XIST directly regulates X-linked and autosomal genes in naive human pluripotent cells.

X chromosome inactivation (XCI) serves as a paradigm for RNA-mediated regulation of gene expression, wherein the long non-coding RNA XIST spreads across the X chromosome in cis to mediate gene silencing chromosome-wide. In female naive human pluripotent stem cells (hPSCs), XIST is in a dispersed configuration, and XCI does not occur, raising questions about XIST's function. We found that XIST spreads across the X chromosome and induces dampening of X-linked gene expression in naive hPSCs. Surprisingly, XIST also targets specific autosomal regions, where it induces repressive chromatin changes and gene expression dampening. Thereby, XIST equalizes X-linked gene dosage between male and female cells while inducing differences in autosomes. The dispersed Xist configuration and autosomal localization also occur transiently during XCI initiation in mouse PSCs. Together, our study identifies XIST as the regulator of X chromosome dampening, uncovers an evolutionarily conserved trans-acting role of XIST/Xist, and reveals a correlation between XIST/Xist dispersal and autosomal targeting.

DDX21 mediates co-transcriptional RNA m6A modification to promote transcription termination and genome stability.

N6-methyladenosine (m6A) is a crucial RNA modification that regulates diverse biological processes in human cells, but its co-transcriptional deposition and functions remain poorly understood. Here, we identified the RNA helicase DDX21 with a previously unrecognized role in directing m6A modification on nascent RNA for co-transcriptional regulation. DDX21 interacts with METTL3 for co-recruitment to chromatin through its recognition of R-loops, which can be formed co-transcriptionally as nascent transcripts hybridize onto the template DNA strand. Moreover, DDX21's helicase activity is needed for METTL3-mediated m6A deposition onto nascent RNA following recruitment. At transcription termination regions, this nexus of actions promotes XRN2-mediated termination of RNAPII transcription. Disruption of any of these steps, including the loss of DDX21, METTL3, or their enzymatic activities, leads to defective termination that can induce DNA damage. Therefore, we propose that the R-loop-DDX21-METTL3 nexus forges the missing link for co-transcriptional modification of m6A, coordinating transcription termination and genome stability.

The lncRNA Malat1 is trafficked to the cytoplasm as a localized mRNA encoding a small peptide in neurons.

Synaptic function in neurons is modulated by local translation of mRNAs that are transported to distal portions of axons and dendrites. The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is broadly expressed across cell types, almost exclusively as a nuclear long noncoding RNA. We found that in differentiating neurons, a portion of Malat1 RNA redistributes to the cytoplasm. Depletion of Malat1 using antisense oligonucleotides (ASOs) stimulates the expression of particular pre- and postsynaptic proteins, implicating Malat1 in their regulation. Neuronal Malat1 is localized in puncta of both axons and dendrites that costain with Staufen1 protein, similar to neuronal RNA granules formed by locally translated mRNAs. Ribosome profiling of cultured mouse cortical neurons identified ribosome footprints within a 5' region of Malat1 containing short open reading frames. The upstream-most reading frame (M1) of the Malat1 locus was linked to the GFP-coding sequence in mouse embryonic stem cells. When these gene-edited cells were differentiated into glutamatergic neurons, the M1-GFP fusion protein was expressed. Antibody staining for the M1 peptide confirmed its presence in wild-type neurons and showed that M1 expression was enhanced by synaptic stimulation with KCl. Our results indicate that Malat1 serves as a cytoplasmic coding RNA in the brain that is both modulated by and modulates synaptic function.

A Prelimbic Cortex-Thalamus Circuit Bidirectionally Regulates Innate and Stress-Induced Anxiety-Like Behavior.

