Changes in Group A Streptococcus emm Types Associated with Invasive Infections in Adults, Spain, 2023.

An increase in invasive group A Streptococcus infection was detected in the northeast of Spain in November 2022. A postpandemic decline in the diversity of circulating emm types involved in invasive group A Streptococcus was observed, along with the emergence of emm49 in this geographic area.

Development of Robust Quantitative Structure-Activity Relationship Models for CYP2C9, CYP2D6, and CYP3A4 Catalysis and Inhibition.

Cytochrome P450 enzymes are responsible for the metabolism of >75% of marketed drugs, making it essential to identify the contributions of individual cytochromes P450 to the total clearance of a new candidate drug. Overreliance on one cytochrome P450 for clearance levies a high risk of drug-drug interactions; and considering that several human cytochrome P450 enzymes are polymorphic, it can also lead to highly variable pharmacokinetics in the clinic. Thus, it would be advantageous to understand the likelihood of new chemical entities to interact with the major cytochrome P450 enzymes at an early stage in the drug discovery process. Typical screening assays using human liver microsomes do not provide sufficient information to distinguish the specific cytochromes P450 responsible for clearance. In this regard, we experimentally assessed the metabolic stability of ∼5000 compounds for the three most prominent xenobiotic metabolizing human cytochromes P450, i.e., CYP2C9, CYP2D6, and CYP3A4, and used the data sets to develop quantitative structure-activity relationship models for the prediction of high-clearance substrates for these enzymes. Screening library included the NCATS Pharmaceutical Collection, comprising clinically approved low-molecular-weight compounds, and an annotated library consisting of drug-like compounds. To identify inhibitors, the library was screened against a luminescence-based cytochrome P450 inhibition assay; and through crossreferencing hits from the two assays, we were able to distinguish substrates and inhibitors of these enzymes. The best substrate and inhibitor models (balanced accuracies ∼0.7), as well as the data used to develop these models, have been made publicly available (https://opendata.ncats.nih.gov/adme) to advance drug discovery across all research groups. SIGNIFICANCE STATEMENT: In drug discovery and development, drug candidates with indiscriminate cytochrome P450 metabolic profiles are considered advantageous, since they provide less risk of potential issues with cytochrome P450 polymorphisms and drug-drug interactions. This study developed robust substrate and inhibitor quantitative structure-activity relationship models for the three major xenobiotic metabolizing cytochromes P450, i.e., CYP2C9, CYP2D6, and CYP3A4. The use of these models early in drug discovery will enable project teams to strategize or pivot when necessary, thereby accelerating drug discovery research.

Dysfunctional voiding behavior and impaired muscle contractility in a rat model of detrusor underactivity.

AIMS: Detrusor underactivity (DU) is an understudied health concern with inadequate clinical management. The pathophysiology of DU is unclear, and current therapies fail to improve symptoms. The current studies characterized voiding function and contractility of bladder and urethral tissues in a novel rat model of DU. METHODS: Female obese prone (OP) and obese resistant (OR) rats were fed a 60 kcal% fat diet at 8 weeks old. A subset of rats (n = 4/strain) underwent uroflowmetry biweekly for 18 weeks in metabolic cages. At 40-56 weeks old, rats (n = 9-10/strain) underwent instrumented cystometry under urethane anesthesia. Following cystometry, bladder and urethral tissues (n = 8-9/strain) were harvested for in vitro assessments of contractility in response to carbachol, electric field stimulation, atropine, alpha, beta-methylene ATP, and caffeine. RESULTS: OP rats exhibited increased urinary frequency (p = 0.0031), decreased voided volume (p = 0.0093), and urine flow rate (p = 0.0064) compared to OR rats during uroflowmetry. Bethanechol (10 mg/kg) did not alter uroflowmetry parameters. During cystometry, OP rats exhibited decreased bladder emptying efficiency (p < 0.0001), decreased pressure to generate a void (p < 0.0001), and increased EUS activity during filling (p = 0.0011). Bladder contractility was decreased in OP rats when exposed to carbachol (p < 0.0003) and ATP (p = 0.0004), whereas middle urethral contractility was increased when exposed to carbachol (p = 0.0014), EFS (p = 0.0289), and caffeine (p = 0.0031). CONCLUSION: Impaired cholinergic and purinergic signaling in the bladder may contribute to poor voiding function in OP rats. In addition, increased urethral activity may engage a guarding reflex to augment continence and exacerbate incomplete emptying.

