Persistent myopathy despite release of partial obstruction: in vivo reversal of dysfunction and transcriptional responses using rapamycin.

Current and potential medical therapy for obstruction-induced myopathic bladder dysfunction (from benign prostatic hyperplasia or posterior urethral valves) focuses on symptoms. The persistent tissue pathology and dysfunction after release of obstruction is often deemed irreversible without any systematic therapeutic approaches. As rapamycin can attenuate bladder smooth muscle hypertrophy and dysfunction during the genesis of partial obstruction in vivo, we tested whether rapamycin could improve persistent function after release of obstruction (de-obstruction or REL). Female Sprague-Dawley rat bladders were partially obstructed (PBO) by suturing around both the urethra and a para-urethral steel rod, then removing the rod. One day prior to release of obstruction (preREL), voiding parameters and residual urine volume of preREL+future rapa, preREL+future veh groups were recorded. Release of obstruction (REL) was performed by suture removal following 6 weeks of PBO. For 4 more weeks after the de-obstruction, REL animals were randomized to rapamycin (REL+rapa) or vehicle (REL+veh). PBO for 6 weeks were used as positive controls. In shams, the urethra was exposed, but no suture tied. Voiding parameters and residual urine volume were measured prior to sacrifice of sham and REL+veh or REL+rapa, and PBO. Rapamycin efficacy was tested by pair-wise comparison of changes in individual voiding data from preREL+future veh or preREL+future rapa versus REL+veh or REL+rapa, respectively, as well as by comparisons of REL+veh to REL+rapa groups. Bladders were weighed and processed for a high-throughput QPCR array, and histopathology. Bladder/body mass ratios with PBO increased significantly and remained higher in the release phase in REL+veh animals. REL+rapa versus REL+veh improved residual volumes and micturition fractions toward sham levels. Three genes encoding extracellular proteins, BMP2, SOD3, and IGFBP7, correlated with functional improvement by Pearson's correlations. The promoters of these genes showed enrichment for several motifs including circadian E-boxes. While obstruction and REL augmented CLOCK and NPAS2 expression above sham levels, rapamycin treatment during release significantly blocked their expression. This experimental design of pharmaco-intervention during the de-obstruction phase revealed a novel pathway dysregulated during the clinically relevant treatment phase of obstructive bladder myopathy.

Estimation of salt intake assessed by 24-h urinary sodium level among adults speaking different dialects from the Chaoshan region of southern China.

OBJECTIVE: Dietary salt intake may vary depending on different lifestyles. We aimed to estimate the different salt intakes and evaluate the knowledge and self-awareness about salt among people speaking the Teochew, Teochew-Hakka and Hakka dialects in the Chaoshan region of southern China. DESIGN: The study followed a cluster sampling of residents in Chaoshan region. General characteristics, lifestyles, health status as well as knowledge and self-awareness related to salt intake were investigated using a questionnaire. Anthropometric variables as well as Na and K excretion in a 24-h urine collection were measured. SETTING: Chaoshan region of China. PARTICIPANTS: Four hundred fifteen adults who spoke only one of these three dialects. RESULTS: The salt intake of adults who spoke the Teochew, Teochew-Hakka and Hakka dialects was 7·19 (interquartile range (IQR) 5·29-10·17), 9·03 (IQR 6·62-11·54) and 10·12 (IQR 7·61-12·82) g/d, respectively, with significant differences between Teochew and Teochew-Hakka speakers and between Teochew and Hakka speakers (both P < 0·05). The Na:K ratio for adults who spoke the three dialects was 3·00 (IQR 2·00-4·11), 3·50 (IQR 2·64-4·82) and 4·52 (IQR 3·35-5·97), respectively, and differed significantly among the groups (all P < 0·05). Multiple linear regression analysis showed increased Na:K ratio associated with hypertension (ß = 0·71, P = 0·043) in Hakka speakers. Knowledge and self-awareness about salt intake were poor in this population. CONCLUSIONS: Salt intake was closely related to lifestyles and was higher than the upper limit (5 g/d) recommended by the WHO in adults of Chaoshan, especially those speaking the Hakka dialect.