Anxiety-related disorders respond to cognitive behavioral therapies, which involved the medial prefrontal cortex (mPFC). Previous studies have suggested that subregions of the mPFC have different and even opposite roles in regulating innate anxiety. However, the specific causal targets of their descending projections in modulating innate anxiety and stress-induced anxiety have yet to be fully elucidated. Here, we found that among the various downstream pathways of the prelimbic cortex (PL), a subregion of the mPFC, PL-mediodorsal thalamic nucleus (MD) projection, and PL-ventral tegmental area (VTA) projection exhibited antagonistic effects on anxiety-like behavior, while the PL-MD projection but not PL-VTA projection was necessary for the animal to guide anxiety-related behavior. In addition, MD-projecting PL neurons bidirectionally regulated remote but not recent fear memory retrieval. Notably, restraint stress induced high-anxiety state accompanied by strengthening the excitatory inputs onto MD-projecting PL neurons, and inhibiting PL-MD pathway rescued the stress-induced anxiety. Our findings reveal that the activity of PL-MD pathway may be an essential factor to maintain certain level of anxiety, and stress increased the excitability of this pathway, leading to inappropriate emotional expression, and suggests that targeting specific PL circuits may aid the development of therapies for the treatment of stress-related disorders.

Improved enzymatic labeling of fluorescent in situ hybridization probes applied to the visualization of retained introns in cells.

Fluorescence in situ hybridization (FISH) is a widely used tool for quantifying gene expression and determining the location of RNA molecules in cells. We present an improved method for FISH probe production that yields high-purity probes with a wide range of fluorophores using standard laboratory equipment at low cost. The method modifies an earlier protocol that uses terminal deoxynucleotidyl transferase to add fluorescently labeled nucleotides to synthetic deoxyoligonucleotides. In our protocol, amino-11-ddUTP is joined to an oligonucleotide pool prior to its conjugation to a fluorescent dye, thereby generating pools of probes ready for a variety of modifications. This order of reaction steps allows for high labeling efficiencies regardless of the GC content or terminal base of the oligonucleotides. The degree of labeling (DOL) for spectrally distinct fluorophores (Quasar, ATTO, and Alexa dyes) was mostly >90%, comparable with commercial probes. The ease and low cost of production allowed the generation of probe sets targeting a wide variety of RNA molecules. Using these probes, FISH assays in C2C12 cells showed the expected subcellular localization of mRNAs and pre-mRNAs for Polr2a (RNA polymerase II subunit 2a) and Gapdh, and of the long noncoding RNAs Malat1 and Neat1 Developing FISH probe sets for several transcripts containing retained introns, we found that retained introns in the Gabbr1 and Noc2l transcripts are present in subnuclear foci separate from their sites of synthesis and partially coincident with nuclear speckles. This labeling protocol should have many applications in RNA biology.

Hundred-Fold Enhancement in the Anomalous Hall Effect Induced by Hydrogenation.

The anomalous Hall effect (AHE) is one of the most fascinating transport properties in condensed matter physics. However, the AHE magnitude, which mainly depends on net spin polarization and band topology, is generally small in oxides and thus limits potential applications. Here, we demonstrate a giant enhancement of AHE in a LaCoO3-induced 5d itinerant ferromagnet SrIrO3 by hydrogenation. The anomalous Hall resistivity and anomalous Hall angle, which are two of the most critical parameters in AHE-based devices, are found to increase to 62.2 µΩ·cm and 3%, respectively, showing an unprecedentedly large enhancement ratio of ∼10000%. Theoretical analysis suggests the key roles of Berry curvature in enhancing AHE. Furthermore, the hydrogenation concomitantly induces the significant elevation of Curie temperature from 75 to 160 K and 40-fold reinforcement of coercivity. Such giant regulation and very large AHE magnitude observed in SrIrO3 could pave the path for 5d oxide devices.

Facilitation of Behavioral and Cortical Emergence from Isoflurane Anesthesia by GABAergic Neurons in Basal Forebrain.

