As a step towards understanding how neuronal maturation is regulated, we studied the developmental switch of response to the neurotransmitter GABA, from excitatory depolarization to inhibitory hyperpolarization. We compared acutely isolated retinal ganglion cells (RGCs) at various developmental stages and RGCs differentiated in vitro from embryonic retinal progenitors for the effects of aging and, independently, of retinal environment age on their GABAA receptor (GABAAR) responses, elicited by muscimol. We found that neurons generated in vitro from progenitors exhibited depolarizing, immature GABA responses, like those of early postnatal RGCs. As progenitor-derived neurons aged from 1 to 3 weeks, their GABA responses matured. Interestingly, signals secreted by the early postnatal retina suppressed acquisition of mature GABA responses. This suppression was not associated with changes in expression of GABAAR or of the chloride co-transporter KCC2, but rather with inhibition of KCC2 dimerization in differentiating neurons. Taken together, these data indicate GABA response maturation depends on release of inhibition by developmentally regulated diffusible signals from the retina.During development, newly-differentiated neurons undergo several morphological and physiological changes to become functional, mature neurons. Physiologic maturation of neuronal cells derived from isolated stem or progenitor cells may provide insight into maturation  Several developmental milestones, including expression of excitable channels5 and maturation of the actions of neurotransmitters8, reflect a shift in function during development and are required before a neuron can operate as a functional unit within a mature neuronal circuit. Reaching such developmental milestones distinguishes a physiologically maturing neuron.In the central nervous system (CNS), neurons are born from stem or progenitor cells, and as the brain develops, the newborn neurons acquire physiological properties that play a major role in their survival and in the development of networks of highly specific synaptic connections10, permitting study of neuronal development under controlled conditions. When retinal and other neurons first arise from neural progenitor cells in vivo, they differ from mature neurons in several critical ways. For example, newly-formed retinal ganglion cells (RGCs) have greater intrinsic capacity for rapid axon growth, which transitions towards dendrite growth ability early in development11. The composition of NMDA receptor subunits also changes as RGCs mature, playing an important role in RGC-bipolar cell synaptogenesis and circuit refinement13. A particularly dramatic developmental landmark is the reversal of responses to the neurotransmitter GABA from excitatory early in development, helping to drive activity, to inhibitory in mature neurons14. This switch is achieved not through modifications of the GABA receptor itself, but through an increase in the chloride gradient produced primarily by action of the chloride co-transporter KCC2, so that chloride moves in rather than out through activated GABAA receptors16. Some studies have indicated that diffusible factors from the superior colliculus17 or GABA itself18 may contribute to this developmental reversal in GABA response. These examples demonstrate the complex developmental changes neurons undergo on their path to maturity and formation of functional neural circuits.Are either stem cells or immature neurons extrinsically signaled to mature physiologically, or are such developmental changes intrinsically programmed in a cell-autonomous fashion? The retina is a favorable part of the CNS to address these questions, as retinal progenitor cells and their progeny can be differentiated and studied in purified or enriched culturesin vivo, very little is known about the developmental maturation of neurons generated from stem cells or progenitor cells in vitro. This has become particularly important given the current interest in investigating stem cells both for disease modeling and cell transplantation therapy. The present experiments were designed to study neurons generated from embryonic retinal progenitors in vitro, particularly their maturation, using the response to GABA receptor activation as one such indicator. These data focus on the cellular and molecular mechanisms associated with the shift from excitation to inhibition, and advance a new model in which intrinsically regulated maturation of GABAA responses in RGC-like neurons is inhibited by diffusible molecules released by the immature retina that work to regulate KCC2 activity by inhibiting dimerization.In contrast to normal development A receptors in neurons19 and was used to assess the GABAA response in progenitor-derived neurons using calcium imaging with fura 2-AM, since Ca2+ levels rise when the neurons depolarize20. First, as a control using cultured RGCs to confirm previous results17, muscimol application elicited a strong elevation of intracellular calcium in purified, cultured RGCs from early postnatal  rats  retinal progenitor cells in serum-free medium containing BDNF (50 ng/ml) and CNTF (10 ng/ml), as described in Methods. By 7 days in culture (7 DIV), chosen as a time point corresponding to an early postnatal age in vivo, cells with neuronal morphology responded to muscimol by elevating intracellular calcium, reflecting a depolarization  or in the presence of a P5 or P14 retinal suspensions in hanging cell inserts . The progenitor derived neurons cultured under the inserts were imaged in similar fashion  as well as decreased fluorescence (hyperpolarization) in response to muscimol application Fig. . The conA receptor response maturation seen in the presence of P5 retinal cells did not appear to be mediated through inhibition of KCC2 expression. We also tested the possibility that downregulation of GABAA receptor in control and P14 retinal cell insert samples caused the loss of GABA response, but qPCR for GABAA receptor subunit alpha 1 showed no significant difference in mRNA expression between conditions, suggesting that the functional differences in responses to muscimol were not due to loss of GABAA receptor expression , which has been shown to be a potential regulator of KCC2 expression and GABA response reversal in cortical neurons33. Such mixed cultures could provide the requisite signals for the newly-formed neurons to mature with time and suggest a useful model system for future experiments targeted at identifying such signals.We observed that after a short time in culture, progenitor-derived neurons exhibit immature GABAin vitro led to an additionally unexpected finding that soluble factors from P5 and not P14 retinal suspensions inhibited the maturation of that response. This difference was apparently not due to differences in the cell viability of P5 vs P14 inserts, as both preparations exhibited similar survival across all conditions. Again, using this model system, future experiments could determine the identity and properties of the inhibitory signal or signals, the specific cells that produced them, and whether bidirectional signaling is required between the P5 retinal cells and the nascent RGCs. Nevertheless, the discovery of an intrinsic developmental switch in GABA receptor physiology for which there is an extrinsic inhibition is novel.The maturation of the GABA response A receptor responses. However, a difference was observed in post-translational modification of KCC2, specifically in dimerization, which is known to influence function34. This supports the hypothesis that the early retinal environment inhibits GABAA response maturation by suppressing formation of functional, dimerized KCC2 co-transporter. Phosphorylation of a key tyrosine residue has previously been shown to be required for KCC2 oligomerization34, so molecules secreted by cells in the early postnatal retinal suspension may disrupt this as yet unidentified signaling pathway. It remains to be determined if these factors mediate a delay in the acquisition of mature responses or can completely inhibit the process, perhaps over longer times in culture. Indeed, maturation of GABAA receptor activity might depend on a balance of molecules that inhibit and facilitate maturation, and it would be interesting to know whether identified signals can induce a reversal of maturation in developmentally mature neurons in vitro or in vivo.Although we first expected KCC2 expression to dictate this maturation timeline, KCC2 expression was upregulated under all culture conditions with sufficient time, including those inhibiting maturation of GABAOur results point to changes in KCC2 underlying the maturation of the response to GABA, but they do not eliminate the possibility that other mechanisms might also participate. For example, there could be a contribution of the NKCC1 transporter, which rises in non-neuronal cells and in neurons, and initially accounts for the lowered Nernst potential for chloride. Another formal possibility would be an action of background synaptic interactions, such that muscimol produces disinhibition, but the uniform results with calcium and voltage-sensitive dye imaging across neurons in preparations showing delayed maturation argue against it.As a technical matter, calcium imaging is an indirect but well-established measure of the voltage response, it permits rapid screening of large numbers of cells, and it is useful for the detection of depolarization whether or not some of the measured calcium is released from intracellular stores. Moreover, the results with voltage indicator dye were consistent with those from calcium imaging, but in future studies it would be of interest to add pharmacological approaches, such as application of thapsigargin to deplete calcium stores in endoplasmic reticulum or 2-Aminoethoxydiphenyl borate (2-APB) to block IP3-mediated calcium release, to determine if some of the calcium rise is from such intracellular release. Direct electrophysiological recordings would also complement these data, but our attempts at recording from the small, immature cells in these cultures proved difficult and unreliable.The fact that cultured neurons, which changed their responses to muscimol, did not exhibit calcium responses in baseline readings, supports the notion that if background synaptic activity were present, it did not provide measurable excitation of one cell by another. This is of interest in part to address the theoretical question of whether cellular heterogeneity and synaptic interactions might contribute to the observed changes in physiology. Not only did all immature neurons depolarize at early stages, but with maturation the transition to hyperpolarization measured with voltage sensitive dyes was consistent with only a subset of cells retaining calcium signals.in vitro from embryonic retinal progenitors. This may guide a choice between ‘immature’ and ‘mature’ neurons for effective cell replacement following in vivo transplantation. Based on available literature on adult born neurons in vivo31, neurons that are depolarized by GABA may be a more viable donor population when synaptic integration is desired. Thus, identification of molecules that inhibit acquisition of mature, inhibitory GABA responses might foster generation of immature stem cell or progenitor cell-derived neurons that are better able to make suitable synaptic connections with host neurons.Stem cell-derived neurons are an often-studied source of donor material for cell replacement, and given the importance of GABA during neuronal development, it might be preferable to use cells with specified GABA response properties, as well as other indicators of physiologic maturation. Our study indicates that the excitatory response to GABA is present transiently in newly-born neurons generated Taken together, our data shed light on the mechanism behind functional maturation of neurons from progenitor cells. We anticipate that understanding the regulation of developmental changes in neurons and being able to influence them under controlled conditions will be critical to generating better disease models and cell replacement strategies.35. Briefly, whole retinas were extracted and dissociated with papain, followed by sequential immunopanning with the Thy1 antibody to yield 99.5% pure RGCs. Purified RGCs were cultured for 3–4 days before calcium imaging. Embryonic retinal progenitors were obtained from E14 rat embryos by papain  digestion of dissected retinas. Approximately 100,000 purified RGCs or retinal progenitors were plated on individual poly-D-lysine- and laminin-coated coverslips in 24 well plates in serum-free RGC growth medium containing sodium pyruvate, N-acetyl