Advances in the study of drug metabolism - symposium report of the 12th Meeting of the International Society for the Study of Xenobiotics (ISSX).

The 12th International Society for the Study of Xenobiotics (ISSX) meeting, held in Portland, OR, USA from July 28 to 31, 2019, was attended by diverse members of the pharmaceutical sciences community. The ISSX New Investigators Group provides learning and professional growth opportunities for student and early career members of ISSX. To share meeting content with those who were unable to attend, the ISSX New Investigators herein elected to highlight the "Advances in the Study of Drug Metabolism" symposium, as it engaged attendees with diverse backgrounds. This session covered a wide range of current topics in drug metabolism research including predicting sites and routes of metabolism, metabolite identification, ligand docking, and medicinal and natural products chemistry, and highlighted approaches complemented by computational modeling. In silico tools have been increasingly applied in both academic and industrial settings, alongside traditional and evolving in vitro techniques, to strengthen and streamline pharmaceutical research. Approaches such as quantum mechanics simulations facilitate understanding of reaction energetics toward prediction of routes and sites of drug metabolism. Furthermore, in tandem with crystallographic and orthogonal wet lab techniques for structural validation of drug metabolizing enzymes, in silico models can aid understanding of substrate recognition by particular enzymes, identify metabolic soft spots and predict toxic metabolites for improved molecular design. Of note, integration of chemical synthesis and biosynthesis using natural products remains an important approach for identifying new chemical scaffolds in drug discovery. These subjects, compiled by the symposium organizers, presenters, and the ISSX New Investigators Group, are discussed in this review.

Inherent steroid 17α,20-lyase activity in defunct cytochrome P450 17A enzymes.

Cytochrome P450 (P450) 17A1 catalyzes the oxidations of progesterone and pregnenolone and is the major source of androgens. The enzyme catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction, and several mechanisms have been proposed for the latter step. Zebrafish P450 17A2 catalyzes only the 17α-hydroxylations. We previously reported high similarity of the crystal structures of zebrafish P450 17A1 and 17A2 and human P450 17A1. Five residues near the heme, which differed, were changed. We also crystallized this five-residue zebrafish P450 17A1 mutant, and the active site still resembled the structure in the other proteins, with some important differences. These P450 17A1 and 17A2 mutants had catalytic profiles more similar to each other than did the wildtype proteins. Docking with these structures can explain several minor products, which require multiple enzyme conformations. The 17α-hydroperoxy (OOH) derivatives of the steroids were used as oxygen surrogates. Human P450 17A1 and zebrafish P450s 17A1 and P450 17A2 readily converted these to the lyase products in the absence of other proteins or cofactors (with catalytically competent kinetics) plus hydroxylated 17α-hydroxysteroids. The 17α-OOH results indicate that a "Compound I" (FeO3+) intermediate is capable of formation and can be used to rationalize the products. We conclude that zebrafish P450 17A2 is capable of lyase activity with the 17α-OOH steroids because it can achieve an appropriate conformation for lyase catalysis in this system that is precluded in the conventional reaction.

Sensory pudendal nerve stimulation increases bladder capacity through sympathetic mechanisms in cyclophosphamide-induced cystitis rats.

AIMS: Interstitial cystitis and bladder pain syndrome is a prevalent health concern with inadequate treatments. Neuromodulation has emerged as a therapeutic option to treat patients refractory to standard care. The objective of this study was to determine the efficacy and mechanism(s) of sensory pudendal nerve stimulation on bladder function in cystitis rats. METHODS: Female rats were administered saline (n = 8) or cyclophosphamide (CYP, 150 mg/kg IP, n = 16) and single-trial cystometry experiments were conducted under urethane anesthesia 48 h after injection. Electrical stimulation (0.02-0.22 mA, 10-20 Hz) was delivered to the sensory branch of the pudendal nerve and its effect on the bladder and external urethral sphincter were measured. Stimulation trials were also conducted following bilateral hypogastric nerve transection (HGNT) or pharmacological inhibition of beta-adrenergic receptors (propranolol, 1 mg/kg IV) to determine the mechanisms of bladder inhibition. RESULTS: CYP-induced cystitis decreased bladder capacity (P = 0.0352) and bladder compliance (P = 0.024) by up to 38% of control. Electrical stimulation of the sensory pudendal nerve increased bladder capacity (P < 0.0001) in control and CYP rats by up to 51-52% of their respective baselines. HGNT did not influence bladder inhibition generated by sensory pudendal nerve stimulation in control rats, whereas HGNT and propranolol decreased the efficacy of electrical stimulation in CYP rats. CONCLUSIONS: Sympathetic reflex activity mediates sensory pudendal nerve stimulation in CYP treated but not control rats. These studies demonstrate an alternative approach to neuromodulation in cystitis and establish mechanistic changes during stimulation that may enable the development of novel therapeutics.

