Notes

Enantioselective fluorination involving 3-indolinone-2-carboxylates with NFSI catalyzed through chiral bisoxazolines.
Thus, the within-gene clustering of pathogenic and benign DNA changes is an important and previously underappreciated feature of the human exome, which can be harnessed to improve the prediction of pathogenicity and disambiguation of DNA variants of uncertain significance.Recurrence risk calculations in autosomal recessive diseases are complicated when the effect of genetic variants and their population frequencies and penetrances are unknown. An example of this is Stargardt disease (STGD1), a frequent recessive retinal disease caused by bi-allelic pathogenic variants in ABCA4. In this cross-sectional study, 1,619 ABCA4 variants from 5,579 individuals with STGD1 were collected and categorized by (1) severity based on statistical comparisons of their frequencies in STGD1-affected individuals versus the general population, (2) their observed versus expected homozygous occurrence in STGD1-affected individuals, (3) their occurrence in combination with established mild alleles in STGD1-affected individuals, and (4) previous functional and clinical studies. We used the sum allele frequencies of these severity categories to estimate recurrence risks for offspring of STGD1-affected individuals and carriers of pathogenic ABCA4 variants. The risk for offspring of an STGD1-affected individual with the "severe|severe" genotype or a "severe|mild with complete penetrance" genotype to develop STGD1 at some moment in life was estimated at 2.8%-3.1% (1 in 36-32 individuals) and 1.6%-1.8% (1 in 62-57 individuals), respectively. The risk to develop STGD1 in childhood was estimated to be 2- to 4-fold lower 0.68%-0.79% (1 in 148-126) and 0.34%-0.39% (1 in 296-252), respectively. In conclusion, we established personalized recurrence risk calculations for STGD1-affected individuals with different combinations of variants. We thus propose an expanded genotype-based personalized counseling to appreciate the variable recurrence risks for STGD1-affected individuals. This represents a conceptual breakthrough because risk calculations for STGD1 may be exemplary for many other inherited diseases.Predictive coding is an important candidate theory of self-supervised learning in the brain. Its central idea is that sensory responses result from comparisons between bottom-up inputs and contextual predictions, a process in which rates and synchronization may play distinct roles. We recorded from awake macaque V1 and developed a technique to quantify stimulus predictability for natural images based on self-supervised, generative neural networks. We find that neuronal firing rates were mainly modulated by the contextual predictability of higher-order image features, which correlated strongly with human perceptual similarity judgments. By contrast, V1 gamma (γ)-synchronization increased monotonically with the contextual predictability of low-level image features and emerged exclusively for larger stimuli. Consequently, γ-synchronization was induced by natural images that are highly compressible and low-dimensional. Natural stimuli with low predictability induced prominent, late-onset beta (β)-synchronization, likely reflecting cortical feedback. Our findings reveal distinct roles of synchronization and firing rates in the predictive coding of natural images.Many mammalian neurons release multiple neurotransmitters to activate diverse classes of postsynaptic ionotropic receptors. Entopeduncular nucleus somatostatin (EP Sst+) projection neurons to the lateral habenula (LHb) release both glutamate and GABA, but it is unclear whether these are packaged into the same or segregated pools of synaptic vesicles. Here, we describe a method combining electrophysiology, spatially patterned optogenetics, and computational modeling designed to analyze the mechanism of glutamate/GABA co-release in mouse brain. We find that the properties of postsynaptic currents elicited in LHb neurons by optogenetically activating EP Sst+ terminals are only consistent with co-packaging of glutamate/GABA into individual vesicles. Furthermore, presynaptic neuromodulators that weaken EP Sst+ to LHb synapses maintain the co-packaging of glutamate/GABA while reducing vesicular release probability. Our approach is applicable to the study of multi-transmitter neurons throughout the brain, and our results constrain the mechanisms of neuromodulation and synaptic integration in LHb.The biophysical properties of existing optogenetic tools constrain the scale, speed, and fidelity of precise optogenetic control. Here, we use structure-guided mutagenesis to engineer opsins that exhibit very high potency while retaining fast kinetics. These new opsins enable large-scale, temporally and spatially precise control of population neural activity. We extensively benchmark these new opsins against existing optogenetic tools and provide a detailed biophysical characterization of a diverse family of opsins under two-photon illumination. This establishes a resource for matching the optimal opsin to the goals and constraints of patterned optogenetics experiments. Finally, by combining these new opsins with optimized procedures for holographic photostimulation, we demonstrate the simultaneous coactivation of several hundred spatially defined neurons with a single hologram and nearly double that number by temporally interleaving holograms at fast rates. These newly engineered opsins substantially extend the capabilities of patterned illumination optogenetic paradigms for addressing neural circuits and behavior.Pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLCs) have shown great potential as an alternative to primary human hepatocytes (PHHs) for in vitro modeling. Several differentiation protocols have been described to direct PSCs toward the hepatic fate. Here, by leveraging recent knowledge of the signaling pathways involved in liver development, we describe a robust, scalable protocol that allowed us to consistently generate high-quality bipotent human hepatoblasts and HLCs from both embryonic stem cells and induced PSC (iPSCs). Although