cysteine, L-glutamine, Sato supplement, insulin, BDNF (brain-derived neurotrophic factor), CNTF (ciliary neurotrophic factor), and forskolin, as described previously36, and neural supplement GS21  was added. The cells were cultured for 7–21 days with media changes every 3 days. After 4–5 days in vitro, RGC-like neurons were generated from the retinal progenitor cells along with some glia-like cells.RGCs from early and late postnatal rats were purified and cultured as publishedHanging cell culture inserts  were prepared using papain-digested retinas from P5 and P14 rats; these were placed within the culture wells hanging over the retinal progenitors. The contents of the hanging inserts were left undisturbed for the duration of culture, while the media were replenished through media changes in the main chamber. Survival of P5 and P14 retinal cell suspensions was determined by dye exclusion. Briefly, cells were detached from the hanging culture inserts by moderate pipetting, diluted 1:1 with Trypan blue and counted using a hemocytometer.2 2, MgCl2 1, glucose 30, and HEPES 25, pH 7.4, containing 1 mg/ml bovine serum albumin. The cells were washed with Tyrode’s solution and placed in the imaging chamber . Calcium imaging was performed on an inverted fluorescence microscope  with a fast switching light source  using MetaFluor software (Molecular Devices) working within a linear range of the camera. Regions of interest (ROIs) were drawn around brightly labeled cell bodies corresponding to cells with neuronal morphology observed under DIC illumination. As a control, in each field of view 1–2 ROIs were also drawn around cells with non-neuronal, glia-like morphology. Calcium responses within these regions of interest were observed as changes in fluorescence ratio (340/380) upon sequential stimulation with muscimol  and KCl (30 mM) in Tyrode’s solution. The muscimol dose was within the reported linear range of dose response curves. Test solutions were replaced with Tyrode’s solution following each stimulus. A response was considered positive if the change in fluorescence ratio exceeded six times the standard deviation in baseline values. With this criterion, cells were scored as responsive or non-responsive to a specific stimulus. More than 25 cells were tested and analyzed in each experiment. Each point on the summary plots corresponds to the fraction of cells in each experiment that responded to muscimol. 100% of the neuronal cells responded to KCl. As controls, 1–2 cells of non-neuronal morphology were also measured; these cells did not respond to KCl.Cover slips with cultured cells were incubated for 30 min at room temperature with 5 µM fura 2-AM  in Tyrode’s solution: (in mM) NaCl 129, KCl 5, CaClA receptor remained active and the Nernst potential for chloride had shifted.Where indicated, to determine that a lack of calcium response was because the chloride current had reversed, the KCC2 inhibitor dihydroindenyl-oxy-alkanoic acid  was added and the response to muscimol retested. DIOA was dissolved in 0.1 N NaOH (20 mM stock) and was added at a final concentration of 20 µM in Tyrode’s solution. The appearance of a response in DIOA was used to confirm that the GABA37. High magnification KCC2 images were acquired on a Zeiss 880 confocal microscope at 63X using Airyscan Fast.Following calcium imaging experiments, coverslips were fixed with 4% paraformaldehyde in 1X PBS. The cells were blocked and permeabilized  before staining for co-transporter KCC2  overnight at 4 °C. Fluorophore-conjugated anti-rabbit secondary antibodies were used to label the primary antibodies and unlabeled controls. Stained samples were imaged on a Zeiss Axio Observer 7 using the same imaging parameters between test groups. KCC2 fluorescence intensity was obtained from non-saturated immunostained images by measuring the mean gray values of over 100 cells per condition per experiment using FijiProgenitor cells were cultured in 24-well plates on coverslips for 14 days. Coverslips were moved to the imaging chamber and washed with Tyrode’s solution. Red FLIPR dye  was reconstituted to a final concentration of 20 µM and added to the imaging chamber. The cells were incubated for 30 mins at 37 °C. Muscimol (100 µM) and KCl (30 mM) were added to the FLIPR dye solution and applied to the cells using gravity perfusion while the fluorescence responses were imaged by taking images every 500 ms. Fluorescence changes were analyzed using Fiji software and values plotted in Excel.37 and normalized to GAPDH expression for analysis.Progenitor cells with and without hanging inserts were cultured in 6-well plates for 7 or 21 days. The cultures were harvested by mechanical dissociation in ice-cold 1X protease inhibitor  in PBS and centrifuged at 16,000 rpm at 4 °C to lyse the cells. The membrane pellet was frozen and processed for protein extraction by treating with 1% DDM  and 1X protease inhibitor in PBS for 1.5 hours on ice. The samples were centrifuged at 16,000 rpm at 4 °C to remove non-solubilized fragments, and the protein concentration in the supernatant was measured by BCA assays. The protein samples and ladder were run on 3–8% Tris-acetate gels without reducing agent at 150 V for 1.5 hours. The proteins were transferred onto Immobilon-P PVDF membranes overnight at 4 °C. Following transfer, the membranes were blocked with 5% milk in 1X Tris-buffered saline with 0.1% Tween-20 (TBST) and immunoblotted for KCC2  and GAPDH . HRP-conjugated secondary antibodies were used before treatment with ECL substrate . Western blots were imaged using ImageQuant LAS 4000. Multiple images were taken with increasing exposure times to ensure a linear working range. Protein expression levels were measured using FijiProgenitor cells were cultured with and without hanging inserts in 12-well plates for 7 and 21 DIV before RNA extraction using the RNeasy mini kit . Following cDNA synthesis from the RNA samples, qPCR reactions were set up using TaqMan gene expression assays for Tuj1 (Hs00801390_s1), KCC2 (Hs00221168_m1) and GABA A1 receptor subunit (Hs00971228_m1) using the CFX Connect™ Real-Time PCR Detection System (BioRad). Experiments were set up in triplicates. qPCR data from three experimental repeats were analyzed using REST 2009 (Qiagen) with Tuj1 as the reference gene. All values were normalized to the ‘no insert’ values for analysis.post-hoc t-tests to determine significance between specific groups. P-values for t-tests have been reported throughout the text. For comparison, the Kruskal-Wallis test on the data comparing the percentage of cells responding to muscimol at 21 DIV under the different conditions found that these data were significant with p-value < 0.0001. Post-hoc Mann-Whitney tests found that (1) the percentage of cells responding to muscimol at 21 DIV in the absence of retinal suspension (no insert) was significantly lower than those in the presence of P5 retinal suspension (P5 insert); p < 0.0001; (2) the percentage of cells responding to muscimol at 21 DIV in the presence of P5 retinal suspension was significantly higher than those in the presence of P14 suspension (P14 insert); p < 0.0001; and (3) the percentage of cells responding at 21 DIV with no retinal suspension was not significantly different from those cultured in the presence of P14 retinal suspension (p = 0.6124).Statistical analyses for significance were performed using one-way ANOVA followed by


Wdr35) drives ongoing, pathological remodelling of the biliary tree, resulting in progressive cyst formation and growth. The development of cystic tissue requires the activation of transforming growth factor-β (TGFβ) signalling, which promotes the expression of a pro-cystic, fibronectin-rich extracellular matrix and which itself is perceived by a changing profile of Integrin receptors on the cystic epithelium. This signalling axis is conserved in liver cysts from patients with either autosomal dominant polycystic kidney disease or autosomal dominant polycystic liver disease, indicating there are common cellular mechanisms for liver cyst growth regardless of the underlying genetic cause. Cyst number and size can be reduced by inhibiting TGFβ or Integrin signalling in vivo and we suggest that our findings represent a therapeutic route for patients with polycystic liver disease, the majority of which would not be amenable to surgery.The formation of multiple cysts in the liver occurs in a number of isolated monogenic diseases or multisystemic syndromes, during which bile ducts develop into fluid-filled biliary cysts. For patients with polycystic liver disease (PCLD), non-surgical treatments are limited and managing life-long abdominal swelling, pain, and increasing risk of cyst rupture and infection is common. We demonstrate here that loss of the primary cilium on postnatal biliary epithelial cells (via the deletion of the cilia gene  PKD1, PKD2 and PKHD1  that are required to build and maintain functional PC are mutated  genes  mutated –17. Acro mutated . Furtherde novo production of a laminin- and fibronectin-rich pro-cystic microenvironment. This newly formed niche promotes both cell-autonomous changes in cell shape and duct-level mechanical rearrangements that converge to drive cyst-fission, a process whereby single, large cysts undergo morphological splitting. This process gives rise to many, smaller polycystic progeny and can be halted by pharmacological inhibition of specific pro-cystic integrin receptors, thereby providing a potential approach to treat patients living with liver cysts.Here, we show that PC loss in adult mouse BECs is sufficient to cause bile duct expansion, driving cyst formation through cysts leads to the formation of renal cysts, though deletion of the same gene in the liver does not automatically lead to hepatic cyst formation, indicating that different tissues are variously sensitive to cilia loss /Intraflagellar Transport 121 (Ift121) gene specifically in postnatal BECs Wdr35 bile ducts, PC protrude from the apical surfaces of BECs and are positive for IFT-B complex (IFT88-positive) and the ciliary membrane-localised protein ARL13B; however in T;Wdr35flox/floxKrt19-CreER mice (abbreviated to Wdr35-/-) after the administration of tamoxifen, BEC PC are largely absent and instead exist as IFT88-positive, shortened rudiments lacking ARL13B and normal PC function, as we have previously described  and Vcam1 (Vascular Cell Adhesion Molecule-1), which were not expressed by +/+Wdr35 cells  and retained a number of BEC-specific markers including EpCAM (on which BECs were sorted), the pan-BEC marker Spp1  pSMAD3S423/S425 was absent. In contrast, in cystic BECs, high nuclear SMAD4 and extensive pSMAD3S423/S425 staining was observed , a sensitive antibody-based proteomic approach biased to detect changes in ECM and cell-adhesion proteins, we found that when compared to freshly isolated ducts, 72 h cultured ducts increased their fibronectin production as they became cystic. Furthermore, the amount of fibronectin produced was inhibited in cultures that had been treated with SIS3 .Fn1, is a target of SMAD3 in vitro  as well as ECM components including collagens and laminins , a canonical ECM receptor which heterodimerises with other α-integrin subunits to sense alterations in the ECM. Analysis of cystic -/-Wdr35 BECs also identified candidate interactions between Itgb1 and Fn1, however this Fn1 domain expanded to include putative -/-Wdr35-specific interactions with Itga2, Itga3, Itgav, Itgb6, Cd44, and Plaur (+/+Wdr35 vs -/-)Wdr35 to define which integrin subunits were most differentially expressed between these groups ((Itgb1) transcripts were universally expressed by BECs whether they were normal or cystic, whereas α2-integrin (Itga2) mRNA was selectively expressed by -/-Wdr35 mutant cells  and kidney (PKD1 and PKD2) disease-related genes, suggesting that the acquisition of integrin-α2β1 mediated-sensing of the microenvironment may be a common mechanism during cyst formation across pathologies regardless of the underlying genetics.As in our mouse model of PCLD, patients with cystic liver diseases have high amounts of ITGA2  and pMLC-/-Wdr35 livers, where some cysts appeared largely spherical in shape, other cysts readily formed septa that protruded into or across the cyst lumen at both 6 and 12 m  cysts in their liver; however, it remains unclear whether each cyst originates clonally from mutant precursor cells (corresponding to many separate originating events) or whether single cysts themselves grow and then structurally divide to produce a polycystic pattern of disease (in which cells of origin are divided between multiple cysts). Indeed, in and 12 m . These rand 12 m .Wdr35-loss are derived from multiple individual clonal