Kinetic processivity of the two-step oxidations of progesterone and pregnenolone to androgens by human cytochrome P450 17A1.

Cytochrome P450 (P450, CYP) 17A1 plays a critical role in steroid metabolism, catalyzing both the 17α-hydroxylation of pregnenolone and progesterone and the subsequent 17α,20-lyase reactions to form dehydroepiandrosterone (DHEA) and androstenedione (Andro), respectively, critical for generating glucocorticoids and androgens. Human P450 17A1 reaction rates examined are enhanced by the accessory protein cytochrome b5 (b5), but the exact role of b5 in P450 17A1-catalyzed reactions is unclear as are several details of these reactions. Here, we examined in detail the processivity of the 17α-hydroxylation and lyase steps. b5 did not enhance reaction rates by decreasing the koff rates of any of the steroids. Steroid binding to P450 17A1 was more complex than a simple two-state system. Pre-steady-state experiments indicated lag phases for Andro production from progesterone and for DHEA from pregnenolone, indicating a distributive character of the enzyme. However, we observed processivity in pregnenolone/DHEA pulse-chase experiments. (S)-Orteronel was three times more inhibitory toward the conversion of 17α-hydroxypregnenolone to DHEA than toward the 17α-hydroxylation of pregnenolone. IC50 values for (S)-orteronel were identical for blocking DHEA formation from pregnenolone and for 17α-hydroxylation, suggestive of processivity. Global kinetic modeling helped assign sets of rate constants for individual or groups of reactions, indicating that human P450 17A1 is an inherently distributive enzyme but that some processivity is present, i.e. some of the 17α-OH pregnenolone formed from pregnenolone did not dissociate from P450 17A1 before conversion to DHEA. Our results also suggest multiple conformations of P450 17A1, as previously proposed on the basis of NMR spectroscopy and X-ray crystallography.

Molecular mechanisms of isocitrate dehydrogenase 1 (IDH1) mutations identified in tumors: The role of size and hydrophobicity at residue 132 on catalytic efficiency.

Isocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate (ICT) to α-ketoglutarate (αKG) in the cytosol and peroxisomes. Mutations in IDH1 have been implicated in >80% of lower grade gliomas and secondary glioblastomas and primarily affect residue 132, which helps coordinate substrate binding. However, other mutations found in the active site have also been identified in tumors. IDH1 mutations typically result in a loss of catalytic activity, but many also can catalyze a new reaction, the NADPH-dependent reduction of αKG to d-2-hydroxyglutarate (D2HG). D2HG is a proposed oncometabolite that can competitively inhibit αKG-dependent enzymes. Some kinetic parameters have been reported for several IDH1 mutations, and there is evidence that mutant IDH1 enzymes vary widely in their ability to produce D2HG. We report that most IDH1 mutations identified in tumors are severely deficient in catalyzing the normal oxidation reaction, but that D2HG production efficiency varies among mutant enzymes up to ∼640-fold. Common IDH1 mutations have moderate catalytic efficiencies for D2HG production, whereas rarer mutations exhibit either very low or very high efficiencies. We then designed a series of experimental IDH1 mutants to understand the features that support D2HG production. We show that this new catalytic activity observed in tumors is supported by mutations at residue 132 that have a smaller van der Waals volume and are more hydrophobic. We report that one mutation can support both the normal and neomorphic reactions. These studies illuminate catalytic features of mutations found in the majority of patients with lower grade gliomas.