DNA Methylation Reduces the Yes-Associated Protein 1/WW Domain Containing Transcription Regulator 1 Pathway and Prevents Pathologic Remodeling during Bladder Obstruction by Limiting Expression of BDNF.

Chronic bladder obstruction and bladder smooth muscle cell (SMC) stretch provide fibrotic and mechanical environments that can lead to epigenetic change. Therefore, we examined the role of DNA methylation in bladder pathology and transcriptional control. Sprague-Dawley female rats underwent partial bladder obstruction by ligation of a silk suture around the proximal urethra next to a 0.9-mm steel rod. Sham operation comprised passing the suture around the urethra. After 2 weeks, rats were randomized to normal saline or DNA methyltransferase inhibitor, 5-aza-2-deoxycytidine (DAC) at 1 mg/kg, three times/week intraperitoneally. After 6 weeks, bladders were weighed and divided for histology and RNA analysis by high-throughput real-time quantitative PCR arrays. DAC treatment during obstruction in vivo profoundly augmented brain-derived neurotrophic factor (BDNF) expression compared with the obstruction with vehicle group, which was statistically correlated with pathophysiologic parameters. BDNF, cysteine rich angiogenic inducer 61 (CYR61), and connective tissue growth factor (CTGF) expression clustered tightly together using Pearson's correlation analysis. Their promoters were associated with the TEA domain family member 1 (TEAD1) and Yes-associated protein 1/WW domain containing transcription regulator 1 pathways. Interestingly, DAC treatment increased BDNF expression in bladder SMCs (P < 0.0002). Stretch-induced BDNF was inhibited by the YAP/WWTR1 inhibitor verteporfin. Verteporfin improved the SMC phenotype (proliferative markers and SMC marker expression), in part by reducing BDNF. Expression of BDNF is limited by DNA methylation and associated with pathophysiologic changes during partial bladder outlet obstruction and SMC phenotypic change in vitro.

Iron binding capacity of dephytinised soy protein isolate hydrolysate as influenced by the degree of hydrolysis and enzyme type.

Soy protein is increasingly used in extended meat products and dairy type products due to the presence of high quality proteins with excellent functional properties. However, it has been shown to inhibit iron bioavailability because of phytic acid present in the protein. This present study investigated the effects of dephytinise from soy protein isolate (SPI) on iron binding capacity and degree of hydrolysis. Also the effects of enzyme type and degree of hydrolysis on iron binding capacity were studied. It was demonstrated that phytase and anion exchange resin could remove effectively the phytate from SPI. The dephytinise would decrease the degree of hydrolysis of SPI. The enzyme type and degree of hydrolysis influenced significantly the iron binding capacity of the hydrolysate. Flavourzyme might be the best choice for producing peptides with iron binding capacity from SPI and middle degree of hydrolysis would be benefitable to this process.

1D Cu(I)-based chiral organic-inorganic hybrid material with second harmonic generation and circular polarized luminescence.

In recent years, organic-inorganic hybrid materials have demonstrated exceptional performance in nonlinear optics, attracting widespread attention. However, there are relatively few examples of coordination compounds synthesized with Cu as the metal center that exhibit excellent nonlinear optical properties. In this study, we successfully synthesized a pair of enantiomers named R/S-Cu2I2 by reacting chiral ligands with CuI. The crystal structure reveals a one-dimensional copper-iodide chain structure built by Cu2I2 clusters, and its ordered arrangement in space provides not only a strong second harmonic generation (SHG) signal (1.24 × KDP) but also a large birefringence (0.15@1064 nm). Under excitation at 395 nm, the crystals exhibit red fluorescence peaked at 675 nm. The CD spectra of R/S-Cu2I2 show a distinct mirror-symmetric Cotton effect, and their CPL signals are corresponding and opposite in the emission range, with a maximum glum of approximately ±2.5 × 10-3. Theoretical calculations using density functional theory were also carried out to enhance our understanding of the correlation between their structures and optical properties.

