Interstitial cell of Cajal-like cells (ICC-LC) exhibit dynamic spontaneous activity but are not functionally innervated in mouse urethra.

Urethral smooth muscle cells (USMC) contract to occlude the internal urethral sphincter during bladder filling. Interstitial cells also exist in urethral smooth muscles and are hypothesized to influence USMC behaviours and neural responses. These cells are similar to Kit+ interstitial cells of Cajal (ICC), which are gastrointestinal pacemakers and neuroeffectors. Isolated urethral ICC-like cells (ICC-LC) exhibit spontaneous intracellular Ca2+ signalling behaviours that suggest these cells may serve as pacemakers or neuromodulators similar to ICC in the gut, although observation and direct stimulation of ICC-LC within intact urethral tissues is lacking. We used mice with cell-specific expression of the Ca2+ indicator, GCaMP6f, driven off the endogenous promoter for Kit (Kit-GCaMP6f mice) to identify ICC-LC in situ within urethra muscles and to characterize spontaneous and nerve-evoked Ca2+ signalling. ICC-LC generated Ca2+ waves spontaneously that propagated on average 40.1 ± 0.7 µm, with varying amplitudes, durations, and spatial spread. These events originated from multiple firing sites in cells and the activity between sites was not coordinated. ICC-LC in urethra formed clusters but not interconnected networks. No evidence for entrainment of Ca2+ signalling between ICC-LC was obtained. Ca2+ events in ICC-LC were unaffected by nifedipine but were abolished by cyclopiazonic acid and decreased by an antagonist of Orai Ca2+ channels (GSK-7975A). Phenylephrine increased Ca2+ event frequency but a nitric oxide donor (DEA-NONOate) had no effect. Electrical field stimulation (EFS, 10 Hz) of intrinsic nerves, which evoked contractions of urethral rings and increased Ca2+ event firing in USMC, failed to evoke responses in ICC-LC. Our data suggest that urethral ICC-LC are spontaneously active but are not regulated by autonomic neurons.

Tas1R3 Dependent and Independent Recognition of Sugars in the Urethra and the Role of Tuft Cells in this Process.

Increased sugar concentrations on mucosal surfaces display risk factors for infections. This study aims to clarify sugar monitoring in the urethra. Urethral tuft cells (UTC) are known sentinels monitoring the urethral lumen for potentially harmful substances and initiating protective mechanisms. Next-generation sequencing (NGS), RT-PCR, and immunohistochemistry show expression of the taste receptor Tas1R3 in murine UTC, a crucial component of the classical sweet detection pathway. Isolated UTC respond to various sugars with an increase of intracellular [Ca2+]. The Tas1R3 inhibitor gurmarin and Tas1R3 deletion reduces these responses. Utilizing mice lacking UTC, glibenclamide, a K+-ATP channel antagonist, and phlorizin, a SGLT1 inhibitor, reveal an additional Tas1R3 independent sweet detection pathway. Inhibition of both pathways abrogates the sugar responses. Rat cystometry shows that intraurethral application of sucrose and glucose increases detrusor muscle activity Tas1R3 dependently. Sugar monitoring in the urethra occurs via two distinct pathways. A Tas1R3 dependent pathway, exclusive to UTC, and a Tas1R3 independent sweet detection pathway, which can be found both in UTC and in other urethral epithelial cells.

Single cell transcriptomic analysis of external genitalia reveals complex and sexually dimorphic cell populations in the early genital tubercle.

