Stress-Induced Metabolic Disorder in Peripheral CD4+ T Cells Leads to Anxiety-like Behavior.

Physical or mental stress leads to neuroplasticity in the brain and increases the risk of depression and anxiety. Stress exposure causes the dysfunction of peripheral T lymphocytes. However, the pathological role and underlying regulatory mechanism of peripheral T lymphocytes in mood disorders have not been well established. Here, we show that the lack of CD4+ T cells protects mice from stress-induced anxiety-like behavior. Physical stress-induced leukotriene B4 triggers severe mitochondrial fission in CD4+ T cells, which further leads to a variety of behavioral abnormalities including anxiety, depression, and social disorders. Metabolomic profiles and single-cell transcriptome reveal that CD4+ T cell-derived xanthine acts on oligodendrocytes in the left amygdala via adenosine receptor A1. Mitochondrial fission promotes the de novo synthesis of purine via interferon regulatory factor 1 accumulation in CD4+ T cells. Our study implicates a critical link between a purine metabolic disorder in CD4+ T cells and stress-driven anxiety-like behavior.

Acetylation Blocks cGAS Activity and Inhibits Self-DNA-Induced Autoimmunity.

The presence of DNA in the cytoplasm is normally a sign of microbial infections and is quickly detected by cyclic GMP-AMP synthase (cGAS) to elicit anti-infection immune responses. However, chronic activation of cGAS by self-DNA leads to severe autoimmune diseases for which no effective treatment is available yet. Here we report that acetylation inhibits cGAS activation and that the enforced acetylation of cGAS by aspirin robustly suppresses self-DNA-induced autoimmunity. We find that cGAS acetylation on either Lys384, Lys394, or Lys414 contributes to keeping cGAS inactive. cGAS is deacetylated in response to DNA challenges. Importantly, we show that aspirin can directly acetylate cGAS and efficiently inhibit cGAS-mediated immune responses. Finally, we demonstrate that aspirin can effectively suppress self-DNA-induced autoimmunity in Aicardi-Goutières syndrome (AGS) patient cells and in an AGS mouse model. Thus, our study reveals that acetylation contributes to cGAS activity regulation and provides a potential therapy for treating DNA-mediated autoimmune diseases.

p53 Represses the Mevalonate Pathway to Mediate Tumor Suppression.

There are still gaps in our understanding of the complex processes by which p53 suppresses tumorigenesis. Here we describe a novel role for p53 in suppressing the mevalonate pathway, which is responsible for biosynthesis of cholesterol and nonsterol isoprenoids. p53 blocks activation of SREBP-2, the master transcriptional regulator of this pathway, by transcriptionally inducing the ABCA1 cholesterol transporter gene. A mouse model of liver cancer reveals that downregulation of mevalonate pathway gene expression by p53 occurs in premalignant hepatocytes, when p53 is needed to actively suppress tumorigenesis. Furthermore, pharmacological or RNAi inhibition of the mevalonate pathway restricts the development of murine hepatocellular carcinomas driven by p53 loss. Like p53 loss, ablation of ABCA1 promotes murine liver tumorigenesis and is associated with increased SREBP-2 maturation. Our findings demonstrate that repression of the mevalonate pathway is a crucial component of p53-mediated liver tumor suppression and outline the mechanism by which this occurs.

G3BP1 promotes DNA binding and activation of cGAS.

Cyclic GMP-AMP synthase (cGAS) is a key sensor responsible for cytosolic DNA detection. Here we report that GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) is critical for DNA sensing and efficient activation of cGAS. G3BP1 enhanced DNA binding of cGAS by promoting the formation of large cGAS complexes. G3BP1 deficiency led to inefficient DNA binding by cGAS and inhibited cGAS-dependent interferon (IFN) production. The G3BP1 inhibitor epigallocatechin gallate (EGCG) disrupted existing G3BP1-cGAS complexes and inhibited DNA-triggered cGAS activation, thereby blocking DNA-induced IFN production both in vivo and in vitro. EGCG administration blunted self DNA-induced autoinflammatory responses in an Aicardi-Goutières syndrome (AGS) mouse model and reduced IFN-stimulated gene expression in cells from a patient with AGS. Thus, our study reveals that G3BP1 physically interacts with and primes cGAS for efficient activation. Furthermore, EGCG-mediated inhibition of G3BP1 provides a potential treatment for cGAS-related autoimmune diseases.

KRAS and YAP1 converge to regulate EMT and tumor survival.

