En masse organoid phenotyping informs metabolic-associated genetic susceptibility to NASH.

Genotype-phenotype associations for common diseases are often compounded by pleiotropy and metabolic state. Here, we devised a pooled human organoid-panel of steatohepatitis to investigate the impact of metabolic status on genotype-phenotype association. En masse population-based phenotypic analysis under insulin insensitive conditions predicted key non-alcoholic steatohepatitis (NASH)-genetic factors including the glucokinase regulatory protein (GCKR)-rs1260326:C>T. Analysis of NASH clinical cohorts revealed that GCKR-rs1260326-T allele elevates disease severity only under diabetic state but protects from fibrosis under non-diabetic states. Transcriptomic, metabolomic, and pharmacological analyses indicate significant mitochondrial dysfunction incurred by GCKR-rs1260326, which was not reversed with metformin. Uncoupling oxidative mechanisms mitigated mitochondrial dysfunction and permitted adaptation to increased fatty acid supply while protecting against oxidant stress, forming a basis for future therapeutic approaches for diabetic NASH. Thus, "in-a-dish" genotype-phenotype association strategies disentangle the opposing roles of metabolic-associated gene variant functions and offer a rich mechanistic, diagnostic, and therapeutic inference toolbox toward precision hepatology. VIDEO ABSTRACT.

Risk SNP-Mediated Promoter-Enhancer Switching Drives Prostate Cancer through lncRNA PCAT19.

The prostate cancer (PCa) risk-associated SNP rs11672691 is positively associated with aggressive disease at diagnosis. We showed that rs11672691 maps to the promoter of a short isoform of long noncoding RNA PCAT19 (PCAT19-short), which is in the third intron of the long isoform (PCAT19-long). The risk variant is associated with decreased and increased levels of PCAT19-short and PCAT19-long, respectively. Mechanistically, the risk SNP region is bifunctional with both promoter and enhancer activity. The risk variants of rs11672691 and its LD SNP rs887391 decrease binding of transcription factors NKX3.1 and YY1 to the promoter of PCAT19-short, resulting in weaker promoter but stronger enhancer activity that subsequently activates PCAT19-long. PCAT19-long interacts with HNRNPAB to activate a subset of cell-cycle genes associated with PCa progression, thereby promoting PCa tumor growth and metastasis. Taken together, these findings reveal a risk SNP-mediated promoter-enhancer switching mechanism underlying both initiation and progression of aggressive PCa.

Biology and Clinical Implications of the 19q13 Aggressive Prostate Cancer Susceptibility Locus.

Genome-wide association studies (GWAS) have identified rs11672691 at 19q13 associated with aggressive prostate cancer (PCa). Here, we independently confirmed the finding in a cohort of 2,738 PCa patients and discovered the biological mechanism underlying this association. We found an association of the aggressive PCa-associated allele G of rs11672691 with elevated transcript levels of two biologically plausible candidate genes, PCAT19 and CEACAM21, implicated in PCa cell growth and tumor progression. Mechanistically, rs11672691 resides in an enhancer element and alters the binding site of HOXA2, a novel oncogenic transcription factor with prognostic potential in PCa. Remarkably, CRISPR/Cas9-mediated single-nucleotide editing showed the direct effect of rs11672691 on PCAT19 and CEACAM21 expression and PCa cellular aggressive phenotype. Clinical data demonstrated synergistic effects of rs11672691 genotype and PCAT19/CEACAM21 gene expression on PCa prognosis. These results provide a plausible mechanism for rs11672691 associated with aggressive PCa and thus lay the ground work for translating this finding to the clinic.

Posttranscriptional regulation of FAN1 by miR-124-3p at rs3512 underlies onset-delaying genetic modification in Huntington's disease.

