion, we have established a novel role for FPR-1 in VSMC contractility and motility, similar to the one observed in sentinel cells of the innate immune system. This discovery is fundamental for vascular immuno-pathophysiology, given that FPR-1 in VSMCs not only functions as an immune system receptor, but it also has an important role for the dynamic plasticity of arteries.

Deletion or pharmacological blockade of TLR4 confers protection against cyclophosphamide-induced mouse cystitis.

Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS) is a chronic inflammatory disease without consistently effective treatment. We investigate the role of toll-like receptor 4 (TLR4) on voiding dysfunction and inflammation in the cyclophosphamide (CYP)-induced mouse cystitis. Male C57BL/6 [wild-type, (WT)] and/or TLR4 knockout (TLR4-/-) mice were treated with an injection of CYP (300 mg/kg, 24 h) or saline (10 ml/kg). The pharmacological blockade of the TLR4 by resatorvid (10 mg/kg) was also performed 1 h prior CYP-injection in WT mice. Urodynamic profiles were assessed by voiding stain on filter paper and filling cystometry. Contractile responses to carbachol were measured in isolated bladders. In CYP-exposed WT mice, mRNA for TLR4, myeloid differentiation primary response 88, and TIR-domain-containing adapter-inducing interferon-ß increased by 45%, 72%, and 38%, respectively ( P < 0.05). In free-moving mice, CYP-exposed mice exhibited a higher number of urinary spots and smaller urinary volumes. Increases of micturition frequency and nonvoiding contractions, concomitant with decreases of intercontraction intervals and capacity, were observed in the filling cystometry of WT mice ( P < 0.05). Carbachol-induced bladder contractions were significantly reduced in the CYP group, which was paralleled by reduced mRNA for M2 and M3 muscarinic receptors. These functional and molecular alterations induced by CYP were prevented in TLR4-/- and resatorvid-treated mice. Additionally, the increased levels of inflammatory markers induced by CYP exposure, myeloperoxidase activity, interleukin-6, and tumor necrosis factor-alpha were significantly reduced by resatorvid treatment. Our findings reveal a central role for the TLR4 signaling pathway in initiating CYP-induced bladder dysfunction and inflammation and thus emphasize that TLR4 receptor blockade may have clinical value for IC/BPS treatment.

Oxidative Stress in Aortas of Patients with Advanced Occlusive and Aneurysmal Diseases.

BACKGROUND: Aortoiliac occlusive disease (AOD) and abdominal aortic aneurysm (AAA) are very important cardiovascular diseases that present different aspects of pathophysiology; however, oxidative stress and inflammatory response seem be relevant in both of them. Our objective was to evaluate oxidative damage and degree of inflammatory infiltrate in aortas of patients surgically treated for AOD and AAA. MATERIALS AND METHODS: Levels of reactive oxygen species (ROS), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, and myeloperoxidase (MPO) expression as well as nitrite levels and superoxide dismutase (SOD) and catalase (CAT) activities were evaluated in aortas of patients with AOD (n = 16) or AAA (n = 14), while the control group was formed by cadaveric organ donors (n = 10). We also analyzed the degree of inflammatory infiltrate in these aortas. RESULTS: There was an increase in ROS levels and NADPH oxidase activity in patients with AOD and AAA when compared with the control group, and the AOD group demonstrated higher ROS production and NADPH oxidase activity and also nitrite levels when compared with the AAA group (P < 0.001). On the other hand, an increase of SOD activity in the AOD group and CAT activity in the AAA group was observed. Inflammatory infiltrate and MPO expression were higher in the AOD group when compared with the control group (P < 0.05). CONCLUSIONS: Oxidative stress is relevant in both AOD and AAA, though AOD presented higher ROS levels and NADPH activity. Increased activities of antioxidant enzymes may be a compensatory phenomenon which occurs in aortas of patients with AOD and AAA. Perhaps, a relationship between oxidative stress and degree of inflammatory infiltrate may exist in the pathophysiology of AOD and AAA.

