Low Confluence Research Articles

Preoperative evaluation of the persistent urogenital sinus in cases with congenital adrenal hyperplasia

BackgroundCongenital adrenal hyperplasia (CAH) is a common cause of urogenital sinus (UGS) anomalies. Delineating the abnormal anatomy of the UGS is crucial prior to the operative repair. Genitography and cystourethroscopy are commonly used by most surgeons to plan for reconstruction. We aim at this study to evaluate the role of these modalities and to compare their accuracy in delineating the abnormal anatomy.MethodsWe did a prospective study of 30 female paediatric patients with persistent UGS secondary to CAH. All cases were evaluated with genitography and cystourethroscopy. We compared the accuracy of each modality in delineating the confluence. The confluence depth, the urethral length proximal to the confluence, the vaginal dimensions, and the depth of the bladder neck were measured in the genitograms.ResultsCystourethroscopy showed the abnormal anatomy in all 30 cases (100%). The genitogram showed the site of confluence in 24 cases out of 30 cases (80%). According to the site of the vaginal opening in relation to the external urethral sphincter in the genitogram, we had 2 groups: the low confluence group (N = 14) and the high confluence group (N = 16). The mean confluence depth in the low group was 9.8 mm vs. 20.5 mm in the high group (p-value < 0.001). To find a cutoff point to differentiate low from high groups, we used the values that resulted from dividing the length of the urethra proximal to the confluence over the depth of the bladder neck. By using the ROC curve, the cutoff point was 0.63 mm (< 0.63 mm high, > / = 0.63 mm low) (p-value < .001).ConclusionWe recommend using both cystourethroscopy and genitography for preoperative evaluation. Cystourethroscopy is superior in identifying the abnormal anatomy, but it lacks precision in taking measurements. Genitography can avoid this drawback and can precisely measure the confluence depth, vaginal dimensions, and length of the proximal urethra. We suggest using this formula (length of the urethra proximal to the confluence/depth of bladder neck) to classify persistent UGS as low (> / = 0.63 mm) and high (< 0.63 mm).

NKX2-5 regulates vessel remodeling in scleroderma-associated pulmonary arterial hypertension.

NKX2-5 is a member of the homeobox-containing transcription factors critical in regulating tissue differentiation in development. Here, we report a role for NKX2-5 in vascular smooth muscle cell phenotypic modulation in vitro and in vascular remodeling in vivo. NKX2-5 is upregulated in scleroderma patients with pulmonary arterial hypertension. Suppression of NKX2-5 expression in smooth muscle cells halted vascular smooth muscle proliferation and migration, enhanced contractility, and blocked the expression of extracellular matrix genes. Conversely, overexpression of NKX2-5 suppressed the expression of contractile genes (ACTA2, TAGLN, CNN1) and enhanced the expression of matrix genes (COL1) in vascular smooth muscle cells. In vivo, conditional deletion of NKX2-5 attenuated blood vessel remodeling and halted the progression to hypertension in a mouse chronic hypoxia model. This study revealed that signals related to injury such as serum and low confluence, which induce NKX2-5 expression in cultured cells, is potentiated by TGF-β and further enhanced by hypoxia. The effect of TGF-β was sensitive to ERK5 and PI3K inhibition. Our data suggest a pivotal role for NKX2-5 in the phenotypic modulation of smooth muscle cells during pathological vascular remodeling and provide proof of concept for therapeutic targeting of NKX2-5 in vasculopathies.

Abstract 4722: Enhanced phenotypic screening: A kinetic, multiplexed approach to analyzing drug effects using Incucyte® Live-Cell Analysis System