External genital organs are among the most recognizable sexually dimorphic characters. The penis and clitoris develop from the embryonic genital tubercle, an outgrowth at the anterior margin of the cloaca that undergoes an extensive period of development in male and female embryos prior to the onset of sexual differentiation. In mice, differentiation into the penis and clitoris begins around embryonic day (E)15.5. Current knowledge of cell types that comprise the genital tubercle is limited to a few studies that have fate mapped derivatives of endoderm, mesoderm, and ectoderm. Here we use single cell transcriptomics to characterize the cell populations in the genital tubercles of male and female mouse embryos at E14.5, approximately 24 â€‹h before the onset of sexual differentiation, and we present the first comprehensive atlas of single-cell gene expression during external genital development. Clustering analyses and annotation using marker genes shows 19 distinct cell populations in E14.5 genital tubercles. Mapping of cell clusters to anatomical locations using in situ gene expression patterns revealed granularity of cellular specializations and positional identities. Although E14.5 precedes sexually dimorphic morphogenesis of the genital tubercle, comparative analysis of males and females identified sexual dimorphisms at the single cell level, including male-specific cell clusters with transcriptional signatures of smooth muscle and bone progenitors, both of which are known to be sexually dimorphic in adult genitalia, as well as immune cells. These results provide a new resource for classification of external genital cell types based on gene expression profiles and reveal sex-specific cellular specializations in the early genital tubercle.

Fabrication of Adipose-Derived Stem Cell-Based Self-Assembled Scaffold under Hypoxia and Mechanical Stimulation for Urethral Tissue Engineering.

Long urethral strictures are often treated with autologous genital skin and buccal mucosa grafts; however, risk of hair ingrowth and donor site morbidity, restrict their application. To overcome this, we introduced a tissue-engineered human urethra comprising adipose-derived stem cell (ASC)-based self-assembled scaffold, human urothelial cells (UCs) and smooth muscle cells (SMCs). ASCs were cultured with ascorbic acid to stimulate extracellular matrix (ECM) production. The scaffold (ECM) was stained with collagen type-I antibody and the thickness was measured under a confocal microscope. Results showed that the thickest scaffold (28.06 ± 0.59 µm) was achieved with 3 × 104 cells/cm2 seeding density, 100 µg/mL ascorbic acid concentration under hypoxic and dynamic culture condition. The biocompatibility assessment showed that UCs and SMCs seeded on the scaffold could proliferate and maintain the expression of their markers (CK7, CK20, UPIa, and UPII) and (α-SMA, MHC and Smootheline), respectively, after 14 days of in vitro culture. ECM gene expression analysis showed that the ASC and dermal fibroblast-based scaffolds (control) were comparable. The ASC-based scaffold can be handled and removed from the plate. This suggests that multiple layers of scaffold can be stacked to form the urothelium (seeded with UCs), submucosal layer (ASCs only), and smooth muscle layer (seeded with SMCs) and has the potential to be developed into a fully functional human urethra for urethral reconstructive surgeries.

Transscrotal stimulation of the testes and epididymides improves urethral sperm collection in domestic cats.

Urethral catheterisation after medetomidine administration is the method of choice for semen collection in cats, but it yields variable results. This study tested whether scrotal manual stimulation can improve urethral sperm collection in domestic cats. The study was performed on 20 male cats, from which two urethral semen samples were collected, one before and one after 2min of transscrotal finger massage of the testes and epididymides. Both sperm samples were assessed for total sperm count and motility using computer-aided sperm analysis, viability and morphology (eosin-nigrosin staining). The transscrotal manual stimulation allowed a significantly higher number of spermatozoa to be obtained (P=0.0015). Viability was similar before and after the stimulation (median 92% and 90.5%), whereas the number of motile (median 60% and 70%) and morphologically normal (median 17% and 30.5%) spermatozoa was higher in the second sample (P=0.03 and P=0.002 respectively), which confirms that transscrotal massage induced the expulsion of a fresh pool of spermatozoa into the urethra. Transscrotal stimulation of the testes and epididymides significantly improves urethral semen collection in domestic cats and can be easily introduced into clinical practice.

Frontiers in urethra regeneration: current state and future perspective.

Despite the positive achievements attained, the treatment of male urethral strictures and hypospadiases still remains a challenge, particularly in cases of severe urethral defects. Complications and the need for additional interventions in such cases are common. Also, shortage of autologous tissue for graft harvesting and significant morbidity in the location of harvesting present problems and often lead to staged treatment. Tissue engineering provides a promising alternative to the current sources of grafts for urethroplasty. Since the first experiments in urethral substitution with tissue engineered grafts, this topic in regenerative medicine has grown remarkably, as many different types of tissue-engineered grafts and approaches in graft design have been suggested and testedin vivo. However, there have been only a few clinical trials of tissue-engineered grafts in urethral substitution, involving hardly more than a hundred patients overall. This indicates that the topic is still in its inception, and the search for the best graft design is continuing. The current review focuses on the state of the art in urethral regeneration with tissue engineering technology. It gives a comprehensive overview of the components of the tissue-engineered graft and an overview of the steps in graft development. Different cell sources, types of scaffolds, assembling approaches, options for vascularization enhancement and preclinical models are considered.