events or because of the physical division of cysts, we crossed T; Wdr35flox/floxK19CreER mice with the Confetti lineage reporter mouse line T; Wdr35flox/flox; R26LSL-Confetti;(K19CreER abbreviated to -/-Wdr35-Confetti), (Wdr35-deletion (and Confetti labelling), livers were surveyed using 3D confocal imaging using the FUnGI protocol(57). In -/-Wdr35-Confetti mice, small, isolated cysts were never completely clonal in colour and were comprised of both labelled and unlabelled cells  surface of cysts, whereas non-cultured ducts were ITGA2-negative , . In thisnfetti), , therebynfetti), . Six moned cells . Where ced cells . These rnegative .TKrt19CreER mice also carrying a LSL-tdTomatoR26 reporter allele (to define which cells had activated Cre) and flox/floxItga2 (or +/+Itga2 as a control). Cre was activated in vivo by administering mice tamoxifen and 7 days after the final dose, ducts were isolated and cultured in Matrigel (from hereon in defined at -/-)Itga2. Immediately after plating (0 h), tdTomato-positive ducts from -/-Itga2 mice had the same dimensions as those from +/+Itga2 animals. After 72 h in culture, the tdTomato-positive cysts forming from -/-Itga2 ducts were smaller than in +/+Itga2 controls  mutation) or liver (PRKCSH (c. 292+1G>C)) disease and treated with TC-I 15 for 48 h demonstrated a reduction in ITGA2 protein , this wWdr35-loss is simply a model of liver cyst formation and within patients there have been a suite of causative mutations identified. Some of these are essential cilia genes (including WDR35), however the most commonly mutated proteins constitute the polycystin complex and many others are involved in protein trafficking and processing within the endoplasmic reticulum. Although we have shown that across a range of genetic mutations common cellular processes are activated, without systematic deletion of these “cystic” genes we are unable to comment on the diverse features that each mutation could infer.There are limitations to this study. PCLD evolves throughout a patient’s lifetime and although we demonstrate that cyst generation is an ongoing process with active remodelling of the biliary epithelium, we are limited in our understanding of cyst-initiating mechanisms and our current study cannot address whether there are earlier opportunities to limit the transformation of healthy tissue into cysts. Moreover, although we have concentrated on the epithelial component of cysts, there are likely a variety of stromal and immune cells that support cystic growth and cyst fission. We did not assess the importance of these in this work and to do so will be essential to build a complete picture of the cystic process. Last, a priori and sample sizes are provided within the figure legends. In experiments where animals or cultures received inhibitors, these were randomized prior to dosing. All animal tissues were collected blinded and analysed without knowledge of to which group they belonged. Complete materials and methods can be found within the supplementary materials.The primary hypothesis of this study was that loss of the primary cilium results in global changes to cholangiocyte signalling to disrupt bile duct homeostasis. Secondary hypotheses regarding the positive roles of TGFβ and integrin signalling in cyst growth were developed as a consequence of the first hypothesis being proven true and tested using pharmacological and genetic inhibition studies. The sample size of this study was not defined JPH Drenth  provided samples from patients with PCLD with known genetics under ethical approval 2012/317. Further samples were provided by the NHS Lothian and University of Edinburgh Academic and Clinical Central Office for Research & Development (ACCORD) biobank under ethical code TGU-LAB-1512. All tissues were collected under informed consent or were archival diagnostic specimens. Human tissue details are summarised in Animals were maintained in SPF environment and studies carried out in accordance with the guidance issued by the Medical Research Council in “Responsibility in the Use of Animals in Medical Research” (July 1993) and licensed by the Home Office under the Animals (Scientific Procedures) Act 1986. Experiments were performed under project license number PFD31D3D4 in facilities at the University of Edinburgh (PEL 60/6025).All experimental groups were analysed for normality using a D’Agostino–Pearson Omnibus test. Groups that were normally distributed were compared with either a two-tailed Student’s t test or a one-way ANOVA with a post hoc correction for multiple testing. Non-parametric data were analysed using a Wilcoxon–Mann–Whitney U test or a Kruskall–Wallis test when comparing multiple non-parametric data. Throughout, P< 0.05 was considered significant. Data are represented as mean with S.E.M. for parametric data or median with S.D. for non-parametric data. All n= are individual animals and N= are technical or experimental replicates and are and defined in the figure legends.All figures were assembled with Adobe Illustrator and graphics were created with BioRender.com.Supplementary Material


The current consensus for the prevention and management of type 2 diabetes mellitus (T2DM) is that high-quality diets and adherence to a healthy lifestyle provide significant health benefits. Remarkably, however, there is little agreement on the proportions of macronutrients in the diet that should be recommended to people suffering from pre-diabetes or T2DM. We herein discuss emerging evidence that underscores the importance of gene-diet interactions in the improvement of glycemic biomarkers in T2DM. We propose that we can achieve better glycemic control in T2DM patients by coupling Mediterranean diets to genetic information as a predictor for optimal diet macronutrient composition in a personalized manner. We provide evidence to support this concept by presenting a case study of a T2DM patient who achieved rapid glycemic control when adhered to a personalized, genetically-guided Mediterranean Diet. Long considered a disease of minor significance, type 2 diabetes mellitus (T2DM) currently represents a major threat to human health with an estimated number of 425 million adult patients and four million deaths globally in 2017. Substantial evidence indicates that T2DM can be largely prevented or managed through adherence to a healthy lifestyle and a high-quality diet  gene variant rs7903146 has attracted significant attention as it is one of the strongest genetic markers of predisposition to diabetes, with the TT genotype increasing disease risk by 40–50% [Even though carbohydrates (CHO) have a major dietary influence on postprandial blood glucose, evidence on total carbohydrate needs and T2DM is largely conflicting . Surprisy 40–50% . Interesy 40–50%  and our MTNR1B) variant rs1387153 is another example of genetic variation influencing T2DM patient responses to CHO. An analysis of 5 cohort studies including more than 28,000 participants has demonstrated that the minor T allele of rs1387153 strongly interacts with CHO in modulating fasting glucose, to the extent that every 1% increase in CHO intake exacerbates the fasting glucose-raising effect of the T allele [The melatonin receptor 1b  variant rs2287161, which has been associated with the regulation of circadian rhythms and linked to depression and sleep disturbance [PCSK7) gene variant rs236918 who were assigned to a high-carbohydrate diet displayed a greater decrease in fasting insulin levels and HOMA-IR than non-carriers [MTNR1B and PCSK7 polymorphisms have been associated with increased risk of diabetes [Cryptochrome 1  gene variant rs780094, which has been explored as a component of polygenic risk for T2DM and dyslipidemia [GCKR variant on both triglyceride and glucose levels [Likewise, the glucokinase regulatory protein (ipidemia  diminishe levels .APOA5) SNP rs964184. It has been reported that compared to rs964184 TT homozygotes, T2DM patients with impaired fasting glucose who carry the C allele of rs964184 could be more susceptible to the adverse effects of a high carbohydrate diet based on refined rice by having an elevation of triglycerides [APOA5 rs964184 C variant, to prevent diabetic hypertriglyceridemia.Another gene polymorphism associated with the response of T2DM patients to particular sources of carbohydrates is apolipoprotein-A5  gene variant rs1440581. Individuals who are homozygous for the T allele of this SNP benefit most by a high-fat diet  in reducing insulin and homeostatic model assessment for β-cell function (HOMA-B) during weight loss whereas the opposite effect is observed for a low fat diet  [PPM1K rs1440581 TT genotype would benefit from an energy-restricted high-fat diet whereas carriers of the C allele could be advised to follow a low-fat diet.Several genes have been identified to impact responses to fat in T2DM. One of them is hydrate) . Similarhydrate) ). TogethGenetic risk scores (GRS), generated by combining the additive effects of several genetic variants, have also been explored to predict optimal dietary fat composition for T2DM patients. For example, a high genetic risk score (GRS) of 14 fasting glucose-associated SNPs could predict health benefit for a diet low in fat . FurtherTCF7L2 rs7903146 polymorphism relative to CC homozygotes [CLOCK rs1801260 [Variations in food sources of fat may play an important role in modulating fasting glucose and other bioclinical variables in T2DM patients. In general, replacing saturated with unsaturated fat in the diet reduces total cholesterol, LDL-C and CVD risk . Surprisozygotes  Likewises1801260  or to abs1801260 . Typically, the average daily level of protein intake for people with diabetes is 1–1.5 g/kg body weight/day or 15–20% of total calories. However, two published meta-analyses have indicated that high protein diets confer several health benefits in diabetes patients ,64.We reason that genetic variation may affect individual responses to different protein percentages in the diet . SeveralDHCR7 variant rs12785878 had a greater improvement of glycaemic parameters when assigned to high-protein diets, whereas improvement was not evident in those who followed a low protein diet [An interaction between vitamin D metabolism-related variants and macronutrient responses in the context of T2DM was demonstrated by the Pounds Lost Trial group; patients with the vitamin D-increasing T allele of the ein diet .A variety of dietary patterns are acceptable for the management of diabetes. Still, the evidence surrounding comparative benefits of different macronutrient ratios or dietary patterns in T2DM patients and the influence of genetics thereof is scarce . GIPR variant rs2287019  rs3014866 variant are more likely to benefit from a low SFA: carbohydrate ratio intake to improve insulin sensitivity, based on HOMA-IR levels [One example of genetic variation influencing responses to mixed dietary patterns is s2287019 . T2DM paR levels .n-3 PUFA and MUFA and low in saturated fat are universally proposed for the prevention and management of T2DM. This concept aligns with the Mediterranean dietary patterns typified by high-quality nutritional elements offering several health benefits. Notably, there is significant variation in the macronutrient distribution among different Mediterranean diets  [Rapidly evolving technologies have offered unparalleled opportunities to assess bioclinical, dietary and genetic data that can improve the management of metabolic diseases such as T2DM. As indicated in this review, high-quality diets rich in an-like) . Likewisan-like) .MTNR1B rs1387153 risk allele T, with a high genetic risk score for diabetes and a high genetic score for coffee consumption was advised to change the macronutrient distribution of his diet from 54% carbohydrate, 32% fat and 16% protein to lower carbohydrate (50%), low fat (25%) and high protein (25%) diet based on Mediterranean high quality foods and olive oil (rich in MUFA and low in saturated fat) [MTNR1B rs10830963 risk allele G which is in linkage disequilibrium with rs1387153 in Greek people) [We propose that prevention and management of T2DM could be improved by combining genetically-guided quantitative and qualitative macronutrient recommendations  with theted fat) ,67,68. S people) ,70,71. TThe emerging prospects of precision nutrition call for re-evaluation of dietary therapies for T2DM taking into account genetic information that could enable stratification of patients towards specific macronutrient intakes. Intervention and observational studies should involve collection of extensive dietary exposure data and, importantly, validation in independent populations to provide solid evidence for interactions between genotype, diet and disease .