Epigenetic insights to pediatric uropathology: Celebrating the fundamental biology vision of Tony Khoury.

INTRODUCTION: Many pediatric urology conditions affect putatively normal tissues or appear too commonly to be based solely on specific DNA mutations. Understanding epigenetic mechanisms in pediatric urology, therefore, has many implications that can impact cell and tissue responses to settings, such as environmental and hormonal influences on urethral development, uropathogenic infections, obstructive stimuli, all of which originate externally or extracellularly. Indeed, the cell's response to external stimuli is often mediated epigenetically. In this commentary, we highlight work on the critical role that epigenetic machinery, such as DNA methyltransferases (DNMTs), Enhancer of Zeste Polycomb Repressive Complex 2 Subunit (EZH2), and others play in regulating gene expression and cellular functions in three urological contexts. DESIGN: Animal and cellular constructs were used to model clinical pediatric uropathology. The hypertrophy, trabeculation, and fibrosis of the chronically obstructed bladder was explored using smooth muscle cell models employing disorganised vs. normal extracellular matrix (ECM), as well as a new animal model of chronic obstructive bladder disease (COBD) which retains its pathologic features even after bladder de-obstruction. Cell models from human and murine hypospadias or genital tubercles (GT) were used to illustrate developmental responses and epigenetic dependency of key developmental genes. Finally, using bladder urothelial and organoid culture systems, we examined activity of epigenetic machinery in response to non uropathogenic vs. uropathogenic E.coli (UPEC). DNMT and EZH2 expression and function were interrogated in these model systems. RESULTS: Disordered ECM exerted a principal mitogenic and epigenetic role for on bladder smooth muscle both in vitro and in CODB in vivo. Key genes, e.g., BDNF and KCNB2 were under epigenetic regulation in actively evolving obstruction and COBD, though each condition showed distinct epigenetic responses. In models of hypospadias, estrogen strongly dysregulated WNT and Hox expression, which was normalized by epigenetic inhibition. Finally, DNA methylation machinery in the urothelium showed specific activation when challenged by uropathogenic E.coli. Similarly, UPEC induces hypermethylation and downregulation of the growth suppressor p16INK4A. Moreover, host cells exposed to UPEC produced secreted factors inducing epigenetic responses transmissible from one affected cell to another without ongoing bacterial presence. DISCUSSION: Microenvironmental influences altered epigenetic activity in the three described urologic contexts. Considering that many obstructed bladders continue to display abnormal architecture and dysfunction despite relief of obstruction similar to after resection of posterior valves or BPH, the epigenetic mechanisms described highlight novel approaches for understanding the underlying smooth muscle myopathy of this crucial clinical problem. Similarly, there is evidence for an epigenetic basis of xenoestrogen on development of hypospadias, and UTI-induced pan-urothelial alteration of epigenetic marks and propensity for subsequent (recurrent) UTI. The impact of mechanical, hormonal, infectious triggers on genitourinary epigenetic machinery activity invite novel avenues for targeting epigenetic modifications associated with these non-cancer diseases in urology. This includes the use of deactivated CRISPR-based technologies for precise epigenome targeting and editing. Overall, we underscore the importance of understanding epigenetic regulation in pediatric urology for the development of innovative therapeutic and management strategies.

Comparison of a novel potentiator of CFTR channel activity to ivacaftor in ameliorating mucostasis caused by cigarette smoke in primary human bronchial airway epithelial cells.