ANO1 in urethral SMCs contributes to sex differences in urethral spontaneous tone.

Stress urinary incontinence (SUI) is more common in women than in men, and sex differences in anatomic structure and physiology have been suggested as causes; however, the underlying cellular and molecular mechanisms remain unclear. The spontaneous tone (STT) of the urethra has been shown to have a fundamental effect on preventing the occurrence of SUI. Here, we investigated whether the urethral STT exhibited sex differences. First, we isolated urethral smooth muscle (USM) and detected STT in female mice and women. No STT was found in male mice or men. Furthermore, caffeine induced increased contractility and intracellular Ca2+ concentration in urethrae from female mice compared with male mice. EACT [an N-aroylaminothiazole, anoctamin-1 (ANO1) activator] elicited increased intracellular Ca2+ concentration and stronger currents in female mice than in male mice. Moreover, ANO1 expression in single USM cells from women and female mice was almost twofold higher than that found in cells from men and male mice. In summary, ANO1 in USM contributes to sex differences in urethral spontaneous tone. This finding may provide new guidance for the treatment of SUI in women and men.

Transcriptomic Analysis of Human Mesenchymal Stem Cell Therapy in Incontinent Rat Injured Urethra.

Periurethral human mesenchymal stem cell (hMSC) injections are associated with functional improvement in animal models of postpartum stress urinary incontinence (SUI). However, limited data exist on the role of hMSCs in modulating gene expression in tissue repair after urethral injury. To this end, we quantified temporal gene expression modulation in hMSCs, and in injured rat urethral tissue, using RNA-seq in an animal model of SUI, over a 3-day period following urethral injury, and local hMSC injection. We injected PKH fluorescent-labeled hMSC into the periurethral space of rats following a 4 h vaginal distention (VD) (three rats per time point). Control rats underwent VD injury only, and all animals were euthanized at 12, 24, 36, 72 h postinjury. Rat urethral and vaginal tissues were frozen and sectioned. Fluorescent labeled hMSCs were distinguished from adjacent, unlabeled rat urethral tissue. RNA was prepared from hMSCs and urethral tissue obtained by laser dissection of frozen tissue sections and sequenced on an Illumina HiSeq 2500. Differentially expressed genes (DEGs) over 72 h were evaluated using a two-group t-test (p < 0.05). Our transcriptional analyses identified candidate genes involved in tissue injury that were broadly sorted by injury and exposure to hMSC throughout the first 72 h of acute phase of injury. DEGs in treated urethra, compared with untreated urethra, were functionally associated with tissue repair, angiogenesis, neurogenesis, and oxidative stress suppression. DEGs included a variety of cytokines, extracellular matrix stabilization and regeneration genes, cytokine signaling modification, cell cycle regulation, muscle differentiation, and stabilization. Moreover, our results revealed DEG changes in hMSCs (PKH-labeled) harvested from injured urethra. The expressions are related to DNA damage repair, transcription activation, stem cell regulation, cell survival, apoptosis, self-renewal, cell proliferation, migration, and injury response. Impact statement Stress urinary incontinence (SUI) affects nearly half of women over 40, resulting in reduced quality of life and increased health care cost. Development of SUI is multifactorial and strongly associated with vaginal delivery. While stem cell therapy in animal models of SUI and limited preliminary clinical trials demonstrate functional improvement of SUI, the role of stem cell therapy in modulating tissue repair is unclear impeding advanced clinical trials. Our work provides a new understanding of the transcriptional mechanisms with which human mesenchymal stem cells improve acute injury repair thus guiding the development of cell-based therapies for women with nonacute established SUI.

