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The intestinal epithelium forms an essential barrier between a host and its microbiota. Protozoa and helminths are members of the gut microbiota of mammals, including humans, yet the many ways that gut epithelial cells orchestrate responses to these eukaryotes remain unclear. Here we show that tuft cells, which are taste-chemosensory epithelial cells, accumulate during parasite colonization and infection. Disruption of chemosensory signaling through the loss of TRMP5 abrogates the expansion of tuft cells, goblet cells, eosinophils, and type 2 innate lymphoid cells during parasite colonization. Tuft cells are the primary source of the parasite-induced cytokine interleukin-25, which indirectly induces tuft cell expansion by promoting interleukin-13 production by innate lymphoid cells. Our results identify intestinal tuft cells as critical sentinels in the gut epithelium that promote type 2 immunity in response to intestinal parasites.

Rett syndrome (RTT) is an X-linked neurological disorder caused by mutations in the methyl-CpG–binding protein 2 (MeCP2) gene. The majority of RTT missense mutations disrupt the interaction of the MeCP2 with DNA or the nuclear receptor corepressor (NCoR)/silencing mediator of retinoic acid and thyroid receptors (SMRT) corepressor complex. Here, we show that the “NCoR/SMRT interaction domain” (NID) of MeCP2 directly contacts transducin beta-like 1 (TBL1) and TBL1 related (TBLR1), two paralogs that are core components of NCoR/SMRT. We determine the cocrystal structure of the MeCP2 NID in complex with the WD40 domain of TBLR1 and confirm by in vitro and ex vivo assays that mutation of interacting residues of TBLR1 and TBL1 disrupts binding to MeCP2. Strikingly, the four MeCP2-NID residues mutated in RTT are those residues that make the most extensive contacts with TBLR1. Moreover, missense mutations in the gene for TBLR1 that are associated with intellectual disability also prevent MeCP2 binding. Our study therefore reveals the molecular basis of an interaction that is crucial for optimal brain function.

In zebrafish, Müller glia (MG) are a source of retinal stem cells that can replenish damaged retinal neurons and restore vision 1 . In mammals, however, MG do not spontaneously re-enter the cell cycle to generate a population of stem or progenitor cells that differentiate into retinal neurons. Nevertheless, the regenerative machinery may exist in the mammalian retina, as retinal injury can stimulate MG proliferation followed by limited neurogenesis 2 – 7 . Therefore, there is still a fundamental question regarding whether MG-derived regeneration can be exploited to restore vision in mammalian retinas. Gene transfer of β-catenin stimulates MG proliferation in the absence of injury in mouse retinas 8 . Here we report that following gene transfer of β-catenin, cell-cycle-reactivated MG can be reprogrammed to generate rod photoreceptors by subsequent gene transfer of transcription factors essential for rod cell fate specification and determination. MG-derived rods restored visual responses in Gnat1 rd17 Gnat2 cpfl3 double mutant mice, a model of congenital blindness 9 , 10 , throughout the visual pathway from the retina to the primary visual cortex. Together, our results provide evidence of vision restoration after de novo MG-derived genesis of rod photoreceptors in mammalian retinas. Müller glia in mature mouse retina can be stimulated to produce rod cells; this treatment restores visual responses in a model of congenital blindness.

ObjectiveWe found that carbonic anhydrase IV (CA4), a member of the carbonic anhydrases, is silenced in colorectal cancer (CRC). We analysed its epigenetic inactivation, biological effects and prognostic significance in CRC.DesignThe biological functions of CA4 were determined by in vitro and in vivo tumorigenicity assays. The CA4 co-operator was identified by immunoprecipitation and mass spectrometry. CA4 downstream effectors and signalling pathways were elucidated by promoter luciferase assay, electrophoretic mobility shift assay and chromatin immunoprecipitation. The clinical impact of CA4 was assessed in 115 patients with CRC.ResultsCA4 was silenced in all nine CRC cell lines and 92.6% of CRC tumours. The promoter hypermethylation contributed to the inactivation of CA4, and it was detected in 75.7% of the patients with CRC. After a median follow-up of 49.3 months, multivariate analysis showed that the patients with CA4 hypermethylation had a recurrence of Stage II/III CRC. The re-expression of CA4 inhibited cell proliferation, induced apoptosis and cell cycle arrest in the G1 phase. CA4 inhibited the activity of the Wnt signalling pathway and mediated the degradation of β-catenin. CA4 interacted with Wilms’ tumour 1-associating protein (WTAP) and induced WTAP protein degradation through polyubiquitination. Moreover, CA4 promoted the transcriptional activity of Wilms’ tumour 1 (WT1), an antagonist of the Wnt pathway, which resulted in the induction of transducin β-like protein 1 (TBL1) and the degradation of β-catenin.ConclusionsCA4 is a novel tumour suppressor in CRC through the inhibition of the Wnt signalling pathway by targeting the WTAP–WT1–TBL1 axis. CA4 methylation may serve as an independent biomarker for the recurrence of CRC.

