Science Bulletin

Chromatin organization plays a central role in regulating genome accessibility and gene expression in eukaryotic cells. However, the inherent flexibility and structural heterogeneity of chromatin pose significant challenges for its structure determination. Here, we use a Nuc-back strategy with cryo-electron tomography (cryo-ET) and subtomogram averaging methods to visualize chromatin at the nucleosome level by averaging nucleosome at moderate-to-high resolution, classifying the fundamental unit of chromatin, i.e., nucleosome, into distinct classes, and linking different nucleosome structures to chromatin architecture. We reveal that nucleosome heterogeneity is a key factor in chromatin flexibility which disrupts interactions between nucleosomes. In addition, this strategy allows for the localization and visualization of chromatin regulators and their structure on chromatin. These results provide a foundation for future research in 3D genome and epigenetic process visualization. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/730377v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@a66d81org.highwire.dtl.DTLVardef@5f5049org.highwire.dtl.DTLVardef@18ff939org.highwire.dtl.DTLVardef@1333704_HPS_FORMAT_FIGEXP M_FIG C_FIG

Voltage-gated potassium channels (Kv) are a large family of potassium channels composed of 40 members across 12 subtypes. The KCNH genes encode 3 subfamilies of voltage-gated potassium channels: Kv10 (EAG, ether a go go), Kv11 (ERG, EAG-related gene), and Kv12 (ELK, EAG-like K). Kv channels play prominent roles in the neuronal and cardiovascular systems. Mutations in Kv channels have been linked to many human diseases, such as epilepsy, heart arrhythmias, and cancers. Significant progress has been made in understanding protein structures, physiological functions, and the pharmacological modifiers. However, the evolutionary history and gene expression of vertebrate KCNH genes during embryonic development remain largely unknown. We systematically identified and cloned 14 kcnh genes in zebrafish. Then, we examined vertebrate KCNH channel evolution by phylogenetic and syntenic analyses. Our data revealed that the three subtypes of the KCNH gene family have already evolved in invertebrates, long before the emergence of vertebrates. The number of vertebrate KCNH genes increased, most likely due to whole-genome duplications (WGDs). In addition, we examined zebrafish kcnh gene expression during early embryogenesis by in situ hybridization. Each subgroups genes showed similar but distinct gene expression domains with some exceptions. Most of them were expressed in neural tissues. Notably, kcnh6a showed robust expression in the developing heart, consistent with its conserved role in cardiac repolarization. Additionally, a few kcnh genes were transiently expressed in nonneural tissues, such as somites and the notochord, suggesting they may have a unique role in embryonic development. Our phylogenetic and developmental analyses of KCNH channels shed light on their evolutionary history and potential roles during embryogenesis, in line with their physiological functions and human channelopathies.

BackgroundMoyamoya disease (MMD) is characterized by progressive arterial stenosis and abnormal collateral formation, but the spatial organization of vessel-wall abnormalities remains incompletely understood. MethodsWe combined Xenium in situ spatial transcriptomics and multiplex immunofluorescence in superficial temporal artery samples from patients with MMD and controls, and performed gain- and loss-of-function experiments in human brain microvascular endothelial cells (HBMECs). Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), tube-formation, Transwell migration, and cell scratch assays were used to assess signaling and endothelial phenotypes. ResultsMMD vascular tissue showed intimal hyperplasia, altered spatial cellular architecture, and enrichment of extracellular matrix- and proteoglycan-related programs, with upregulation of sulfatase 1 (SULF1). In HBMECs, SULF1 knockdown reduced, whereas SULF1 overexpression enhanced, vascular endothelial growth factor A165 (VEGF-A165)-induced vascular endothelial growth factor receptor 2 (VEGFR2), extracellular signal-regulated kinase 1/2 (ERK1/2), and protein kinase B (AKT) phosphorylation, migration, tube formation, and angiogenesis- and adhesion-related gene expression. Heparinase III attenuated the signaling effects associated with SULF1 overexpression. ConclusionThese findings suggest that SULF1-associated extracellular matrix alterations may contribute to local vessel-wall remodeling and enhanced endothelial responsiveness in MMD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/721514v1_ufig1.gif" ALT="Figure 1"> View larger version (81K): org.highwire.dtl.DTLVardef@143db87org.highwire.dtl.DTLVardef@1a9d9org.highwire.dtl.DTLVardef@1362215org.highwire.dtl.DTLVardef@f7c5a3_HPS_FORMAT_FIGEXP M_FIG C_FIG