Background: Cystic Fibrosis causing mutations in the gene CFTR , reduce the activity of the CFTR channel protein, and leads to mucus aggregation, airway obstruction and poor lung function. A role for CFTR in the pathogenesis of other muco-obstructive airway diseases such as Chronic Obstructive Pulmonary Disease (COPD) has been well established. The CFTR modulatory compound, Ivacaftor (VX-770), potentiates channel activity of CFTR and certain CF-causing mutations and has been shown to ameliorate mucus obstruction and improve lung function in people harbouring these CF-causing mutations. A pilot trial of Ivacaftor supported its potential efficacy for the treatment of mucus obstruction in COPD. These findings prompted the search for CFTR potentiators that are more effective in ameliorating cigarette-smoke (CS) induced mucostasis. Methods: A novel small molecule potentiator (SK-POT1), previously identified in CFTR binding studies, was tested for its activity in augmenting CFTR channel activity using patch clamp electrophysiology in HEK-293 cells, a fluorescence-based assay of membrane potential in Calu-3 cells and in Ussing chamber studies of primary bronchial epithelial cultures. Addition of cigarette smoke extract (CSE) to the solutions bathing the apical surface of Calu-3 cells and primary bronchial airway cultures was used to model COPD. Confocal studies of the velocity of fluorescent microsphere movement on the apical surface of CSE exposed airway epithelial cultures, were used to assess the effect of potentiators on CFTR-mediated mucociliary movement. Results: We showed that SK-POT1, like VX-770, was effective in augmenting the cyclic AMP-dependent channel activity of CFTR. SK-POT-1 enhanced CFTR channel activity in airway epithelial cells previously exposed to CSE and ameliorated mucostasis on the surface of primary airway cultures. Conclusion: Together, this evidence supports the further development of SK-POT1 as an intervention in the treatment of COPD.

Rapamycin attenuates bladder hypertrophy during long-term outlet obstruction in vivo: tissue, matrix and mechanistic insights.

PURPOSE: Previous molecular studies showed that the mTOR inhibitor rapamycin prevents bladder smooth muscle hypertrophy in vitro. We investigated the effect of rapamycin treatment in vivo on bladder smooth muscle hypertrophy in a rat model of partial bladder outlet obstruction. MATERIALS AND METHODS: A total of 48 female Sprague-Dawley® rats underwent partial bladder outlet obstruction and received daily subcutaneous injections of rapamycin (1 mg/kg) or vehicle commencing 2 weeks postoperatively. A total of 36 rats underwent sham surgery and received rapamycin or vehicle. Rats were sacrificed 3, 6 and 12 weeks after surgery. Before sacrifice, voiding was observed in a metabolic cage for 24 hours. Bladder-to-body weight in gm bladder weight per kg body weight and post-void residual urine were assessed. We evaluated Col1a1, Col3a1, Eln and Mmp7 mRNA expression and histology. Two-factor ANOVA and the post hoc t test were applied. RESULTS: Bladder outlet obstruction caused a significant increase in bladder weight in all obstructed groups. Three weeks postoperatively (1 week of treatment) there was no difference in the bladder-to-body weight ratio in the obstructed group. However, at 6 and 12 weeks (4 and 10 weeks of treatment, respectively) the bladder-to-body weight ratio of rats with obstruction plus rapamycin was significantly lower than that of rats with obstruction plus vehicle. Post-void residual urine volume after 6 and 12 weeks of obstruction was lower in obstructed rats with rapamycin compared to that in obstructed rats with vehicle. Rapamycin decreased the obstruction induced expression of Col1a1, Col3a1, Eln and Mmp7. CONCLUSIONS: Rapamycin prevents mechanically induced hypertrophy in cardiovascular smooth muscle. In vivo mTOR inhibition may attenuate obstruction induced detrusor hypertrophy and help preserve bladder function.

Host-Guest Assembly-Crosslinked Nanoemulsions with Dual-Antimicrobial Mechanism for Treating Multidrug-Resistant Bacterial Biofilms.