Urethral luminal epithelia are castration-insensitive cells of the proximal prostate.

BACKGROUND: Castration-insensitive epithelial progenitors capable of regenerating the prostate have been proposed to be concentrated in the proximal region based on facultative assays. Functional characterization of prostate epithelial populations isolated with individual cell surface markers has failed to provide a consensus on the anatomical and transcriptional identity of proximal prostate progenitors. METHODS: Here, we use single-cell RNA sequencing to obtain a complete transcriptomic profile of all epithelial cells in the mouse prostate and urethra to objectively identify cellular subtypes. Pan-transcriptomic comparison to human prostate cell types identified a mouse equivalent of human urethral luminal cells, which highly expressed putative prostate progenitor markers. Validation of the urethral luminal cell cluster was performed using immunostaining and flow cytometry. RESULTS: Our data reveal that previously identified facultative progenitors marked by Trop2, Sca-1, KRT4, and PSCA are actually luminal epithelial cells of the urethra that extend into the proximal region of the prostate, and are resistant to castration-induced androgen deprivation. Mouse urethral luminal cells were identified to be the equivalent of previously identified human club and hillock cells that similarly extend into proximal prostate ducts. Benign prostatic hyperplasia (BPH) has long been considered an "embryonic reawakening," but the cellular origin of the hyperplastic growth concentrated in the periurethral region is unclear. We demonstrate an increase in urethral luminal cells within glandular nodules from BPH patients. Urethral luminal cells are further increased in patients treated with a 5-α reductase inhibitor. CONCLUSIONS: Our data demonstrate that cells of the proximal prostate that express putative progenitor markers, and are enriched by castration in the proximal prostate, are urethral luminal cells and that these cells may play an important role in the etiology of human BPH.

Longitudinal muscular column in the prostatic urethral wall: Its form, shape, and possible function based on mathematical simulation in ejaculation.

BACKGROUND: The shape and function of the longitudinal muscular column (LMC) of the prostate have not been established in detail. The present study was undertaken to elucidate the roles of the LMC of the posterior wall of the prostatic urethra (PSU) in the emission phase of ejaculation by investigating the form and muscular arrangement of the LMC. METHODS: Prostates and urinary bladders were obtained from 14 Korean adult cadavers. Nine specimens were histologically analyzed using hematoxylin and eosin, Masson's trichrome, and Verhoeff-van Gieson staining. Two specimens were scanned using microcomputed tomography (micro-CT), and all scanned images were reconstructed into a three-dimensional model. RESULTS: At the proximal level of the prostate, the ejaculatory ducts (EDs) and prostatic utricle (PU) together were surrounded by circular smooth-muscle fibers. However, at the seminal colliculus (SC) where the EDs and PU opened, they were mainly surrounded by an abundance of longitudinal fibers. The longitudinal fibers posterior to the EDs and PU formed a distinctive LMC in the posterior urethral wall. In histologic sections and micro-CT images, the LMC extended distally from the level of the SC to the level of the membranous urethra (MBU). We simulated a potential mechanism of LMC using a mathematical model of its movements. CONCLUSIONS: Comprehensive analyses based on in-depth assessment of histologic characteristics and micro-CT images demonstrated extension of the LMC from the level of the SC to the level of the MBU, enabling a better understanding of ejaculation physiology involving the LMC. These results suggest that the LMC in the posterior wall of the PSU is a critical component of ejaculation by facilitating the ejection of seminal vesicle fluid into the PSU via well-coordinated contractions.

Preparation and Characterization of Nano-Laponite/PLGA Composite Scaffolds for Urethra Tissue Engineering.