General anesthesia shares many similarities with natural sleep in behavior and electroencephalogram (EEG) patterns. The latest evidence suggests that general anesthesia and sleep-wake behavior may share overlapping neural substrates. The GABAergic neurons in the basal forebrain (BF) have recently been demonstrated to play a key role in controlling wakefulness. It was hypothesized that BF GABAergic neurons may participate in the regulation of general anesthesia. Here, using in vivo fiber photometry, we found that the activity of BF GABAergic neurons was generally inhibited during isoflurane anesthesia, having obviously decreased during the induction of anesthesia and being gradually restored during the emergence from anesthesia, in Vgat-Cre mice of both sexes. Activation of BF GABAergic neurons with chemogenetic and optogenetic approaches decreased sensitivity to isoflurane, delayed induction, and accelerated emergence from isoflurane anesthesia. Optogenetic activation of BF GABAergic neurons decreased EEG δ power and the burst suppression ratio (BSR) during 0.8% and 1.4% isoflurane anesthesia, respectively. Similar to the effects of activating BF GABAergic cell bodies, photostimulation of BF GABAergic terminals in the thalamic reticular nucleus (TRN) also strongly promoted cortical activation and behavioral emergence from isoflurane anesthesia. Collectively, these results showed that the GABAergic BF is a key neural substrate for general anesthesia regulation that facilitates behavioral and cortical emergence from general anesthesia via the GABAergic BF-TRN pathway. Our findings may provide a new target for attenuating the depth of anesthesia and accelerating emergence from general anesthesia.SIGNIFICANCE STATEMENT The basal forebrain (BF) is a key brain region controlling sleep-wake behavior. Activation of GABAergic neurons in the BF potently promotes behavioral arousal and cortical activity. Recently, many sleep-wake-related brain structures have been reported to participate in the regulation of general anesthesia. However, it is still unclear what role BF GABAergic neurons play in general anesthesia. In this study, we aim to reveal the role of BF GABAergic neurons in behavioral and cortical emergence from isoflurane anesthesia and elucidate the underlying neural pathways. Understanding the specific role of BF GABAergic neurons in isoflurane anesthesia would improve our understanding of the mechanisms of general anesthesia and may provide a new strategy for accelerating emergence from general anesthesia.

Mature astrocytes as source for astrocyte repopulation after deletion in the medial prefrontal cortex: Implications for depression.

The adult brain retains a high repopulation capacity of astrocytes after deletion, and both mature astrocytes in the neocortex and neural stem cells in neurogenic regions possess the potential to generate astrocytes. However, the origin and the repopulation dynamics of the repopulating astrocytes after deletion remain largely unclear. The number of astrocytes is reduced in the medial prefrontal cortex (mPFC) of patients with depression, and selective elimination of mPFC astrocytes is sufficient to induce depression-like behaviors in rodents. However, whether astrocyte repopulation capacity is impaired in depression is unknown. In this study, we used different transgenic mouse lines to genetically label different cell types and demonstrated that in the mPFC of normal adult mice of both sexes, mature astrocytes were a major source of the repopulating astrocytes after acute deletion induced by an astrocyte-specific toxin, L-alpha-aminoadipic acid (L-AAA), and astrocyte regeneration was accomplished within two weeks accompanied by reversal of depression-like behaviors. Furthermore, re-ablation of mPFC astrocytes post repopulation led to reappearance of depression-like behaviors. In adult male mice subjected to 14-day chronic restraint stress, a well-validated mouse model of depression, the number of mPFC astrocytes was reduced; however, the ability of mPFC astrocytes to repopulate after L-AAA-induced deletion was largely unaltered. Our study highlights a potentially beneficial role for repopulating astrocytes in depression and provides novel therapeutic insights into enhancing local mature astrocyte generation in depression.

Desymmetrization-Addition Reaction of Cyclopropenes to Imines via Synergistic Photoredox and Cobalt Catalysis.

Herein, we designed a reaction for the desymmetrization-addition of cyclopropenes to imines by leveraging the synergy between photoredox and asymmetric cobalt catalysis. This protocol facilitated the synthesis of a series of chiral functionalized cyclopropanes with high yield, enantioselectivity, and diastereoselectivity (44 examples, up to 93% yield and >99% ee). A possible reaction mechanism involving cyclopropene desymmetrization by Co-H species and imine addition by Co-alkyl species was proposed. This study provides a novel route to important chiral cyclopropanes and extends the frontier of asymmetric metallaphotoredox catalysis.

Synthesis of Chiral Endocyclic Allenes and Alkynes via Pd-Catalyzed Asymmetric Higher-Order Dipolar Cycloaddition.