Supplementing wildtype copies of functionally defective genes with adeno-associated virus (AAV) is a strategy being explored clinically for various retinal dystrophies. However, the low cargo limit of this vector allows its use in only a fraction of patients with mutations in relatively small pathogenic genes. To overcome this issue, we developed a single AAV platform that allows local replacement of a mutated sequence with its wildtype counterpart, based on combined CRISPR-Cas9 and micro-homology-mediated end-joining (MMEJ). In blind mice, the mutation replacement rescued approximately 10% of photoreceptors, resulting in an improvement in light sensitivity and an increase in visual acuity. These effects were comparable to restoration mediated by gene supplementation, which targets a greater number of photoreceptors. This strategy may be applied for the treatment of inherited disorders caused by mutations in larger genes, for which conventional gene supplementation therapy is not currently feasible. Replacing mutant genes with wildtype copies using adeno-associated virus (AAV) has been explored for the treatment of inherited retinopathies, but the low cargo limit restricts its use. Here the authors describe a single AAV platform that allows local replacement of a mutated sequence with its wildtype counterpart, based on combined CRISPR-Cas9 and micro-homology-mediated end joining.

The light responses of rod and cone photoreceptors have been studied electrophysiologically for decades, largely with ex vivo approaches that disrupt the photoreceptors’ subretinal microenvironment. Here we report the use of optical coherence tomography (OCT) to measure light-driven signals of rod photoreceptors in vivo. Visible light stimulation over a 200-fold intensity range caused correlated rod outer segment (OS) elongation and increased light scattering in wild-type mice, but not in mice lacking the rod G-protein alpha subunit, transducin (Gαt), revealing these responses to be triggered by phototransduction. For stimuli that photoactivated one rhodopsin per Gαt the rod OS swelling response reached a saturated elongation of 10.0 ± 2.1%, at a maximum rate of 0.11% s−1. Analyzing swelling as osmotically driven water influx, we find the H₂O membrane permeability of the rod OS to be (2.6 ± 0.4) × 10−5 cm·s−1, comparable to that of other cells lacking aquaporin expression. Application of Van’t Hoff’s law reveals that complete activation of phototransduction generates a potentially harmful 20% increase in OS osmotic pressure. The increased backscattering from the base of the OS is explained by a model combining cytoplasmic swelling, translocation of dissociated G-protein subunits from the disc membranes into the cytoplasm, and a relatively higher H₂O permeability of nascent discs in the basal rod OS. Translocation of phototransduction components out of the OS may protect rods from osmotic stress, which could be especially harmful in disease conditions that affect rod OS structural integrity.

Though rod and cone photoreceptors use similar phototransduction mechanisms, previous model calculations have indicated that the most important differences in their light responses are likely to be differences in amplification of the G-protein cascade, different decay rates of phosphodiesterase (PDE) and pigment phosphorylation, and different rates of turnover of cGMP in darkness. To test this hypothesis, we constructed TrUx;GapOx rods by crossing mice with decreased transduction gain from decreased transducin expression, with mice displaying an increased rate of PDE decay from increased expression of GTPase-activating proteins (GAPs). These two manipulations brought the sensitivity of TrUx;GapOx rods to within a factor of 2 of WT cone sensitivity, after correcting for outer-segment dimensions. These alterations did not, however, change photoreceptor adaptation: rods continued to show increment saturation though at a higher background intensity. These experiments confirm model calculations that rod responses can mimic some (though not all) of the features of cone responses after only a few changes in the properties of transduction proteins.

