Insights into phage-bacteria interaction in cold seep Gigantidas platifrons through metagenomics and transcriptome analyses.

Viruses are crucial for regulating deep-sea microbial communities and biogeochemical cycles. However, their roles are still less characterized in deep-sea holobionts. Bathymodioline mussels are endemic species inhabiting cold seeps and harboring endosymbionts in gill epithelial cells for nutrition. This study unveiled a diverse array of viruses in the gill tissues of Gigantidas platifrons mussels and analyzed the viral metagenome and transcriptome from the gill tissues of Gigantidas platifrons mussels collected from a cold seep in the South Sea. The mussel gills contained various viruses including Baculoviridae, Rountreeviridae, Myoviridae and Siphovirdae, but the active viromes were Myoviridae, Siphoviridae, and Podoviridae belonging to the order Caudovirales. The overall viral community structure showed significant variation among environments with different methane concentrations. Transcriptome analysis indicated high expression of viral structural genes, integrase, and restriction endonuclease genes in a high methane concentration environment, suggesting frequent virus infection and replication. Furthermore, two viruses (GP-phage-contig14 and GP-phage-contig72) interacted with Gigantidas platifrons methanotrophic gill symbionts (bathymodiolin mussels host intracellular methanotrophic Gammaproteobacteria in their gills), showing high expression levels, and have huge different expression in different methane concentrations. Additionally, single-stranded DNA viruses may play a potential auxiliary role in the virus-host interaction using indirect bioinformatics methods. Moreover, the Cro and DNA methylase genes had phylogenetic similarity between the virus and Gigantidas platifrons methanotrophic gill symbionts. This study also explored a variety of viruses in the gill tissues of Gigantidas platifrons and revealed that bacteria interacted with the viruses during the symbiosis with Gigantidas platifrons. This study provides fundamental insights into the interplay of microorganisms within Gigantidas platifrons mussels in deep sea.

Bayesian network analysis of factors influencing type 2 diabetes, coronary heart disease, and their comorbidities.

OBJECTIVE: Bayesian network (BN) models were developed to explore the specific relationships between influencing factors and type 2 diabetes mellitus (T2DM), coronary heart disease (CAD), and their comorbidities. The aim was to predict disease occurrence and diagnose etiology using these models, thereby informing the development of effective prevention and control strategies for T2DM, CAD, and their comorbidities. METHOD: Employing a case-control design, the study compared individuals with T2DM, CAD, and their comorbidities (case group) with healthy counterparts (control group). Univariate and multivariate Logistic regression analyses were conducted to identify disease-influencing factors. The BN structure was learned using the Tabu search algorithm, with parameter estimation achieved through maximum likelihood estimation. The predictive performance of the BN model was assessed using the confusion matrix, and Netica software was utilized for visual prediction and diagnosis. RESULT: The study involved 3,824 participants, including 1,175 controls, 1,163 T2DM cases, 982 CAD cases, and 504 comorbidity cases. The BN model unveiled factors directly and indirectly impacting T2DM, such as age, region, education level, and family history (FH). Variables like exercise, LDL-C, TC, fruit, and sweet food intake exhibited direct effects, while smoking, alcohol consumption, occupation, heart rate, HDL-C, meat, and staple food intake had indirect effects. Similarly, for CAD, factors with direct and indirect effects included age, smoking, SBP, exercise, meat, and fruit intake, while sleeping time and heart rate showed direct effects. Regarding T2DM and CAD comorbidities, age, FBG, SBP, fruit, and sweet intake demonstrated both direct and indirect effects, whereas exercise and HDL-C exhibited direct effects, and region, education level, DBP, and TC showed indirect effects. CONCLUSION: The BN model constructed using the Tabu search algorithm showcased robust predictive performance, reliability, and applicability in forecasting disease probabilities for T2DM, CAD, and their comorbidities. These findings offer valuable insights for enhancing prevention and control strategies and exploring the application of BN in predicting and diagnosing chronic diseases.

LENAS: Learning-Based Neural Architecture Search and Ensemble for 3-D Radiotherapy Dose Prediction.

Radiation therapy treatment planning requires balancing the delivery of the target dose while sparing normal tissues, making it a complex process. To streamline the planning process and enhance its quality, there is a growing demand for knowledge-based planning (KBP). Ensemble learning has shown impressive power in various deep learning tasks, and it has great potential to improve the performance of KBP. However, the effectiveness of ensemble learning heavily depends on the diversity and individual accuracy of the base learners. Moreover, the complexity of model ensembles is a major concern, as it requires maintaining multiple models during inference, leading to increased computational cost and storage overhead. In this study, we propose a novel learning-based ensemble approach named LENAS, which integrates neural architecture search with knowledge distillation for 3-D radiotherapy dose prediction. Our approach starts by exhaustively searching each block from an enormous architecture space to identify multiple architectures that exhibit promising performance and significant diversity. To mitigate the complexity introduced by the model ensemble, we adopt the teacher-student paradigm, leveraging the diverse outputs from multiple learned networks as supervisory signals to guide the training of the student network. Furthermore, to preserve high-level semantic information, we design a hybrid loss to optimize the student network, enabling it to recover the knowledge embedded within the teacher networks. The proposed method has been evaluated on two public datasets: 1) OpenKBP and 2) AIMIS. Extensive experimental results demonstrate the effectiveness of our method and its superior performance to the state-of-the-art methods. Code: github.com/hust-linyi/LENAS.

TLR4/Rac1/NLRP3 Pathway Mediates Amyloid-ß-Induced Neuroinflammation in Alzheimer's Disease.