How important is the α1-adrenoceptor in primate and rodent proximal urethra? Sex differences in the contribution of α1-adrenoceptor to urethral contractility.

Urethral smooth muscle (USM) contributes to urinary continence by contracting during the urine storage phase, which is mainly mediated by activation of postjunctional α1-adrenoceptors. Males and females show differences in the functioning of the lower urinary tract and the most common urinary tract symptoms (LUTS). LUTS in men typically occur in association with bladder outlet obstruction, whereas in women urinary urge-incontinence symptoms are more common. Therefore, this study aimed to evaluate sex differences in α1-adrenoceptor subtype expression and their importance in proximal urethra contraction in the mouse (C57BL6/J) and marmoset (Callithrix jacchus). Contractile responses to phenylephrine, norepinephrine, potassium chloride (KCl), and electrical-field stimulation (EFS) were evaluated. Phenylephrine, norepinephrine, KCl, and EFS produced markedly greater contractions in male mice and marmoset USM compared with females. The sex differences remained unchanged by Nω-nitro-l-arginine (l-NAME; nitric oxide synthase inhibitor), atropine (muscarinic receptor antagonist), and PPADS (P2X1-purinoceptor antagonist). Additionally, selective α1A (but not α1B- and α1D-)-adrenoceptor antagonists significantly reduced phenylephrine-induced USM contractions. qRT-PCR for α1A-, B-, and D-adrenoceptor subtypes revealed a marked presence of the α1A-adrenoceptor subtype in male USM, but not females. Male mouse urethra also exhibited a higher tyrosine hydroxylase mRNA expression. Histomorphometric analysis showed a greater USM area in male than female mice. In conclusion, male mouse and marmoset proximal USM shows strong α1A- adrenoceptor-induced contractions and abundant α1A-adrenoceptor expression, whereas α1A-adrenoceptor-mediated mechanisms are much less important in females. The differential expression of α1-adrenoceptors in the proximal urethra may contribute to the higher incidence of urinary incontinence in women and obstructed voiding in men.

Long-term treatment with the beta-3 adrenoceptor agonist, mirabegron ameliorates detrusor overactivity and restores cyclic adenosine monophosphate (cAMP) levels in obese mice.

AIMS: To evaluate the effects of the beta-3 adrenoceptor agonist, mirabegron in a mouse model of detrusor overactivity induced by obesity. METHODS: C57BL/6 male mice were fed with standard chow or high-fat diet for 12 weeks. Lean and obese mice were treated orally with mirabegron (10 mg/kg/day) from the last 2 weeks of diet. Cystometric evaluations, functional assays, protein expression for phosphodiesterase type 4 (PDE4), and cyclic adenosine monophosphate (cAMP) measurement were carried out. RESULTS: In obese mice the body weight, epididymal fat mass, fasting glucose, and low-density lipoprotein (LDL) levels were higher (P < 0.001) than in the lean mice. A reduction of 34% and 54% and an increase of 35% in the epididimal fat, LDL, and HDL levels (P < 0.05), respectively, were observed in the obese group treated with mirabegron, whereas no changes were seen in the lipid profile from lean mice. Obese group showed irregular micturition pattern, characterized by significant increases in frequency and non-void contractions. Carbachol, potassium chloride, and electrical-field stimulation induced detrusor smooth muscle (DSM) contractions, which were greater in bladders from obese mice than from lean mice. Two-week treatment with mirabegron restored all the contractile response alterations in the DSM. Basal intracellular levels of cAMP were reduced (68%), whereas PDE4 protein expression was increased (54%) in bladder from obese mice. Mirabegron restored the cAMP levels in obese bladder, without changing the PDE4 expression. CONCLUSION: Mirabegron was able to completely restore the urinary alterations seen in the bladder from obese mice.