Recombinant adeno-associated virus (rAAV) vectors are pivotal for gene therapy; however, the encapsidation of residual DNA, particularly plasmid backbone sequences, pose significant safety risks. Recent studies have identified the p5 promoter, which contains a Rep-binding element and a terminal resolution site (TRS), as a cryptic origin of replication that facilitates packaging of upstream sequences. In this study, we investigated the effect of p5 TRS modifications on impurity DNA levels in a single-plasmid All-in-One (AiO) AAV production system. Wild-type p5 (p5wt) promoted significant packaging of upstream plasmid backbone DNA, especially when the backbone was positioned between p5wt and the inverted terminal repeat. Introducing mutations or deletions in the p5 TRS significantly reduced encapsidation of plasmid-derived sequences, including kanamycin resistance genes, and improved the ratio of full to partial particles, as seen with the p5{Delta}loop variant. Furthermore, the p5{Delta}loop-AiO system showed higher rAAV yields than both conventional triple-transfection methods and previously reported p5-spacer variants. Thus, our findings suggest a robust vector design strategy for minimizing DNA impurities, thereby enhancing the safety and efficacy of AAV-based gene therapy.

Adeno-associated virus (AAV) vectors are widely used in gene therapy, whereas low manufacturing efficiency and a large proportion of empty capsids are major obstacles. This study focused on the Yin Yang 1 (YY1) binding motif (YY1-motif) and investigated the effect of its presence or insertion at upstream of the Replicase (Rep)/Capsid Cap) gene on AAV vector production. We found that the YY1-motif incidentally presented in a Rep/Cap plasmid was associated with high vector production. We then designed several modified Rep/Cap (RC2) constructs. The YY1-motif insertion at the upstream of Rep/Cap gene increased vector yield in a repeat-number-dependent manner, and similar effects were not observed with other promoters insertion. Furthermore, the insertion of the YY1-motif reduced the amount of Cap protein per the same amount of full particle in supernatants on multiple serotypes, indicating the improvement in the empty/full capsid ratio. The YY1-motif insertion did not affect the AAV vector infectivity. These results denote that the YY1-motif has a universal regulatory function that optimizes the Rep/Cap expression balance, and simultaneously improves the production efficiency and full particle formation of AAV vectors. This finding could contribute to the development of highly efficient and high-quality AAV manufacturing processes.

Background: RSV is a major cause of severe lung infections in young children, with over 95% of deaths occurring in poorer countries. Bangladesh has high rates of RSV illness in children but lacks genetic data from after the COVID-19 pandemic. New vaccines and antibody treatments are now available, making local genetic information essential. Objectives: We sequenced complete RSV genomes from Bangladeshi patients to study virus types, genetic changes, and protein mutations, and shared our data openly. Methods: From August 2024 to December 2025, we took 59 RSV-positive samples with high virus levels from hospital patients and sequenced their full genomes using Oxford Nanopore technology. Results: Among 11,874 patients, 1,390 (11.7%) had RSV, mostly RSV-A (94.6%). We obtained 49 good-quality full genomes from the 59 samples (83% success): 43 RSV-A (ON1 type, five sub-lineages) and 6 RSV-B (BA9 type). We found S276N in 35% of RSV-A and S389P in all RSV-B, but neither stops current antibody treatments. All RSV-A viruses gained a new sugar attachment site on their F protein, and most RSV-B viruses gained one too. We uploaded all 49 genomes to GISAID for public use. Conclusion: This work shows we can do full RSV genome sequencing in Bangladesh. The viruses here still match the targets of new vaccines and antibodies, which is reassuring. Our findings provide a foundation for planning RSV prevention in Bangladesh and South Asia.