TCF4 gene. The mutant intronic CUG RNA is present at one–two copies per cell, posing a challenge to understand how a rare RNA can cause disease. Late-onset FECD is a uniquely advantageous model for studying how RNA triggers disease because: (i) Affected tissue is routinely removed during surgery; (ii) The expanded CTG mutation is one of the most prevalent disease-causing mutations, making it possible to obtain pre-symptomatic tissue from eye bank donors to probe how gene expression changes precede disease; and (iii) The affected tissue is a homogeneous single cell monolayer, facilitating accurate transcriptome analysis. Here, we use RNA sequencing (RNAseq) to compare tissue from individuals who are pre-symptomatic (Pre_S) to tissue from patients with late stage FECD (FECD_REP). The abundance of mutant repeat intronic RNA in Pre_S and FECD_REP tissue is elevated due to increased half-life in a corneal cells. In Pre_S tissue, changes in splicing and extracellular matrix gene expression foreshadow the changes observed in advanced disease and predict the activation of the fibrosis pathway and immune system seen in late-stage patients. The absolute magnitude of splicing changes is similar in pre-symptomatic and late stage tissue. Our data identify gene candidates for early drivers of disease and biomarkers that may represent diagnostic and therapeutic targets for FECD. We conclude that changes in alternative splicing and gene expression are observable decades prior to the diagnosis of late-onset trinucleotide repeat disease.How genetic defects trigger the molecular changes that cause late-onset disease is important for understanding disease progression and therapeutic development. Fuchs’ endothelial corneal dystrophy (FECD) is an RNA-mediated disease caused by a trinucleotide CTG expansion in an intron within the  An inherent paradox of inherited late-onset degenerative disease is the fact that the genetic basis for disease exists years before symptoms arise. At some point the genetic defect begins to trigger a cascade of cellular changes that result in disease, but how is the cascade initiated? Unfortunately, the search for early drivers of many genetic diseases that involve mutant RNA is hindered by lack of accessible pre-symptomatic tissue to identify early changes in gene expression that occur years prior to diagnosis. Corneal tissue provides an accessible source of both pre- and post-symptomatic tissue to explore the molecular origins of inherited late-onset disease.Corneal diseases represent one of the leading causes of vision loss and blindness globally . InheritIn FECD, the post-mitotic endothelium undergoes premature senescence and apoptosis . DescemeTCF4 gene   that possess the CTG expanded repeat; (vii) The availability of tissue from four different cohorts  . FECD haALS/FTD) –C; (ii) ALS/FTD) ; (iii) THere, we use the advantages gained from analyzing the corneal endothelial monolayer to evaluate transcriptomic data from four cohorts of corneal endothelial tissue: control, Pre_S, FECD_REP and FECD_NR. We identify extensive and large magnitude changes in RNA splicing that are shared between Pre_S and FECD-REP cohorts but are not observed in FECD_NR samples. Levels of gene expression are changed in Pre_S samples relative to controls and suggest early triggering of the fibrosis pathway prior to clinical observation of disease symptoms. In late stage disease, pathways related to fibrosis and activation of immune system are shared by FECD_REP and FECD_NR but mitochondrial dysfunction is more pronounced in the FECD_REP cohort. These results lay a basis for understanding the onset of FECD and other trinucleotide repeat diseases and provide potential targets for therapeutic design.The study was conducted in compliance with the tenets of the Declaration of Helsinki and with the approval of the institutional review board of the University of Texas Southwestern Medical Center (UTSW). Subjects underwent a complete eye examination including slit lamp biomicroscopy by a cornea fellowship-trained ophthalmologist. Patients underwent endothelial keratoplasty for FECD severity Krachmer grade 5  or 6  assessed by slit lamp microscopy . After sCorneal endothelial samples from post-mortem donor corneas preserved in Optisol GS corneal storage media  were obtained from the eye bank of Transplant Services at UT Southwestern. Certified eye bank technicians screened the donor corneal endothelium with slit lamp biomicroscopy and Cellchek EB-10 specular microscopy . Endothelium-Descemet's membrane monolayers from donor corneas were micro-dissected and stored as previously described . DNA froTCF4 gene was genotyped using a combination of short tandem repeat (STR) and triplet repeat primed polymerase chain reaction (TP-PCR) assays as we have previously described  by homogenization in QIAzol lysis reagent, chloroform extraction and isolation with NucleoSpin RNA XS . RNA quantity and quality were determined by Bioanalyzer 2100. RNA libraries were prepared for each tissue sample with high RIN (> 5.0), using the TruSeq RNA sample Prep kit version 2 . For TruSeq stranded total RNAseq, ribosomal transcripts were depleted from total RNA, using Ribo-Zero Gold RNA removal kit followed by replacement of deoxythymidine triphosphate (dTTP) with deoxyuridine triphosphate (dUTP) during reverse transcription in the second strand synthesis, using TruSeq stranded total library preparation kit. The resulting libraries were minimally amplified to enrich for fragments using adapters on both ends and then quantified for sequencing at eight samples/flow cell by using a NextSeq 500/550 (Illumina) sequencer (PE 150).Whole transcriptomic sequencing data from each tissue sample was analyzed using an analysis pipeline that includes STAR for initial mapping and Cufflinks (v2.21) for gene and isoform differential analysis, among other publicly available programs. For gene/isoform differential analysis, the minimum expression level of 1.5 FPKM and a false discovery rate (FDR) < 0.05 were chosen as the threshold. The meta gene pathway analysis was carried out with IPA (Qiagen). The binary alignment map files from STAR were analyzed using rMATS (v.4.0) that quantitates the expression level of alternatively spliced genes between groups. To find the most significant events, we used stringent filtering criteria within rMATS to perform pairwise comparisons among four groups: percentage of spliced in (PSI) changes > 0.15; FDR < 0.001. For PSI, rMATS calculates a value for every differential splicing event, providing a range from 0 to 1, with 0 being completely excluded and 1 being uniformly included in the splicing products. Alternative splicing events were also compared to those obtained in tibialis anterior muscle of myotonic dystrophy type one patients (DM1). DM1 raw data were obtained from Gene Expression Omnibus  and analyzed similarly as the FECD data (visit DMseq.org for more information).2 in Minimal Essential Media Eagle (MEM)  supplemented with 15% heat inactivated fetal bovine serum (Sigma) and 0.5% MEM nonessential amino acids (Sigma).FECD corneal endothelial cell lines (F35T and F45SV) and a control corneal endothelial cells (W4056–17-001579) were cultured as described  The C9 aTCF4 RNA levels were analyzed by qPCR.Endothelial or fibroblast cells were seeded in 6-well plate at 90% confluence. At the next day, actinomycin D was added into the wells at 5 μg/ml final concentrations. Cells were harvest using TRIzol agent (Sigma) at different time point. The RPL19 mRNA.Total RNA was extracted from control, Pre_S or FECD corneal endothelial tissues  by NucleSamples were homogeneous endothelial cell monolayers surgically removed from patients or dissected from donor tissue Figures  and 2A. TCF4 gene was obtained from pre-symptomatic eye bank donors (Pre_S) and from FECD patients after transplant surgery (FECD_REP). Tissue that was negative for the expanded CTG mutation within TCF4 was obtained from eye bank donors (Control) and from patients with non-CTG related FECD (FECD_NR). Genotype data indicated that all samples had normal numbers of CTG repeats within the DMPK gene  with attention to changes in alternative splicing, differential gene expression and pathway analysis Figure . Tissue e Figure .TCF4 gene within the general Caucasian population  was performed on tissue samples from nine non-FECD/non-expanded repeat donors (Control), six pre-symptomatic donors with the expanded repeat (Pre_S), six patients with the expanded repeat and FECD (FECD_REP) and four patients with FECD who did not have the expanded CTG repeat mutation (FECD_NR) Figure  Supplem. Only saClustering analysis of overall gene expression patterns revealed that samples from control and Pre_S donors were closer to one another than to samples from patients with FECD_REP or FECD_NR late stage disease Figure . This reTCF4 gene showed a striking difference between representative samples from individuals with the expanded CTG repeat mutation (FECD_REP and Pre_S) and individuals who lack the mutation (control and FECD_NR)  within the ) Figure . SamplesTCF4 mRNA from the four cohorts was not significantly different making haploinsufficiency of gene product less likely as the mechanism of disease , FECD_REP patient skin fibroblasts (VVM84), control (without CUG expanded repeat) corneal endothelium (W4056) or control skin fibroblasts (C9)   Figure  and C. Rh Figure  top. Likh Figure , middle.By contrast, we observe a striking ∼20-fold increase in the half-life of the upstream region of intron 2 in FECD corneal cells (F35T and F45SV) that possess the expanded CTG mutation and have detectable foci Figure , bottom.TCF4 from Pre_S or FECD_REP tissue relative to samples from individuals in the control or FECD_NR cohorts who lack the expanded CTG repeat  . PreviouMPK gene ,34.We used RNAseq to evaluate splicing changes between control tissue and the Pre_S, FECD_REP and FECD_NR tissue cohorts Figure . To clasRegardless of which tissue was analyzed, the primary changes in alternative splicing relative to control tissue were increases in the number of skipped exons (SE) events Figure . FECD_REThree hundred and thirteen of the alternative splicing events in Pre_S tissue involved exon skipping . HeatmapWhile not nearly as frequent, other forms of alternative splicing events, alternative 5′ splice site (A5SS), alternative 3′-splice site (A3SS), mutually exclusive exon (MXE) and retained intron (RI), also showed that the Pre_S cohort was more similar to the FECD_REP group than the FECD_NR cohort . Taken tWe then separated the top 25 skipped exon events in Pre_S tissue, which are shared with FECD_REP for evaluation. As with the overall group of 313 events, the splicing of these genes was much more like FECD_REP tissue than to control or FECD_NR tissue Figure . Of the MBNL1 and MBNL2. Splicing of these genes was also changed in tissue from a myotonic dystrophy mouse model . SYNE1 encodes nesprin-1 which links the nuclear membrane to the actin cytoskeleton and may also affect cell morphology. MBNL1 and MBNL2 were chosen because potential alterations in their expression might feed back into even greater changes in splicing.We used reverse-transcription PCR to evaluate changes of splicing for six genes, 2 Figure . INF2 prThe changes in splicing predicted by global RNAseq analysis were confirmed by visual inspection of genes using Sashimi plots Figure  and valiWe also carried out a differential alternative splicing analysis between Pre_S and FECD_REP tissues directly, rather than using the Control as the reference as described above. Although there are hundreds of significant skipped