Multi-omics analysis reveals drivers of loss of ß-cell function after newly diagnosed autoimmune type 1 diabetes: An INNODIA multicenter study.

AIMS: Heterogeneity in the rate of ß-cell loss in newly diagnosed type 1 diabetes patients is poorly understood and creates a barrier to designing and interpreting disease-modifying clinical trials. Integrative analyses of baseline multi-omics data obtained after the diagnosis of type 1 diabetes may provide mechanistic insight into the diverse rates of disease progression after type 1 diabetes diagnosis. METHODS: We collected samples in a pan-European consortium that enabled the concerted analysis of five different omics modalities in data from 97 newly diagnosed patients. In this study, we used Multi-Omics Factor Analysis to identify molecular signatures correlating with post-diagnosis decline in ß-cell mass measured as fasting C-peptide. RESULTS: Two molecular signatures were significantly correlated with fasting C-peptide levels. One signature showed a correlation to neutrophil degranulation, cytokine signalling, lymphoid and non-lymphoid cell interactions and G-protein coupled receptor signalling events that were inversely associated with a rapid decline in ß-cell function. The second signature was related to translation and viral infection was inversely associated with change in ß-cell function. In addition, the immunomics data revealed a Natural Killer cell signature associated with rapid ß-cell decline. CONCLUSIONS: Features that differ between individuals with slow and rapid decline in ß-cell mass could be valuable in staging and prediction of the rate of disease progression and thus enable smarter (shorter and smaller) trial designs for disease modifying therapies as well as offering biomarkers of therapeutic effect.

A robotic system for automated chemical synthesis of therapeutic agents.

The development of novel compounds for tissue-specific targeting and imaging is often impeded by a lack of lead compounds and the availability of reliable chemistry. Automated chemical synthesis systems provide a potential solution by enabling reliable, repeated access to large compound libraries for screening. Here we report an integrated solid-phase combinatorial chemistry system created using commercial and customized robots. Our goal is to optimize reaction parameters, such as varying temperature, shaking, microwave irradiation, aspirating and dispensing large-sized solid beads, and handling different washing solvents for separation and purification. This automated system accommodates diverse chemical reactions such as peptide synthesis and conventional coupling reactions. To confirm its functionality and reproducibility, 20 nerve-specific contrast agents for biomedical imaging were systematically and repeatedly synthesized and compared to other nerve-targeted agents using molecular fingerprinting and Uniform Manifold Approximation and Projection, which lays the foundation for creating reliable and reproductive chemical libraries in bioimaging and nanomedicine.

Intestinal epigenomic alterations are associated with a dysregulated nutrient absorption phenotype in obesity.

Obesity is an epidemic with myriad health effects, but little is understood regarding individual obese phenotypes and how they may respond to therapy. Epigenetic changes associated with obesity have been detected in blood, liver, pancreas, and adipose tissues. Previous work found that dietary glucose hyperabsorption occurs in some obese subjects, but detailed transcriptional or epigenomic features of the intestine associated with this phenotype are unknown. This study evaluated differentially expressed genes and relative chromatin accessibility in intestinal organoids established from donors classified as lean, obese, or obese hyperabsorptive by body mass index and glucose transport assays. Transcriptomic analysis indicated that obese hyperabsorptive subjects have significantly upregulated dietary nutrient absorption proteins and downregulated type I interferon targets. Chromatin accessibility and transcription factor footprinting suggested that enhanced binding of HNF4G promotes the obese hyperabsorption phenotype. Quantitative PCR assessment in a larger subject cohort suggested that intestinal epithelial expression of CUBN, GIP, and SLC2A5 have high correlation with hyperabsorption. The obese hyperabsorption phenotype is characterized by transcriptional changes that support increased nutrient uptake and may be driven by differentially accessible chromatin. Recognizing unique intestinal phenotypes in obesity provides new perspective in considering therapeutic targets and options to manage the disease.

Amoebicidal effect of chlorine dioxide gas against pathogenic Naegleria fowleri and Acanthamoeba polyphaga.