Cancer cells that express oncogenic alleles of RAS typically require sustained expression of the mutant allele for survival, but the molecular basis of this oncogene dependency remains incompletely understood. To identify genes that can functionally substitute for oncogenic RAS, we systematically expressed 15,294 open reading frames in a human KRAS-dependent colon cancer cell line engineered to express an inducible KRAS-specific shRNA. We found 147 genes that promoted survival upon KRAS suppression. In particular, the transcriptional coactivator YAP1 rescued cell viability in KRAS-dependent cells upon suppression of KRAS and was required for KRAS-induced cell transformation. Acquired resistance to Kras suppression in a Kras-driven murine lung cancer model also involved increased YAP1 signaling. KRAS and YAP1 converge on the transcription factor FOS and activate a transcriptional program involved in regulating the epithelial-mesenchymal transition (EMT). Together, these findings implicate transcriptional regulation of EMT by YAP1 as a significant component of oncogenic RAS signaling.

p53-dependent Nestin regulation links tumor suppression to cellular plasticity in liver cancer.

The p53 tumor suppressor coordinates a series of antiproliferative responses that restrict the expansion of malignant cells, and as a consequence, p53 is lost or mutated in the majority of human cancers. Here, we show that p53 restricts expression of the stem and progenitor-cell-associated protein nestin in an Sp1/3 transcription-factor-dependent manner and that Nestin is required for tumor initiation in vivo. Moreover, loss of p53 facilitates dedifferentiation of mature hepatocytes into nestin-positive progenitor-like cells, which are poised to differentiate into hepatocellular carcinomas (HCCs) or cholangiocarcinomas (CCs) in response to lineage-specific mutations that target Wnt and Notch signaling, respectively. Many human HCCs and CCs show elevated nestin expression, which correlates with p53 loss of function and is associated with decreased patient survival. Therefore, transcriptional repression of Nestin by p53 restricts cellular plasticity and tumorigenesis in liver cancer.

A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing.

CRISPR-Cas9 genome editing has transformed biotechnology and therapeutics. However, in vivo applications of some Cas9s are hindered by large size (limiting delivery by adeno-associated virus [AAV] vectors), off-target editing, or complex protospacer-adjacent motifs (PAMs) that restrict the density of recognition sequences in target DNA. Here, we exploited natural variation in the PAM-interacting domains (PIDs) of closely related Cas9s to identify a compact ortholog from Neisseria meningitidis-Nme2Cas9-that recognizes a simple dinucleotide PAM (N4CC) that provides for high target site density. All-in-one AAV delivery of Nme2Cas9 with a guide RNA targeting Pcsk9 in adult mouse liver produces efficient genome editing and reduced serum cholesterol with exceptionally high specificity. We further expand our single-AAV platform to pre-implanted zygotes for streamlined generation of genome-edited mice. Nme2Cas9 combines all-in-one AAV compatibility, exceptional editing accuracy within cells, and high target site density for in vivo genome editing applications.

High-throughput identification of regulatory elements and functional assays to uncover susceptibility genes for nasopharyngeal carcinoma.

Large-scale genetic association studies have identified multiple susceptibility loci for nasopharyngeal carcinoma (NPC), but the underlying biological mechanisms remain to be explored. To gain insights into the genetic etiology of NPC, we conducted a follow-up study encompassing 6,907 cases and 10,472 controls and identified two additional NPC susceptibility loci, 9q22.33 (rs1867277; OR = 0.74, 95% CI = 0.68-0.81, p = 3.08 × 10-11) and 17q12 (rs226241; OR = 1.42, 95% CI = 1.26-1.60, p = 1.62 × 10-8). The two additional loci, together with two previously reported genome-wide significant loci, 5p15.33 and 9p21.3, were investigated by high-throughput sequencing for chromatin accessibility, histone modification, and promoter capture Hi-C (PCHi-C) profiling. Using luciferase reporter assays and CRISPR interference (CRISPRi) to validate the functional profiling, we identified PHF2 at locus 9q22.33 as a susceptibility gene. PHF2 encodes a histone demethylase and acts as a tumor suppressor. The risk alleles of the functional SNPs reduced the expression of the target gene PHF2 by inhibiting the enhancer activity of its long-range (4.3 Mb) cis-regulatory element, which promoted proliferation of NPC cells. In addition, we identified CDKN2B-AS1 as a susceptibility gene at locus 9p21.3, and the NPC risk allele of the functional SNP rs2069418 promoted the expression of CDKN2B-AS1 by increasing its enhancer activity. The overexpression of CDKN2B-AS1 facilitated proliferation of NPC cells. In summary, we identified functional SNPs and NPC susceptibility genes, which provides additional explanations for the genetic association signals and helps to uncover the underlying genetic etiology of NPC development.