Abstract Phenotypic screening is a valuable tool for analysis of the complex mix of effects that drug treatment has on cells. Traditionally image-based screening uses fixed cells stained with reagents at a pre-chosen end point, however our technique combines kinetic, label-free readouts with non-perturbing fluorescence reporters to enable information-rich quantification of compound effects. A549 lung cancer cells were treated with a library of over 800 FDA-approved drugs while images were acquired in the Incucyte® Live-Cell Analysis System every two hours for four days. Cell growth and viability were measured using label-free readouts of confluence and dead cell count while cell cycle, cell death and Akt pathways were investigated using multiplexed fluorescent readouts. Cytotoxicity was measured using label-free Incucyte® AI Cell Health Analysis Software Module which uses two neural networks to segment individual cells and classify them as live or dead. Assay robustness was quantified using the time course of positive and negative controls (camptothecin and vehicle, respectively). Z’ reached a maximal value &amp;gt;0.9 between 48h and 72h; at 72h 4.4% of the compounds within the library induced &amp;gt;50% cell death. Correlation between % Live cells and % Confluence indicated 3 types of effect: cytotoxic (low viability, low confluence), cytostatic (high viability, low confluence), and none (high viability, high confluence). Morphological changes were observed in a multitude of wells with 29 compounds inducing cell enlargement &amp;gt;1800 µm2, suggesting induction of senescence. Hits from fluorescent readouts were identified as the compounds which perturbed the quantified readout more than 3 standard deviations away from the mean value of the vehicle. Mechanisms of cell death were examined by correlative analysis of the total dead cell population (label-free cell death identification), caspase-active (caspase 3/7-reagent positive) and Annexin V positive cells. This enabled rapid identification of compounds which induced apoptosis by caspase-independent pathways, including the serotonin-selective reuptake inhibitor Sertraline. Kinetic plots of cell cycle stage (% S|G2|M or G1) revealed contrasting mechanisms. Mycophenolic acid induced cell cycle arrest, observed as a consistent increase in the percentage of cells in G1 over time. However flumazenil displayed repeated, transient peaks in the percentage of cells in S|G2|M over the same 72h period, indicating synchronisation behavior. Incucyte® Live-Cell Analysis System as a phenotypic screening platform provides valuable kinetic data from cells within the physiologically relevant environment of a standard incubator. Label-free readouts yield direct cellular information and can be combined with fluorescent data to provide vastly more information than a single endpoint readout. Citation Format: Kirsty McBain, Gillian Lovell, Jasmine Trigg, Nicola Bevan, Daniel Appledorn. Enhanced phenotypic screening: A kinetic, multiplexed approach to analyzing drug effects using Incucyte® Live-Cell Analysis System [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4722.

Polyethylene glycol precipitation is an efficient method to obtain extracellular vesicle-depleted fetal bovine serum.

Mesenchymal stromal/stem cell derived-extracellular vesicles (MSC-EVs) have gained interest as drug delivery nanoparticles, having immunoregulatory and potentiating tissue repair property. To maintain growth of MSCs and obtain pure MSC-derived EVs, the culture media should contain fetal bovine serum (FBS) devoid of EVs, as the presence of FBS EVs confounds the properties of MSC-EVs. Therefore, we tested three methods: 18h ultracentrifugation (UC) and ultrafiltration (UF), which are common FBS EV depletion methods in current MSC-EV research, and polyethylene glycol (PEG) precipitation to obtain three EV depleted FBS (EVdFBS) batches, and compared them to FBS and commercial (Com) EVdFBS on human adipose stem cell (hADSC) growth, differentiation, enrichment of EVs in hADSC supernatant and their biological function on collagen metabolism. Our comparative study showed UC and UF vary in terms of depletion efficiency and do not completely deplete EVs and affects the growth-promoting quality of FBS. Specifically, FBS EV depletion was comparable between PEG (95.6%) and UF (96.6%) but less by UC (82%), as compared to FBS. FBS protein loss was markedly different among PEG (47%), UF (87%), and UC (51%), implying the ratio of EV depletion over protein loss was PEG (2.03), UF (1.11), and UC (1.61). A significant decrease of TGFβ/Smad signaling, involving in MSC growth and physiology, was observed by UF. After 96 hours of exposure to 5% FBS or 5% four different EVdFBS cell growth media, the osteogenesis ability of hADSCs was not impaired but slightly lower mRNA expression level of Col2a observed in EVdFBS media during chondrogenesis. In consistent with low confluency of hADSCs observed by optical microscope, cell proliferation in response to 5% UF EVdFBS media was inhibited significantly. Importantly, more and purer ADSCs EVs were obtained from ADSCs cultured in 5% PEG EVdFBS media, and they retained bioactive as they upregulated the expression of Col1a1, TIMP1 of human knee synovial fibroblast. Taken together, this study showed that PEG precipitation is the most efficient method to obtain EV depleted FBS for growth of MSCs, and to obtain MSC EVs with minimal FBS EV contamination.