Implication of Rho-kinase and soluble guanylyl cyclase enzymes in prostate smooth muscle dysfunction in middle-aged rats.

AIMS: Aging is highly associated with benign prostate hyperplasia (BPH). We investigated here the alterations of the contractile and relaxant machinery in prostates of middle-aged rats, focusing on the Rho-kinase, nitric oxide (NO)-soluble guanylyl cyclase (sGC), α1- and ß-adrenoceptor pathways. METHODS: Male Wistar young (3.5-month old) and middle-aged rats (10-month old) were used. Quantitative image analysis of prostates and functional assays evaluating the prostate contractions and relaxations were employed. Measurement of [3 H]-noradrenaline efflux, western blotting for α1 and ß1 sGC subunits, and cyclic nucleotide levels were carried out. RESULTS: Prostates of middle-aged rats showed significant increases in lumen and smooth muscle cells, but no alterations in the relative prostate weight were observed. In vivo, noradrenaline (10-7 -10-4 g/kg) produced greater prostatic contractions in middle-aged compared with control rats. Likewise, the in vitro contractions to phenylephrine (1 nM-100 µM) and α,ß-methylene ATP (1-10 µM) were greater in middle-aged rats. Electrical-field stimulation (EFS, 1-32 Hz) promoted higher [3 H]-noradrenaline efflux and prostate contractions in middle-aged rats. Reduced expressions of α1 and ß1 sGC subunits and diminished NO-mediated prostate relaxations in middle-age were observed. Isoproterenol-induced relaxations and cAMP levels were reduced in prostates of middle-aged rats. The Rho-kinase inhibitor fasudil (50 mg/kg, 2 weeks) normalized the prostate hypercontractility in middle-age rats. CONCLUSIONS: Prostate hypercontractility in middle-aging is associated with increased release of noradrenaline and Rho-kinase pathway, as well as with impairments of NO-sGC and ß-adrenoceptor pathways. Middle-aged rats are suitable to explore the enhanced prostatic tone in the absence of prostate overgrowth. Neurourol. Urodynam. 36:589-596, 2017. © 2016 Wiley Periodicals, Inc.

Curcumin attenuates memory deficits and the impairment of cholinergic and purinergic signaling in rats chronically exposed to cadmium.

This study investigated the protective effect of curcumin on memory loss and on the alteration of acetylcholinesterase and ectonucleotidases activities in rats exposed chronically to cadmium (Cd). Rats received Cd (1 mg/kg) and curcumin (30, 60, or 90 mg/kg) by oral gavage 5 days a week for 3 months. The animals were divided into eight groups: vehicle (saline/oil), saline/curcumin 30 mg/kg, saline/curcumin 60 mg/kg, saline/curcumin 90 mg/kg, Cd/oil, Cd/curcumin 30 mg/kg, Cd/curcumin 60 mg/kg, and Cd/curcumin 90 mg/kg. Curcumin prevented the decrease in the step-down latency induced by Cd. In cerebral cortex synaptosomes, Cd-exposed rats showed an increase in acetylcholinesterase and NTPDase (ATP and ADP as substrates) activities and a decrease in the 5'-nucleotidase activity. Curcumin was not able to prevent the effect of Cd on acetylcholinesterase activity, but it prevented the effects caused by Cd on NTPDase (ATP and ADP as substrate) and 5'-nucleotidase activities. Increased acetylcholinesterase activity was observed in different brain structures, whole blood and lymphocytes of the Cd-treated group. In addition, Cd increased lipid peroxidation in different brain structures. Higher doses of curcumin were more effective in preventing these effects. These findings show that curcumin prevented the Cd-mediated memory impairment, demonstrating that this compound has a neuroprotective role and is capable of modulating acetylcholinesterase, NTPDase, and 5'-nucleotidase activities. Finally, it highlights the possibility of using curcumin as an adjuvant against toxicological conditions involving Cd exposure. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 70-83, 2017.