Although the "RNA world" hypothesis suggests that RNA played a crucial role in the origin of life [7], the functional framework of RNA in prebiotic protein synthesis and the mechanisms of genetic code formation during the prebiotic period remain poorly understood. Here, using the prebiotic "primordial soup" as a model, we reconstructed the detailed steps that would yield a protein with a stable ordered amino-acid sequence in the "primordial soup" at the prebiotic period. In the "primordial soup", a large number of medium- to large-sized biomolecule-like substances--such as RNA-like and protein-like molecules of various sizes and shapes, as well as related polymers like amino-acid-RNA-like etc.--did generate and accumulate. Moreover, protein-like and RNA-like molecules formed even more intricate complexes. These complexes bound free mRNA-like molecules through complementary base pairing. Subsequently, with an extremely low probability, two adjacent amino-acid-RNA-like molecules became bound to this free mRNA-like molecule, and their amino acids underwent a condensation reaction by the complexes, producing peptides and eventually proteins or polypeptides. This free mRNA-like molecule exhibits a certain flexible structure, whereas the super-large complexes formed by protein-like and RNA-like molecules (which possess certain activities) and the amino-acid-RNA molecules exhibit relatively rigid structures. Long-term evolution and mutual selection led to the emergence of proteins with stable amino acid sequences and moderate catalytic activity. In this way, the nucleotide information embedded in such mRNA-like molecules indirectly express through protein synthesis--a process we term the "A Co-Adaptation Flexible-Rigid Docking Model", where flexible mRNA-like molecules dock onto rigid complexes to enable ordered peptide formation. Finally, we show how trinucleotide codons emerge naturally from the flexible-rigid docking constraints.

Gene therapy has been the subject of extensive research following the advent of gene-editing technologies. Genetic disorders with difficult-to-target tissues, such as cystic fibrosis (CF), still face many challenges in developing efficacious gene therapy. The potential universal approach of gene replacement involves inserting a functional CFTR gene after generating DNA double strand breaks using gene editors such as CRISPR/Cas9. However, this strategy has not achieved clinical significance, as CRISPR/Cas9-mediated integration of CFTR is limited primarily by the infrequent activity of the homology-directed repair (HDR) pathway. To circumvent this limitation and improve CFTR transgene integration and expression, we explored a method of adding a second integration site, which we termed the dual-locus-targeting method. Using a helper-dependent adenoviral vector (HDAd)-delivered CRISPR/Cas9 system in porcine epithelial cells, we found that sequential delivery of two vectors, one targeting the CFTR locus and the other the genomic safe harbour site GGTA1, enhanced the integration efficiency of lacZ and CFTR donor genes to 16.5% and 3.4%, respectively. These results demonstrated a potential strategy to improve the efficacy of CFTR replacement for the development of a universal and permanent gene therapy treatment for CF lung disease. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/731381v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1774590org.highwire.dtl.DTLVardef@1782915org.highwire.dtl.DTLVardef@1d13b12org.highwire.dtl.DTLVardef@17d3f93_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundMetazoan adenosine-to-inosine (A-to-I) mRNA editing temporospatially diversifies the neuronal transcriptome and proteome. The limited read length from next-generation sequencing (NGS) constrains the quantification of the potentially differential editing levels across different splicing isoforms, restricting our understanding of the extent to which RNA editing contributes to molecular diversity and its interplay with splicing. MethodsWe employed reverse transcription nested PCR (RT-nPCR) and developed a novel interfering-Primer PCR (iPrimer PCR) technique to distinguish different transcripts of any gene. We selected multiple essential genes exhibiting RNA editing in coding sequences (CDSs) or untranslated regions (UTRs) for isoform-specific amplification and Sanger sequencing. ResultsNine different Adar isoforms together with pre-mRNA had distinct editing levels at the S>G auto-recoding site, which was predicted to have isoform-specific effects on catalytic activities. Although pre-mRNA editing might exert isoform-dependent promotion/suppression of splicing, closely located editing sites, such as those in neuronal genes qvr and stj, still exhibited high correlation in editing levels due to co-editing. iPrimer strategy further discovered differential recoding levels between the long/short 3UTR isoforms of gene jef. ConclusionsWe provide the first comprehensive solution for isoform-specific PCR amplification of any gene, enabling quantification of RNA editing level of different isoforms. Our results offer insights into how RNA editing interplays with splicing, and highlight its complicated role in expanding molecular diversity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/725286v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1ebc82org.highwire.dtl.DTLVardef@1ea365dorg.highwire.dtl.DTLVardef@1971aceorg.highwire.dtl.DTLVardef@160d053_HPS_FORMAT_FIGEXP M_FIG C_FIG We developed isoform-specific PCR followed by Sanger sequencing, and achieved the quantification of differential RNA editing levels in different transcripts of a gene.