M6A RNA MODIfiCATION AND A DRIVER OF CELLULAR PLASTICITY AND INTRA-TUMOURAL HETEROGENEITY IN GLIOBLASTOMA

Abstract AIMS The immense cellular plasticity of glioblastoma is a barrier to successful therapy. N6-methyladenosine (m6A) methylation of RNA is increasingly recognised as a key driver of cellular differentiation. Crucially, it is a reversible and dynamic modification that alters cellular phenotype via the regulation of mRNA metabolism. We hypothesise that m6A methylation mediates glioblastoma plasticity, regulating transcriptional networks and thus influencing cellular adaptation under different microenvironmental conditions. Our aims are to assess whether total m6A levels and m6A-regulatory gene expression are responsive to change in oxygenation levels and cellular confluency. Furthermore, we aim to characterise the spatial distribution of m6A in 3D cell culture models. METHOD We have investigated m6A levels in patient-derived cell lines from the invasive region of adult glioblastoma. Mass spectrometry and anti-m6A immunofluorescence confocal microscopy was used to quantify total m6A levels. We have compared total m6A levels in normoxic versus hypoxic culture conditions and in low versus high confluency 2D cell culture, including cell migration during a scratch assay. We have measured expression levels of key m6A-regulatory genes using rtPCR. RESULTS Total m6A levels and regulatory gene expression were not significantly different in normoxic versus hypoxic conditions. However, m6A levels were decreased in high confluency versus low confluency conditions. We will further characterise the spatial distribution of m6A in 3D cell culture models using immunofluorescence confocal microscopy. CONCLUSIONS We demonstrate a degree of variability in m6A levels in response to micro-environmental conditions, such as cellular confluency. These findings indicate a role for m6A in tumoral heterogeneity and cellular adaptation.

Glucose and cell confluency regulation of HDGF and beta-catenin nuclear localization

Introduction: Diabetes mellitus (DM) is the seventh leading cause of death in the US and diabetic kidney disease (DKD)is the cause of nearly 50% of individuals receiving dialysis. Kidney proximal tubule (PT) cells are sensitive to injury under diabetic conditions of high glucose. Maladaptive repair can cause fibrosis and loss of PT function contributing to end-stage renal disease. PT cell proliferation re-establishes the monolayer responsible for PT functionality. Preliminary lab findings showed increased abundance and nuclear localization of Hepatoma-derived growth factor (HDGF), known to regulate proliferation, in cells grown at low (&lt;40%) confluency in high glucose supplemented growth media. This change associated with altered nuclear co-localization with β-catenin (ß-Cat), involved in renal fibrosis, suggesting crosstalk between both factors may be involved in PT cell responses in DKD. Hypothesis: Cell confluency and glucose concentrations regulate the spatial and absolute abundance/degradation of HDGF altering cell signaling pathways in PT cells. Methods: Cultured human kidney PT cells (HK-11 cell line) were grown to low and high confluency under normal (5mM) and high (25mM) glucose conditions. RNA was collected via column purification for analysis of cell signaling. RNASeq and bioinformatic analysis. revealed transcript abundances and respective statistical significances between normal and high glucose conditions grown to low confluency. MetaCore and STRING were utilized to examine fold change differences of the transcriptome and affected pathways. To determine regulation of protein abundance, HK-11 cells were cultured in normal glucose media to 80-90% confluency and treated with no agent (control), DMSO, MG132 (proteasome inhibitor), and chloroquine (lysosome inhibitor), and abundance of HDGF and β-Cat activation analyzed by immunoblot. Results: HDGF transcripts from conditions of normal and high glucose grown to low confluency were found in relatively equal abundance (q ≤ 1). Pathway analyses revealed genes associated with multiple pathways including nuclear signaling, ER stress, proliferation and ubiquitination exhibited differential transcript abundance at statistically significant values. Immunoblot analysis suggested that HDGF is degraded via the lysosome. Conclusions: HDGF regulation by diabetes-like high glucose does not occur at the level of transcription and but through lysosomal degradation. ß-Cat nuclear localization is increased under conditions of high glucose and low cellular confluency. To confirm HDGF degradation and ß-Cat activation, more replicates are needed for statistical analysis. Future studies will determine the role of β-cat activation on HDGF abundance and nuclear localization. NIH T35-DK072923, P20-GM103436, NIH P20 GM113226, P50 AA024337, P30 ES030283 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Influences of Momentum Ratio on Transverse Dispersion for Intermediate-Field Mixing Downstream of Channel Confluence