Many Mendelian disorders, such as Huntington's disease (HD) and spinocerebellar ataxias, arise from expansions of CAG trinucleotide repeats. Despite the clear genetic causes, additional genetic factors may influence the rate of those monogenic disorders. Notably, genome-wide association studies discovered somewhat expected modifiers, particularly mismatch repair genes involved in the CAG repeat instability, impacting age at onset of HD. Strikingly, FAN1, previously unrelated to repeat instability, produced the strongest HD modification signals. Diverse FAN1 haplotypes independently modify HD, with rare genetic variants diminishing DNA binding or nuclease activity of the FAN1 protein, hastening HD onset. However, the mechanism behind the frequent and the most significant onset-delaying FAN1 haplotype lacking missense variations has remained elusive. Here, we illustrated that a microRNA acting on 3'-UTR (untranslated region) SNP rs3512, rather than transcriptional regulation, is responsible for the significant FAN1 expression quantitative trait loci signal and allelic imbalance in FAN1 messenger ribonucleic acid (mRNA), accounting for the most significant and frequent onset-delaying modifier haplotype in HD. Specifically, miR-124-3p selectively targets the reference allele at rs3512, diminishing the stability of FAN1 mRNA harboring that allele and consequently reducing its levels. Subsequent validation analyses, including the use of antagomir and 3'-UTR reporter vectors with swapped alleles, confirmed the specificity of miR-124-3p at rs3512. Together, these findings indicate that the alternative allele at rs3512 renders the FAN1 mRNA less susceptible to miR-124-3p-mediated posttranscriptional regulation, resulting in increased FAN1 levels and a subsequent delay in HD onset by mitigating CAG repeat instability.

Extended haplotype with rs41524547-G defines the ancestral origin of SCA10.

Spinocerebellar ataxia type 10 (SCA10) is a rare autosomal dominant ataxia caused by a large expansion of the (ATTCT)n repeat in ATXN10. SCA10 was described in Native American and Asian individuals which prompted a search for an expanded haplotype to confirm a common ancestral origin for the expansion event. All patients with SCA10 expansions in our cohort share a single haplotype defined at the 5'-end by the minor allele of rs41524547, located ~35 kb upstream of the SCA10 expansion. Intriguingly, rs41524547 is located within the miRNA gene, MIR4762, within its DROSHA cleavage site and just outside the seed sequence for mir4792-5p. The world-wide frequency of rs41524547-G is less than 5% and found almost exclusively in the Americas and East Asia-a geographic distribution that mirrors reported SCA10 cases. We identified rs41524547-G(+) DNA from the 1000 Genomes/International Genome Sample Resource and our own general population samples and identified SCA10 repeat expansions in up to 25% of these samples. The reduced penetrance of these SCA10 expansions may be explained by a young (pre-onset) age at sample collection, a small repeat size, purity of repeat units, or the disruption of miR4762-5p function. We conclude that rs41524547-G is the most robust at-risk SNP allele for SCA10, is useful for screening of SCA10 expansions in population genetics studies and provides the most compelling evidence to date for a single, prehistoric origin of SCA10 expansions sometime prior to or during the migration of individuals across the Bering Land Bridge into the Americas.

High-throughput functional dissection of noncoding SNPs with biased allelic enhancer activity for insulin resistance-relevant phenotypes.

Most of the single-nucleotide polymorphisms (SNPs) associated with insulin resistance (IR)-relevant phenotypes by genome-wide association studies (GWASs) are located in noncoding regions, complicating their functional interpretation. Here, we utilized an adapted STARR-seq to evaluate the regulatory activities of 5,987 noncoding SNPs associated with IR-relevant phenotypes. We identified 876 SNPs with biased allelic enhancer activity effects (baaSNPs) across 133 loci in three IR-relevant cell lines (HepG2, preadipocyte, and A673), which showed pervasive cell specificity and significant enrichment for cell-specific open chromatin regions or enhancer-indicative markers (H3K4me1, H3K27ac). Further functional characterization suggested several transcription factors (TFs) with preferential allelic binding to baaSNPs. We also incorporated multi-omics data to prioritize 102 candidate regulatory target genes for baaSNPs and revealed prevalent long-range regulatory effects and cell-specific IR-relevant biological functional enrichment on them. Specifically, we experimentally verified the distal regulatory mechanism at IRS1 locus, in which rs952227-A reinforces IRS1 expression by long-range chromatin interaction and preferential binding to the transcription factor HOXC6 to augment the enhancer activity. Finally, based on our STARR-seq screening data, we predicted the enhancer activity of 227,343 noncoding SNPs associated with IR-relevant phenotypes (fasting insulin adjusted for BMI, HDL cholesterol, and triglycerides) from the largest available GWAS summary statistics. We further provided an open resource (http://www.bigc.online/fnSNP-IR) for better understanding genetic regulatory mechanisms of IR-relevant phenotypes.