Hypertension is commonly defined as a binary condition despite substantial heterogeneity in diagnosis, treatment, and blood pressure (BP) control. We propose a three-axis state model integrating diagnosis status, treatment intensity, and BP control to better characterize hypertension phenotypes. The framework generates 27 possible states that can be condensed into seven clinically meaningful groups. We applied the model to 5,129,584 Japanese adults using the National Database of Health Insurance Claims and Specific Health Checkups. Hierarchical cluster analysis, sensitivity analysis excluding patients with cardiovascular diseases other than hypertension, and validation against antihypertensive medication use were performed. Overall, 64% of participants were classified as normotensive, whereas 36% belonged to hypertension-related groups, including 11% with unrecognized hypertension and 7% with diagnosed but untreated hypertension. Agreement with data-driven hierarchical cluster analysis was substantial (weighted {kappa}=0.87). The group distribution remained largely unchanged in the sensitivity analysis, supporting the robustness of the proposed classification. Hypertension diagnosis also showed high validity, with a sensitivity of 96.5%, specificity of 91.8%, and substantial agreement with antihypertensive medication use ({kappa}=0.78). This three-axis framework provides a robust and clinically interpretable approach for characterizing hypertension phenotypes, enabling systematic identification of care gaps and supporting research, clinical decision-making, and population health management.

The human cerebral cortex folds into a stereotyped shape during gestation. Different principles govern the large and small scales of the final brain geometry. Here, I show that the fetal cerebrum can be described as a band limited spherical harmonic Fourier object which entire gyrification process collapses to a single one-dimensional curve, in which the maximum harmonic degree acts as a developmental coordinate. The closed form descriptor predicts gestational age with mean absolute error 0.13 and 0.38 weeks across fetal brain atlases, exceeding the published learning-based state of the art by a factor of three to seven. The same descriptor, applied to single subjects in the FeTA pathological dataset, can classify the per subject distance from the normative trajectory and discriminate pathological from neurotypical fetuses. The result is a single closed form, zero-training-cost descriptor that simultaneously dates the fetal brain and detects atypical development.

CRISPR-Cas9 technology is a powerful tool extensively used for genome editing in mouse and many other species. Streptococcus pyogenes Cas9 efficiently cuts both alleles in mouse zygotes leaving many edited embryos without a functional protein that might be needed to sustain development, to survive postnatally or to reproduce, thus complicating its overwhelmingly advantageous use in making gene modifications. About 25% of mouse genes are essential for embryonic development and another 7% are necessary for fertility, thus for these genes it is desirable to maintain a functional allele to establish viable lines from CRISPR-Cas9 edited mouse embryos. However, exclusive monoallelic editing is challenging to achieve with current CRISPR methods. Controlling the activity of Cas9 in genome editing is an ongoing research field focused on developing new methods to curtail its damage caused by excess of on-target and off-target editing. In this study we describe a novel and a simple method, we termed das-CRISPR, for dCas allele sequestration in combination with CRISPR system, that allows monoallelic editing of targeted allele in mouse and in cultured cell lines. This method incorporates the use of a nuclease deficient deadCas9 (dCas9) present at higher levels than an active Cas9, both complexed with the same single guide RNA (sgRNA) sequence. We showed the delivery of the two proteins as ribonucleoprotein complexes (RNP) into mouse zygotes leads to the generation of viable and fertile mice carrying lethal mutations in an essential gene. We found that greater amounts of dCas9 RNPs bind and protect a target site while the lower amount of functional Cas9 RNPs accessed the unoccupied target site resulting in higher frequency of monoallelic gene editing, compared to using just Cas9 alone. We also showed this method can mitigate and control the activity of Cas9 in mouse NIH3T3 cells in culture to achieve monoallelic editing. This method is a versatile approach to controlling excessive Cas9 activity on-target and off-target both in vitro and in vivo.