This study aims to analyze the influences of momentum ratio (Mr) and confluence angle (α) on the transverse dispersion in an urban scale confluence channel from the numerical simulation results using the Environmental Fluid Dynamics Code model. By changing the momentum flux and confluence angle from the simulation results, the analysis focused on the relations between the vertical variations of transverse velocity and transverse dispersion. The high momentum tributary aligned the mixing interface toward the outer bank and created a strong helical motion, which transported the contaminated water along the channel bed and inflows into the recirculation zone. The high momentum ratio induced the large vertical shear in transverse velocity with a strong helical motion and increased the transverse dispersion. However, the helical motion persistence rapidly decreased as the flow reached downstream and led to a decrease in the transverse dispersion for the large confluence angle. Thus, the transverse dispersion coefficient increased with a high momentum ratio and low confluence angle, and the dimensionless transverse dispersion coefficient was in the range of 0.39-0.67, which is observed in meandering channels, for Mr > 1 and α = 45°.

Staged feminizing genitoplasty with total urogenital sinus mobilization

Background: The diagnosis and treatment of individuals with Disorders of Sexual Development is a complex process and requires a multidisciplinary approach. Each individual should be evaluated objectively and the treatment process of each individual has its own unique differences. The aim of this study is to discuss the effectiveness of staged feminizing genitoplasty operations using the total urogenital mobilization technique. Material and methods: Hospital records of patients who underwent vaginoplasty using the TUM technique between 2014 and 2019 by a single surgeon in a pediatric surgery center of a tertiary pediatric hospital were retrospectively reviewed. Results: All ten patients who underwent TUM had 46 XX chromosomes. The TUM procedure for vaginoplasty was performed in all patients by the author. A clitorolabioplasty had been previously performed in six patients with congenital adrenal hyperplasia. The follow-up period of the patients was 1-6 years. No serious complications developed in any of the patients. Neither infection nor tissue necrosis occurred. A second surgical procedure was not required for urinary fistula, vaginal stenosis, urinary incontinence, or early suture dehiscence. Conclusion: This study shows that vaginoplasty can be performed in a single session in infancy for patients with low confluence and a short Urogenital Sinus (UGS). Apart from this, postponing vaginoplasty surgeries to the adolescence period and performing UGS mobilization surgery seem to carry successful results.

Cell Confluence Modulates TRPV4 Channel Activity in Response to Hypoxia.

Transient receptor potential vanilloid 4 (TRPV4) is a polymodal Ca2+-permeable channel involved in various hypoxia-sensitive pathophysiological phenomena. Different tools are available to study channel activity, requiring cells to be cultured at specific optimal densities. In the present study, we examined if cell density may influence the effect of hypoxia on TRPV4 activity. Transiently TRPV4-transfected HEK293T cells were seeded at low or high densities corresponding to non-confluent or confluent cells, respectively, on the day of experiments, and cultured under in vitro normoxia or hypoxia. TRPV4-mediated cytosolic Ca2+ responses, single-channel currents, and Ca2+ influx through the channel were measured using Ca2+ imaging/microspectrofluorimetric assay, patch-clamp, and Bioluminescence Resonance Energy Transfer (BRET), respectively. TRPV4 plasma membrane translocation was studied using confocal microscopy, biotinylation of cell surface proteins, and BRET. Our results show that hypoxia exposure has a differential effect on TRPV4 activation depending on cell confluence. At low confluence levels, TRPV4 response is increased in hypoxia, whereas at high confluence levels, TRPV4 response is strongly inhibited, due to channel internalization. Thus, cell density appears to be a crucial parameter for TRPV4 channel activity.