The pathogenic free-living amoebae, Naegleria fowleri and Acanthamoeba polyphaga, are found in freshwater, soil, and unchlorinated or minimally chlorinated swimming pools. N. fowleri and A. polyphaga are becoming problematic as water leisure activities and drinking water are sources of infection. Chlorine dioxide (ClO2) gas is a potent disinfectant that is relatively harmless to humans at the concentration used for disinfection. In this study, we examined the amoebicidal effects of ClO2 gas on N. fowleri and A. polyphaga. These amoebae were exposed to ClO2 gas from a ready-to-use product (0.36 ppmv/h) for 12, 24, 36, and 48 h. Microscopic examination showed that the viability of N. fowleri and A. polyphaga was effectively inhibited by treatment with ClO2 gas in a time-dependent manner. The growth of N. fowleri and A. polyphaga exposed to ClO2 gas for 36 h was completely inhibited. In both cases, the mRNA levels of their respective actin genes were significantly reduced following treatment with ClO2 gas. ClO2 gas has an amoebicidal effect on N. fowleri and A. polyphaga. Therefore, ClO2 gas has been proposed as an effective agent for the prevention and control of pathogenic free-living amoeba contamination.

SETD2 loss in renal epithelial cells drives epithelial-to-mesenchymal transition in a TGF-ß-independent manner.

Histone-lysine N-methyltransferase SETD2 (SETD2), the sole histone methyltransferase that catalyzes trimethylation of lysine 36 on histone H3 (H3K36me3), is often mutated in clear cell renal cell carcinoma (ccRCC). SETD2 mutation and/or loss of H3K36me3 is linked to metastasis and poor outcome in ccRCC patients. Epithelial-to-mesenchymal transition (EMT) is a major pathway that drives invasion and metastasis in various cancer types. Here, using novel kidney epithelial cell lines isogenic for SETD2, we discovered that SETD2 inactivation drives EMT and promotes migration, invasion, and stemness in a transforming growth factor-beta-independent manner. This newly identified EMT program is triggered in part through secreted factors, including cytokines and growth factors, and through transcriptional reprogramming. RNA-seq and assay for transposase-accessible chromatin sequencing uncovered key transcription factors upregulated upon SETD2 loss, including SOX2, POU2F2 (OCT2), and PRRX1, that could individually drive EMT and stemness phenotypes in SETD2 wild-type (WT) cells. Public expression data from SETD2 WT/mutant ccRCC support the EMT transcriptional signatures derived from cell line models. In summary, our studies reveal that SETD2 is a key regulator of EMT phenotypes through cell-intrinsic and cell-extrinsic mechanisms that help explain the association between SETD2 loss and ccRCC metastasis.

Effects of pupil size in video head-impulse tests.

The results of video head impulse tests (video-HITs) may be confounded by data artifacts of various origins, including pupil size and eyelid obstruction of the pupil. This study aimed to determine the effect of these factors on the results of video-HITs. We simulated ptosis by adopting pharmacological dilatation of the pupil in 21 healthy participants (11 women; age 24-58 years). Each participant underwent video-HITs before and after pupillary dilatation using 0.5% tropicamide. We assessed the changes in the vestibulo-ocular reflex (VOR) gain, corrective saccade amplitude, and frequency of eyelid flicks. After pupillary dilatation, the VOR gain decreased for both right (RAC; 1.12 [Formula: see text] 0.12 vs. 1.01 [Formula: see text] 0.16, p = 0.011) and left anterior canals (LACs; 1.15 [Formula: see text] 0.13 vs. 0.96 [Formula: see text] 0.14, p < 0.001), and right posterior canal (RPC, 1.10 [Formula: see text] 0.13 vs. 0.98 [Formula: see text] 0.09, p = 0.001). The corrective saccade amplitudes also decreased significantly for all four vertical canals. The frequency of eyelid flicks, however, did not change. The changes of VOR gain were positively correlated with the lid excursion in RPC (r = 0.629, p = 0.002) and LPC (r = 0.549, p = 0.010). Our study indicates that eyelid position and pupil size should be considered when interpreting the results of video-HITs, especially for the vertical canals. Pupils should be shrunk in a very well-lit room, and artifacts should be prevented by taping or lifting the eyelids as required during video-HITs.

Retention of Intrinsic Photophysical Properties of Porphyrin Building Blocks in 3D Organic Frameworks through Magic Angle Alignment.