While adeno-associated virus (AAV) vectors have shown therapeutic benefit in clinical applications, noted challenges include low transduction efficiencies, poor cellular targeting, and vector related adverse events. Recently, it was demonstrated that a rationally designed synthetic inverted terminal repeat (SynITR) altered the AAV vector-induced DNA damage response and abrogated apoptosis in human embryonic stem cells. To explore the utility of AAV-SynITR for diverse gene therapy applications, vector production, transduction, and the cellular response were evaluated in various contexts. Regarding production, SynITR preparations exhibited comparable titers to wtITR in a serotype/transgene-independent manner. Despite slightly decreased transduction efficiency in various cell lines, intravenous administration of AAV8 vectors showed SynITR enhanced transduction in a tissue-specific manner in liver (>7-fold) and kidney and pancreas (>2-fold) at equivalent vector copy numbers; however, no differences were observed in muscle/heart/spleen tissues. Interestingly, persistent {gamma}H2AX, a marker of aging/chronic inflammation, was abundant in the liver and spleen following wtITR (but not SynITR) transduction. In human corneas, SynITR enhanced transduction up to 16-fold over wtITRs. These data demonstrate that SynITRs elicit tissue-specific transduction enhancement and alter the cellular stress response. Importantly, the SynITRs offer an alternative context to elucidate wtITR biology for targeted, enhanced, and potentially safer human gene therapy.

Heterogeneous nuclear ribonucleoprotein U (HNRNPU) deficiency is a rare genetic cause of neurodevelopmental disorders (NDDs) lacking targeted therapies. Here, we developed a transcriptomic-guided compound prioritization pipeline using Connectivity Map (CMap) analysis on multi-model transcriptomic signatures from HNRNPU-deficient human cells and mouse models. Ten compounds were selected through manual curation and functionally screened in patient-derived HNRNPU-deficient neuroepithelial stem (NES) cells with earlier observed cellular phenotypes. Two of the compounds, AS601245 and Lenalidomide, significantly reduced the elevated neural progenitor population during differentiation, and their combination further decreased primary cilia incidence, indicating partial rescue of the patient-specific cellular phenotypes. To understand the mechanisms underlying the partial rescue, we employed proteome integral solubility alteration (PISA) and expression proteomics. PISA assay identified TMEM150C and GSK3A as proximal targets of combined treatment. Additionally, we observed reversal of multiple biological pathways including downregulation of Wnt signalling and upregulation of mitochondrial pathways and transmembrane proteins. Altogether, we established a computational-experimental pipeline for transcriptomic-guided drug repurposing for a monogenic NDD, and demonstrated that the network-level modulation partially rescues the delayed neural differentiation in HNRNPU-deficient neural cells.

MicroRNA (miRNAs) are small non-coding RNAs that regulate gene expression by binding to the target messenger RNA (mRNA), whose versatility has inspired RNA-interference (RNAi)-based drug designs. However, off-target effects lead to unintended gene silencing and toxicity. Existing methods suffer from experimental data scarcity and fail to effectively integrate target specificity into designing de novo small interference RNAs (siRNA). To overcome the above challenges, we present SO_SCPLOWPECIC_SCPLOWMO_SCPLOWIC_SCPLOWR, a specificity-guided generative framework that synthesizes target-conditioned miRNAs. By training on a large experimental data containing 2.2M miRNA-mRNA pairs, SO_SCPLOWPECIC_SCPLOWMO_SCPLOWIC_SCPLOWR minimizes off-target effects with enhanced on-target potency. As a result, SO_SCPLOWPECIC_SCPLOWMO_SCPLOWIC_SCPLOWR-generated miRNAs bind more strongly to the target mRNAs than the observed miRNAs and much less so to off-target mRNAs. We tested SO_SCPLOWPECIC_SCPLOWMO_SCPLOWIC_SCPLOWR on mRNA targets for liver disease, for which 6 FDA-approved siRNA-based drugs were available. SO_SCPLOWPECIC_SCPLOWMO_SCPLOWIC_SCPLOWR recovers binding regions that correspond to FDA-approved siRNA drugs across 3 targets, and demonstrates greater structural specificity for on-target mRNAs than for off-target mRNAs. Together, SO_SCPLOWPECIC_SCPLOWMO_SCPLOWIC_SCPLOWR offers an AI solution to synthesize miRNA-inspired and target-specific siRNA sequences towards RNAi-based drug design.