exon events identified between Pre_S and FECD_REP, out of 132 skipped exon events shared between Pre_S/Control and FECD_REP/Control comparison, only five of them are significantly different between Pre_S and FECD_REP . This meP-value generated by Cuffdiff, one of the programs within Cufflinks suite, as the determining metric. Changes with a P-value < 0.05 were deemed significant and included in our analysis. Analysis by DeSeq2, an alternate program for evaluating RNAseq data, produced similar results when compared with the output of CuffDiff  Figure  and B. TPre_S tissue had 215 genes with significantly altered expression levels relative to control tissue. Only five changes in gene expression were uniquely shared between Pre_S and FECD_NR tissue, compared to 73 shared changes with FECD_REP Figure . The cloVolcano plots allow a global overview of individual gene expression changes. They are useful for visualizing patterns of changes and identifying ‘outlier’ genes that combine highly significant changes in gene expression with higher fold changes. The fold change among top genes are less in Pre_S than in FECD_REP or FECD_NR and the identity of top genes differ Figure . This fiCOCH) (FN1) (THBS2)  ,39, fibrH) (FN1)  and thro (THBS2) .FN1, COL4A2, COCH, CTGF, MSI1, LUM, KDR and SOD3. Four of these genes, FN1, COL4A2, CTGF, KDR, were within the fibrosis pathway. COCH and LUM encode ECM proteins, MSI1 encodes a RNA binding protein/splicing factor and SOD3 protein is related to oxidative stress. The observed changes in gene expression confirm our RNAseq results  to confirm the changes in the level of gene expression detected by RNAseq in both Pre_S and FECD tissue Figure . qPCR mes Figure .To elucidate the potential impact of changes in the expression of individual genes on physiologic processes, we applied Ingenuity Pathway Analysis (IPA) to our RNAseq data. Overwhelmingly, the top common canonical pathway was hepatic fibrosis/hepatic stellate cell activation  Figure . InvolveFN1, one of the highest differentially expressed genes in Pre_S tissue  and four members of the collagen alpha chain family including COL1A2 which is also abundant in liver fibrosis  and class II major histocompatibility (MHC) proteins (>250-fold) both required for these cells to activate helper T cells implicate the immune system in both FECD groups in late-stage disease , significant numbers of pre-symptomatic samples can be obtained from individual donors positive for the CTG expansion. These tissues, together with FECD_REP, FECD_NR and control tissues ..TCF4 intystrophy . Unlike insitu hybridization (FISH) as RNA foci. These RNA foci are a hallmark of both Pre_S and FECD_REP corneal endothelium . Wh. WhinsitConsistent with our published clinical FECD cohort at our institution , the meaTCF4 repeat expansion to become a FECD patient? We can only speculate at this stage. It is possible that the gene expression changes observed in pre-symptomatic individuals increase the likelihood for dysfunction in individual cells. Eventually, this dysfunction crosses a molecular threshold and leads triggers the fibrosis pathway in individual cells. As patients age, more cells are affected, producing the findings of FECD visible to the ophthalmologist and the symptoms related to loss of vision noticed by patients.What does it take for an individual with a mutant C9orf72 ALS/FTD  copies per cell in disease tissue . It is l ALS/FTD . We finds Figure . This enAlthough we observed stabilization of the mutant intronic RNA in two patient-derived corneal cell lines and not in skin fibroblasts harboring the expansion, additional studies in other cell types are warranted to test the hypothesis that there is a corneal cell-specific mechanism that stabilizes the expanded CUG repeat.TCF4 and other microsatellite expansions are associated with intron retention   protein family. MBNL proteins are splicing factors and their sequestration reduces the concentration of free MBNL in cell nuclei and affects splicing. Overexpression of MBNL1 can reverse RNA missplicing and myotonia in a DM1 mouse model . MBNL isPK gene)  and FECDPK gene) ,53 tissuPK gene) .We have recently reported that relatively little MBNL protein is in the nuclei of FECD_REP cells and human tissue . Using qMany of the alterations in splicing observed in FECD_REP tissue also define gene expression in presymptomatic tissue, Pre_S Figures  and 5. TThe similarity of alternative splicing changes between symptomatic FECD_REP and presymptomatic Pre_S tissue is much greater than that between Pre_S and FECD_NR samples. The data suggest that there are fundamental differences in the origins of the two forms of FECD. While their origins differ, late-stage FECD_REP and FECD_NR converge at a common set of clinical findings.We note that the FECD_NR cohort shares gene splicing changes with both the Pre_S  and FECDThe data also suggest that splicing changes and perturbation of ECM genes seen in FECD_REP late stage disease tissue begin to be observed long before symptoms are observed during standard clinical evaluation. The molecular trigger, mutant RNA and early molecular changes, altered splicing and observable RNA foci, co-exist in cells that appear to have a normal phenotype. The changes in the magnitude of RNA splicing between control tissue and either Pre_S or late stage FECD_REP samples are similar. This observation suggests that the mutant RNA triggers the splicing changes in key genes independent of the progression of disease.The finding that splicing is an early trigger has important implications for the development of agents to treat FECD. It is reasonable to expect that such agents would be most effective when administered early in disease progression prior to production of ECM with degeneration of the corneal endothelium and activation of the immune system. During drug development it should be possible to monitor the changes in alternative splicing and expression of ECM biomarkers caused by expression of the mutant trinucleotide repeat and rank drug candidates by their ability to return splicing to a more normal state—agents that reverse the splicing defect would be promising development candidates. Monitoring splicing of key genes would offer a rapid and definitive assay for screening compounds. We have previously shown that synthetic oligonucleotides complementary to the CUG repeat can reverse the splicing defect .Recently, Fautsch and colleagues reported that some individuals without previously noted guttae who possessed the expanded repeat but developed ‘non-FECD corneal edema’ do not show changes in alternative splicing . These rAs seen in many other unstable microsatellite diseases, we reported a significant positive correlation between the clinical severity of FECD and the triplet repeat length in peripheral leukocytes . PreviouIn the clinic, late stage FECD is a disease of cellular degeneration and aberrant ECM deposition Figure . PreviouCOCH) and fibronectin (FN1) with >16- and >32-fold change increases respectively. Cochlin is a secretory ECM protein originally identified in the cochlear cells of the inner ear , another common age-related degenerative disorder  accession number is GSE142538.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE142538.gkaa422_Supplemental_FileClick here for additional data file.


Rationale: Construction of functional vascularized three-dimensional tissues has been a longstanding objective in the field of tissue engineering. The efficacy of using a tissue expander capsule as an induced vascular bed to prefabricate functional vascularized smooth muscle tissue flaps for bladder reconstruction in a rabbit model was tested.Methods: Skin tissue expanders were inserted into the groin to induce vascularized capsule pouch formation. Smooth muscle cells and endothelial progenitor cells were harvested and cocultured to form pre-vascularized smooth muscle cell sheet. Then repeated transplantation of triple-layer cell sheet grafts onto the vascularized capsular tissue was performed at 2-day intervals to prefabricate functional vascularized smooth muscle tissue flaps. Bladder muscular wall defects were created and repaired by six-layer cell sheet graft (sheet only), capsule flap (capsule only) and vascularized capsule prelaminated with smooth muscle cell sheet (sheet plus capsule). The animals were followed for 3 months after implantation and their bladders were explanted serially.Results: Bladder capacity and compliance were maintained in sheet plus capsule group throughout the 3 months. Tissue bath stimulation demonstrated that contractile responses to carbachol and KCl among the three groups revealed a significant difference (p < 0.05). Histologically, inflammation was evident in the capsule only group at 1 month and fibrosis was observed in sheet only group at 3 months. The vessel density in capsule only and sheet plus capsule group were significantly higher than in the sheet only group at each time point (p < 0.05). Comparison of the smooth muscle content among the three groups revealed a significant difference (p < 0.05).Conclusion: These results proved that the capsule may serve as an induced vascular bed for vascularized smooth muscle tissue flap prefabrication. The prefabricated functional vascularized smooth muscle tissue flap has the potential for reliable bladder reconstruction and may create new opportunities for vascularization in 3-D tissue engineering. Tissue engineering is one of the important technologies of regenerative medicine which constitutes and restructures tissue-like construction by the living cells of patient. Since Langer and Vacanti bioengineered cartilage tissue in the shape of an ear by seeding cell suspensions into a three-dimensional 3-D) biodegradable scaffold, scaffold-based tissue engineering has rapidly spread around the world -D biodegNonetheless, despite these progresses, new problems still challenge the field. One major barrier impedes effective reconstruction of cell-dense tissues, such as the heart, kidney, and liver Compared with scaffold-based tissue engineering, cell sheet technology features an increased concentration of cells, more uniform cell distribution, and the absence of immunological interference caused by scaffold materials. The preserved 3-D structure of the ECM in cell sheets provides a nutritive and structural microenvironment for cell adhesion and growth, as well as regulating metabolism, differentiation, and regeneration. Moreover, cell sheets can be easily transferred onto damaged organs and 3-D tissues may be engineered by stacking 2-D cell sheets in vitro bioreactor systems in vitro bioreactors can be designed to mimic in vivo microenvironments, duplicating the true in vivo conditions under ex vivo circumstances is difficult. Furthermore, it does not consider the functional elements of the regenerative environment, including immune, nervous, and hormonal systems, which play crucial roles in tissue regeneration and organ development. An emerging trend to circumvent these problems follows the in vivo bioreactor principle, which uses the body as a bioreactor to cultivate the traditional triad  and to leverage the body's own self-regenerative capacity to regenerate new tissue Another long-standing critical obstacle in tissue engineering is the formation of perfusable blood vessels in 3-D tissue constructs for oxygen and nutrient supplies and waste removal in vivo bioreactor for thick vascularized smooth muscle tissue flap prefabrication and could also be used as an induced vascular bed for buccal mucosa-lined flap prefabrication for tubularized urethral reconstruction In our previous