Exon Junction Complex Shapes the Transcriptome by Repressing Recursive Splicing.

Recursive splicing (RS) starts by defining an "RS-exon," which is then spliced to the preceding exon, thus creating a recursive 5' splice site (RS-5ss). Previous studies focused on cryptic RS-exons, and now we find that the exon junction complex (EJC) represses RS of hundreds of annotated, mainly constitutive RS-exons. The core EJC factors, and the peripheral factors PNN and RNPS1, maintain RS-exon inclusion by repressing spliceosomal assembly on RS-5ss. The EJC also blocks 5ss located near exon-exon junctions, thus repressing inclusion of cryptic microexons. The prevalence of annotated RS-exons is high in deuterostomes, while the cryptic RS-exons are more prevalent in Drosophila, where EJC appears less capable of repressing RS. Notably, incomplete repression of RS also contributes to physiological alternative splicing of several human RS-exons. Finally, haploinsufficiency of the EJC factor Magoh in mice is associated with skipping of RS-exons in the brain, with relevance to the microcephaly phenotype and human diseases.

Altered Hierarchical Gradients of Intrinsic Neural Timescales in Mild Cognitive Impairment and Alzheimer's Disease.

Alzheimer's disease (AD) is a devastating neurodegenerative disease that affects millions of seniors in the United States. Resting-state functional magnetic resonance imaging (rs-fMRI) is widely used to study neurophysiology in AD and its prodromal condition, mild cognitive impairment (MCI). The intrinsic neural timescale (INT), which can be estimated through the magnitude of the autocorrelation of neural signals from rs-fMRI, is thought to quantify the duration that neural information is stored in a local circuit. Such heterogeneity of the timescales forms a basis of the brain functional hierarchy and captures an aspect of circuit dynamics relevant to excitation/inhibition balance, which is broadly relevant for cognitive functions. Given that, we applied rs-fMRI to test whether distinct changes of INT at different hierarchies are present in people with MCI, those progressing to AD (called Converter), and AD patients of both sexes. Linear mixed-effect model was implemented to detect altered hierarchical gradients across populations followed by pairwise comparisons to identify regional differences. High similarities between AD and Converter were observed. Specifically, the inferior temporal, caudate, and pallidum areas exhibit significant alterations in both AD and Converter. Distinct INT-related pathological changes in MCI and AD were found. For AD/Converter, neural information is stored for a longer time in lower hierarchical areas, while higher levels of hierarchy seem to be preferentially impaired in MCI leading to a less pronounced hierarchical gradient. These results inform that the INT holds great potential as an additional measure for AD prediction, even a stable biomarker for clinical diagnosis.

Estrogen receptor alpha and NFATc1 bind to a bone mineral density-associated SNP to repress WNT5B in osteoblasts.

Genetic factors and estrogen deficiency contribute to the development of osteoporosis. The single-nucleotide polymorphism (SNP) rs2887571 is predicted from genome-wide association studies (GWASs) to associate with osteoporosis but has had an unknown mechanism. Analysis of osteoblasts from 110 different individuals who underwent joint replacement revealed that the genotype of rs2887571 correlates with WNT5B expression. Analysis of our ChIP-sequencing data revealed that SNP rs2887571 overlaps with an estrogen receptor alpha (ERα) binding site. Here we show that 17ß-estradiol (E2) suppresses WNT5B expression and further demonstrate the mechanism of ERα binding at the enhancer containing rs2887571 to suppress WNT5B expression differentially in each genotype. ERα interacts with NFATc1, which is predicted to bind directly at rs2887571. CRISPR-Cas9 and ChIP-qPCR experiments confirm differential regulation of WNT5B between each allele. Homozygous GG has a higher binding affinity for ERα than homozygous AA and results in greater suppression of WNT5B expression. Functionally, WNT5B represses alkaline phosphatase expression and activity, decreasing osteoblast differentiation and mineralization. Furthermore, WNT5B increases interleukin-6 expression and suppresses E2-induced expression of alkaline phosphatase during osteoblast differentiation. We show that WNT5B suppresses the differentiation of osteoblasts via receptor tyrosine kinase-like orphan receptor 1/2 (ROR1/2), which activates DVL2/3/RAC1/CDC42/JNK/SIN3A signaling and inhibits ß-catenin activity. Together, our data provide mechanistic insight into how ERα and NFATc1 regulate the non-coding SNP rs2887571, as well as the function of WNT5B on osteoblasts, which could provide alternative therapeutic targets for osteoporosis.