Construction of three-dimensional (3D) frameworks maintaining intrinsic photophysical properties of monomeric building blocks is difficult and challenging due to the existence of various molecular interactions, such as metal-organic and π-π interactions. A 3D hydrogen-bonded organic framework (YSH-1Zn) with permanent porosity was constructed using a porphyrin having six carboxylic acid groups (1Zn). Brunauer-Emmett-Teller surface area measurement indicated that YSH-1Zn has a porous structure with a surface area of 392 m2/g. Single-crystal X-ray diffraction analysis revealed that 1Zn creates a 5-fold interwoven 3D network structure adopting a monoclinic system with a space group of P21/c. Each 1Zn within a single crystal exhibits parallel alignment with a slip-stack angle of 54.6°, in good agreement with the magic angle. Although the center-to-center distance of the nearest zinc atoms in YSH-1Zn is only 5.181 Å, the UV/vis absorption and fluorescence emission of YSH-1Zn are not different from those of 1Zn, indicating the absence of an interaction between excitons. Due to the magic angle alignment of 1Zn, the fluorescence lifetime, decay profiles, and quantum yield remained uniform even in the solid state.

BMAL1 modulates senescence programming via AP-1.

Cellular senescence and circadian dysregulation are biological hallmarks of aging. Whether they are coordinately regulated has not been thoroughly studied. We hypothesize that BMAL1, a pioneer transcription factor and master regulator of the molecular circadian clock, plays a role in the senescence program. Here, we demonstrate BMAL1 is significantly upregulated in senescent cells and has altered rhythmicity compared to non-senescent cells. Through BMAL1-ChIP-seq, we show that BMAL1 is uniquely localized to genomic motifs associated with AP-1 in senescent cells. Integration of BMAL1-ChIP-seq data with RNA-seq data revealed that BMAL1 presence at AP-1 motifs is associated with active transcription. Finally, we showed that BMAL1 contributes to AP-1 transcriptional control of key features of the senescence program, including altered regulation of cell survival pathways, and confers resistance to drug-induced apoptosis. Overall, these results highlight a previously unappreciated role of the core circadian clock component BMAL1 on the molecular phenotype of senescent cells.

Hypoxia-Inducible Factor 1α Stabilization Restores Epigenetic Control of Nitric Oxide Synthase 1 Expression and Reverses Gastroparesis in Female Diabetic Mice.

BACKGROUND & AIMS: Although depletion of neuronal nitric oxide synthase (NOS1)-expressing neurons contributes to gastroparesis, stimulating nitrergic signaling is not an effective therapy. We investigated whether hypoxia-inducible factor 1α (HIF1A), which is activated by high O2 consumption in central neurons, is a Nos1 transcription factor in enteric neurons and whether stabilizing HIF1A reverses gastroparesis. METHODS: Mice with streptozotocin-induced diabetes, human and mouse tissues, NOS1+ mouse neuroblastoma cells, and isolated nitrergic neurons were studied. Gastric emptying of solids and volumes were determined by breath test and single-photon emission computed tomography, respectively. Gene expression was analyzed by RNA-sequencing, microarrays, immunoblotting, and immunofluorescence. Epigenetic assays included chromatin immunoprecipitation sequencing (13 targets), chromosome conformation capture sequencing, and reporter assays. Mechanistic studies used Cre-mediated recombination, RNA interference, and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated epigenome editing. RESULTS: HIF1A signaling from physiological intracellular hypoxia was active in mouse and human NOS1+ myenteric neurons but reduced in diabetes. Deleting Hif1a in Nos1-expressing neurons reduced NOS1 protein by 50% to 92% and delayed gastric emptying of solids in female but not male mice. Stabilizing HIF1A with roxadustat (FG-4592), which is approved for human use, restored NOS1 and reversed gastroparesis in female diabetic mice. In nitrergic neurons, HIF1A up-regulated Nos1 transcription by binding and activating proximal and distal cis-regulatory elements, including newly discovered super-enhancers, facilitating RNA polymerase loading and pause-release, and by recruiting cohesin to loop anchors to alter chromosome topology. CONCLUSIONS: Pharmacologic HIF1A stabilization is a novel, translatable approach to restoring nitrergic signaling and treating diabetic gastroparesis. The newly recognized effects of HIF1A on chromosome topology may provide insights into physioxia- and ischemia-related organ function.