Plant essential oils have good antimicrobial properties, but their poor stability and compatibility in aqueous solutions greatly limit their practical application. To address this issue, a dynamically crosslinked nanoemulsion based on host-guest assembly was developed in this study. First, a ß-cyclodextrin-functionalized quaternary ammonium surfactant (ß-CD-QA) and adamantane-terminated polyethylene glycol (APA) crosslinker were first synthesized. Then, the oil-in-water host-guest crosslinked nanoemulsions (HGCTNs) were formed by incorporating tea tree essential oils (TTO) as a natural antimicrobial agent. The results showed that HGCTNs significantly improved the stability of the essential oil nanoemulsions and extended their shelf life. Furthermore, HGCTNs demonstrated effective antimicrobial properties against both Gram-negative/positive bacterioplankton and bacterial biofilms. The results of antibacterial experiments showed that the dynamically crosslinked HGCTNs exhibit superior antibacterial efficacy, with a minimum inhibitory concentration (MIC) of 12.5 v/v % (0.13 µL/mL TTO) and could eradicate the biofilms. The electrical conductivity of the bacterial solution gradually increased within 5 h of treatment with the nanoemulsions, indicating that the HGCTNs have a slow-release effect of TTO and sustainable antibacterial ability. The antimicrobial mechanism can be attributed to the synergistic antibacterial action of the ß-CD-QA surfactant containing a quaternary ammonium moiety and TTO, which are stabilized by nanoemulsions.

Spontaneous urinary bladder regeneration after subtotal cystectomy increases YAP/WWTR1 signaling and downstream BDNF expression: Implications for smooth muscle injury responses.

Rodents have the capacity for spontaneous bladder regeneration and bladder smooth muscle cell (BSMC) migration following a subtotal cystectomy (STC). YAP/WWTR1 and BDNF (Brain-derived neurotrophic factor) play crucial roles in development and regeneration. During partial bladder outlet obstruction (PBO), excessive YAP/WWTR1 signaling and BDNF expression increases BSMC hypertrophy and dysfunction. YAP/WWTR1 and expression of BDNF and CYR61 were examined in models of regeneration and wound repair. Live cell microscopy was utilized in an ex vivo model of STC to visualize cell movement and division. In Sprague-Dawley female rats, STC was performed by resection of the bladder dome sparing the trigone, followed by closure of the bladder. Smooth muscle migration and downstream effects on signaling and expression were also examined after scratch wound of BSMC with inhibitors of YAP and BDNF signaling. Sham, PBO and incision (cystotomy) were comparators for the STC model. Scratch wound in vitro increased SMC migration and expression of BDNF, CTGF and CYR61 in a YAP/WWTR1-dependent manner. Inhibition of YAP/WWTR1 and BDNF signaling reduced scratch-induced migration. BDNF and CYR61 expression was elevated during STC and PBO. STC induces discrete genes associated with endogenous de novo cell regeneration downstream of YAP/WWTR1 activation.

SRSF3/AMOTL1 splicing axis promotes the tumorigenesis of nasopharyngeal carcinoma through regulating the nucleus translocation of YAP1.

Dysregulation of serine/arginine splicing factors (SRSFs) and abnormal alternative splicing (AS) have been widely implicated in various cancers but scarcely investigated in nasopharyngeal carcinoma (NPC). Here we examine the expression of 12 classical SRSFs between 87 NPC and 10 control samples, revealing a significant upregulation of SRSF3 and its association with worse prognosis in NPC. Functional assays demonstrate that SRSF3 exerts an oncogenic function in NPC progression. Transcriptome analysis reveals 1,934 SRSF3-regulated AS events in genes related to cell cycle and mRNA metabolism. Among these events, we verify the generation of a long isoform of AMOTL1 (AMOTL1-L) through a direct bond of the SRSF3 RRM domain with the exon 12 of AMOTL1 to promote exon inclusion. Functional studies also reveal that AMOTL1-L promotes the proliferation and migration of NPC cells, while AMOTL1-S does not. Furthermore, overexpression of AMOTL1-L, but not -S, significantly rescues the inhibitory effects of SRSF3 knockdown. Additionally, compared with AMOTL1-S, AMOTL1-L has a localization preference in the intracellular than the cell membrane, leading to a more robust interaction with YAP1 to promote nucleus translocation. Our findings identify SRSF3/AMOTL1 as a novel alternative splicing axis with pivotal roles in NPC development, which could serve as promising prognostic biomarkers and therapeutic targets for NPC.