not yet fully mature, such HLCs were more similar to adult PHHs than were cells obtained with previously described protocols, showing good potential as a physiologically representative alternative to PHHs for in vitro modeling. PSC-derived hepatoblasts effectively generated with this protocol could differentiate into mature hepatocytes and cholangiocytes within syngeneic liver organoids, thus opening the way for representative human 3D in vitro modeling of liver development and pathophysiology.Amyotrophic lateral sclerosis is a fatal disease pathologically typified by motor and cortical neurodegeneration as well as microgliosis. The FUS P525L mutation is highly penetrant and causes ALS cases with earlier disease onset and more aggressive progression. To date, how P525L mutations may affect microglia during ALS pathogenesis had not been explored. In this study, we engineered isogenic control and P525L mutant FUS in independent human iPSC lines and differentiated them into microglia-like cells. We report that the P525L mutation causes FUS protein to mislocalize from the nucleus to cytoplasm. Homozygous P525L mutations perturb the transcriptome profile in which many differentially expressed genes are associated with microglial functions. Specifically, the dysregulation of several chemoreceptor genes leads to altered chemoreceptor-activated calcium signaling. However, other microglial functions such as phagocytosis and cytokine release are not significantly affected. Our study underscores the cell-autonomous effects of the ALS-linked FUS P525L mutation in a human microglia model.Neural stem cells (NSCs) in the adult brain are a source of neural cells for brain injury repair. selleck chemicals llc We investigated whether their capacity to generate new neurons and glia is determined by thyroid hormone (TH) during development because serum levels peak during postnatal reorganization of the main NSC niche, the subventricular zone (SVZ). Re-analysis of mouse transcriptome data revealed increased expression of TH transporters and deiodinases in postnatal SVZ NSCs, promoting local TH action, concomitant with a burst in neurogenesis. Inducing developmental hypothyroidism reduced NSC proliferation, disrupted expression of genes implicated in NSC determination and TH signaling, and altered the neuron/glia output in newborns. Three-month-old adult mice recovering from developmental hypothyroidism had fewer olfactory interneurons and underperformed on short-memory odor tests, dependent on SVZ neurogenesis. Our data provide readouts permitting comparison with adverse long-term events following thyroid disruptor exposure and ideas regarding the etiology of prevalent neurodegenerative diseases in industrialized countries.The extracellular matrix (ECM) provides essential cues to promote endothelial specification during tissue development in vivo; correspondingly, ECM is considered essential for endothelial differentiation outside of the body. However, systematic studies to assess the precise contribution of individual ECM proteins to endothelial differentiation have not been conducted. Further, the multi-component nature of differentiation protocols makes it challenging to study the underlying mechanisms by which the ECM contributes to cell fate. In this study, we determined that Laminin 411 alone increases endothelial differentiation of induced pluripotent stem cells over collagen I or Matrigel. The effect of ECM was shown to be independent of vascular endothelial growth factor (VEGF) binding capacity. We also show that ECM-guided endothelial differentiation is dependent on activation of focal adhesion kinase (FAK), integrin-linked kinase (ILK), Notch, and β-catenin pathways. Our results indicate that ECM contributes to endothelial differentiation through multiple avenues, which converge at the expression of active β-catenin.Regeneration of amputated digit tips relies on mesenchymal progenitor cells and their differentiation into replacement bone and tissue stroma. Natural killer (NK) cells have well-characterized roles in antigen-independent killing of virally infected, pre-tumorous, or stressed cells; however, the potential for cytotoxic activity against regenerative progenitor cells is unclear. We identified NK cell recruitment to the regenerating digit tip, and NK cytotoxicity was observed against osteoclast and osteoblast progenitors. Adoptive cell transplants of spleen NK (SpNK) or thymus NK (ThNK) donor cells into immunodeficient mice demonstrated ThNK cell-induced apoptosis with a reduction in osteoclasts, osteoblasts, and proliferative cells, resulting in inhibition of regeneration. Adoptive transfer of NK cells deficient in NK cell activation genes identified that promotion of regeneration by SpNK cells requires Ncr1, whereas inhibition by ThNK cells is mediated via Klrk1 and perforin. Successful future therapies aimed at enhancing regeneration will require a deeper understanding of progenitor cell protection from NK cell cytotoxicity.Cell-based therapies face challenges, including poor cell survival, immune rejection, and integration into pathologic tissue. We conducted an open-label phase 1/2a clinical trial to assess the safety and preliminary efficacy of a subretinal implant consisting of a polarized monolayer of allogeneic human embryonic stem cell-derived retinal pigmented epithelium (RPE) cells in subjects with geographic atrophy (GA) secondary to dry age-related macular degeneration. Postmortem histology from one subject with very advanced disease shows the presence of donor RPE cells 2 years after implantation by immunoreactivity for RPE65 and donor-specific human leukocyte antigen (HLA) class I molecules. Markers of RPE cell polarity and phagocytosis suggest donor RPE function. Further histologic examination demonstrated CD34+ structures beneath the implant and CD4+, CD68+, and FoxP3+ cells in the tissue. Despite significant donor-host HLA mismatch, no clinical signs of retinitis, vitreitis, vasculitis, choroiditis, or serologic immune response were detected in the deceased subject or any other subject in the study.
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