Transplanted rod precursor cells restore visual function, from electrophysiology to behaviour, after transplantation into a mouse model of congenital night blindness. Photoreceptor restoration Previous work has shown that retinal precursor cells can be transplanted successfully into degenerating retinas in mice, but evidence for improvement of vision has been lacking. Now Pearson et al . take a step forward in demonstrating the feasibility of this strategy for restoring vision. Using an improved transplantation protocol for introducing rod precursor cells into the retinas of mice that lack rods, the authors demonstrate that the transplanted cells integrate into and position correctly in the existing network, and that visual function, from electrophysiology to behaviour, is enhanced in the transplant recipients. The study provides important support for the further development of stem-cell therapy for retinal degeneration. Cell transplantation is a potential strategy for treating blindness caused by the loss of photoreceptors. Although transplanted rod-precursor cells are able to migrate into the adult retina and differentiate to acquire the specialized morphological features of mature photoreceptor cells 1 , the fundamental question remains whether transplantation of photoreceptor cells can actually improve vision. Here we provide evidence of functional rod-mediated vision after photoreceptor transplantation in adult Gnat1 −/− mice, which lack rod function and are a model of congenital stationary night blindness 2 . We show that transplanted rod precursors form classic triad synaptic connections with second-order bipolar and horizontal cells in the recipient retina. The newly integrated photoreceptor cells are light-responsive with dim-flash kinetics similar to adult wild-type photoreceptors. By using intrinsic imaging under scotopic conditions we demonstrate that visual signals generated by transplanted rods are projected to higher visual areas, including V1. Moreover, these cells are capable of driving optokinetic head tracking and visually guided behaviour in the Gnat1 −/− mouse under scotopic conditions. Together, these results demonstrate the feasibility of photoreceptor transplantation as a therapeutic strategy for restoring vision after retinal degeneration.

Despite different aetiologies, age-related macular degeneration and most inherited retinal disorders culminate in the same final common pathway, the loss of photoreceptors. There are few treatments and none reverse the loss of vision. Photoreceptor replacement by transplantation is proposed as a broad treatment strategy applicable to all degenerations. Recently, we demonstrated restoration of vision following rod-photoreceptor transplantation into a mouse model of stationary night-blindness, raising the critical question of whether photoreceptor replacement is equally effective in different types and stages of degeneration. We present a comprehensive assessment of rod-photoreceptor transplantation across six murine models of inherited photoreceptor degeneration. Transplantation is feasible in all models examined but disease type has a major impact on outcome, as assessed both by the morphology and number of integrated rod-photoreceptors. Integration can increase (Prph2⁺/∆³⁰⁷), decrease (Crb1rd⁸/rd⁸ Gnat1⁻/⁻, Rh⁻/⁻), or remain constant (PDE6βrd¹/rd¹, Prph2rd²/rd²) with disease progression, depending upon the gene defect, with no correlation with severity. Robust integration is possible even in late-stage disease. Glial scarring and outer limiting membrane integrity, features that change with degeneration, significantly affect transplanted photoreceptor integration. Combined breakdown of these barriers markedly increases integration in a model with an intact outer limiting membrane, strong gliotic response, and otherwise poor transplantation outcome (Rho⁻/⁻), leading to an eightfold increase in integration and restoration of visual function. Thus, it is possible to achieve robust integration across a broad range of inherited retinopathies. Moreover, transplantation outcome can be improved by administering appropriate, tailored manipulations of the recipient environment.

The progression of hepatocellular carcinoma (HCC) is coincident with aberrant splicing of numerous tumor‐related genes. Identification of the tumor‐specific splice variants that facilitate HCC metastasis may provide a more comprehensive insight into the mechanisms of HCC metastasis. Through RNA sequencing and bioinformatic analyses, PPM1G was identified as a biomarker associated with HCC metastasis. Our data mapped a transcriptome‐wide landscape of alternative splicing events modulated by PPM1G in HCC. Notably, we characterized the exon six‐skipping transcript of TBL1X as an onco‐splice variant regulated by PPM1G. Experimental validation revealed the enrichment of TBL1X‐S in response to PPM1G overexpression. Moreover, mRNA stability analyses revealed that PPM1G prolonged the half‐life of the TBL1X‐S transcript. Both PPM1G and TBL1X‐S exhibited metastasis‐promoting phenotypes, with PPM1G‐driven metastasis in HCC being partially dependent on TBL1X‐S. Mechanistically, different TBL1X splice variants showed varying affinities for ZEB1, with TBL1X‐S significantly enhancing ZEB1 activation and repressing CDH1 transcription, potentially accelerating the epithelial‐mesenchymal transition (EMT) process. In conclusion, our study highlights the biological role of PPM1G and TBL1X‐S in tumor metastasis. The PPM1G/TBL1X‐S signaling axis presents a new view for investigating liver cancer metastasis mechanisms. In this study, we highlight the biological role of the PPM1G and TBL1X‐S variants in tumor metastasis. The PPM1G/TBL1X‐S signaling axis presents a new view for investigating liver cancer metastasis mechanisms.