A Pd-catalyzed asymmetric higher-order dipolar cycloaddition between allenyl carbonates and azadienes is achieved by exploiting novel alkylidene-π-allyl-Pd dipoles. This research provides a modular platform for the synthesis of challenging chiral endocyclic allenes bearing a medium-sized heterocyclic motif and a centrally chiral stereocenter in good yields with high enantio- and diastereoselectivities (29 examples, up to 97% yield, 97:3 er and >19:1 dr). Experimental and computational studies elucidate the possible reaction mechanism and the observed stereochemical results. Based on the mechanistic understanding, a new π-propargyl-Pd dipole was designed to further extend the success of the higher order dipolar cycloaddition strategy to the synthesis of 10-membered endocyclic alkynes from propargyl carbonates and azadienes (13 examples, up to 98% yield and 94.5:5.5 er).

Enantioselective Cyanofunctionalization of Aromatic Alkenes via Radical Anions.

Alkene radical ions constitute an integral and unique class of reactive intermediates for the synthesis of valuable compounds because they have both unpaired spins and charge. However, relatively few synthetic applications of alkene radical anions have emerged due to a dearth of generally applicable and mild radical anion generation approaches. Precise control over the chemo- and stereoselectivity in alkene radical anion-mediated processes represents another long-standing challenge due to their high reactivity. To overcome these issues, here, we develop a new redox-neutral strategy that seamlessly merges photoredox and copper catalysis to enable the controlled generation of alkene radical anions and their orthogonal enantioselective cyanofunctionalization via distonic-like species. This new strategy enables highly regio-, chemo-, and enantioselective hydrocyanation, deuterocyanation, and cyanocarboxylation of alkenes without stoichiometric reductants or oxidants under visible light irradiation. This protocol provides a new blueprint for the exploration of the transformation potential of alkene radical anions.

PTEN Deficiency Facilitates Exosome Secretion and Metastasis in Cholangiocarcinoma by Impairing TFEB-mediated Lysosome Biogenesis.

BACKGROUND & AIMS: In eukaryotes, the ubiquitin-proteasome system and the autophagy-lysosome pathway are essential for maintaining cellular proteostasis and associated with cancer progression. Our previous studies have demonstrated that phosphatase and tensin homolog (PTEN), one of the most frequently mutated genes in human cancers, limits proteasome abundance and determines chemosensitivity to proteasome inhibitors in cholangiocarcinoma (CCA). However, whether PTEN regulates the lysosome pathway remains unclear. METHODS: We tested the effects of PTEN on lysosome biogenesis and exosome secretion using loss- and gain-of-function strategies in CCA cell lines. Using in vitro dephosphorylation assays, we explored the regulatory mechanism between PTEN and the key regulator of lysosome biogenesis, transcription factor EB (TFEB). Using the migration assays, invasion assays, and trans-splenic liver metastasis mouse models, we evaluated the function of PTEN deficiency, TFEB-mediated lysosome biogenesis, and exosome secretion on tumor metastasis. Moreover, we investigated the clinical significance of PTEN expression and exosome secretion by retrospective analysis. RESULTS: PTEN facilitated lysosome biogenesis and acidification through its protein phosphatase activity to dephosphorylate TFEB at Ser211. Notably, PTEN deficiency increased exosome secretion by reducing lysosome-mediated degradation of multi-vesicular bodies, which further facilitated the proliferation and invasion of CCA. TFEB agonist curcumin analog C1 restrained the metastatic phenotype caused by PTEN deficiency in mouse models, and we highlighted the correlation between PTEN deficiency and exosome secretion in clinical cohorts. CONCLUSIONS: In CCA, PTEN deficiency impairs lysosome biogenesis to facilitate exosome secretion and cancer metastasis in a TFEB phosphorylation-dependent manner.

Tracking pre-mRNA maturation across subcellular compartments identifies developmental gene regulation through intron retention and nuclear anchoring.