Maternal exposure to 4-vinylcyclohexene diepoxide during pregnancy induces subfertility and birth defects of offspring in mice.

4-vinylcyclohexene diepoxide (VCD), widely used in industry, is a hazardous compound that can cause premature ovarian failure, but whether maternal VCD exposure affects the health and reproduction of offspring is unknown. Here we focused on the effects of VCD on fertility and physical health of F1 and F2 offspring in mice. The pregnant mice were injected intraperitoneally with different dosages of VCD once every day from 6.5 to 18.5 days post-coitus (dpc). We showed that maternal exposure to VCD during pregnancy significantly reduced the litter size and ovarian reserve, while increasing microtia occurrences of F1 mice. The cytospread staining showed a significant inhibition of meiotic prophase I progression from the zygotene stage to the pachytene stage. Mechanistically, the expression level of DNA damage marker (γ-H2AX) and BAX/BCL2 ratios were significantly increased, and RAD51 and DMC1 were extensively recruited to DNA double strand breaks sites in the oocytes of offspring from VCD-exposed mothers. Overall, our results provide solid evidence showing that maternal exposure to VCD during pregnancy has intergenerational deleterious effects on the offspring.

Single-Cell Analysis Reveals Malignant Cells Reshape the Cellular Landscape and Foster an Immunosuppressive Microenvironment of Extranodal NK/T-Cell Lymphoma.

Extranodal natural killer/T-cell lymphoma (NKTCL) is an aggressive type of lymphoma associated with Epstein-Barr virus (EBV) and characterized by heterogeneous tumor behaviors. To better understand the origins of the heterogeneity, this study utilizes single-cell RNA sequencing (scRNA-seq) analysis to profile the tumor microenvironment (TME) of NKTCL at the single-cell level. Together with in vitro and in vivo models, the study identifies a subset of LMP1+ malignant NK cells contributing to the tumorigenesis and development of heterogeneous malignant cells in NKTCL. Furthermore, malignant NK cells interact with various immunocytes via chemokines and their receptors, secrete substantial DPP4 that impairs the chemotaxis of immunocytes and regulates their infiltration. They also exhibit an immunosuppressive effect on T cells, which is further boosted by LMP1. Moreover, high transcription of EBV-encoded genes and low infiltration of tumor-associated macrophages (TAMs) are favorable prognostic indicators for NKTCL in multiple patient cohorts. This study for the first time deciphers the heterogeneous composition of NKTCL TME at single-cell resolution, highlighting the crucial role of malignant NK cells with EBV-encoded LMP1 in reshaping the cellular landscape and fostering an immunosuppressive microenvironment. These findings provide insights into understanding the pathogenic mechanisms of NKTCL and developing novel therapeutic strategies against NKTCL.

Viscoelastic Notch Signaling Hydrogel Induces Liver Bile Duct Organoid Growth and Morphogenesis.

Cholangiocyte organoids can be used to model liver biliary disease; however, both a defined matrix to emulate cholangiocyte self-assembly and the mechano-transduction pathways involved therein remain elusive. A series of defined viscoelastic hyaluronan hydrogels to culture primary cholangiocytes are designed and it is found that by mimicking the stress relaxation rate of liver tissue, cholangiocyte organoid growth can be induced and expression of Yes-associated protein (YAP) target genes could be significantly increased. Strikingly, inhibition of matrix metalloproteinases (MMPs) does not significantly affect organoid growth in 3D culture, suggesting that mechanical remodeling of the viscoelastic microenvironment-and not MMP-mediated degradation-is the key to cholangiocyte organoid growth. By immobilizing Jagged1 to the hyaluronan, stress relaxing hydrogel, self-assembled bile duct structures form in organoid culture, indicating the synergistic effects of Notch signaling and viscoelasticity. By uncovering critical roles of hydrogel viscoelasticity, YAP signaling, and Notch activation, cholangiocyte organogenesis is controlled, thereby paving the way for their use in disease modeling and/or transplantation.