Effects of substance P on human cerebral microvascular endothelial cell line hCMEC/D3 are mediated exclusively through a truncated NK-1 receptor and depend on cell confluence

The neuropeptide substance P (SP) mediates pain transmission, immune modulation, vasodilation and neurogenic inflammation. Its role in the peripheral nervous system has been well characterised. However, its actions on the blood-brain barrier (BBB) are less clear and warrant further study. The aim of this study was to characterise the effect of SP on the brain microvascular endothelial cells using the immortalized human brain microvascular endothelial cell line hCMEC/D3. As part of our studies, we have evaluated changes in expression, at mRNA and protein levels, of genes involved in the function of the blood-brain barrier such as occludin, induced by exposure to SP. We show that the effect of SP is dependent on cell confluence status. Thus, at low confluence but not at full confluence, SP treatment reduced occludin expression. The expression of the SP receptor, neurokinin-1 receptor (NK-1R) (the truncated form of the receptor expressed exclusively in this cell line) was also modulated in a similar pattern. SP treatment stimulated extracellular signal-regulated kinase (Erk2) phosphorylation which was not associated to changes in Interleukin-6 (IL-6), Interleukin-8 (IL-8), or Intercellular Adhesion Molecule 1 (ICAM-1) protein expression. In addition, SP treatment effectively recovered nitric oxide production on cells exposed to tumour necrosis factor alpha (TNF-α). SP did not trigger intracellular calcium release in hCMEC/D3 cells. We conclude that hCMEC/D3 cells are partially responsive to SP, that the effects are mediated through the truncated form of the receptor and are dependent on the confluence status of these cells.

Modeling the influence of cell–cell contact and TGF-β signaling on the epithelial mesenchymal transition in MCF7 breast carcinoma cells

The epithelial mesenchymal transition (EMT) is a process by which cells lose their adhesive nature and gain the migratory properties associated with mesenchymal cells. This transition allows cells to migrate away from a primary tumor while maintaining their newly acquired invasive behavior, suggesting that there is a bistable switch between the epithelial and mesenchymal phenotypes. In recent experimental work, we found evidence of this bistability in the MCF7 breast carcinoma cell line (Gasior et al., 2019). Underlying the complex processes governing EMT, we identify a feedback loop between E-cadherin, a protein involved in cellular adhesion, and Slug, a transcription factor that is upregulated during EMT. Here, we present a simple mathematical model that examines the relationship between E-cadherin and Slug in response to pro-epithelial and pro-mesenchymal factors, cell–cell contact and TGF-β, respectively. We hypothesize that cell–cell contact is a critical component in the transition from the epithelial to the mesenchymal phenotype and that it is possible to initiate EMT with the loss of cell–cell contact or the activation of the TGF-β signaling pathway. We propose a reversible bistable switch in response to a loss of cell–cell contact but an irreversible bistable switch when the cell is exposed to TGF-β. Taken together, this model shows that acquiring and retaining invasive behavior by cells with high levels of cell–cell contact is not impossible but, instead, depends on the cooperation between the two switches. The predictions of this model for E-cadherin and Slug levels were compared against relative gene expression data from our recent experiments with MCF7 cells (Gasior et al., 2019). Our model works well to predict E-cadherin and Slug mRNA expression in low confluence experiments, while also highlighting issues that arise when comparing experimental results to theoretical predictions.

Early Recognition of the PCL/Fibrous Carbon Nanocomposites Interaction with Osteoblast-like Cells by Raman Spectroscopy.

Poly(ε-caprolactone) (PCL) is a biocompatible resorbable material, but its use is limited due to the fact that it is characterized by the lack of cell adhesion to its surface. Various chemical and physical methods are described in the literature, as well as modifications with various nanoparticles aimed at giving it such surface properties that would positively affect cell adhesion. Nanomaterials, in the form of membranes, were obtained by the introduction of multi-walled carbon nanotubes (MWCNTs and functionalized nanotubes, MWCNTs-f) as well as electro-spun carbon nanofibers (ESCNFs, and functionalized nanofibers, ESCNFs-f) into a PCL matrix. Their properties were compared with that of reference, unmodified PCL membrane. Human osteoblast-like cell line, U-2 OS (expressing green fluorescent protein, GFP) was seeded on the evaluated nanomaterial membranes at relatively low confluency and cultured in the standard cell culture conditions. The attachment and the growth of the cell populations on the polymer and nanocomposite samples were monitored throughout the first week of culture with fluorescence microscopy. Simultaneously, Raman microspectroscopy was also used to track the dependence of U-2 OS cell development on the type of nanomaterial, and it has proven to be the best method for the early detection of nanomaterial/cell interactions. The differentiation of interactions depending on the type of nanoadditive is indicated by the ν(COC) vibration range, which indicates the interaction with PCL membranes with carbon nanotubes, while it is irrelevant for PCL with carbon nanofibers, for which no changes are observed. The vibration range ω(CH2) indicates the interaction for PCL with carbon nanofibers with seeded cells. The crystallinity of the area ν(C=O) increases for PCL/MWCNTs and for PCL/MWCNTs-f, while it decreases for PCL/ESCNFs and for PCL/ESCNFs-f with seeded cells. The crystallinity of the membranes, which is determined by Raman microspectroscopy, allows for the assessment of polymer structure changes and their degradability caused by the secretion of cell products into the ECM and the differentiation of interactions depending on the carbon nanostructure. The obtained nanocomposite membranes are promising bioactive materials.