Activation of soluble guanylyl cyclase by BAY 58-2667 improves bladder function in cyclophosphamide-induced cystitis in mice.

Activators of soluble guanylyl cyclase (sGC) interact directly with its prosthetic heme group, enhancing the enzyme responsiveness in pathological conditions. This study aimed to evaluate the effects of the sGC activator BAY 58-2667 on voiding dysfunction, protein expressions of α1 and ß1 sGC subunits and cGMP levels in the bladder tissues after cyclophosphamide (CYP) exposure. Female C57BL/6 mice (20-25 g) were injected with CYP (300 mg/kg ip) to induce cystitis. Mice were pretreated or not with BAY 58-2667 (1 mg/kg, gavage), given 1 h before CYP injection. The micturition patterns and in vitro bladder contractions were evaluated at 24 h. In freely moving mice, the CYP injection produced reduced the micturition volume and increased the number of urine spots. Cystometric recordings in CYP-injected mice revealed significant increases in basal pressure, voiding frequency, and nonvoiding contractions (NVCs), along with decreases in bladder capacity, intercontraction interval, and compliance. BAY 58-2667 significantly prevented the micturition alterations observed in both freely moving mice and cystometry and normalized the reduced in vitro carbachol-induced contractions in the CYP group. Reduced protein expressions of α1 and ß1 sGC subunits and of cGMP levels were observed in the CYP group, all of which were prevented by BAY 58-2667. CYP exposure significantly increased reactive-oxygen species (ROS) generation in both detrusor and urothelium, and this was normalized by BAY 58-2667. The increased myeloperoxidase and cyclooxygenase-2 activities in the bladders of the CYP group remained unchanged by BAY 58-2667. Activators of sGC may constitute a novel and promising therapeutic approach for management of interstitial cystitis.

The beta-3 adrenoceptor agonist, mirabegron relaxes isolated prostate from human and rabbit: new therapeutic indication?

BACKGROUND: Alpha1 (α1)-blockers, 5-alpha reductase and phosphodiesterase type-5 inhibitors are pharmacological classes currently available for benign prostatic hyperplasia (BPH) treatment. Mirabegron, a beta-3 adrenoceptor (ß3-AR) agonist has been approved for the therapy of overactive bladder and may constitute a new therapeutic option for BPH treatment. This study is aimed to evaluate the in vitro effects of mirabegron in human and rabbit prostatic smooth muscle. METHODS: In rabbit prostate, electrical field stimulation (EFS)-induced contraction and concentration-response curve (CRC) to mirabegron in phenylephrine pre-contracted tissues were carried out. The potency (pEC50 ) and maximal response (Emax ) values were determined. In human prostate, CRC to phenylephrine was carried out in the absence and presence of mirabegron. Immunohistochemistry analysis for ß3-AR was also carried out. RESULTS: In human prostate, immunohistochemistry analysis revealed the presence of ß3-AR on the transition zone and mirabegron reduced by 42% the phenylephrine-induced contractions. In rabbit prostate, mirabegron produced concentration-dependent relaxations (pEC50 : 6.01 ± 0.12; Emax : 106 ± 3%), which were fully resistant to the blockade of ß1-AR and ß2-AR. The ß3-AR blocker L748,337 caused a six-fold rightward shift in mirabegron-induced relaxations. Mirabegron (10 µM) reduced by 63% the EFS-induced contractions. Inhibitors of nitric oxide (L-NAME) and of soluble guanylate cyclase (ODQ) along with a cocktail of K+ channel blockers (apamin, charybdotoxin, glibenclamide, tetraethylammonium) all failed to significantly affect the mirabegron-induced rabbit relaxations. CONCLUSION: Mirabegron relaxes prostatic smooth muscle, providing an experimental support for the clinical investigation of its combination with an α1-blockers or PDE5 inhibitors in the treatment of BPH. Prostate 75:440-447, 2015. © 2014 Wiley Periodicals, Inc.