Mechanism of 17α,20-Lyase and New Hydroxylation Reactions of Human Cytochrome P450 17A1: 18O LABELING AND OXYGEN SURROGATE EVIDENCE FOR A ROLE OF A PERFERRYL OXYGEN.

Cytochrome P450 (P450) reactions can involve C-C bond cleavage, and several of these are critical in steroid and sterol biosynthesis. The mechanisms of P450s 11A1, 17A1, 19A1, and 51A1 have been controversial, in the context of the role of ferric peroxide (FeO2 (-)) versus perferryl (FeO(3+), compound I) chemistry. We reinvestigated the 17α-hydroxyprogesterone and 17α-hydroxypregnenolone 17α,20-lyase reactions of human P450 17A1 and found incorporation of one (18)O atom (from (18)O2) into acetic acid, consonant with proposals for a ferric peroxide mechanism (Akhtar, M., Lee-Robichaud, P., Akhtar, M. E., and Wright, J. N. (1997) J. Steroid Biochem. Mol. Biol. 61, 127-132; Akhtar, M., Wright, J. N., and Lee-Robichaud, P. (2011) J. Steroid Biochem. Mol. Biol. 125, 2-12). However, the reactions were supported by iodosylbenzene (a precursor of the FeO(3+) species) but not by H2O2 We propose three mechanisms that can involve the FeO(3+) entity and that explain the (18)O label in the acetic acid, two involving the intermediacy of an acetyl radical and one a steroid 17,20-dioxetane. P450 17A1 was found to perform 16-hydroxylation reactions on its 17α-hydroxylated products to yield 16,17α-dihydroxypregnenolone and progesterone, suggesting the presence of an active perferryloxo active species of P450 17A1 when its lyase substrate is bound. The 6ß-hydroxylation of 16α,17α-dihydroxyprogesterone and the oxidation of both 16α,17α-dihydroxyprogesterone and 16α,17α-dihydroxypregnenolone to 16-hydroxy lyase products were also observed. We provide evidence for the contribution of a compound I mechanism, although contribution of a ferric peroxide pathway in the 17α,20-lyase reaction cannot be excluded.

The effects of neuromodulation in a novel obese-prone rat model of detrusor underactivity.

Obesity is a global epidemic associated with an increased risk for lower urinary tract dysfunction. Inefficient voiding and urinary retention may arise in late-stage obesity when the expulsive force of the detrusor smooth muscle cannot overcome outlet resistance. Detrusor underactivity (DUA) and impaired contractility may contribute to the pathogenesis of nonobstructive urinary retention. We used cystometry and electrical stimulation of peripheral nerves (pudendal and pelvic nerves) to characterize and improve bladder function in urethane-anesthetized obese-prone (OP) and obese-resistant (OR) rats following diet-induced obesity (DIO). OP rats exhibited urinary retention and impaired detrusor contractility following DIO, reflected as increased volume threshold, decreased peak micturition pressure, and decreased voiding efficiency (VE) compared with OR rats. Electrical stimulation of the sensory branch of the pudendal nerve did not increase VE, whereas patterned bursting stimulation of the motor branch of the pudendal nerve increased VE twofold in OP rats. OP rats required increased amplitude of electrical stimulation of the pelvic nerve to elicit bladder contractions, and maximum evoked bladder contraction amplitudes were decreased relative to OR rats. Collectively, these studies characterize a novel animal model of DUA that can be used to determine pathophysiology and suggest that neuromodulation is a potential management option for DUA.

Purinergic signalling underlies transforming growth factor-ß-mediated bladder afferent nerve hyperexcitability.