Improved CRISPR/Cas9 gene editing in primary human myoblasts using low confluency cultures on Matrigel

BackgroundCRISPR/Cas9 is an invaluable tool for studying cell biology and the development of molecular therapies. However, delivery of CRISPR/Cas9 components into some cell types remains a major hurdle. Primary human myoblasts are a valuable cell model for muscle studies, but are notoriously difficult to transfect. There are currently no commercial lipofection protocols tailored for primary myoblasts, and most generic guidelines simply recommend transfecting healthy cells at high confluency. This study aimed to maximize CRISPR/Cas9 transfection and editing in primary human myoblasts.MethodsSince increased cell proliferation is associated with increased transfection efficiency, we investigated two factors known to influence myoblast proliferation: cell confluency, and a basement membrane matrix, Matrigel. CRISPR/Cas9 editing was performed by delivering Cas9 ribonucleoprotein complexes via lipofection into primary human myoblasts, cultured in wells with or without a Matrigel coating, at low (~ 40%) or high (~ 80%) confluency.ResultsCells transfected at low confluency on Matrigel-coated wells had the highest levels of transfection, and were most effectively edited across three different target loci, achieving a maximum editing efficiency of 93.8%. On average, editing under these conditions was >4-fold higher compared to commercial recommendations (high confluency, uncoated wells).ConclusionThis study presents a simple, effective and economical method of maximizing CRISPR/Cas9-mediated gene editing in primary human myoblasts. This protocol could be a valuable tool for improving the genetic manipulation of cultured human skeletal muscle cells, and potentially be adapted for use in other cell types.

Rare Portal Venous Anomaly in a Living Liver Donor: A Case Report

Knowledge of the anatomy of the portal system is essential for safe liver resection. We report a very rare anatomic anomaly of the portal system in a living liver donor. A 24-year-old female living liver donor was found to have anomalies of the portal system on preoperative contrast-enhanced computed tomography. The ventral branch of the right anterior segment arose from the transverse portion of the left portal vein. The gallbladder and round ligament were positioned normally. Intraoperative cholangiography for evaluation of biliary anatomy revealed very low confluence of the right and left hepatic ducts. All the bile ducts from the right lobe merged into the right hepatic duct. A right lobe graft was performed, including the ventral area of the right anterior segment. The portal branch of the ventral area of the right anterior segment could be transected extrahepatically. In the recipient operation, each of the right main portal branches, including the right posterior segment branch and the dorsal branch of the right anterior segment, and the ventral branch of the right anterior segment, were anastomosed to the right and left branches of the portal vein, respectively, of the recipient. The transected right hepatic duct of the graft was anastomosed with the recipient's common hepatic duct. Sixteen years after the liver transplant, the recipient continues to do well and has good portal flow.