SAFB associates with nascent RNAs and can promote gene expression in mouse embryonic stem cells.

Scaffold attachment factor B (SAFB) is a conserved RNA-binding protein that is essential for early mammalian development. However, the functions of SAFB in mouse embryonic stem cells (ESCs) have not been characterized. Using RNA immunoprecipitation followed by RNA-seq (RIP-seq), we examined the RNAs associated with SAFB in wild-type and SAFB/SAFB2 double-knockout ESCs. SAFB predominantly associated with introns of protein-coding genes through purine-rich motifs. The transcript most enriched in SAFB association was the lncRNA Malat1, which also contains a purine-rich region in its 5' end. Knockout of SAFB/SAFB2 led to differential expression of approximately 1000 genes associated with multiple biological processes, including apoptosis, cell division, and cell migration. Knockout of SAFB/SAFB2 also led to splicing changes in a set of genes that were largely distinct from those that exhibited changes in expression level. The spliced and nascent transcripts of many genes whose expression levels were positively regulated by SAFB also associated with high levels of SAFB, implying that SAFB binding promotes their expression. Reintroduction of SAFB into double-knockout cells restored gene expression toward wild-type levels, an effect again observable at the level of spliced and nascent transcripts. Proteomics analysis revealed a significant enrichment of nuclear speckle-associated and RS domain-containing proteins among SAFB interactors. Neither Xist nor Polycomb functions were dramatically altered in SAFB/2 knockout ESCs. Our findings suggest that among other potential functions in ESCs, SAFB promotes the expression of certain genes through its ability to bind nascent RNA.

Association of the C allele of rs479200 in the EGLN1 gene with COVID-19 severity in Indian population: a novel finding.

The present study investigated two single nucleotide polymorphisms (SNPs)-rs479200 and rs516651 in the host EGLN1/PHD2 gene for their association with COVID-19 severity. A retrospective cohort of 158 COVID-19 patients from the Indian population (March 2020 to June 2021) was enrolled. Notably, the frequency of C allele (0.664) was twofold higher than T allele (0.336) in severe COVID-19 patients. Here, we report a novel finding that the C allele of rs479200 in the EGLN1 gene imparts a high risk of severe COVID-19 (odds ratio-6.214 (1.84-20.99) p = 0.003; 9.421 (2.019-43.957) p = 0.004), in additive inheritance model (adjusted and unadjusted, respectively).

The GE296_RS03820 and GE296_RS03830 genes are involved in capsular polysaccharide biosynthesis in Riemerella anatipestifer.