Asymmetric gradient orbital interaction of hetero-diatomic active sites for promoting C - C coupling.

Diatomic-site catalysts (DACs) garner tremendous attention for selective CO2 photoreduction, especially in the thermodynamical and kinetical mechanism of CO2 to C2+ products. Herein, we first engineer a novel Zn-porphyrin/RuCu-pincer complex DAC (ZnPor-RuCuDAC). The heteronuclear ZnPor-RuCuDAC exhibits the best acetate selectivity (95.1%), while the homoatomic counterparts (ZnPor-Ru2DAC and ZnPor-Cu2DAC) present the best CO selectivity. In-situ spectroscopic measurements reveal that the heteronuclear Ru-Cu sites easily appear C1 intermediate coupling. The in-depth analyses confirm that due to the strong gradient orbital coupling of Ru4d-Cu3d resonance, two formed *CO intermediates of Ru-Cu heteroatom show a significantly weaker electrostatic repulsion for an asymmetric charge distribution, which result from a side-to-side absorption and narrow dihedral angle distortion. Moreover, the strongly overlapped Ru/Cu-d and CO molecular orbitals split into bonding and antibonding orbitals easily, resulting in decreasing energy splitting levels of C1 intermediates. These results collectively augment the collision probability of the two *CO intermediates on heteronuclear DACs. This work first provides a crucial perspective on the symmetry-forbidden coupling mechanism of C1 intermediates on diatomic sites.

Characterization and Optimization of Multiomic Single-Cell Epigenomic Profiling.

The snATAC + snRNA platform allows epigenomic profiling of open chromatin and gene expression with single-cell resolution. The most critical assay step is to isolate high-quality nuclei to proceed with droplet-base single nuclei isolation and barcoding. With the increasing popularity of multiomic profiling in various fields, there is a need for optimized and reliable nuclei isolation methods, mainly for human tissue samples. Herein we compared different nuclei isolation methods for cell suspensions, such as peripheral blood mononuclear cells (PBMC, n = 18) and a solid tumor type, ovarian cancer (OC, n = 18), derived from debulking surgery. Nuclei morphology and sequencing output parameters were used to evaluate the quality of preparation. Our results show that NP-40 detergent-based nuclei isolation yields better sequencing results than collagenase tissue dissociation for OC, significantly impacting cell type identification and analysis. Given the utility of applying such techniques to frozen samples, we also tested frozen preparation and digestion (n = 6). A paired comparison between frozen and fresh samples validated the quality of both specimens. Finally, we demonstrate the reproducibility of scRNA and snATAC + snRNA platform, by comparing the gene expression profiling of PBMC. Our results highlight how the choice of nuclei isolation methods is critical for obtaining quality data in multiomic assays. It also shows that the measurement of expression between scRNA and snRNA is comparable and effective for cell type identification.

Titration-based normalization of antibody amount improves consistency of ChIP-seq experiments.

Chromatin immunoprecipitation (ChIP) is an antibody-based approach that is frequently utilized in chromatin biology and epigenetics. The challenge in experimental variability by unpredictable nature of usable input amounts from samples and undefined antibody titer in ChIP reaction still remains to be addressed. Here, we introduce a simple and quick method to quantify chromatin inputs and demonstrate its utility for normalizing antibody amounts to the optimal titer in individual ChIP reactions. For a proof of concept, we utilized ChIP-seq validated antibodies against the key enhancer mark, acetylation of histone H3 on lysine 27 (H3K27ac), in the experiments. The results indicate that the titration-based normalization of antibody amounts improves assay outcomes including the consistency among samples both within and across experiments for a broad range of input amounts.

Near-infrared fluorescence lifetime imaging of amyloid-ß aggregates and tau fibrils through the intact skull of mice.