Inhibition of DNA methylation during chronic obstructive bladder disease (COBD) improves function, pathology and expression.

Partial bladder outlet obstruction due to prostate hyperplasia or posterior urethral valves, is a widespread cause of urinary dysfunction, patient discomfort and also responsible for immense health care costs. Even after removal or relief of obstruction, the functional and pathologic aspects of obstruction remain as a chronic obstructive bladder disease (COBD). Epigenetic changes, such as DNA methylation, contribute to the persistent character of many chronic diseases, and may be altered in COBD. We tested whether candidate genes and pathways and the pathophysiology of COBD were affected by a hypomethylating agent, decitabine (DAC). COBD was created in female Sprague-Dawley rats by surgical ligation of the urethra for 6 weeks, followed by removal of the suture. Sham ligations were performed by passing the suture behind the urethra. After removal of the obstruction or sham removal, animals were randomized to DAC treatment (1 mg/kg/3-times/week intraperitoneally) or vehicle (normal saline). Bladder function was non-invasively tested using metabolic cages, both one day prior to de-obstruction at 6 weeks and prior to sacrifice at 10 weeks. Residual volume and bladder mass were measured for each bladder. Bladders were examined by immunostaining as well as qPCR. The effects of DNA methyltransferase (DNMT)-3A knockout or overexpression on smooth muscle cell (SMC) function and phenotype were also examined in bladder SMC and ex vivo culture. Residual volumes of the DAC treated group were not significantly different from the NS group. Compared to COBD NS, COBD DAC treatment helped preserve micturition volume with a significant recovery of the voiding efficiency (ratio of the maximum voided volume/maximum bladder capacity) by one third (Fig. 1, p > 0.05). Brain-derived neurotrophic factor (BDNF) variants 1 and 5 were upregulated by COBD and significantly reduced by DAC treatment. Deposition of collagen in the COBD bladder was reduced by DAC, but gross hypertrophy remained. In bladder SMC, DNMT3A overexpression led to a loss of contractile function and phenotype. In bladders, persistently altered by COBD, inhibition of DNA-methylation enhances functional recovery, unlike treatment during partial obstruction, which exacerbates obstructive pathology. The underlying mechanisms may relate to the gene expression changes in BDNF and their effects on signaling in the bladder.

Generation of functional ciliated cholangiocytes from human pluripotent stem cells.

The derivation of mature functional cholangiocytes from human pluripotent stem cells (hPSCs) provides a model for studying the pathogenesis of cholangiopathies and for developing therapies to treat them. Current differentiation protocols are not efficient and give rise to cholangiocytes that are not fully mature, limiting their therapeutic applications. Here, we generate functional hPSC-derived cholangiocytes that display many characteristics of mature bile duct cells including high levels of cystic fibrosis transmembrane conductance regulator (CFTR) and the presence of primary cilia capable of sensing flow. With this level of maturation, these cholangiocytes are amenable for testing the efficacy of cystic fibrosis drugs and for studying the role of cilia in cholangiocyte development and function. Transplantation studies show that the mature cholangiocytes generate ductal structures in the liver of immunocompromised mice indicating that it may be possible to develop cell-based therapies to restore bile duct function in patients with biliary disease.

High-Throughput Functional Analysis of CFTR and Other Apically Localized Proteins in iPSC-Derived Human Intestinal Organoids.

Induced Pluripotent Stem Cells (iPSCs) can be differentiated into epithelial organoids that recapitulate the relevant context for CFTR and enable testing of therapies targeting Cystic Fibrosis (CF)-causing mutant proteins. However, to date, CF-iPSC-derived organoids have only been used to study pharmacological modulation of mutant CFTR channel activity and not the activity of other disease-relevant membrane protein constituents. In the current work, we describe a high-throughput, fluorescence-based assay of CFTR channel activity in iPSC-derived intestinal organoids and describe how this method can be adapted to study other apical membrane proteins. Specifically, we show how this assay can be employed to study CFTR and ENaC channels and an electrogenic acid transporter in the same iPSC-derived intestinal tissue. This phenotypic platform promises to expand CF therapy discovery to include strategies that target multiple determinants of epithelial fluid transport.