Robotic-assisted excision of a prostatic utricle cyst in a 12-month boy with proximal hypospadia and 45X0/ 46XY karyotype

Prostatic utricle cysts result from incomplete regression of Mullerian duct structures and occur most frequently in males with perineal or peno-scrotal hypospadias. Utricular cysts may present with various signs and symptoms including urinary tract infection, pain and post-void incontinence, a palpable abdominal mass or recurrent epididymitis. We present a 12 months boy we treated due to proximal hypospadias in two stages, in association to a dysplastic undescended testis that was excised. The karyotype of the patient was 45X0/46XY and after successful hypospadias treatment, the mother complained of purulent discharge in the diapers. A sonogram showed a large cyst behind the bladder with heterogeneous content. The diagnosis of utricle cyst was confirmed by a NMR with detailed anatomy and low confluence of the neck of the cyst to the urethra. We performed a robotic excision of the cyst consisting of careful dissection between bladder and rectum and a safe ligation of the communication of it to the urethra. Patient outcome was uneventful. To the best of our knowledge this is the youngest patient with utricle cyst treated with robotic approach reported in the literature.

Early Feminizing Genitoplasty in Girls with Congenital Adrenal Hyperplasia (CAH)—Analysis of Unified Surgical Management

Aim: To analyze a single-centre experience in feminizing genitoplasty in virilized girls with congenital adrenal hyperplasia (CAH). Methods: Review of medical records of all 46, XX CAH patients undergoing single stage feminizing genitoplasty between 2003 and 2018 was performed. Results: A total of 31 girls aged from 4 months to 10 years were included in the study. The majority (n = 26/31, 84%) were operated before 2 years of age (median 8 months). External virilization was rated as Prader 3 (n = 7/31), Prader 4 (n = 21/31) and Prader 5 (n = 3/31). The urethrovaginal confluence location was low in 19 and high in 12 girls with a percentage distribution similar in Prader 4 and 5 (p > 0.05) but significantly different in Prader 3 (p = 0.017). The follow-up ranged from 12 months to 15 years. All parents assessed the cosmetic result as satisfactory. Perioperative complications occurred in two patients and included rectal injury (n = 1/31) and prolonged bleeding (n = 1/31). Three patients developed late complications including labial atheromas (n = 2/31) and vaginal stricture requiring surgical dilatation (n = 1/31). Low confluence did not decrease the risk of complications. Conclusions: Early feminizing genitoplasty in girls with congenital adrenal hyperplasia, irrespective of virilization severity, gives satisfactory cosmetic results and is characterized by low and acceptable surgical risk. Nevertheless, the most important determinant of the effectiveness of such management would be future patients’ satisfaction.

Persistent cloaca: persistence of the challenge

BackgroundPersistent cloaca or cloacal anomalies represent a special category of anorectal anomalies affecting the female sex with a reported incidence of about one in 25,000 live birth.The study included 34 cases of cloaca that were managed at our unit between 2003 through 2017. We retrospectively reviewed patients’ records that included clinical presentation, investigations, operative data, and follow-up notes.Anatomically, we stratified cloaca into three types according to the level of urogenital confluence. A low confluence (type 1) was defined by being at or below the level of the lower border of pubic symphysis with a short common channel (11 cases). A high confluence (type 3) was defined by being at or above the level of the upper border of pubic symphysis (9 cases). Between the low and high types, we defined an intermediate type (type 2) where the urogenital confluence was behind the mid-portion of pubic symphysis (14 cases).ResultsRenal anomalies were common association: solitary kidney in seven, pelvic kidney in two, and urinary tract dilatation (hydroureteronephrosis) in 12 cases. At follow-up, chronic renal insufficiency was detected in seven casesThe prognosis for urinary continence was excellent in low confluence (type 1) cloaca. On the other hand, urinary incontinence was common among type 3 (high confluence) cloaca (62.5%).ConclusionRenal anomalies represent a common association with cloaca and a major cause of morbidity. Efforts should be directed to preserve renal function during the initial management, and to preserve the continence potential following the definitive repair.Level of evidenceThis is a case series with no comparison group (level IV).

Influences of riverbed morphology on patterns and magnitudes of hyporheic water exchange within a natural river confluence