KEY POINTS: The sensory components of the urinary bladder are responsible for the transduction of bladder filling and are often impaired with neurological injury or disease. Elevated extracellular ATP contributes, in part, to bladder afferent nerve hyperexcitability during urinary bladder inflammation or irritation. Transforming growth factor-ß1 (TGF-ß1) may stimulate ATP release from the urothelium through vesicular exocytosis mechanisms with minimal contribution from pannexin-1 channels to increase bladder afferent nerve discharge. Bladder afferent nerve hyperexcitability and urothelial ATP release with CYP-induced cystitis is decreased with TGF-ß inhibition. These results establish a causal link between an inflammatory mediator, TGF-ß, and intrinsic signalling mechanisms of the urothelium that may contribute to the altered sensory processing of bladder filling. ABSTRACT: The afferent limb of the micturition reflex is often compromised following bladder injury, disease and inflammatory conditions. We have previously demonstrated that transforming growth factor-ß (TGF-ß) signalling contributes to increased voiding frequency and decreased bladder capacity with cystitis. Despite the functional presence of TGF-ß in bladder inflammation, the precise mechanisms of TGF-ß mediating bladder dysfunction are not yet known. Thus, the present studies investigated the sensory components of the urinary bladder that may underlie the pathophysiology of aberrant TGF-ß activation. We utilized bladder-pelvic nerve preparations to characterize bladder afferent nerve discharge and the mechanisms of urothelial ATP release with distention. Our findings indicate that bladder afferent nerve discharge is sensitive to elevated extracellular ATP during pathological conditions of urinary bladder inflammation or irritation. We determined that TGF-ß1 may increase bladder afferent nerve excitability by stimulating ATP release from the urothelium via vesicular exocytosis mechanisms with minimal contribution from pannexin-1 channels. Furthermore, blocking aberrant TGF-ß signalling in cyclophosphamide-induced cystitis with TßR-1 inhibition decreased afferent nerve hyperexcitability with a concomitant decrease in urothelial ATP release. Taken together, these results establish a role for purinergic signalling mechanisms in TGF-ß-mediated bladder afferent nerve activation that may ultimately facilitate increased voiding frequency. The synergy between intrinsic urinary bladder signalling mechanisms and an inflammatory mediator provides novel insight into bladder dysfunction and supports new avenues for therapeutic intervention.

Structural and kinetic basis of steroid 17α,20-lyase activity in teleost fish cytochrome P450 17A1 and its absence in cytochrome P450 17A2.

Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progesterone (Prog) and pregnenolone (Preg) to glucocorticoids and androgens. In mammals, a single enzyme, P450 17A1, catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction with both Prog and Preg. Teleost fish contain two 17A P450s; zebrafish P450 17A1 catalyzes both 17α-hydroxylation and lyase reactions with Prog and Preg, and P450 17A2 is more efficient in pregnenolone 17α-hydroxylation but does not catalyze the lyase reaction, even in the presence of cytochrome b5. P450 17A2 binds all substrates and products, although more loosely than P450 17A1. Pulse-chase and kinetic spectral experiments and modeling established that the two-step P450 17A1 Prog oxidation is more distributive than the Preg reaction, i.e. 17α-OH product dissociates more prior to the lyase step. The drug orteronel selectively blocked the lyase reaction of P450 17A1 but only in the case of Prog. X-ray crystal structures of zebrafish P450 17A1 and 17A2 were obtained with the ligand abiraterone and with Prog for P450 17A2. Comparison of the two fish P450 17A-abiraterone structures with human P450 17A1 (DeVore, N. M., and Scott, E. E. (2013) Nature 482, 116-119) showed only a few differences near the active site, despite only ∼50% identity among the three proteins. The P450 17A2 structure differed in four residues near the heme periphery. These residues may allow the proposed alternative ferric peroxide mechanism for the lyase reaction, or residues removed from the active site may allow conformations that lead to the lyase activity.

Isotope-Labeling Studies Support the Electrophilic Compound I Iron Active Species, FeO(3+), for the Carbon-Carbon Bond Cleavage Reaction of the Cholesterol Side-Chain Cleavage Enzyme, Cytochrome P450 11A1.

The enzyme cytochrome P450 11A1 cleaves the C20-C22 carbon-carbon bond of cholesterol to form pregnenolone, the first 21-carbon precursor of all steroid hormones. Various reaction mechanisms are possible for the carbon-carbon bond cleavage step of P450 11A1, and most current proposals involve the oxoferryl active species, Compound I (FeO(3+)). Compound I can either (i) abstract an O-H hydrogen atom or (ii) be attacked by a nucleophilic hydroxy group of its substrate, 20R,22R-dihydroxycholesterol. The mechanism of this carbon-carbon bond cleavage step was tested using (18)O-labeled molecular oxygen and purified P450 11A1. P450 11A1 was incubated with 20R,22R-dihydroxycholesterol in the presence of molecular oxygen ((18)O2), and coupled assays were used to trap the labile (18)O atoms in the enzymatic products (i.e., isocaproaldehyde and pregnenolone). The resulting products were derivatized and the (18)O content was analyzed by high-resolution mass spectrometry. P450 11A1 showed no incorporation of an (18)O atom into either of its carbon-carbon bond cleavage products, pregnenolone and isocaproaldehyde . The positive control experiments established retention of the carbonyl oxygens in the enzymatic products during the trapping and derivatization processes. These results reveal a mechanism involving an electrophilic Compound I species that reacts with nucleophilic hydroxy groups in the 20R,22R-dihydroxycholesterol intermediate of the P450 11A1 reaction to produce the key steroid pregnenolone.