The purpose of this study was to construct a biomimetic urethral repair substitute. The nano-Laponite/polylactic acid-glycolic acid copolymer (PLGA) fiber scaffolds were produced to replicate the natural human urethra tissue microenvironment. PLGA (molar ratio 50:50) and Laponite were used in this study as raw materials. The nano-Laponite/PLGA scaffolds were fabricated via electrospinning technology. After preparing the material, the microstructural and mechanical properties of the nano-Laponite/PLGA scaffold were tested via scanning electron microscopy and electronic universal testing. The effects of different amounts of Laponite on the degradation of the nano-Laponite/PLGA scaffold were studied. Human umbilical vein endothelial cells (HUVECs) were co-cultured with PLGA and nano-Laponite/PLGA scaffolds for 24, 48, or 72 h. Scanning electron microscopy results illustrated that the microstructure of the scaffold fabricated by electrospinning was similar to that of the natural extracellular matrix. When the electrospinning liquid contained 10% Laponite, the nano-Laponite/PLGA stress-strain curve illustrated that the scaffold has strong elastic deformation ability. HUVECs exhibited good growth on the nano-Laponite/PLGA scaffold. When the scaffold contained 1% Laponite, the cell proliferation rate in the CCK-8 test was significantly better than that for the other three materials, displaying good cell culture characteristics. The 1% nano-Laponite/PLGA composite scaffold can be used as a suitable urethral repair material, but its performance requires further development and research.

Development of Male External Urethral Sphincter and Tissue-Resident Stem/Progenitor Cells in Rats.

Stress urinary incontinence (SUI) after prostate surgery is primarily caused by urethral sphincter damage. There are few effective therapeutic approaches for male SUI due to both insufficient study of the structure of the external urethral sphincter (EUS) and incomplete understanding of the resident EUS stem/progenitor cells. The goals of this study were to localize and to determine the distribution of tissue-resident stem/progenitor cells in the male EUS throughout EUS development and to understand the anatomic temporal patterns of the EUS. Newborn Sprague Dawley rats were intraperitoneally injected with the thymidine analogue, 5-ethynyl-2-deoxyuridine (EdU), and the EUS was harvested at five time points (1, 2, 3, 4, and 8 weeks postinjection). The tissue was then processed for EdU staining and immunofluorescence staining for stem cell markers Ki67 and proliferating cell nuclear antigen. We counted the EdU+ label-retaining cells (LRCs) at each time point and colocalized with each stem cell marker, also we isolated and cultured the cells in vitro. The results revealed that the number of EdU+ LRCs in each EUS cross-section decreased over time and that the LRCs were located immediately under the basal membrane of laminin, densely adherent to the muscle fibers. In addition, the thickness of the striated muscle layer developed much faster than the smooth muscle layer during EUS development. By 4 weeks, the structure of the EUS layers was well differentiated. The EUS resident stem/progenitor cells were isolated with MACS® MicroBeads system, and myogenesis was confirmed. In this study, we defined both the time-course development of the EUS and the distribution of resident stem/progenitor cells. This information is crucial for forthcoming studies regarding male micturition and for development of novel therapeutic approaches for postoperative male SUI.

Role of the Gonococcal Neisserial Heparin Binding Antigen in Microcolony Formation, and Serum Resistance and Adherence to Epithelial Cells.

The sexually transmitted infection gonorrhoea is on the rise worldwide and an increased understanding of the mechanisms of colonization and pathogenesis of Neisseria gonorrhoeae is required to aid development of new treatment and prevention strategies. In the current study, we investigate the neisserial heparin-binding antigen (NHBA) of N. gonorrhoeae and confirm its role in binding to several glycans, including heparin, and identify interactions of NHBA with both gonococcal and host cells. Furthermore, we report that a gonococcal nhba mutant displays decreased cell aggregation and microcolony formation, as well as reduced survival in human serum and reduced adherence to human cervical and urethral epithelial cells, relative to the wild-type strain. These data indicate that the gonococcal NHBA contributes to several aspects of the colonization and survival of N. gonorrhoeae and may be a target for new antimicrobial or vaccines.

Regenerative potential of human dental pulp stem cells in the treatment of stress urinary incontinence: In vitro and in vivo study.