Genome-wide profiling of prime editor off-target sites in vitro and in vivo using PE-tag.

Prime editors have a broad range of potential research and clinical applications. However, methods to delineate their genome-wide editing activities have generally relied on indirect genome-wide editing assessments or the computational prediction of near-cognate sequences. Here we describe a genome-wide approach for the identification of potential prime editor off-target sites, which we call PE-tag. This method relies on the attachment or insertion of an amplification tag at sites of prime editor activity to allow their identification. PE-tag enables genome-wide profiling of off-target sites in vitro using extracted genomic DNA, in mammalian cell lines and in the adult mouse liver. PE-tag components can be delivered in a variety of formats for off-target site detection. Our studies are consistent with the high specificity previously described for prime editor systems, but we find that off-target editing rates are influenced by prime editing guide RNA design. PE-tag represents an accessible, rapid and sensitive approach for the genome-wide identification of prime editor activity and the evaluation of prime editor safety.

Quasi-open Cu(I) sites for efficient CO separation with high O2/H2O tolerance.

Carbon monoxide (CO) separation relies on chemical adsorption but suffers from the difficulty of desorption and instability of open metal sites against O2, H2O and so on. Here we demonstrate quasi-open metal sites with hidden or shielded coordination sites as a promising solution. Possessing the trigonal coordination geometry (sp2), Cu(I) ions in porous frameworks show weak physical adsorption for non-target guests. Rational regulation of framework flexibility enables geometry transformation to tetrahedral geometry (sp3), generating a fourth coordination site for the chemical adsorption of CO. Quantitative breakthrough experiments at ambient conditions show CO uptakes up to 4.1 mmol g-1 and CO selectivity up to 347 against CO2, CH4, O2, N2 and H2. The adsorbents can be completely regenerated at 333-373 K to recover CO with a purity of >99.99%, and the separation performances are stable in high-concentration O2 and H2O. Although CO leakage concentration generally follows the structural transition pressure, large amounts (>3 mmol g-1) of ultrahigh-purity (99.9999999%, 9N; CO concentration < 1 part per billion) gases can be produced in a single adsorption process, demonstrating the usefulness of this approach for separation applications.

NLRP3 Phosphorylation Is an Essential Priming Event for Inflammasome Activation.

Many infections and stress signals can rapidly activate the NLRP3 inflammasome to elicit robust inflammatory responses. This activation requires a priming step, which is thought to be mainly for upregulating NLRP3 transcription. However, recent studies report that the NLRP3 inflammasome can be activated independently of transcription, suggesting that the priming process has unknown essential regulatory steps. Here, we report that JNK1-mediated NLRP3 phosphorylation at S194 is a critical priming event and is essential for NLRP3 inflammasome activation. We show that NLRP3 inflammasome activation is disrupted in NLRP3-S194A knockin mice. JNK1-mediated NLRP3 S194 phosphorylation is critical for NLRP3 deubiquitination and facilitates its self-association and the subsequent inflammasome assembly. Importantly, we demonstrate that blocking S194 phosphorylation prevents NLRP3 inflammasome activation in cryopyrin-associated periodic syndromes (CAPS). Thus, our study reveals a key priming molecular event that is a prerequisite for NLRP3 inflammasome activation. Inhibiting NLRP3 phosphorylation could be an effective treatment for NLRP3-related diseases.

Bright Nonblinking Photoluminescence with Blinking Lifetime from a Nanocavity-Coupled Quantum Dot.

Colloidal quantum dots (QDs) are excellent luminescent nanomaterials for many optoelectronic applications. However, photoluminescence blinking has limited their practical use. Coupling QDs to plasmonic nanostructures shows potential in suppressing blinking. However, the underlying mechanism remains unclear and debated, hampering the development of bright nonblinking dots. Here, by deterministically coupling a QD to a plasmonic nanocavity, we clarify the mechanism and demonstrate unprecedented single-QD brightness. In particular, we report for the first time that a blinking QD could obtain nonblinking photoluminescence with a blinking lifetime through coupling to the nanocavity. We show that the plasmon-enhanced radiative decay outcompetes the nonradiative Auger process, enabling similar quantum yields for charged and neutral excitons in the same dot. Meanwhile, we demonstrate a record photon detection rate of 17 MHz from a colloidal QD, indicating an experimental photon generation rate of more than 500 MHz. These findings pave the way for ultrabright nonblinking QDs, benefiting diverse QD-based applications.