Temporal lobe epilepsy (TLE) is a complex neurological disorder characterized by spontaneous recurrent seizures and its underlying mechanism remains elusive. This study aimed to investigate the role of cystine-knot AMPAR modulating protein 44 (CKAMP44) in the pathological process of TLE and its potential as a therapeutic target using kainic acid (KA)-induced epilepsy mouse model of TLE. Our results showed that CKAMP44 protein and mRNA expression was significantly increased and primarily localized to neurons during the chronic phase of TLE. Nkx2-1 regulated the transcription of CKAMP44 in the hippocampus brain tissues of KA-induced TLE mice. Inhibition of CKAMP44 suppressed seizure susceptibility and severity in the KA-induced epilepsy mice via behavioral and local field potential monitoring. Furthermore, inhibition of CKAMP44 decreased frequency and amplitudes of spontaneous excitatory postsynaptic currents indicating that the excitatory synaptic transmission was reduced in an in vitro epilepsy model. Mechanistically, inhibition of CKAMP44 specifically upregulated the membrane surface expression of GluA1 and the phosphorylation level of GluA1-ser831 by downregulating protein phosphatase 3 regulatory subunit B(PPP3r2) expression. Overexpression of PPP3r2 downregulated the phosphorylation level and surface expression of GluA1, which ultimately exacerbated the seizure activity suppressed by CKAMP44 knockdown. Collectively, our results indicate that CKAMP44 may be a potential therapeutic target for the treatment of TLE.

Tomato yellow leaf curl virus (TYLCV) is a devastating viral pathogen threatening agricultural crops globally. In this study, we identified a novel TYLCV isolate (TYLCV-YN6244), which caused viral epidemic in resistant tomato cultivars at Yuanmo county, Yunnan Province of China. We determined the complete genome of TYLCV-YN6244 and found it encoded six viral proteins characteristic of Geminivirus. We identified its V2 protein as a potent viral suppressor of RNA silencing (VSR), and generated infectious clone of wildtype TYLCV-YN6244, or V2-defective TYLCV-YN6244 (TYLCV-YN6244-{Delta}V2) in which V2 was deleted. Both of infectious clones were capable of systemically infecting tobacco and tomato. However, TYLCV-YN6244 but not TYLCV-YN6244-{Delta}V2 could cause disease symptoms in wildtype tobacco or tomato plants, and viral accumulation was drastically reduced in plants infected with TYLCV-YN6244-{Delta}V2 compared to TYLCV-YN6244 while the efficiency of virus-derived small interfering RNAs (vsiRNAs) biogenesis was conversely increased in plants infected with TYLCV-YN6244-{Delta}V2. Surprisingly, small RNA profiling indicated that 21nt and 22nt rather than 24nt vsiRNAs were predominantly produced in tomato plants infected with either TYLCV-YN6244 or TYLCV-YN6244-{Delta}V2. Furthermore, transcriptome analyses revealed that TYLCV-YN6244 or TYLCV-YN6244-{Delta}V2 infection differentially modulated metabolism and defense-related pathways in tomato, probably underlying distinct viral pathogenicity and disease symptoms induced in plants. Overall, our research not only identified a novel pathogenic TYLCV isolate but also characterized molecular biology and host response in tomato with infectious clones firstly developed, with implications in untangling virus-host interaction for developing novel resistance in crop tomato.

Objectives; The role of nuclear receptor subfamily 4, group A, member 3 (NR4A3) in hepatic steatosis, inflammation, and insulin resistance (IR) within the context of metabolic dysfunction-associated steatotic liver disease (MASLD) remains largely underexplored. Consequently, this study aimed to examine NR4A3's impact on MASLD and the potential underlying mechanisms. Methods; We aimed to elucidate the functional role of NR4A3 in MASLD through its knockdown in cell culture and animal models. To establish the cell culture model of MASLD, LO2 cells were treated with free fatty acids (FFAs), while male C57BL/6 mice were fed a high-fat diet (HFD) to create the animal model. NR4A3 knockdown was achieved using specific short hairpin RNA (NR4A3-shRNA) in the mice model and three small interfering RNAs (NR4A3-siRNAs) in the cell culture model. The lipids content, fatty acid synthesis, inflammatory factors, and IR were then assessed with and without NR4A3 knockdown. Furthermore, the underlying mechanism through which NR4A3 exerts its influence was explored by analyzing the interaction between NR4A3 and activating transcription factor 3 (ATF3). Results: In the cell culture experiments, the knockdown of NR4A3 significantly decreased the lipids content, fatty acid synthesis, and inflammatory factors in the LO2 cells treated with FFAs in the NR4A3-shRNA group compared with those in the NC-shRNA control group. In the animal model experiments, NR4A3 knockdown in the HFD male C57BL/6 mice significantly ameliorated HFD-induced hepatic steatosis, inflammation, and IR. Mechanistically, the knockdown of NR4A3 downregulated the expression and transcriptional activity of ATF3, resulting in an impaired ATF3 function. ATF3 overexpression significantly reversed lipid accumulation decline and reduced inflammation after NR4A3 knockdown. Conclusion: The downregulation of NR4A3 alleviates MASLD by modulating ATF3, suggesting this may be a promising therapeutic target.