OBJECTIVES: To evaluate the regenerative potential of human dental pulp stem cells (hDPSCs) in an animal model of stress urinary incontinence (SUI). SUI, an involuntary leakage of urine, is due to physical stress involving an increase in bladder pressure and a damage of external urethral sphincter affecting muscles and nerves. Conventional therapies can only relieve the symptoms. Human DPSCs are characterized by peculiar stemness and immunomodulatory properties and might provide an alternative tool for SUI therapy. MATERIALS AND METHODS: In vitro phase: hDPSCs were induced towards the myogenic commitment following a 24 hours pre-conditioning with 5-aza-2'-deoxycytidine (5-Aza), then differentiation was evaluated. In vivo phase: pudendal nerve was transected in female rats to induce stress urinary incontinence; then, pre-differentiated hDPSCs were injected in the striated urethral sphincter. Four weeks later, urethral sphincter regeneration was assayed through histological, functional and immunohistochemical analyses. RESULTS: Human DPSCs were able to commit towards myogenic lineage in vitro and, four weeks after cell injection, hDPSCs engrafted in the external urethral sphincter whose thickness was almost recovered, committed towards myogenic lineage in vivo, promoted vascularization and an appreciable recovery of the continence. Moreover, hDPSCs were detected within the nerve, suggesting their participation in repair of transected nerve. CONCLUSIONS: These promising data and further investigations on immunomodulatory abilities of hDPSCs would allow to make them a potential tool for alternative therapies of SUI.

LIM homeodomain transcription factor Isl1 affects urethral epithelium differentiation and apoptosis via Shh.

Urethral hypoplasia, including failure of urethral tube closure, is one of the common phenotypes observed in hereditary human disorders, the mechanism of which remains unclear. The present study was thus designed to study the expression, functions, and related mechanisms of the LIM homeobox transcription factor Isl1 throughout mouse urethral development. Results showed that Isl1 was highly expressed in urethral epithelial cells and mesenchymal cells of the genital tubercle (GT). Functional studies were carried out by utilizing the tamoxifen-inducible Isl1-knockout mouse model. Histological and morphological results indicated that Isl1 deletion caused urethral hypoplasia and inhibited maturation of the complex urethral epithelium. In addition, we show that Isl1-deleted mice failed to maintain the progenitor cell population required for renewal of urethral epithelium during tubular morphogenesis and exhibited significantly increased cell death within the urethra. Dual-Luciferase reporter assays and yeast one-hybrid assays showed that ISL1 was essential for normal urethral development by directly targeting the Shh gene. Collectively, results presented here demonstrated that Isl1 plays a crucial role in mouse urethral development, thus increasing our potential for understanding the mechanistic basis of hereditary urethral hypoplasia.

The effects of microenergy acoustic pulses on animal model of obesity-associated stress urinary incontinence. Part 2: In situ activation of pelvic floor and urethral striated muscle progenitor cells.

AIM: To investigate the possibility and mechanism of microenergy acoustic pulses (MAP) for activating tissue resident stem/progenitor cells within pelvic and urethral muscle and possible mechanism. METHODS: The female Zucker Lean and Zucker Fatty rats were randomly divided into four groups: ZL control, ZLMAP, ZF control, and ZFMAP. MAP was applied at 0.033 mJ/mm2 , 3 Hz for 500 pulses, and the urethra and pelvic floor muscles of each rat was then harvested for cell isolation and flow cytometry assay. Freshly isolated cells were analyzed by flow cytometry for Pax-7, Int-7α, H3P, and EdU expression. Meanwhile, pelvic floor muscle-derived stem cells (MDSCs) were harvested through magnetic-activated cell sorting, MAP was then applied to MDSCs to assess the mechanism of stem cell activation. RESULTS: Obesity reduced EdU-label-retaining cells and satellite cells in both pelvic floor muscle and urethra, while MAP activated those cells and enhanced cell proliferation, which promoted regeneration of striated muscle cells of the pelvic floor and urethral sphincter. Activation of focal adhesion kinase (FAK)/AMP-activated protein kinase (AMPK) /Wnt/ß-catenin signaling pathways by MAP is the potential mechanism. CONCLUSIONS: MAP treatment activated tissue resident stem cells within pelvic floor and urethral muscle in situ via activating FAK-AMPK and Wnt/ß-catenin signaling pathway.

Glycointeractome of Neisseria gonorrhoeae: Identification of Host Glycans Targeted by the Gonococcus To Facilitate Adherence to Cervical and Urethral Epithelial Cells.