Acoustic firearm discharge detection and classification in an enclosed environment.

Two different signal processing algorithms are described for detection and classification of acoustic signals generated by firearm discharges in small enclosed spaces. The first is based on the logarithm of the signal energy. The second is a joint entropy. The current study indicates that a system using both signal energy and joint entropy would be able to both detect weapon discharges and classify weapon type, in small spaces, with high statistical certainty.

The effects of tempol on cyclophosphamide-induced oxidative stress in rat micturition reflexes.

We hypothesized that cyclophosphamide- (CYP-) induced cystitis results in oxidative stress and contributes to urinary bladder dysfunction. We determined (1) the expression of oxidative stress markers 3-nitrotyrosine (3-NT), reactive oxygen species (ROS)/reactive nitrogen species (RNS), inflammatory modulators, neuropeptides calcitonin gene-related peptide (CGRP), substance P (Sub P), and adenosine triphosphate (ATP) that contribute to the inflammatory process in the urinary tract and (2) the functional role of oxidative stress in urinary bladder dysfunction with an antioxidant, Tempol, (1 mM in drinking water) combined with conscious cystometry. In CYP-treated (4 hr or 48 hr; 150 mg/kg, i.p.) rats, ROS/RNS and 3-NT significantly (P ≤ 0.01) increased in urinary bladder. CYP treatment increased ATP, Sub P, and CGRP expression in the urinary bladder and cystometric fluid. In CYP-treated rats, Tempol significantly (P ≤ 0.01) increased bladder capacity and reduced voiding frequency compared to CYP-treated rats without Tempol. Tempol significantly (P ≤ 0.01) reduced ATP expression, 3-NT, and ROS/RNS expression in the urinary tract of CYP-treated rats. These studies demonstrate that reducing oxidative stress in CYP-induced cystitis improves urinary bladder function and reduces markers of oxidative stress and inflammation.

Bladder sensory physiology: neuroactive compounds and receptors, sensory transducers, and target-derived growth factors as targets to improve function.

Urinary bladder dysfunction presents a major problem in the clinical management of patients suffering from pathological conditions and neurological injuries or disorders. Currently, the etiology underlying altered visceral sensations from the urinary bladder that accompany the chronic pain syndrome, bladder pain syndrome (BPS)/interstitial cystitis (IC), is not known. Bladder irritation and inflammation are histopathological features that may underlie BPS/IC that can change the properties of lower urinary tract sensory pathways (e.g., peripheral and central sensitization, neurochemical plasticity) and contribute to exaggerated responses of peripheral bladder sensory pathways. Among the potential mediators of peripheral nociceptor sensitization and urinary bladder dysfunction are neuroactive compounds (e.g., purinergic and neuropeptide and receptor pathways), sensory transducers (e.g., transient receptor potential channels) and target-derived growth factors (e.g., nerve growth factor). We review studies related to the organization of the afferent limb of the micturition reflex and discuss neuroplasticity in an animal model of urinary bladder inflammation to increase the understanding of functional bladder disorders and to identify potential novel targets for development of therapeutic interventions. Given the heterogeneity of BPS/IC and the lack of consistent treatment benefits, it is unlikely that a single treatment directed at a single target in micturition reflex pathways will have a mass benefit. Thus, the identification of multiple targets is a prudent approach, and use of cocktail treatments directed at multiple targets should be considered.

Expression and function of transforming growth factor-ß isoforms and cognate receptors in the rat urinary bladder following cyclophosphamide-induced cystitis.