studies, we have prefabricated a vascularized capsule pouch and proved that it may serve as an For the 3-stage animal experiments, 40 New Zealand white male rabbits  weighing 2.5-3.0 kg  were used . The SMCs were refed every 2 days until they were 80-90% confluent, usually 6-8 days. SMCs at passages 3 to 5 were harvested for identification or coculture. Myosin heavy chain 11  and alpha-smooth muscle actin  were used for immunofluorescence staining and flow cytometry.SMCs and EPCs were cultured as previously described 6 cells/cm2 and cultured in microvascular endothelial cell growth medium 2 . The medium was half refreshed every day until typical cell colonies formed, usually 10-12 days. EPCs at passages 3 to 5 were used for further experiments.Peripheral blood (20 mL) was obtained via cardiac puncture and EPCs were isolated from other components of the peripheral blood by density-gradient centrifugation with Histopaque-1083, according to the manufacturer's instructions. The cells were then seeded on 24-well plates pre-coated with fibronectin  at a density of 1×105/cm2, EPCs: 1×105/cm2), EPCs (7×105/cm2), or SMCs (7×105/cm2), after being cultured for 7 days, was collected and centrifuged to remove debris and contaminating cells and levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and transforming growth factor-β (TGF-β) in the supernatants were measured using ELISA kits following the manufacturer's instructions. On day 7, CD31 expression within the cocultured cells was detected. Then the cocultured cells were transferred to a biosafety cabinet and a circular scratch was made around the edge of the dish. From the edge to the center, the confluent cell layer was pushed centripetally with a pipette tip and an intact sheet was harvested using mechanical methods  and 8 rabbits with 16 unlabeled prefabricated constructs were harvested for morphological and physiological analyses 1 month after transplantation . The rabbits were then killed with overdoses of pentobarbital.Twenty-four rabbits were prepared as described for muscle biopsy In the sheet only group, a six-layer cell sheet construct with a mean diameter of 20.0 mm was grafted onto the urothelial diverticulum and secured with 6-0 PDS sutures. In the capsule only group, the vascularized capsule (1 month after full inflation) was isolated on its SCI vascular pedicle. After making a small hole in the abdominal wall, the pedicle capsule flap was then pulled from the groin space to the abdominal cavity and wrapped directly onto the bladder diverticulum. In sheet plus capsule group, 6-layer cell sheets were transplanted onto the capsule vascular bed at 2-day intervals as previously described and the prefabricated capsule smooth muscle composite tissue flap (1 month after incubation) was transposed as a pedicled flap into the abdominal cavity toward the bladder muscular wall defect in a similar manner to the capsule only group  was the threshold pressure (pressure that triggered voiding) minus the resting bladder pressure. ∆V was the maximal bladder capacity (mL).Bladder cystometry was performed according to previously described methods -6 to 1 × 10-4 M. Atropine 10-4 M was used as a competitive antagonist Four animals in each group were humanely euthanized 1 and 3 months post-implantation and the regenerated tissue molds harvested from each original implantation site were excised from the native bladders. One half was stored at -80 °C and the second half was placed in oxygenated Krebs solution at 37 °C for organ bath contractility studies. Briefly, regenerated muscle molds were cut into 8 × 3 mm longitudinal tissue strips that were suspended in a modified Krebs solution. The muscle strips were attached to a force transducer, stretched to a preload tension of 1.0 g, and equilibrated for 30 min. Then 80 μM of KCl was used to depolarize the tissue. Dose-response cholinergic contractions were assessed with carbachol at a dosage ranging from 1 × 10TM cDNA Synthesis kit . The sequence of primers for rabbit Myh 11 was designed as follows: forward, 5'- GCGATTCGGCAAGTTCATCC-3'; reverse, 5'-TGGAAGGTCCTCTCCTCTCG-3'. Reverse transcription was performed at 37 °C for 15 min and qPCR was conducted for 5 min at 95 °C, followed by 40 cycles of 20 s at 95 °C, 20 s at 65 °C, and 20 s at 75 °C. The relative expression level of corresponding mRNA was analyzed using the comparative Ct (∆∆Ct) method and normalised by housekeeping gene GAPDH Except for histological analysis, the residual samples were flash-frozen in liquid nitrogen and pulverized using a mortar. Total RNA was extracted using an RNeasy Mini kit and the cDNA was synthesized from 1 μg of total RNA using an iScriptAll of the samples were fixed in 4% paraformaldehyde and embedded in paraffin. Then 5 μm serial sections were generated and evaluated by hematoxylin and eosin (H&E) and immunohistochemistry staining p values <0.05.All of the statistical analyses were conducted using GraphPad Prism 6.0 software . The data were presented as the mean ± SD. Bladder volumes and pressures were recorded as continuous integers. Isometric contractions were recorded as maximal contraction (grams) per individual tissue and normalized to the total tissue weight (g/100 mg). The analysis of variance and Kruskal-Wallis test were used to determine the differences among the three groups at each time points. Differences were considered statistically significant at Bladder SMCs were successfully isolated and exhibited a characteristic spindle morphology at passage 3 Figure A. ImmunoThe functional characteristics of the EPCs were identified via ac-LDL endocytosis and UEA-1 binding. The results showed that the EPCs incorporated DiI-Ac-LDL and bound UEA-1 Figure D. ImmunoP < 0.01) than those in the EPCs and SMCs monoculture group  maintained normal bladder capacities and compliance throughout the study  showed no evident responses to KCl and carbachol stimulation at 1 month, but demonstrated mild responses to KCl and carbachol at 3 months. At 1 and 3 months, the cell-seeded composites group (sheet plus capsule) displayed similar contractile responses to carbachol and KCl, which were reversible with atropine Figure B. Comparp < 0.05; Figure p <0.05; Figure RT-PCR was used to test the gene expression level of the smooth muscles and H&E and IHC staining were used to assess the amount of smooth muscle and neovascularization in the epithelial lower layer. In the sheet only group, disorganized smooth muscle tissue was observed 1 month after surgery. At 3 months, the bladder muscular wall defects were filled with dense fibrous tissues and there was little evidence of smooth muscle and neovascularization surrounding the urothelium. In the capsule only group, scattered discrete smooth muscle tissue and progressive infiltration of inflammatory cells were identified on the retrieval bladder constructs 1 month after surgery. At 3 months, the smooth muscle became more organized and intensive and vascular proliferation was evident beneath the urothelium. In the sheet plus capsule group, bundles of intensive organized smooth muscle tissues with minimal inflammatory cells were observed on the retrieved bladder muscular wall constructs 1 and 3 months after surgery, which could be clearly distinguished from the sheet only and capsule only groups Figure A. ComparThe field of tissue engineering has progressed rapidly, but poor vascularization remains a major obstacle in bioengineering cell-dense tissues, limiting the viable size of constructs due to hypoxia, nutrient insufficiency, and waste accumulation. Prior classical vascularization strategies focused on stimulating vascular ingrowth into tissue constructs, which can be achieved by incorporating growth factors or endothelial cells Cell sheet engineering, which has a major advantage of allowing the retrieval of intact cell layers along with their naturally organized extracellular matrix (ECM), has emerged as a promising strategy for generating pre-vascularized cell-dense tissues without requiring any scaffold materials The capsule induction technique provides several essential advantages for vascularization in tissue engineering. The silicone block or expander  creates a spherical capsule pouch and the induced foreign body reaction provokes a predictable neovascularisation in the capsule tissue Previous studies proved that fibrin glue is an essential supplement for seeding autologous cells onto induced vascularized capsules Autologous SMC sheet grafting has the potential for reliable bladder reconstruction. The retention, survival, and engraftment of transplanted cells in cell sheet therapy are largely influenced by the degree of vascularization in the transplanted area, and lack of sufficient vasculature limits the viable tissue thickness to 3 layers Bladder compliance represents the bladder's ability to expand while maintaining a safe low bladder pressure and a well-organized muscular layer can improve bladder compliance. Poorly compliant bladders do not stretch and require high pressures to expand. As they fill under high bladder pressures, these bladders can cause kidney damage. According to Figures in vivo tissue engineering may be applied to many other tissue types in addition to the smooth muscle tissue demonstrated herein and may facilitate the discovery of new biological and medical techniques as well as lead to further advances in tissue engineering and its potential therapeutic applications.In the clinical replacement of many internal organs, direct multistep transplantation of bioengineered tissues may be challenging because each individual procedure has a relatively high risk of complications. Alternatively, ectopic fabrication of viable thick tissues and their transplantation into target organs may alleviate complications Although this study's results are encouraging, it had some limitations. The major disadvantage lies in the multiple surgical interventions and long development cycles required to fabricate vascularized smooth muscle tissue flaps. Furthermore, bladder wall reconstruction always requires vascularized composite tissue that contains smooth muscle bundles and epithelial lining, so the production of a consistent bilayer structure appears more applicable for bladder augmentation. In our subsequent series studies, we are attempting to fabricate bionic vascularized composite tissue that contains smooth muscle bundles and epithelial lining for bladder augmentation. Moreover, the bladder muscular wall defects and number of experimental animals that were evaluated per time point is low and the results only represent short-term success. A larger sample with more bladder wall defects and a longer-term follow-up would be more convincing. In addition, we merely explored the possibility of three-layered SMC sheets, while the maximum layer of SMC sheets that could be successfully transplanted on the axial capsule vascular bed at a single time was not investigated. When the thickness limitation is overcome, cell sheet engineering will provide groundbreaking treatment options for injured tissues or organs.in vivo tissue engineering and the clinical repair of various damaged organs.The present step-by-step polysurgery approach based on cell sheet integration and capsule induction technique appears feasible for fabricating viable thick vascularized tissues with perfusable blood vessels. This prefabricated flap may be safely transposed with its new pedicle for bladder muscular wall reconstruction. This technique may create new opportunities for Supplementary video/movie S1.Click here for additional data file.Supplementary video/movie S2.Click here for additional data file.