Disrupting actin filaments enhances glucose-stimulated insulin secretion independent of the cortical actin cytoskeleton.

Just under the plasma membrane of most animal cells lies a dense meshwork of actin filaments called the cortical cytoskeleton. In insulin-secreting pancreatic ß cells, a long-standing model posits that the cortical actin layer primarily acts to restrict access of insulin granules to the plasma membrane. Here we test this model and find that stimulating ß cells with pro-secretory stimuli (glucose and/or KCl) has little impact on the cortical actin layer. Chemical perturbations of actin polymerization, by either disrupting or enhancing filamentation, dramatically enhance glucose-stimulated insulin secretion. Using scanning electron microscopy, we directly visualize the cortical cytoskeleton, allowing us to validate the effect of these filament-disrupting chemicals. We find the state of the cortical actin layer does not correlate with levels of insulin secretion, suggesting filament disruptors act on insulin secretion independently of the cortical cytoskeleton.

Isomeric Porous Cu(I) Triazolate Frameworks Showing Periodic and Aperiodic Flexibility for Efficient CO Separation.

Guest-induced (crystal-to-crystal) transformation, i.e., periodic flexibility, is a typical feature of molecule-based crystalline porous materials, but its role for adsorptive separation is controversial. On the other hand, aperiodic flexibility is rarely studied. This work reports a pair of isomeric Cu(I) triazolate frameworks, namely, α-[Cu(fetz)] (MAF-2Fa) and ß-[Cu(fetz)] (MAF-2Fb), which show typical periodic and aperiodic flexibility for CO chemical adsorption, respectively. Quantitative mixture breakthrough experiments show that, while MAF-2Fa exhibits high adsorption capacity at high pressures but negligible adsorption below the threshold pressure and with leakage concentrations of 3-8%, MAF-2Fb exhibits relatively low adsorption capacity at high pressures but no leakage (residual CO concentration <1 ppb). Tandem connection of MAF-2Fa and MAF-2Fb can combine their advantages of high CO adsorption capacities at high and low pressures, respectively. MAF-2Fa and MAF-2Fb can both keep the separation performances unchanged at high relative humidities, but only MAF-2Fb shows a unique coadsorption behavior at a relative humidity of 82%, which can be used to improve purification performances.

Molecular-Sieving Separation of Methanol/Benzene Azeotrope by a Flexible Metal-Organic Framework.

Separation of methanol/benzene azeotrope mixtures is very challenging not only by the conventional distillation technique but also by adsorbents. In this work, we design and synthesize a flexible Ca-based metal-organic framework MAF-58 consisting of cheap raw materials. MAF-58 shows selective methanol-induced pore-opening flexibility. Although the opened pores are large enough to accommodate benzene molecules, MAF-58 shows methanol/benzene molecular sieving with ultrahigh experimental selectivity, giving 5.1 mmol g-1 high-purity (99.99%+) methanol and 2.0 mmol g-1 high-purity (99.97%+) benzene in a single adsorption/desorption cycle. Computational simulations reveal that the preferentially adsorbed, coordinated methanol molecules act as the gating component to selectively block the diffusion of benzene, offering a new gating adsorption mechanism.

Multiomics characterization of mouse hepatoblastoma identifies yes-associated protein 1 target genes.

BACKGROUND AND AIMS: Hepatoblastoma (HB) is the most common primary liver malignancy in childhood and lacks targeted therapeutic options. We previously engineered, to our knowledge, the first yes-associated protein 1 (YAP1) S127A -inducible mouse model of HB, demonstrating tumor regression and redifferentiation after YAP1 withdrawal through genome-wide enhancer modulation. Probing accessibility, transcription, and YAP1 binding at regulatory elements in HB tumors may provide more insight into YAP1-driven tumorigenesis and expose exploitable vulnerabilities in HB. APPROACH AND RESULTS: Using a multiomics approach, we integrated high-throughput transcriptome and chromatin profiling of our murine HB model to identify dynamic activity at candidate cis -regulatory elements (cCREs). We observed that 1301 of 305,596 cCREs exhibit "tumor-modified" (TM) accessibility in HB. We mapped 241 TM enhancers to corresponding genes using accessibility and histone H3K27Ac profiles. Anti-YAP1 cleavage under targets and tagmentation in tumors revealed 66 YAP1-bound TM cCRE/gene pairs, 31 of which decrease expression after YAP1 withdrawal. We validated the YAP1-dependent expression of a putative YAP1 target, Jun dimerization protein 2 (JDP2), in human HB cell lines using YAP1 and LATS1/2 small interfering RNA knockdown. We also confirmed YAP1-induced activity of the Jdp2 TM enhancer in vitro and discovered an analogous human enhancer in silico. Finally, we used transcription factor (TF) footprinting to identify putative YAP1 cofactors and characterize HB-specific TF activity genome wide. CONCLUSIONS: Our chromatin-profiling techniques define the regulatory frameworks underlying HB and identify YAP1-regulated gene/enhancer pairs. JDP2 is an extensively validated target with YAP1-dependent expression in human HB cell lines and hepatic malignancies.