Numerous proinflammatory cytokines have been implicated in the reorganization of lower urinary tract function following cyclophosphamide (CYP)-induced cystitis. The present study investigated the functional profile of three pleiotropic transforming growth factor-ß (TGF-ß) isoforms and receptor (TßR) variants in the normal and inflamed (CYP-induced cystitis) rat urinary bladder. Our findings indicate that TGF-ß (1, 2, and 3) and TßR (1, 2, and 3) transcript and protein expression were regulated to varying degrees in the urothelium or detrusor smooth muscle following intermediate (48 h; 150 mg/kg ip) or chronic (75 mg/kg ip; once every 3 days for 10 days), but not acute (4 h; 150 mg/kg ip), CYP-induced cystitis. Conscious, open-outlet cystometry was performed to determine whether aberrant TGF-ß signaling contributes to urinary bladder dysfunction following intermediate (48 h) CYP-induced cystitis. TßR-1 inhibition with SB505124 (5 µM) significantly (p ≤ 0.001) decreased voiding frequency and increased bladder capacity (2.5-fold), void volume (2.6-fold), and intercontraction intervals (2.5-fold) in CYP-treated (48 h) rats. Taken together, these results provide evidence for 1) the involvement of TGF-ß in lower urinary tract neuroplasticity following urinary bladder inflammation, 2) a functional role of TGF-ß signaling in the afferent limb of the micturition reflex, and 3) urinary bladder TßR-1 as a viable target to reduce voiding frequency with cystitis.

Effect of peeling, cutting, or shredding of lettuce, carrot, or potato on the efficacy of chlorine disinfection.

Minimally processed vegetables are washed and subsequently disinfected by immersion in water solutions with antimicrobials which reduce the initial pathogenic or spoilage microbial load. Chlorine remains one of the most widely used disinfectants for vegetables and hence the importance of studying its properties. The aim of this study was to evaluate the effect of peeling, cutting, and shredding on the effectiveness of chlorine (200 ppm) as a disinfectant in lettuce, carrot, and potato. Three independent repetitions of each experiment were completed, and data was statistically analyzed. Results showed that the maintenance of the chlorine concentration in the disinfectant solution, over time, depended on the vegetables' preliminary processing technique (whole, peeled, cut, or shredded) (p < 0.05). In general, the disinfection treatments studied reduced Escherichia coli by 1-8 logs. The addition of chlorine in the disinfectant solution allowed greater reduction in E. coli than using water immersions (p < 0.05) and disinfection times longer than 5 min did not improve these microbiological reductions (p>0.05). The vegetables' subdivision (whole, peeled, cut, or shredded) can affect both E coli's reduction and the vegetables' residual chlorine concentration. No trend was observed in terms of sensory differences and their relationship to the vegetables' processing and disinfection. These results suggest that each facility must validate its disinfection processes, according to the conditions established on site and reduction goals related to initial microbial counts, vegetables' quality, processing operations, and other important aspects.

Antibiotic Susceptibility and Clarithromycin Resistance Determinants in Helicobacter pylori in the Northeast of Spain: A One-Year Prospective Study.

Helicobacter pylori is one of the most widespread infections, and it is reaching alarming resistance levels worldwide. The recommended first-line empirical treatment differs according to the local rate of clarithromycin resistance. Macrolide resistance is mainly associated with three point mutations in the 23S rRNA gene. The aim of this study was to describe the antibiotic susceptibility of H. pylori in our healthcare area and the main mechanisms involved in clarithromycin resistance. Gastric biopsies (n = 641) were collected and cultured in a one-year prospective study. Antibiotic susceptibility testing was performed by gradient diffusion. A multiplex real-time PCR test (AllplexTMH.pylori & ClariR Assay, Seegene) was used to detect the most frequent mutations associated with clarithromycin resistance. Overall, 141 isolates were available for antibiotic susceptibility testing. The highest resistance rates were detected in metronidazole and levofloxacin. The rate of clarithromycin resistance was 12.1%, and the associated mutations were A2143G and A2142G. More than half of the clarithromycin-resistant isolates presented high MIC values (>256 mg/L). Tetracycline resistance was not detected, suggesting that therapies that contain tetracycline could be a suitable option. The low clarithromycin resistance rate coupled with the high rates of metronidazole resistance may support the recovery of the classical triple therapy in our healthcare area.