Steps of mRNA maturation are important gene regulatory events that occur in distinct cellular locations. However, transcriptomic analyses often lose information on the subcellular distribution of processed and unprocessed transcripts. We generated extensive RNA-seq data sets to track mRNA maturation across subcellular locations in mouse embryonic stem cells, neuronal progenitor cells, and postmitotic neurons. We find disparate patterns of RNA enrichment between the cytoplasmic, nucleoplasmic, and chromatin fractions, with some genes maintaining more polyadenylated RNA in chromatin than in the cytoplasm. We bioinformatically defined four regulatory groups for intron retention, including complete cotranscriptional splicing, complete intron retention in the cytoplasmic RNA, and two intron groups present in nuclear and chromatin transcripts but fully excised in cytoplasm. We found that introns switch their regulatory group between cell types, including neuronally excised introns repressed by polypyrimidine track binding protein 1 (PTBP1). Transcripts for the neuronal gamma-aminobutyric acid (GABA) B receptor, 1 (Gabbr1) are highly expressed in mESCs but are absent from the cytoplasm. Instead, incompletely spliced Gabbr1 RNA remains sequestered on chromatin, where it is bound by PTBP1, similar to certain long noncoding RNAs. Upon neuronal differentiation, Gabbr1 RNA becomes fully processed and exported for translation. Thus, splicing repression and chromatin anchoring of RNA combine to allow posttranscriptional regulation of Gabbr1 over development. For this and other genes, polyadenylated RNA abundance does not indicate functional gene expression. Our data sets provide a rich resource for analyzing many other aspects of mRNA maturation in subcellular locations and across development.

Linking watershed formation with the phylogenetic distribution of a soil microscopic fungus in Yunnan Province, China.

BACKGROUND: Phylogeographic studies have gained prominence in linking past geological events to the distribution patterns of biodiversity, primarily in mountainous regions. However, such studies often focus on plant taxa, neglecting the intricate biogeographical patterns of microbes, particularly soil microbial communities. This article explores the spatial distribution of the nematode-trapping fungus Arthrobotrys oligospora, a widespread microorganism, in a tectonically active region at the southeastern edge of the Qinghai-Tibetan Plateau. By analysing the genetic variation of this fungus alongside the historical structure of major river watersheds, we sought to uncover potential connections between the two. Our study involved sampling 149 strains from 116 sites across six major watersheds in the region. RESULTS: The resulting haplotype network revealed five distinct clusters, each corresponding closely to a specific watershed. These clusters exhibited high haplotype diversity and low nucleotide diversity, supporting the notion of watershed-based segregation. Further analysis of haplotypes shared across watersheds provided evidence for three proposed past river connections. In particular, we found numerous shared haplotypes between the Yangtze and Mekong basins, as well as between the Yangtze and the Red basins. Evidence for a Irrawaddy-Salween-Red and a Yangtze-Pearl-Red river connections were also portrayed in our mapping exercise. CONCLUSIONS: These findings emphasize the crucial role of historical geomorphological events in shaping the biogeography of microbial biodiversity, alongside contemporary biotic and abiotic factors. Watershed perimeters emerged as effective predictors of such patterns, suggesting their suitability as analytical units for regional-scale studies. Our study also demonstrates the potential of microorganisms and phylogeographic approaches to complement traditional geological analyses, providing a more comprehensive understanding of past landscape structure and its evolution.

A long-term experiment reveals no trade-off between seed persistence and seedling emergence.

Information on seed persistence and seedling emergence from the soil seed bank is critical for understanding species coexistence and predicting community dynamics. However, quantifying seed persistence in the soil is challenging; thus, its association with other life-history traits is poorly known on a broad scale. Using germination phenology for 349 species in a 42-yr experiment, we quantified the persistence-emergence correlations and their associations with intrinsic regeneration traits using Bayesian phylogenetic multilevel models. We showed no trade-off between seed persistence and seedling emergence. Physically dormant seeds were more persistent but exhibited lower emergence than nondormant seeds. Monocarpic species had both higher persistence and emergence than polycarpic species. Seed mass posed a marginal proxy for persistence, while emergence almost doubled from the smallest to the largest seeds. This study challenges the traditional assumption and is the first demonstration of noncorrelation between persistence and emergence, probably owing to the complexity of regenerative strategies. Species with short persistence and low emergence would be the most vulnerable for in situ conservation. Our analyses of this unique, long-term dataset provide a strong incentive for further experimental studies and a rich data resource for future syntheses.