Riemerella anatipestifer is a pathogenic bacterium that causes duck serositis and meningitis, leading to significant harm to the duck industry. To escape from the host immune system, the meningitis-causing bacteria must survive and multiply in the bloodstream, relying on specific virulence factors such as capsules. Therefore, it is essential to study the genes involved in capsule biosynthesis in R. anatipestifer. In this study, we successfully constructed gene deletion mutants Δ3820 and Δ3830, targeting the GE296_RS03820 and GE296_RS03830 genes, respectively, using the RA-LZ01 strain as the parental strain. The growth kinetics analysis revealed that these two genes contribute to bacterial growth. Transmission and scanning electron microscopy (TEM and SEM) and silver staining showed that Δ3820 and Δ3830 produced the altered capsules and compounds of capsular polysaccharides (CPSs). Serum resistance test showed the mutants also exhibited reduced C3b deposition and decreased resistance serum killing. In vivo, Δ3820 and Δ3830 exhibited markedly declining capacity to cross the blood-brain barrier, compared to RA-LZ01. These findings indicate that the GE296_RS03820 and GE296_RS03830 genes are involved in CPSs biosynthesis and play a key role in the pathogenicity of R. anatipestifer. Furthermore, Δ3820 and Δ3830 mutants presented a tendency toward higher survival rates from RA-LZ01 challenge in vivo. Additionally, sera from ducklings immunized with the mutants showed cross-immunoreactivity with different serotypes of R. anatipestifer, including 1, 2, 7 and 10. Western blot and SDS-PAGE assays revealed that the altered CPSs of Δ3820 and Δ3830 resulted in the exposure of some conserved proteins playing the key role in the cross-immunoreactivity. Our study clearly demonstrated that the GE296_RS03820 and GE296_RS03830 genes are involved in CPS biosynthesis in R. anatipestifer and the capsule is a target for attenuation in vaccine development.

Altered resting-state brain function in endurance athletes.

Previous research has confirmed significant differences in regional brain activity and functional connectivity between endurance athletes and non-athletes. However, no studies have investigated the differences in topological efficiency of the brain functional network between endurance athletes and non-athletes. Here, we compared differences in regional activities, functional connectivity, and topological properties to explore the functional basis associated with endurance training. The results showed significant correlations between Regional Homogeneity in the motor cortex, visual cortex, cerebellum, and the training intensity parameters. Alterations in functional connectivity among the motor cortex, visual cortex, cerebellum, and the inferior frontal gyrus and cingulate gyrus were significantly correlated with training intensity parameters. In addition, the graph theoretical analysis results revealed a significant reduction in global efficiency among athletes. This decline is mainly caused by decreased nodal efficiency and nodal local efficiency of the cerebellar regions. Notably, the sensorimotor regions, such as the precentral gyrus and supplementary motor areas, still exhibit increased nodal efficiency and nodal local efficiency. This study not only confirms the improvement of regional activity in brain regions related to endurance training, but also offers novel insights into the mechanisms through which endurance athletes undergo changes in the topological efficiency of the brain functional network.

Dynamic multilayer functional connectivity detects preclinical and clinical Alzheimer's disease.

Increasing evidence suggests that patients with Alzheimer's disease present alterations in functional connectivity but previous results have not always been consistent. One of the reasons that may account for this inconsistency is the lack of consideration of temporal dynamics. To address this limitation, here we studied the dynamic modular organization on resting-state functional magnetic resonance imaging across different stages of Alzheimer's disease using a novel multilayer brain network approach. Participants from preclinical and clinical Alzheimer's disease stages were included. Temporal multilayer networks were used to assess time-varying modular organization. Logistic regression models were employed for disease stage discrimination, and partial least squares analyses examined associations between dynamic measures with cognition and pathology. Temporal multilayer functional measures distinguished all groups, particularly preclinical stages, overcoming the discriminatory power of risk factors such as age, sex, and APOE ϵ4 carriership. Dynamic multilayer functional measures exhibited strong associations with cognition as well as amyloid and tau pathology. Dynamic multilayer functional connectivity shows promise as a functional imaging biomarker for both early- and late-stage Alzheimer's disease diagnosis.

Aberrant auditory metabolite levels and topological properties are associated with cognitive decline in presbycusis patients.

Presbycusis has been reported as related to cognitive decline, but its underlying neurophysiological mechanism is still unclear. This study aimed to investigate the relationship between metabolite levels, cognitive function, and node characteristics in presbycusis based on graph theory methods. Eighty-four elderly individuals with presbycusis and 63 age-matched normal hearing controls underwent magnetic resonance spectroscopy, functional magnetic resonance imaging scans, audiological assessment, and cognitive assessment. Compared with the normal hearing group, presbycusis patients exhibited reduced gamma-aminobutyric acid and glutamate levels in the auditory region, increased nodal characteristics in the temporal lobe and precuneus, as well as decreased nodal characteristics in the superior occipital gyrus and medial orbital. The right gamma-aminobutyric acid levels were negatively correlated with the degree centrality in the right precuneus and the executive function. Degree centrality in the right precuneus exhibited significant correlations with information processing speed and executive function, while degree centrality in the left medial orbital demonstrated a negative association with speech recognition ability. The degree centrality and node efficiency in the superior occipital gyrus exhibited a negative association with hearing loss and speech recognition ability, respectively. These observed changes indicate alterations in metabolite levels and reorganization patterns at the brain network level after auditory deprivation.