In-band pumped Kerr-lens mode-locked Tm,Ho-codoped calcium aluminate laser.

We report on a Kerr-lens mode-locked Tm,Ho-codoped calcium aluminate laser with in-band pumping of the Tm ions by a spatially single-mode 1678 nm Raman fiber laser. The structurally disordered CaGdAlO4 host crystal is also codoped also with the passive Lu ion for additional inhomogeneous line broadening. The Tm,Ho,Lu:CaGdAlO4 laser generates soliton pulses as short as 79 fs at a central wavelength of 2073.6 nm via soft-aperture Kerr-lens mode-locking. The corresponding average output power amounts to 91 mW at a pulse repetition rate of ∼86 MHz. The average output power can be scaled to 842 mW at the expense of slightly longer pulses of 155 fs at 2045.9 nm, which corresponds to a peak power of ∼58 kW. To the best of our knowledge, this represents the first demonstration of an in-band pumped Kerr-lens mode-locked Tm,Ho solid-state laser at ∼2 µm.

Defocus-integration interferometric scattering microscopy for speckle suppression and enhancing nanoparticle detection on a substrate.

Direct optical detection and imaging of single nanoparticles on a substrate in wide field underpin vast applications across different research fields. However, speckles originating from the unavoidable random surface undulations of the substrate ultimately limit the size of the decipherable nanoparticles by the current optical techniques, including the ultrasensitive interferometric scattering microscopy (iSCAT). Here, we report a defocus-integration iSCAT to suppress the speckle noise and to enhance the detection and imaging of single nanoparticles on an ultra-flat glass substrate and a silicon wafer. In particular, we discover distinct symmetry properties of the scattering phase between the nanoparticle and the surface undulations that cause the speckles. Consequently, we develop the defocus-integration technique to suppress the speckles. We experimentally achieve an enhancement of the signal-to-noise ratio by 6.9 dB for the nanoparticle detection. We demonstrate that the technique is generally applicable for nanoparticles of various materials and for both low and high refractive index substrates.

Serum uric acid levels and health outcomes in CKD: a prospective cohort study.

BACKGROUND: Hyperuricemia is prevalent in individuals with chronic kidney disease (CKD). Elevated serum uric acid (SUA) concentrations have been considered an independent risk factor for the onset of CKD. However, the relationship between SUA concentrations and long-term health outcomes among patients with CKD remains unclear. METHODS: We performed a prospective cohort study with nationally representative sample to investigate the relationship between SUA concentrations and mortality risk including all-cause, cardiovascular disease (CVD) and cancer mortality, among patients with CKD. The weighted restricted cubic spline analyses combined with the multivariate-adjusted Cox proportional hazard models were used to test the nonlinearity of relationship. RESULTS: The 6642 patients participating in National Health and Nutrition Examination Survey 1999-2018 were enrolled. During 656 885 person-months of follow-up time, 2619 all-cause deaths were recorded, including 1030 CVD deaths and 458 cancer deaths. Our study presented J-shaped non-linear relationships between SUA concentrations and all-cause and CVD mortality with inflection points at 311.65 µmol/L and 392.34 µmol/L, respectively. When SUA concentration was higher than those inflection points, every increase of 50 µmol/L SUA was associated with 11.7% and 17.0% greater multivariable-adjusted hazard ratio of all-cause and CVD mortality, respectively. In addition, a negative linear correlation with cancer mortality was detected. CONCLUSION: These findings suggested that maintaining appropriate SUA concentrations may improve long-term health outcomes among CKD patients. The corresponding inflection points of J-shaped non-linear relationships were 311.65 and 392.34 µmol/L for all-cause and CVD mortality. Further clinical trials are required to investigate uric acid-lowering targets.