Non-invasive methods for the in vivo detection of hallmarks of Alzheimer's disease can facilitate the study of the progression of the disease in mouse models and may enable its earlier diagnosis in humans. Here we show that the zwitterionic heptamethine fluorophore ZW800-1C, which has peak excitation and emission wavelengths in the near-infrared optical window, binds in vivo and at high contrast to amyloid-ß deposits and to neurofibrillary tangles, and allows for the microscopic imaging of amyloid-ß and tau aggregates through the intact skull of mice. In transgenic mouse models of Alzheimer's disease, we compare the performance of ZW800-1C with that of the two spectrally similar heptamethine fluorophores ZW800-1A and indocyanine green, and show that ZW800-1C undergoes a longer fluorescence-lifetime shift when bound to amyloid-ß and tau aggregates than when circulating in blood vessels. ZW800-1C may prove advantageous for tracking the proteinic aggregates in rodent models of amyloid-ß and tau pathologies.

MacroH2A histone variants modulate enhancer activity to repress oncogenic programs and cellular reprogramming.

Considerable efforts have been made to characterize active enhancer elements, which can be annotated by accessible chromatin and H3 lysine 27 acetylation (H3K27ac). However, apart from poised enhancers that are observed in early stages of development and putative silencers, the functional significance of cis-regulatory elements lacking H3K27ac is poorly understood. Here we show that macroH2A histone variants mark a subset of enhancers in normal and cancer cells, which we coined 'macro-Bound Enhancers', that modulate enhancer activity. We find macroH2A variants localized at enhancer elements that are devoid of H3K27ac in a cell type-specific manner, indicating a role for macroH2A at inactive enhancers to maintain cell identity. In following, reactivation of macro-bound enhancers is associated with oncogenic programs in breast cancer and their repressive role is correlated with the activity of macroH2A2 as a negative regulator of BRD4 chromatin occupancy. Finally, through single cell epigenomic profiling of normal mammary stem cells derived from mice, we show that macroH2A deficiency facilitates increased activity of transcription factors associated with stem cell activity.

In situ health monitoring of multiscale structures and its instantaneous verification using mechanoluminescence and dual machine learning.

Extensive changes in the legal, commercial and technical requirements in engineering fields have necessitated automated real-time structural health monitoring (SHM) and instantaneous verification. An integrated system with mechanoluminescence (ML) and dual artificial intelligence (AI) modules with subsidiary finite element method (FEM) simulation is designed for in situ SHM and instantaneous verification. The ML module detects the exact position of a crack tip and evaluates the significance of existing cracks with a plastic stress-intensity factor (PSIF; K P ). ML fields and their corresponding K p M L values are referenced and verified using the FEM simulation and bidirectional generative adversarial network (GAN). Well-trained forward and backward GANs create fake FEM and ML images that appear authentic to observers; a convolutional neural network is used to postulate precise PSIFs from fake images. Finally, the reliability of the proposed system to satisfy existing commercial requirements is validated in terms of tension, compact tension, AI, and instrumentation.

Glucocorticoids unmask silent non-coding genetic risk variants for common diseases.

Understanding the function of non-coding genomic sequence variants represents a challenge for biomedicine. Many diseases are products of gene-by-environment interactions with complex mechanisms. This study addresses these themes by mechanistic characterization of non-coding variants that influence gene expression only after drug or hormone exposure. Using glucocorticoid signaling as a model system, we integrated genomic, transcriptomic, and epigenomic approaches to unravel mechanisms by which variant function could be revealed by hormones or drugs. Specifically, we identified cis-regulatory elements and 3D interactions underlying ligand-dependent associations between variants and gene expression. One-quarter of the glucocorticoid-modulated variants that we identified had already been associated with clinical phenotypes. However, their affected genes were 'unmasked' only after glucocorticoid exposure and often with function relevant to the disease phenotypes. These diseases involved glucocorticoids as risk factors or therapeutic agents and included autoimmunity, metabolic and mood disorders, osteoporosis and cancer. For example, we identified a novel breast cancer risk gene, MAST4, with expression that was repressed by glucocorticoids in cells carrying the risk genotype, repression that correlated with MAST4 expression in breast cancer and treatment outcomes. These observations provide a mechanistic framework for understanding non-coding genetic variant-chemical environment interactions and their role in disease risk and drug response.

Vitamin C epigenetically controls osteogenesis and bone mineralization.