P < 5 × 10−4) using a transmission disequilibrium test (TDT) in all the families. Moreover, TDT result confirmed a SCZ susceptibility locus on 3p21.1, encompassing the multigenetic region NEK4-ITIH1-ITIH3-ITIH4. Through several different strategies to predict the potential pathogenic genes in silico, we revealed 4 previous discovered susceptibility genes  and 4 novel susceptibility loci  in Han Chinese SCZ patients. In summary, we identified a list of putative candidate genes for SCZ using a family-based WES approach, thus improving our understanding of the pathology of SCZ and providing critical clues to future functional validation.Schizophrenia (SCZ) is a highly heritable psychiatric disorder that affects approximately 1% of population around the world. However, early relevant studies did not reach clear conclusions of the genetic mechanisms of SCZ, suggesting that additional susceptibility loci that exert significant influence on SCZ are yet to be revealed. So, in order to identify novel susceptibility genes that account for the genetic risk of SCZ, we performed a systematic family-based study using whole exome sequencing (WES) in 65 Han Chinese families. The analysis of 51 SCZ trios with both unaffected parents identified 22 exonic and 1 splice-site de novo mutations (DNMs) on a total of 23 genes, and showed that 12 genes carried rare protein-altering compound heterozygous mutations in more than one trio. In addition, we identified 26 exonic or splice-site single nucleotide polymorphisms (SNPs) on 18 genes with nominal significance ( The hallmark of SCZ is characterized by positive symptoms  and negative symptoms . Over decades, mounting evidences have strongly suggested that SCZ has a significant genetic component, indicated by its heritability estimates of 60–80%3. Currently, SCZ has been widely regarded as a highly complex genetic disorder that is caused by multiple genetic variants in cooperation with environmental risk factors4. In addition, common variants only confer a relatively small influence on SCZ risk5 and affect highly heterogeneous in different populations6. Therefore, an appropriate approach that accounts for the high heritability and strong familial aggregation of SCZ is essential to study the genetic predisposition to SCZ7.Schizophrenia (SCZ) is a severe psychiatric disorder that has a global prevalence of approximately 1%8. In the past several years, a number of GWASs have identified over 100 genetic loci associated with SCZ risk16. Such studies to detect susceptibility genes require a very large sample size since the influences of individual susceptibility variants on SCZ risk may be small17. In addition, despite specific rare CNVs are now recognized as established risk factors for SCZ susceptibility26, these rare CNVs are usually potent risk factors of several other different psychiatric disorders and they often involve with many genes7. Recently, de novo mutations (DNMs) have been shown to be a highly successful new approach to study SCZ risk using family-based WES34. These results give us new options to explore the possible pathogeneses of SCZ.Many approaches have been applied to study the genetic factors that are associated with susceptibility to SCZ, including linkage analysis, genome wide association study (GWAS), copy number variant (CNV) analysis strategy and whole exome sequencing (WES)36. However, the roles of these mutations in other mental disease are still not completely understood.Unlike traditional case-control studies, family-based studies can exclude some confounding factors, such as fractional environmental factors bias and population structure difference. More important, family-based studies allow for directly observation of DNMs, compound heterozygous mutations and transmission disequilibrium mutations in probands. In particular, specific compound heterozygosity has been demonstrated to underlie autism spectrum disordersIn this study, we implemented family-based WES analysis in 65 Han Chinese families with one SCZ offspring to identify DNM or inherited variants that are associated with susceptibility to SCZ. The overall experimental design is presented in Fig. A total of 65 SCZ offsprings  and their biological parents were recruited from psychiatric hospitals in Shanghai, China. We ascertained the diagnosis based on DSM-IV criteria and consensus clinical judgments of two fully qualified senior psychiatric physicians. In our recruited samples, there were 51 SCZ trios , 13 families with one schizophrenic parent and 1 family with both schizophrenic parents. All subjects in study were of Han Chinese origin.37.This study was approved by the Ethical Committee of Bio-X Institutes of Shanghai Jiao Tong University. All subjects or their legal guardians provided written informed consent to their participation before enrollment of this study in accordance with the guidelines laid out in the Declaration of HelsinkiXT Target Enrichment System Human All Exon V5 + UTRs  according to the manufacture’s protocols. After that, libraries were assessed for sequencing using Agilent 2100 Bioanalyzer High Sensitivity DNA chip . Next, trusted high-level libraries were sequenced on Illumina Hiseq 2500 .Genomic DNA was extracted from peripheral blood using the QIAamp DNA Mini Kit . Whole-exome capture library was performed using Agilent SureSelecthttp://bio-bwa.sourceforge.net/) tool. Then, the mapped reads were processed by Picard  to detect the polymerase chain reaction (PCR) duplicates. After marking the duplicates, Genome Analysis Toolkit  was then used to perform local realignment and base quality score recalibration (BQSR) to realign insertion-deletions (indels) and correct for the base quality scores from BAM files. GATK HaplotypeCaller tool was used to call the variants. The called variants  file of every sample were merged using GenotypeGVCFs and then filtered using VariantRecalibrator tool in GATK.The raw sequencing reads (in fastq format) were mapped to the human reference genome (hg19) with the Burrows-Wheeler Aligner  in GATK. In addition, all the detected DNMs must not been described in any reported database. The exonic or splice site DNMs with high confidence were considered as candidates for subsequent validation and analysis.The 51 SCZ trios with both healthy parents were used to identify DNMs since DNMs are more likely to account for sporadic forms of the disease38. We follow this definition to screen protein-altering (including nonsynonymous and stop loss/gain) compound heterozygous mutations in the 51 trios. Heterozygous mutations that had minor allele frequency (MAF) < 0.01 in East Asian population in 1000 Genomes Project database  and their hit genes appeared in more than one trio were selected as heterozygous mutations candidates.Compound heterozygosity was defined as an individual carrying two heterozygous genotypes from one maternal and one paternal copy of different locations within the same gene39 was used to annotate all the DNMs and compound heterozygous mutations. The annotations included the predicted functional consequence according to RefSeq transcripts, whether they overlapped a segmental duplication, predicted effect on protein function and corresponding allele frequencies in multiple public databases .ANNOVAR softwarehttps://www.dnastar.com/) software. PCR was performed using the AmpliTaq Gold DNA Polymerase  following the manufacturer’s instructions. The products of PCR were then sequenced using a 3730XL DNAnalyzer  and Mutation Surveyor software40 was used for mutation detection analysis.The selected candidate DNMs and compound heterozygous mutations as described below were validated by Sanger sequencing. Primers were designed using DNAStar (http://zzz.bwh.harvard.edu/plink/plink2.shtml). Samples with call rate less than 95% and high heterozygosity rate (deviation from triple standard deviations) were removed from further study. In addition, we validated the sex of each individual by concordance between self-reported sex and sex determined by genotyping. Principal component analysis (PCA) was conducted based on the passed QC variants using PLINK software. SNPs with a call rates less than 95%, difference in missing genotype rate between cases and controls greater than 5%, minor allele frequency below 0.01 or significant deviation from expected Hardy-Weinberg equilibrium (P < 1.0 × 10−6) were removed from further family-based association analysis.Transmission disequilibrium test (TDT) was performed to test the family-based association between common single nucleotide polymorphisms (SNPs) and SCZ susceptibility. Quality control (QC) filters were applied to all the 65 family members. For data cleaning, systematic stepwise quality filtering of raw genotyping data was done using PLINK software  analysis was then performed to assess the intolerance of variations in the candidate genes, therefore inferring their pathogenic impacts in SCZ. RVIS was a statistics that rank the genes of interest according to their tolerance to functional genetic variation by comparing the number of observed variants in the gene to the genome wide expectation given the number of neutral variation the gene has. At last, we systematically investigated the networks of functional interactions among the candidate genes using a web-based tool called Association Network Integration Algorithm (GeneMANIA) (http://genemania.org/). The results were visualized in Cytoscape . The hub genes were defined as: the top 3 genes that appeared most according all the 12 cytoHubba ranking methods using Cytoscape software. Subsequently, the Molecular Complex Detection (MCODE) algorithm in Cytoscape software was used to screen modules. An MCODE score > 3 and a node number > 3 were taken as the criteria to define a core module.We used several different strategies to predict the potential pathogenic genes in SCZ. First, we explored whether the candidate genes had been previously identified in other independent GWAS or genetic association studies on SCZ. Then, a residual variation intolerance score . R package  and Haploview tool (https://www.broadinstitute.org/haploview/) was used to visualize TDT results using Q-Q and Manhattan plot respectively. The linkage disequilibrium analysis of significant SNPs locating at 3q21.1 was through Haploview tool.TDT was performed to test the family-based single maker association using PLINK software. In order to obtain the candidate variant associations, Pearson chi-square test was performed to evaluate significance of genotype frequency distributions. The power and pre-specified effect size was calculated by G-power software (https://submit.ncbi.nlm.nih.gov/subs/sra/). Accession codes for the exome-sequenced samples is reported in Supplementary Table Among all subjects, an average of ~72.2 million reads was available with high enough depth . The whole-exome capture was relatively efficient, covering ~81% of the target region at minimal 20× coverage  from the following validation. Direct PCR amplification and Sanger sequencing of each gene with candidate DNMs were performed on all 51 recruited SCZ trios. In total, 23 (74%) candidate mutations were validated as true DNMs in the SCZ probands. They were mapped to the 23 coding sequences (CDS) of the genes of 19 SCZ patients . This previous report had also suggested that there was no significant elevation of DNMs in SCZ patients compared to neutral expectation 31.Using the t-test P = 0.17). We found that the rare protein-altering compound heterozygous mutations in titin (TTN) gene were present in 14 trios. The result of standard Sanger sequencing of SCZ probands and parental DNA was used to validate 13 genes (including TTN gene), which were found to contain rare protein-altering compound heterozygous mutations in more than one trio (Supplementary Table MUC17) gene was found to be a false positive. In addition, considering the special profile of TTN gene , we did not consider it a valuable candidate gene associated with SCZ.We identified 171 genes with rare protein-altering compound heterozygous mutations from 51 SCZ trios (3.35 genes per proband). On average of each proband, 3.26 genes with rare protein-altering compound heterozygous mutations were detected in 23 male probands and 4 genes with rare protein-altering compound heterozygous mutations were found in 24 female probands  gene downstream region . In addition, 4 other SNPs with significant associations  were also found to be in the REPIN1 gene. Finally, our results supported earlier discoveries of the important role of 3p21.1 loci in NEK4-ITIH1-ITIH3-ITIH4 region in SCZ by finding a number of significant SNPs in this specific multigenetic region41. Another genetic association study also found 3p21.1  as risk loci for bipolar disorder (BD), SCZ and psychosis42. We extracted the 18 significant SNPs locating at 3q21.1 and made a linkage disequilibrium analysis. The results showed that they have a strong linkage relationship, as shown in Supplementary Fig. TDT were performed using PLINK software on all 65 SCZ families to investigate the genetic associations in SCZ. A