NTPDase and 5'-nucleotidase activities in synaptosomes of rabbits experimentally infected with BoHV-5.

Bovine herpesvirus type 5 (BoHV-5) is the causative agent of herpetic meningoencephalitis in cattle. The purinergic system is described as a modulator of the immune response and neuroinflammation. These functions are related to the extracellular nucleotides concentration. NTPDase and 5'-nucleotidase are enzymes responsible for controlling the extracellular concentration of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine (ADO). The aim of this study is to determinate the ectonucleotidase activity in cortical synaptosomes and synaptosomes from the hippocampus of rabbits experimentally infected with BoHV-5. Rabbits were divided into four groups, two control groups (non-inoculated animals), and two infected groups (inoculated with BoHV-5). The infected groups received 0.5 ml of BoHV-5 suspension with 10(7.5)TCID50 of viral strain SV-507/99, per paranasal sinuses, and the control groups received 0.5 ml of minimum essential media per paranasal sinuses. Animals were submitted to euthanasia on days 7 and 12 post-inoculation (p.i.); cerebral cortex and hippocampus were collected for the synaptosomes isolation and posterior determination of the ectonucleotidase activities. The results showed a decrease (P < 0.05) in ectonucleotidase activity in synaptosomes from the cerebral cortex of infected rabbits, whereas an increased (P < 0.05) ectonucleotidase activity was observed in synaptosomes from the hippocampus. These differences may be related with the heterogeneous distribution of ectonucleotidases in the different brain regions and also with the viral infectivity. Therefore, it is possible to speculate that BoHV-5 replication results in changes in ectonucleotidase activity in the brain, which may contribute to the neurological signs commonly observed in this disease.

Soluble guanylyl cyclase (sGC) degradation and impairment of nitric oxide-mediated responses in urethra from obese mice: reversal by the sGC activator BAY 60-2770.

Obesity has emerged as a major contributing risk factor for overactive bladder (OAB), but no study examined urethral smooth muscle (USM) dysfunction as a predisposing factor to obesity-induced OAB. This study investigated the USM relaxant machinery in obese mice and whether soluble guanylyl cyclase (sGC) activation with BAY 60-2770 [acid 4-({(4-carboxybutyl) [2-(5-fluoro-2-{[4-(trifluoromethyl) biphenyl-4-yl] methoxy} phenyl) ethyl] amino} methyl) benzoic] rescues the urethral reactivity through improvement of sGC-cGMP (cyclic guanosine monophosphate) signaling. Male C57BL/6 mice were fed for 12 weeks with a high-fat diet to induce obesity. Separate groups of animals were treated with BAY 60-2770 (1 mg/kg per day for 2 weeks). Functional assays and measurements of cGMP, reactive-oxygen species (ROS), and sGC protein expression in USM were determined. USM relaxations induced by NO (acidified sodium nitrite), NO donors (S-nitrosoglutathione and glyceryl trinitrate), and BAY 41-2272 [5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine] (sGC stimulator) were markedly reduced in obese compared with lean mice. In contrast, USM relaxations induced by BAY 60-2770 (sGC activator) were 43% greater in obese mice (P < 0.05), which was accompanied by increases in cGMP levels. Oxidation of sGC with ODQ [1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one] (10 µM) potentiated BAY 60-2770-induced USM responses in the lean group. Long-term oral BAY 60-2770 administration fully prevented the impairment of USM relaxations in obese mice. Reactive-oxygen species (ROS) production was enhanced, but protein expression of ß1 second guanylate cyclase subunit was reduced in USM from obese mice, both of which were restored by BAY 60-2770 treatment. In conclusion, impaired USM relaxation in obese mice is associated with ROS generation and down-regulation of sGC-cGMP signaling. Prevention of sGC degradation by BAY 60-2770 ameliorates the impairment of urethral relaxations in obese mice.