Glucose-oxygen coupling can serve as a biomarker for neuroinflammation-related genetic variants.

The single-nucleotide polymorphism rs3197999 in the macrophage-stimulating protein 1 gene is a missense variant. Studies have indicated that macrophage-stimulating protein 1 mediates neuronal loss and synaptic plasticity damage, and overexpression of the macrophage-stimulating protein 1 gene leads to the excessive activation of microglial cells, thereby resulting in an elevation of cerebral glucose metabolism. Traditional diagnostic models may be disrupted by neuroinflammation, making it difficult to predict the pathological status of patients solely based on single-modal images. We hypothesize that the macrophage-stimulating protein 1 rs3197999 single-nucleotide polymorphism may lead to imbalances in glucose and oxygen metabolism, thereby influencing cognitive resilience and the progression of Alzheimer's disease. In this study, we found that among 121 patients with mild cognitive impairment, carriers of the macrophage-stimulating protein 1 rs3197999 risk allele showed a significant reduction in the coupling of glucose and oxygen metabolism in the dorsolateral prefrontal cortex region. However, the rs3197999 variant did not induce significant differences in glucose metabolism and neuronal activity signals. Furthermore, the rs3197999 risk allele correlated with a higher rate of increase in clinical dementia score, mediated by the coupling of glucose and oxygen metabolism.

Resting-state neural correlates of individual differences in ignored experience and its deleterious effect.

Uncovering the neural mechanisms of ostracism experience (including its subclasses of excluded and ignored experiences) is important. However, the resting-state functional brain substrates responsible for individual differences in ostracism experience and its negative effects remain largely undefined. This study explored these issues in a sample of 198 Chinese college students by assessing the amplitude of low-frequency fluctuations and functional connectivity. The findings indicated a positive correlation between ignored experience and the amplitude of low-frequency fluctuations in the right superior frontal gyrus and the functional connectivity between the right superior frontal gyrus and left cerebellum posterior lobe. Additionally, a negative correlation was found between ignored experience and the functional connectivity between the right superior frontal gyrus and the bilateral insula as well as the bilateral inferior parietal lobule. Moreover, the mediation analysis demonstrated that the effects of the functional connectivities of right superior frontal gyrus-left cerebellum posterior lobe and right superior frontal gyrus-right inferior parietal lobule on revenge intention were mediated by ignored experience. Our study offers novel insights into the neural correlates of both individual variations in ignored experience and its typical deleterious effect. These results could deepen our understanding of individual differences in negative experiences and inspire the development of targeted interventions for social stress from the perspective of the brain.

Polymorphism of IL-12/IL-23 axis is associated with coronary heart disease.

IL12B encodes the shared p40 subunit (IL-12p40) of IL-12 and IL-23, which have diverse immune functions and are closely related to the occurrence and development of atherosclerosis (AS). However, the exact role of IL12B in coronary heart disease (CHD) was still unknown. A case-control association analysis was performed between five single nucleotide polymorphisms (SNPs) of IL12B (rs1003199, rs3212219, rs2569254, rs2853694 and rs3212227) and CHD in Chinese Han population (1824 patients with CHD vs. 2784 controls). Logistic regression analyses were used to study the relationships between SNPs and CHD, while multiple linear regression analyses were used to test the association between the SNP and the severity of CHD. In addition, the plasma IL12B concentration of CHD patients were detected by ELISA. We detected a significant association between one of the SNPs, rs2853694-G and CHD (padj = 2.075 × 10-5 , OR, 0.773 [95% CI, 0.686-0.870]). Stratified analysis showed that rs2853694 was associated with CHD in both male and female populations and was significantly associated with both early- and late-onset CHD. In addition, rs2853694 is also related to the different types of CHD including clinical-CHD and anatomical-CHD. Moreover, there are significant differences in serum IL12B concentrations between rs2853694-TT carriers and rs2853694-GT carriers in CHD patients (p = 0.010). A common variant of IL12B was found significantly associated with CHD and its subgroups. As a shared subunit of IL-12 and IL-23, IL-12p40 may play a key role in IL-12/IL-23 axis mediated AS, which is expected to be an effective therapeutic target for CHD.