Neisseria gonorrhoeae is a significant threat to global health for which a vaccine and novel treatment options are urgently needed. Glycans expressed by human cells are commonly targeted by pathogens to facilitate interactions with the host, and thus characterization of these interactions can aid identification of bacterial receptors that can be exploited as vaccine and/or drug targets. Using glycan array analysis, we identified 247 specific interactions between N. gonorrhoeae and glycans representative of those found on human cells. Interactions included those with mannosylated, fucosylated, and sialylated glycans, glycosaminoglycans (GAGs), and glycans terminating with galactose (Gal), N-acetylgalactosamine (GalNAc), and N-acetylglucosamine (GlcNAc). By investigating the kinetics of interactions with selected glycans, we demonstrate that whole-cell N. gonorrhoeae has a high affinity for mannosylated glycans (dissociation constant [KD ], 0.14 to 0.59 µM), which are expressed on the surface of cervical and urethral epithelial cells. Using chromatography coupled with mass spectrometric (MS) analysis, we identified potential mannose-binding proteins in N. gonorrhoeae Pretreatment of cells with mannose-specific lectin (concanavalin A) or free mannose competitor (α-methyl-d-mannopyranoside) substantially reduced gonococcal adherence to epithelial cells. This suggests that N. gonorrhoeae targets mannosyl glycans to facilitate adherence to host cells and that mannosides or similar compounds have the potential to be used as a novel treatment option for N. gonorrhoeaeIMPORTANCE Multidrug-resistant strains of Neisseria gonorrhoeae are emerging worldwide, and novel treatment and prevention strategies are needed. Glycans are ubiquitously expressed by all human cells and can be specifically targeted by pathogens to facilitate association with host cells. Here we identify and characterize the N. gonorrhoeae host-glycan binding profile (glycointeractome), which revealed numerous interactions, including high-affinity binding to mannosyl glycans. We identify gonococcal potential mannose-binding proteins and show that N. gonorrhoeae uses mannosyl glycans expressed on the surface of cervical and urethral epithelia to facilitate adherence. Furthermore, a mannose-binding lectin or a mannoside compound was able to reduce this adherence. By characterizing the glycointeractome of N. gonorrhoeae, we were able to elucidate a novel mechanism used by this important pathogen to interact with human cells, and this interaction could be exploited to develop novel therapeutics to treat antibiotic-resistant gonorrhea.

Underactive bladder in aging rats is associated with a reduced number of serotonin-expressing cells in the urethra and is improved by serotonin application to the urethra.

The aim of this study was to determine whether aging-related detrusor underactivity (DU) involves a decrease in 5-hydroxytryptamine (5-HT-positive)-expressing urethral cells and whether 5-HT stimulation of urethral sensory fibers improves detrusor function. Cystometries were performed in young (6 months) and aged (18-24 months) female Wistar rats. Aged rats with voiding contractions (VC) that were 2SD below the mean of those in young rats were considered to have DU. Bladder voiding efficiency (BVE) was calculated during saline or 5-HT solution cystometries. Rats were perfusion-fixed with a fixative solution (paraformaldehyde, PFA 4%) through the circulatory system and the urethra sectioned to count the number of 5-HT-immunoreactive (IR) cells. Isovolumetric cystometry was performed while irrigating the urethra with saline or 5µM-HT solution. Two-tailed unpaired t tests were used to determine the significance of differences. In aged DU rats, the mean (±SD) VC frequency was 0.24 ± 0.07 per minute, with an amplitude of 15 ± 3 cm H2 O. The mean (±SD) number of 5-HT-IR cells in the urethra of aged DU and young rats was 90 ± 11 and 182 ± 25, respectively (P < 0.01). 5-HT improved the mean (±SD) BVE of aged DU rats from 49 ± 3% to 78 ± 2% (P < 0.001). In isovolumetric cystometries, detrusor pressure during irrigation of the urethra with saline was 18 ± 1 cm H2 O, compared with 39 ± 2 cm H2 O during irrigation with 5-HT solution (P < 0.05). In rats, DU associated with aging is accompanied by a decrease in the number of 5-HT-positive cells. The results suggest that decreased 5-HT availability decreases urethral sensory fiber excitation, leading to a decrease the number of effective VC.