River confluences are pivotal junctions that have vital influences on hydraulic characteristics and sediment particle distributions as well as on hyporheic water exchange. However, hyporheic water exchange is poorly understood because of the complicated river morphology within a river confluence. Therefore, the river confluence between the Juehe River and Haohe River located in arid and semi-arid areas of northwestern China was selected to evaluate the hyporheic water exchange processes, and the temperatures of different depth sediment were measured on July 11th and 12th, 2016 by an instrument equipped with five temperature sensors. The patterns and magnitudes of hyporheic water exchange were estimated using a one-dimensional heat transport model coupled with temperature data. Meanwhile, terrain points elevations and sediment particle distributions in this river confluence were collected to determine the confluence riverbed morphology. Furthermore, the river confluence morphology effects on hyporheic water exchange were investigated in this study. The different riverbed morphology and hydrodynamic zone locations were observed, which were controlled by the planform geometry with low river confluence flux momentum during the test period. Meanwhile, the upwelling flow dominated in the test area, and the downwelling occurred at three of the 23 total test points, which was induced by the complicated riverbed morphology and hydraulic characteristics. Additionally, the magnitudes of the upwelling flow were significantly influenced by the sediment heterogeneity subjected to the erosional and depositional processes. Hyporheic water exchange is a vital hydrological process in arid and semi-arid areas, where it significantly impacts the ecological environment of rivers. This study provides a valuable guideline for water quality and quantity management in arid and semi-arid areas.

Aligning Synthetic Hippocampal Neural Circuits via Self-Rolled-Up Silicon Nitride Microtube Arrays.

Directing neurons to form predetermined circuits with the intention of treating neurological disorders and neurodegenerative diseases is a fundamental goal and current challenge in neuroengineering. Until recently, only neuronal aggregates were studied and characterized in culture, which can limit information gathered to populations of cells. In this study, we use a substrate constructed of arrays of strain-induced self-rolled-up membrane 3D architectures. This results in changes in the neuronal architecture and altered growth dynamics of neurites. Hippocampal neurons from postnatal rats were cultured at low confluency (∼250 cells mm-2) on an array of transparent rolled-up microtubes (μ-tubes; 4-5 μm diameter) of varying topographical arrangements. Neurite growth on the μ-tubes was characterized and compared to controls in order to establish a baseline for alignment imposed by the topography. Compared to control substrates, neurites are significantly more aligned toward the 0° reference on the μ-tube array. Pitch (20-60 and 100 μm) and μ-tube length (30-80 μm) of array elements were also varied to investigate their impact on neurite alignment. We found that alignment was improved by the gradient pitch arrangement and with longer μ-tubes. Application of this technology will enhance the ability to construct intentional neural circuits through array design and manipulation of individual neurons and can be adapted to address challenges in neural repair, reinnervation, and neuroregeneration.

Evaluation of urogenital sinus surgery in disorder of sex development

To explore the indication and effectiveness of urogenital sinus surgery in feminizing genitoplasty of disorder of sex development (DSD). A retrospective analysis was made on clinical data from 22 patients with DSD who underwent one stage feminizing genitoplasty between October 2010 and December 2015. The patients' age ranged from 1 year and 2 months to 21 years, with the median age of 2 years and 1 month. According to the Prader classification criteria, the appearance of vulvas were rated as grade Ⅰ in 7 cases, grade Ⅱ in 6 cases, grade Ⅲ in 8 cases, and grade Ⅳ in 1 case. Cystoscopy was applied before feminizing genitoplasty in all patients. Low confluence of vagina and urethra was found in 19 patients, while high confluence was found in 3 patients. The mean length of urogenital sinus was 1.6 cm (range, 0.5-3.0 cm). The mean length of water-filled vagina was 4.4 cm (range, 3.5-5.5 cm). Cervix was detected at the end of vagina in 16 patients, meanwhile absence of cervix was detected in 6 patients. The same procedures of clitoroplasty and labioplasty were used in all patients. Three procedures of urogenital sinus surgery were applied, as the "cut-back" vaginoplasty in 6 patients, the "flap" vaginoplasty in 11 patients, and the partial urogenital sinus mobilization (PUM) in 5 patients. All procedures were completed successfully and the incisions healed by stage Ⅰ. All patients were followed up 12-74 months, with the average of 30.5 months. The outcome of appearance evaluation was excellent in 13 patients (59.1%), good in 6 patients (27.3%), and poor in 3 patients (13.6%). Urinary incontinence, post-void residual, urinary infection, and urethrovaginal fistula were not found in 17 toilet trained patients. Urogenital sinus surgery is the most critical step in feminizing genitoplasty of DSD. It can be finished in one stage procedure with clitoroplasty and labioplasty before puberty. If thoroughly evaluation before surgery is completed and the principle of different procedures is handled, the outcome will be satisfactory.