Association Between Home Renovation and Sleeping Problems Among Children Aged 6-18 Years: A Nationwide Survey in China.

BACKGROUND: Although the indoor environment has been proposed to be associated with childhood sleep health, to our knowledge no study has investigated the association between home renovation and childhood sleep problems. METHODS: The study included 186,470 children aged 6-18 years from the National Chinese Children Health Study (2012-2018). We measured childhood sleeping problems via the Chinese version of the Sleep Disturbance Scale for Children (C-SDSC). Information on home renovation exposure within the recent 2 years was collected via parent report. We estimated associations between home renovation and various sleeping problems, defined using both continuous and categorized (binary) C-SDSC t-scores, using generalized mixed models. We fitted models with city as a random effect variable, and other covariates as fixed effects. RESULTS: Out of the overall participants, 89,732 (48%) were exposed to recent home renovations. Compared to the unexposed group, children exposed to home renovations had higher odds of total sleep disorder (odd ratios [OR] = 1.3; 95% confidence interval [CI] = 1.2, 1.4). Associations varied when we considered different types of home renovation materials. Children exposed to multiple types of home renovation had higher odds of sleeping problems. We observed similar findings when considering continuous C-SDSC t-scores. Additionally, sex and age of children modified the associations of home renovation exposure with some of the sleeping problem subtypes. CONCLUSIONS: We found that home renovation was associated with higher odds of having sleeping problems and that they varied when considering the type of renovation, cumulative exposure, sex, and age differences.

Tailoring whispering-gallery fields in optical black hole cavities.

The ability to confine light has great significance in both fundamental science and practical applications. Optical black hole (OBH) cavities show intriguing zero radiation loss and strong field confinement. In this work, we systematically explore the whispering gallery mode (WGM) in a group of generalized OBH cavities, featuring bound states and strong field confinement. The field confinement in generalized OBH cavities is revealed to be enhanced with the increase of index-modulation factors, resulting from the increase of a potential barrier. Furthermore, we reveal the anomalous external resonant modes, exhibiting fascinating field enhancement in the low-index region far beyond the cavity boundary. These anomalous WGMs are attributed to the potential bending effect and above-barrier resonance. Our work may shed light on tailoring WGM fields in gradient-index cavities and find potential applications in light coupling and optical sensing.

Geodesic conformal gradient device based on a torus.

In recent years, optical fields on non-Euclidean geometry have become a hot topic. The geodesic conformal transformation theory, linking curved surfaces with planar gradient refractive indices, holds unique advantages in controlling curved optical fields. However, this theory has not yet addressed surfaces with non-trivial topology with a certain genus. In this work, we design a gradient planar device based on the geodesic conformal transformation theory for toroidal surfaces, which can achieve Gaussian beam focusing. Unlike traditional angle-preserving geodesic theories, the non-zero genus results in the one-to-two discontinuous boundaries in the device, and we utilize inversion transformations to rectify this drawback.

Multiple hyperbolic waves.

This study presents a conceptual design for a hyperbolic material utilizing transformation optics. This material is designed to produce multiple hyperbolic wave fields or polaritons excited by a point source. The design dictates key parameters including branch number, propagation range, and overall propagation direction of deflection. Through this approach, the hyperbolic material demonstrates new effects compared to traditional hyperbolic materials. These advancements offer possibilities for the design and applications of photonic devices in other degrees of freedom.

Mirrored transformation optics.

A mirrored transformation optics (MTO) approach is presented to overcome the material mismatch in transformation optics. It makes good use of the reflection behavior and introduces a mirrored medium to offset the phase discontinuities. Using this approach, a high-performance planar focusing lens of transmission type is designed, which has a larger concentration ratio than the other focusing lens obtained by the generalized Snell's law. The MTO will not change any functionality of the original lens and has promising potential applications in imaging and light energy harvesting.