total of 136,894 SNPs remained after QC procedure and were included in the family-based association analyses. Combined asymptotic test ies Fig. . This an16 have shown that T-SNARE domain containing 1 (TSNARE1) and polybromo 1 (PBRM1) had a high correlation with SCZ. Previous genetic association studies44 have also revealed that oligodendrocyte transcription factor 2 (OLIG2) influences susceptibility to SCZ. Next, our results showed that PBRM1 had the lowest RVIS score of −2.287 and a percentile of 2.23%, suggesting it is amongst the 2.23% most intolerant of human genes  showing the most closely related to other nodes. In addition, 1 core module of this network for the candidate genes was structured, including genes: KAT5, CXXC1, ARHGEF17, REPIN1, TIAM1, TLE2.Firstly, we compared our results with earlier studies to explore the potential pathogenic effects of the 23 candidate genes that harbored the validated DNMs, 12 candidate genes with genuine rare protein-altering compound heterozygous mutations and 18 candidate genes with significant exonic SNPs identified by TDT analysis as described above. Several recent GWASsAlthough we have been studying SCZ for decades and also made some progress, our understanding of the etiology of SCZ is still not very clear and an open question to be addressed. Accumulating data had indicated that genetic factors play a key role in SCZ mechanism, enabled by a large number of studies on genetic variations. Despite the significant increase in statistical power granted by these large-scale analyses, most existing genetic association studies only explained a fraction of SCZ onset or its heritability in families. Therefore, we took advantage of next-generation sequencing (NGS) technology and applied family-based WES to Chinese SCZ families in order to reveal the novel genetic associations to SCZ risk. Compared to traditional GWAS and candidate gene studies, WES is a more cost effective approach by having less bias and higher efficiency. In this study, we identified 23 DNMs and 12 genes with rare compound heterozygous mutations appearing more than one trio among the 51 SCZ trios that were with both healthy parents. It was noted that these 23 tag genes contained more nonsynonymous DNMs than synonymous DNMs. We also revealed 26 exonic or splice site SNPs showed statistical significance using TDT in all 65 SCZ families.46. We found a de novo missense mutation p.G717E of mannosyl Alpha-1,6-Glycoprotein 6-Beta-N-Acetylglucosaminyltransferase B (MGAT5B) was identified in one SCZ family trio. This variant was estimated to be probably damaging, indicated by its Polyphen2 prediction score of 1. MGAT5B encodes a glycosyltransferases, which plays an important role in the nervous system by catalyzing the formation of a variety of glycoconjugates47. Toll like receptor 5 (TLR5) is the only previously reported gene that has been found to be associated with onset of SCZ in similar trio-based study using WES data in Bulgaria31. This gene encodes a member of the toll-like receptor (TLR) family, which plays a crucial role in pathogen recognition and activation of innate immune responses48. In addition, the relationship of neuronal and immunological pathways featured among top hits in recent psychiatric GWAS49, suggesting that TLR5 we identified in this study may exert substantial influence on SCZ risk. At last, OLIG2, a gene known to be associated with SCZ susceptibility, has also been found to be a part of the genetic network underlying oligodendrocyte function in earlier investigations44.Studying DNMs is most effective in which the selective pressure is extremely strong and the effect size for those DNMs is large50. In addition, the exploration of genes harboring rare missense mutations in different sporadic SCZ is an alternate way forward to resolve SCZ’s complexity51. Thus, we payed close attention to those genes that harboring rare compound heterozygous mutations in multiple trios. Our analysis of rare compound heterozygous mutations revealed that the genetic alterations in TSNARE1, SCO-Spondin (SSPO) genes may associated with onset of SCZ. The function of the TSNARE1 gene is not well-understood, but a recent study suggested it may have a vertebrate-specific function in intracellular protein transport and synaptic vesicle exocytosis52. Moreover, a study showed that altering the expression of TSNARE1 affects neurodevelopment in zebrafish53. The subcommissural organ (SCO) is a brain gland located in the roof of the third ventricle that releases glycoproteins into the cerebrospinal fluid, and there is evidence suggesting that serotonin (5HT) participates in the regulation of the SCO secretory activity in the rat55. Another study provided evidence of the statement that the behavioral data of processing speed revealed association with a locus that included SSPO56.It is also important to identify rare variations in SCZ patients, since they usually carry a large effect sizeSTAB1) might contribute to schizophrenia susceptibility previously identified by pathway analysis57. Another study suggested that STAB1 is a new candidate gene for bipolar disorder combining of gene through expression and GWAS data58. Most noteworthy, we confirmed the importance of the NEK4-ITIH1-ITIH3-ITIH4 region locating in 3p21.1  in the pathogenesis of psychiatric disease59. The linkage disequilibrium analysis results showed that significant SNPs in this region have a strong linkage relationship. Inter-Alpha-Trypsin Inhibitor Heavy (ITIH) molecules belong to a family of serine protease inhibitors, and had been implicated in SCZ60, inflammatory diseases62 and carcinogenesis63.Furthermore, we made full use of the characteristics of the family through the TDT method to find the susceptibility gene of SCZ. In this way, we found one gene stabilin 1  that were found to be associated with SCZ by performing WES on 65 Han Chinese SCZ families. Our findings of novel genetic markers will deepen the current knowledge of SCZ pathophysiology. In addition, we used a variety of strategies to comprehensively assess the genetic susceptibility to SCZ, supporting that SCZ does show strong heterogeneity and involve multiple genes and pathways. Further studies are warranted to validate our discoveries of the candidate gene markers and reveal their polygenic effects with other downstream target genes in the SCZ pathophysiology.In summary, we provide a list of putative candidate genes (including Supplementary FiguresSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Table 6


Stroke prevention with left atrial appendage (LAA) occlusion is an important alternative treatment for high-risk atrial fibrillation (AF) patients with contraindications to oral anticoagulation (OAC).2DS2-VASc and HAS-BLED scores of 4 (interquartile range [IQR]: 3–5) and 2 (IQR: 2–3), respectively. Contraindications to lifelong OAC in this cohort were prior intracranial hemorrhage in six patients , prior major gastrointestinal hemorrhage requiring emergency hospital admission and blood transfusion in three patients (two on warfarin and one while not on anticoagulation), severe unexplained anemia on dabigatran in one patient, and failed OAC in one patient. At the time of imaging, none of the patients were receiving OAC, and four (36.4%) patients were on a single antiplatelet agent. There were no significant differences between patients who had documented LAA thrombus compared with those without in terms of age (p = 0.80), sex category (p = 0.74), AF type (p = 0.11), comorbidities , and CHA2DS2-VASc score (p = 0.69).This was a retrospective cohort study of 83 patients who underwent work-up for LAA occlusion at our center between January 2015 and December 2017. LAA thrombi were documented in 11 (13.3%) patients  on preprocedural imaging  . All pap = 0.73). One patient  was prescribed for each patient. The choice of anticoagulation was based on evidence of potential favorable characteristics of apixaban compared with other OAC in terms of efficacy and safety profile.The 10 patients with thrombus resolution underwent successful LAA occlusion with no periprocedural complications. Apixaban was replaced by dual-antiplatelet therapy with aspirin and clopidogrel for 6 weeks postprocedure followed by single (or no) antiplatelet therapy. The decision to discontinue long-term antiplatelet therapy in three patients was based on an individualized approach after accounting for their high bleeding risk. All patients underwent cardiac imaging with transesophageal echocardiography or cardiac computed tomographic angiography at 6 to 8 weeks postprocedure; no device-related thrombus was observed in any patient. Across a median follow-up of 2.5 years (IQR: 0.6–3.3 years), one patient suffered a transient ischemic attack and another had an episode of severe epistaxis despite not being on antiplatelet or OAC therapy at the time. During this same period, four (40%) patients died.Given the low number of patients, our study was underpowered and should therefore be interpreted with caution. Furthermore, the incidence of LAA thrombus in our study may not reflect those of the general AF population as many of our high-risk patients were not on anticoagulation therapy at baseline. It is also worth noting that we adopted a strategy of minimizing the exposure to OAC in this high-risk cohort by performing LAA occlusion without delay in patients with a confirmed resolution of LAA thrombi.Our experience suggests that short-term treatment with apixaban may be effective and safe for aiding the resolution of documented LAA thrombus in high-risk patients who are ineligible for lifelong OAC therapy. This strategy allows the LAA occlusion procedure to be undertaken safely, thereby potentially reducing the long-term risk of thromboembolism.


VLa > VLpv > VLpd, whereas the pallidal median connectivity was VLa ~ VLpv > VLpd in the atlas-based approach and VLpv > VLa > VLpd in the manual approach.The ventrolateral thalamic nucleus (VL), as part of the ‘motor thalamus’, is main relay station of cerebellar and pallidal projections. It comprises anterior (VLa) and posterior (VLpd and VLpv) subnuclei. Though the fibre architecture of cerebellar and pallidal projections to of the VL nucleus has already been focus in a numerous amount of in vitro studies mainly in animals, probabilistic tractography now offers the possibility of an in vivo comparison in healthy humans. In this study we performed a (a) qualitative and (b) quantitative examination of VL-cerebellar and VL-pallidal pathways and compared the probability distributions between both projection fields in the VL after an (I) atlas-based and (II) manual-based segmentation procedure. Both procedures led to high congruent results of cerebellar and pallidal connectivity distributions: the maximum of pallidal projections was located in anterior and medial parts of the VL nucleus, whereas cerebellar connectivity was more located in lateral and posterior parts. The median connectivity for cerebellar connections in both approaches  was The online version of this article (10.1007/s00429-020-02076-9) contains supplementary material, which is available to authorized users. The ventrolateral (VL) thalamic nucleus is main relay station of cerebellar and basal ganglia (BG) projections to the cortex  and output nuclei of the basal ganglia  project to the thalamus with a considerable amount of overlap, as shown in the monkey and human species  native German speakers  with a mean age of 25 (± 4.4) years, without history or signs of neurological disease, were included in the MR analysis. Informed consent was obtained from all individual participants included in the study. For overview of methods please see Supplementary Material.VLa > VLpv > VLpd), whereas the pallidal median connectivity values differed between the atlas-based (VLa ~ VLpv > VLpd) and the manual (VLpv > VLa > VLpd) approach.Interestingly, atlas-based segmentation procedure revealed a 41% SD 2.70) bigger VL masks on the left side and 42% (SD 2.28) bigger VL masks on the right side than the manual outlining procedure; differences were mainly located in top and bottom border regions. In the VL both, manual and atlas-based segmentation, yielded to highly congruent specific patterns in the qualitative analysis of fibre architecture: (1) pallidal connectivity had a maximum in more anterior and medial parts of the VL, whereas cerebellar connectivity was more located in lateral and posterior parts  We found high connectivity values to the VLa and VLp regions for both projection systems, with an anterior-posteriorly increasing gradient for cerebellar projections and a posterior-anteriorly increasing gradient for cerebellar projections. Our results fit well with the current anatomical knowledge [see Fig. 6 in Sakai et al. ]. The paThe ventrolateral thalamus is one of the main target regions in stereotactic treatments for movement disorders like essential tremor  or post-mortem analyses [for detailed overview please see Hintzen et al. ]. DiffusSupplementary file1 (DOCX 170 kb)Below is the link to the electronic supplementary material.