BackgroundRecent studies have established an association between traumatic brain injury (TBI) and cardiometabolic diseases (CMDs). However, the influence of TBI on the sequential progression from a healthy state to CMD, subsequent to cardiometabolic multimorbidity (CMM), and ultimately to mortality remains unclear. MethodsA total of 366,616 participants free of CMD at baseline were derived from the UK Biobank (UKB). CMM was defined as the co-occurrence of [≥]2 CMD, including diabetes mellitus (DM), ischemic heart disease (IHD), and stroke. Cox proportional hazards models and multi-state models were utilized to evaluate the association of TBI with disease transitions from a healthy state to CMM and subsequent mortality. ResultsDuring a median follow-up of 16.91 years, 54,224 participants developed at least one CMD, among whom 7,562 progressed to CMM. Furthermore, 32,785 cases of mortality were documented. In multi-state models, the hazard ratios (HRs) with corresponding 95% confidence intervals (CIs) for transitions from a healthy state to IHD, DM, stroke, and mortality were 1.91(95% CI: 1.77-2.05), 1.89 (95% CI: 1.71-2.09), and 4.73 (95% CI: 4.39-5.09), respectively. For sequential transitions from IHD, DM, and stroke to CMM, the HRs (95% CIs) were 2.67 (95% CI: 2.34-3.04), 3.29 (95% CI: 2.78-3.89), and 1.41 (95% CI: 1.15-1.72), respectively. Additionally, in Cox proportional hazards models, the HRs (95% CIs) for incident CMM and mortality among individuals with TBI were 3.98 (95% CI: 3.63-4.36) and 2.57 (95% CI: 2.44-2.71), respectively. ConclusionThis study found that TBI was associated with increased risk of progression from a healthy state to CMD, and subsequently to CMM and mortality, highlighting the importance of comprehensive management of TBI in cardiometabolic health. What is Known; What the Study AddsO_ST_ABSWhat is KnownC_ST_ABSTraumatic brain injury (TBI) is associated with an elevated risk of developing multiple cardiometabolic diseases (CMDs). What the Study AddsThis study performed a systematic analysis of the relationships between TBI and multiple CMDs, providing valuable clinical references for the prevention and management of the onset and progression of cardiometabolic diseases and cardiometabolic multimorbidity (CMM) among patients with TBI.

BackgroundThe optic nerve serves as a vital conduit for visual signaling, and its degeneration in optic neuropathy results in irreversible vision loss. It is also a widely used model for studying central nervous system (CNS) injury and repair. Although adeno-associated virus (AAV) and lentivirus are extensively applied in CNS research, their transduction efficiency and cell-type specificity within the optic nerve remain poorly characterized. This study aimed to identify the most effective viral vector, serotype, and promoter for direct gene delivery to the adult rat optic nerve. MethodsSprague-Dawley rats (7-10 weeks) received intra-optic nerve injections of lentiviral or AAV vectors encoding GFP under different promoters (CAG, CMV, or GFAP). Two to three weeks post-injection, optic nerves were collected for immunohistochemistry with markers of oligodendrocytes (Olig2), astrocytes (GFAP, Sox9), and microglia (IBA1). Transduction efficiency and cell-type specificity were assessed using confocal microscopy. ResultsAAV2, AAV5, and lentivirus showed minimal transduction, with only sparse GFP-positive cells observed near injection sites. In contrast, AAV-PHP.eB carrying the CAG promoter yielded robust and widespread GFP expression near the injection site. Quantitative analysis revealed that approximately 90% of transduced cells were Olig2-positive oligodendrocytes, indicating strong tropism for this glial population. ConclusionAAV-PHP.eB driven by the CAG promoter enables efficient gene delivery to the optic nerve, with a predominant tropism for oligodendrocytes. This targeted intra-optic nerve injection approach offers a reliable platform for manipulating oligodendrocytes and investigating mechanisms of CNS development, injury, and repair relevant to both optic neuropathies and other CNS diseases.