LIMK1 depletion enhances fasudil-dependent inhibition of urethral fibroblast proliferation and migration.

LIM kinase 1 (LIMK1) is an important regulator of the cell cytoskeleton. This study aimed to examine the role of LIMK1 in mediating the effects of the Rho kinase (ROCK) inhibitor fasudil. In vitro cultures of urethral fibroblasts were divided into LIMK1 knockdown (LIMK1 KD) and LIMK1 control (LIMK1 NC) experimental groups. Each group was incubated with fasudil (50 µmol/L) with or without transforming growth factor ß1 (10 ng/mL) for 24 hours. Wound healing and Transwell assays were performed to determine cell migration. Flow cytometry was used to determine apoptosis. LIMK1, collagen I, collagen III, phospho-myosin light chain (p-MLC), alpha smooth muscle actin (α-SMA), and phospho-Cofilin (p-Cofilin) expression was examined by Western blot analysis. The expression of LIMK1 was further validated in human urethral scar tissues. Transwell and wound healing assays revealed that the cells of the LIMK1 KD group exhibited significantly attenuated migration, when compared with those of the LIMK1 NC group ( P < 0.05). Cell migration was also attenuated in the LIMK1 KD group treated with fasudil ( P < 0.05). Flow cytometry analysis revealed that apoptosis was higher in the LIMK1 KD group than that in LIMK1 NC group ( P < 0.05). Apoptosis was also enhanced in the LIMK1 KD group treated with fasudil ( P < 0.05). Western blot analysis demonstrated that LIMK1, collagen I, collagen III, p-MLC, α-SMA, and p-Cofilin expression was significantly attenuated in both the fasudil-treated and untreated LIMK1 KD groups ( P < 0.05). LIMK1 was positively expressed in human urethral scar tissues while it was negatively expressed in normal urethra tissues. In conclusion, loss of LIMK1 expression inhibits the Rho/ROCK pathway-dependent proliferation and migration via downregulation of collagen I, collagen III, p-Cofilin, and α-SMA. LIMK1 loss can also enhance the inhibitory effects of fasudil on the proliferation and migration of urethral fibroblasts.

Epithelial DNA methyltransferase-1 regulates cell survival, growth and maturation in developing prostatic buds.

DNA methyltransferase 1 (DNMT1) is required for embryogenesis but roles in late forming organ systems including the prostate, which emerges from the urethral epithelium, have not been fully examined. We used a targeted genetic approach involving a Shhcre recombinase to demonstrate requirement of epithelial DNA methyltransferase-1 (Dnmt1) in mouse prostate morphogenesis. Dnmt1 mutant urethral cells exhibit DNA hypomethylation, DNA damage, p53 accumulation and undergo cell cycle arrest and apoptosis. Urethral epithelial cells are disorganized in Dnmt1 mutants, leading to impaired prostate growth and maturation and failed glandular development. We evaluated oriented cell division as a mechanism of bud elongation and widening by demonstrating that mitotic spindle axes typically form parallel or perpendicular to prostatic bud elongation axes. We then deployed a ShhcreERT allele to delete Dnmt1 from a subset of urethral epithelial cells, creating mosaic mutants with which to interrogate the requirement for cell division in specific prostatic bud epithelial populations. DNMT1- cell distribution within prostatic buds is not random as would be expected in a process where DNMT1 was not required. Instead, replication competent DNMT1 + cells primarily accumulate in prostatic bud margins and tips while replication impeded DNMT1- cells accumulate in prostatic bud cores. Together, these results highlight the role of DNMT1 in regulating epithelial bud formation by maintaining cell cycle progression and survival of rapidly dividing urethral epithelial cells, which can be extended to the study of other developing epithelial organs. In addition, our results show that prostatic buds consist of two epithelial cell populations with distinct molecular and functional characteristics that could potentially contribute to specialized lineages in the adult prostate.