A new platform for high-throughput therapy testing on iPSC-derived lung progenitor cells from cystic fibrosis patients.

For those people with cystic fibrosis carrying rare CFTR mutations not responding to currently available therapies, there is an unmet need for relevant tissue models for therapy development. Here, we describe a new testing platform that employs patient-specific induced pluripotent stem cells (iPSCs) differentiated to lung progenitor cells that can be studied using a dynamic, high-throughput fluorescence-based assay of CFTR channel activity. Our proof-of-concept studies support the potential use of this platform, together with a Canadian bioresource that contains iPSC lines and matched nasal cultures from people with rare mutations, to advance patient-oriented therapy development. Interventions identified in the high-throughput, stem cell-based model and validated in primary nasal cultures from the same person have the potential to be advanced as therapies.

One-Step Formation of Protein-Based Tubular Structures for Functional Devices and Tissues.

Tubular biological structures consisting of extracellular matrix (ECM) proteins and cells are basic functional units of all organs in animals and humans. ECM protein solutions at low concentrations (5-10 milligrams per milliliter) are abundantly used in 3D cell culture. However, their poor "printability" and minute-long gelation time have made the direct extrusion of tubular structures in bioprinting applications challenging. Here, this limitation is overcome and the continuous, template-free conversion of low-concentration collagen, elastin, and fibrinogen solutions into tubular structures of tailored size and radial, circumferential and axial organization is demonstrated. The approach is enabled by a microfabricated printhead for the consistent circumferential distribution of ECM protein solutions and lends itself to scalable manufacture. The attached confinement accommodates minute-long residence times for pH, temperature, light, ionic and enzymatic gelation. Chip hosted ECM tubular structures are amenable to perfusion with aqueous solutions and air, and cyclic stretching. Predictive collapse and reopening in a crossed-tube configuration promote all-ECM valves and pumps. Tissue level function is demonstrated by factors secreted from cells embedded within the tube wall, as well as endothelial or epithelial barriers lining the lumen. The described approaches are anticipated to find applications in ECM-based organ-on-chip and biohybrid structures, hydraulic actuators, and soft machines.

The CF Canada-Sick Kids Program in individual CF therapy: A resource for the advancement of personalized medicine in CF.

BACKGROUND: Therapies targeting certain CFTR mutants have been approved, yet variations in clinical response highlight the need for in-vitro and genetic tools that predict patient-specific clinical outcomes. Toward this goal, the CF Canada-Sick Kids Program in Individual CF Therapy (CFIT) is generating a "first of its kind", comprehensive resource containing patient-specific cell cultures and data from 100 CF individuals that will enable modeling of therapeutic responses. METHODS: The CFIT program is generating: 1) nasal cells from drug naïve patients suitable for culture and the study of drug responses in vitro, 2) matched gene expression data obtained by sequencing the RNA from the primary nasal tissue, 3) whole genome sequencing of blood derived DNA from each of the 100 participants, 4) induced pluripotent stem cells (iPSCs) generated from each participant's blood sample, 5) CRISPR-edited isogenic control iPSC lines and 6) prospective clinical data from patients treated with CF modulators. RESULTS: To date, we have recruited 57 of 100 individuals to CFIT, most of whom are homozygous for F508del (to assess in-vitro: in-vivo correlations with respect to ORKAMBI response) or heterozygous for F508del and a minimal function mutation. In addition, several donors are homozygous for rare nonsense and missense mutations. Nasal epithelial cell cultures and matched iPSC lines are available for many of these donors. CONCLUSIONS: This accessible resource will enable development of tools that predict individual outcomes to current and emerging modulators targeting F508del-CFTR and facilitate therapy discovery for rare CF causing mutations.