ER stress increases expression of intracellular calcium channel RyR1 to modify Ca2+ homeostasis in pancreatic beta cells.

Pancreatic beta cells maintain glucose homeostasis by secreting pulses of insulin in response to a rise in plasma glucose. Pulsatile insulin secretion occurs as a result of glucose-induced oscillations in beta-cell cytosolic Ca2+. The endoplasmic reticulum (ER) helps regulate beta-cell cytosolic Ca2+, and ER stress can lead to ER Ca2+ reduction, beta-cell dysfunction, and an increased risk of type 2 diabetes. However, the mechanistic effects of ER stress on individual calcium channels are not well understood. To determine the effects of tunicamycin-induced ER stress on ER inositol 1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) and their involvement in subsequent Ca2+ dysregulation, we treated INS-1 832/13 cells and primary mouse islets with ER stress inducer tunicamycin (TM). We showed TM treatment increased RyR1 mRNA without affecting RyR2 mRNA and decreased both IP3R1 and IP3R3 mRNA. Furthermore, we found stress reduced ER Ca2+ levels, triggered oscillations in cytosolic Ca2+ under subthreshold glucose conditions, and increased apoptosis and that these changes were prevented by cotreatment with the RyR1 inhibitor dantrolene. In addition, we demonstrated silencing RyR1-suppressed TM-induced subthreshold cytosolic Ca2+ oscillations, but silencing RyR2 did not affect these oscillations. In contrast, inhibiting IP3Rs with xestospongin-C failed to suppress the TM-induced cytosolic Ca2+ oscillations and did not protect beta cells from TM-induced apoptosis although xestospongin-C inclusion did prevent ER Ca2+ reduction. Taken together, these results show changes in RyR1 play a critical role in ER stress-induced Ca2+ dysfunction and beta-cell apoptosis.

Precise detection of awareness in disorders of consciousness using deep learning framework.

Diagnosis of disorders of consciousness (DOC) remains a formidable challenge. Deep learning methods have been widely applied in general neurological and psychiatry disorders, while limited in DOC domain. Considering the successful use of resting-state functional MRI (rs-fMRI) for evaluating patients with DOC, this study seeks to explore the conjunction of deep learning techniques and rs-fMRI in precisely detecting awareness in DOC. We initiated our research with a benchmark dataset comprising 140 participants, including 76 unresponsive wakefulness syndrome (UWS), 25 minimally conscious state (MCS), and 39 Controls, from three independent sites. We developed a cascade 3D EfficientNet-B3-based deep learning framework tailored for discriminating MCS from UWS patients, referred to as "DeepDOC", and compared its performance against five state-of-the-art machine learning models. We also included an independent dataset consists of 11 DOC patients to test whether our model could identify patients with cognitive motor dissociation (CMD), in which DOC patients were behaviorally diagnosed unconscious but could be detected conscious by brain computer interface (BCI) method. Our results demonstrate that DeepDOC outperforms the five machine learning models, achieving an area under curve (AUC) value of 0.927 and accuracy of 0.861 for distinguishing MCS from UWS patients. More importantly, DeepDOC excels in CMD identification, achieving an AUC of 1 and accuracy of 0.909. Using gradient-weighted class activation mapping algorithm, we found that the posterior cortex, encompassing the visual cortex, posterior middle temporal gyrus, posterior cingulate cortex, precuneus, and cerebellum, as making a more substantial contribution to classification compared to other brain regions. This research offers a convenient and accurate method for detecting covert awareness in patients with MCS and CMD using rs-fMRI data.