Vitamin C deficiency disrupts the integrity of connective tissues including bone. For decades this function has been primarily attributed to Vitamin C as a cofactor for collagen maturation. Here, we demonstrate that Vitamin C epigenetically orchestrates osteogenic differentiation and function by modulating chromatin accessibility and priming transcriptional activity. Vitamin C regulates histone demethylation (H3K9me3 and H3K27me3) and promotes TET-mediated 5hmC DNA hydroxymethylation at promoters, enhancers and super-enhancers near bone-specific genes. This epigenetic circuit licenses osteoblastogenesis by permitting the expression of all major pro-osteogenic genes. Osteogenic cell differentiation is strictly and continuously dependent on Vitamin C, whereas Vitamin C is dispensable for adipogenesis. Importantly, deletion of 5hmC-writers, Tet1 and Tet2, in Vitamin C-sufficient murine bone causes severe skeletal defects which mimic bone phenotypes of Vitamin C-insufficient Gulo knockout mice, a model of Vitamin C deficiency and scurvy. Thus, Vitamin C's epigenetic functions are central to osteoblastogenesis and bone formation and may be leveraged to prevent common bone-degenerating conditions.

Utricular dysfunction in patients with orthostatic hypotension.

PURPOSE: To delineate the association between otolithic dysfunction and orthostatic hypotension (OH). METHODS: We retrospectively reviewed the medical records of 382 patients who presented with orthostatic dizziness at a tertiary dizziness center between July 2017 and December 2021. Patients were included for analyses when they had completed ocular (oVEMP) and/or cervical vestibular-evoked myogenic potentials (cVEMP), and head-up tilt table test with a Finometer (n = 155). We compared the results between the patients with OH (n = 38) and those with NOI (normal head-up tilt table test despite orthostatic intolerance, n = 117). RESULTS: Thirty-eight patients with OH were further categorized as either classic (n = 30), delayed (n = 7), or initial (n = 1) types. Multivariable logistic regression showed that OH was associated with high baseline systolic BP (p = 0.046), presence of heart failure (p = 0.016), and unilateral oVEMP abnormalities (p = 0.016). n1 latency of oVEMP were negatively correlated with the maximal changes of systolic blood pressure (BP) in 15 s ([Formula: see text]SBP15s, p = 0.013), 3 min ([Formula: see text]SBP3min, p = 0.005) and 10 min ([Formula: see text]SBP10min, p = 0.002). In contrast, the n1-p1 amplitude was positively correlated with [Formula: see text]SBP15s (p = 0.029). Meanwhile, p13 latency of cVEMP was negatively correlated with [Formula: see text]SBP10min (p = 0.018). CONCLUSIONS: Our study provides evidence of utricular dysfunction related to OH.

Glucocorticoids mediate transcriptome-wide alternative polyadenylation: Potential mechanistic and clinical implications.

Alternative polyadenylation (APA) is a common genetic regulatory mechanism that generates distinct 3' ends for RNA transcripts. Changes in APA have been associated with multiple biological processes and disease phenotypes. However, the role of hormones and their drug analogs in APA remains largely unknown. In this study, we investigated transcriptome-wide the impact of glucocorticoids on APA in 30 human B-lymphoblastoid cell lines. We found that glucocorticoids could regulate APA for a subset of genes, possibly by changing the expression of 142 RNA-binding proteins, some with known APA-regulating properties. Interestingly, genes with glucocorticoid-mediated APA were enriched in viral translation-related pathways, while genes with glucocorticoid-mediated expression were enriched in interferon and interleukin pathways, suggesting that glucocorticoid-mediated APA might result in functional consequences distinct from gene expression. For example, glucocorticoids, a pharmacotherapy for severe COVID-19, were found to change the APA but not the expression of LY6E, an important antiviral inhibitor in coronavirus diseases. Glucocorticoid-mediated APA was also cell-type-specific, suggesting an action of glucocorticoids that may be unique to immune regulation. We also observed evidence for genotype-dependent glucocorticoid-mediated APA (referred to as pharmacogenomic-alterative polyadenylation quantitative trait loci), providing potential functional mechanisms for a series of common genetic variants that had previously been associated with immune disorders, but without a clear mechanism. In summary, this study reports a series of observations regarding the impact of glucocorticoids on APA, raising the possibility that this mechanism might have implications for both disease pathophysiology and drug therapy.