Background: Neuroinflammation plays a crucial part in the initial onset and progression of Alzheimer's disease (AD). NLRP3 inflammasome was demonstrated to get involved in amyloid-ß (Aß)-induced neuroinflammation. However, the mechanism of Aß-triggered activation of NLRP3 inflammasome remains poorly understood. Objective: Based on our previous data, the study aimed to identify the downstream signals that bridge the activation of TLR4 and NLRP3 inflammasome associated with Aß. Methods: BV-2 cells were transfected with TLR4siRNA or pretreated with a CLI-095 or NSC23766, followed by Aß1-42 treatment. APP/PS1 mice were injected intraperitoneally with CLI-095 or NSC23766. NLRP3 inflammasome and microglia activation was detected with immunostaining and western blot. G-LISA and Rac1 pull-down activation test were performed to investigate the activation of Rac1. Real-time PCR and ELISA were used to detect the inflammatory cytokines. Aß plaques were assessed by western blotting and immunofluorescence staining. Morris water maze test was conducted to determine the spatial memory in mice. Results: Rac1 and NLRP3 inflammasome were activated by Aß in both in vitro and in vivo experiments. Inhibition of TLR4 reduced the activity of Rac1 and NLRP3 inflammasome induced by Aß1-42. Furthermore, inhibition of Rac1 blocked NLRP3 inflammasome activation mediated by TLR4. Blocking the pathway by CLI095 or NSC23766 suppressed Aß1-42-triggered activation of microglia, reduced the expression of pro-inflammatory mediators and ameliorated the cognition deficits in APP/PS1 mice. Conclusions: Our study demonstrated that TLR4/Rac1/NLRP3 pathway mediated Aß-induced neuroinflammation, which unveiled a novel pathway and key contributors underlying the pathogenic mechanism of Aß.

Clarification of the infection pattern of Xanthomonas citri subsp. citri on citrus fruit by artificial inoculation.

BACKGROUND: Citrus canker is a significant bacterial disease caused by Xanthomonas citri subsp. citri (Xcc) that severely impedes the healthy development of the citrus industry. Especially when citrus fruit is infected by Xcc, it will reduce or even lost its commercial value. However, due to the prolonged fruiting cycle and intricate structure, much less research progress had been made in canker disease on fruit than on leaf. In fact, limited understanding has been achieved on canker development and the response to Xcc infection in fruit. RESULTS: Herein, the progression of canker disease on sweet orange fruit was tracked in the field. Results indicated that typical lesions initially appear on the sepal, style residue, nectary disk, epicarp, and peduncle of young fruits after petal fall. The susceptibility of fruits to Xcc infection diminished as the fruit developed, with no new lesions forming at the ripening stage. The establishment of an efficient method for inoculating Xcc on fruit as well as the artificial inoculation throughout the fruit's developmental cycle clarified this infection pattern. Additionally, microscopic observations during the infection process revealed that Xcc invasion caused structural changes on the surface and cross-section of the fruit. CONCLUSIONS: An efficient system for inoculation on citrus fruit with Xcc was established, by which it can serve for the evaluation of citrus germplasm for canker disease resistance and systematic research on the interactions between Xcc and citrus fruits.

CircVDAC3 sequesters microRNA-592 and elevates EIF4E3 expression to inhibit the progression of gastric cancer.

BACKGROUND: Accumulating evidence has shown that circular RNAs (circRNAs) are involved in gastric cancer (GC) tumorigenesis. However, specific functional circRNAs in GC remain to be discovered, and their underlying mechanisms remain to be elucidated. METHODS: CircRNAs that were differentially expressed between GC tissues and controls were analyzed using a circRNA microarray dataset. The expression of circVDAC3 in GC was determined using quantitative real-time PCR (qRT-PCR), and the structural features of circVDAC3 were validated. Cell function assays and animal experiments were conducted to explore the effects of circVDAC3 on GC. Finally, bioinformatics analysis, fluorescent in situ hybridization, and dual luciferase assays were used to analyze the downstream mechanisms of circVDAC3. RESULTS: Our results showed that circVDAC3 was downregulated in GC and inhibited the proliferation and metastasis of GC cells. Mechanistically, circVDAC3 acts as a competing endogenous RNA (ceRNA) of miR-592 and deregulates the repression of EIF4E3 by miR-592. EIF4E3 is downregulated in GC and overexpression of miR-592 or knockdown of EIF4E3 in circVDAC3-overexpressing cells weakens the anticancer effect of circVDAC3. CONCLUSION: Our study provides evidence that circVDAC3 affects the growth and metastasis of GC cells via the circVDAC3/miR-592/EIF4E3 axis. Our findings offer valuable insights into the mechanisms underlying GC tumorigenesis and suggest novel therapeutic strategies.

The effects of loving-kindness meditation on doctors' communication anxiety, trust, calling and defensive medicine practice.

OBJECTIVE: The study investigated the effects of loving-kindness meditation (LKM) on doctors' communication anxiety, trust, calling, and defensive medicine practice. METHODS: This study recruited 94 doctors from a hospital in China, randomized them to an LKM group (n = 47), and waited for the control group (n = 47). The experimental group accepted an 8-week LKM interference while the waiting for the control group underwent no interference. Researchers measured four major variable factors (communication anxiety, trust, calling, and defensive medicine practice) before and after the LKM intervention. RESULTS: In the experimental group, trust, and calling were significantly higher, and communication anxiety, and defensive medicine practice were significantly lower than in the control group. In the control group, there were no noticeable differences in any of the four variables between the pre-test and post-test. CONCLUSIONS: The results of this study demonstrate that LKM may help to improve trust, and calling, and reduce communication anxiety and defensive medicine practice. The finding of LKM's effect extends the understanding of the integrative effects of positive psychology on the decrease of defensive medicine practice. TRIAL REGISTRATION: ChiCTR2300074568. Registered in Chinese Clinical Trial Registry (ChiCTR), 9 August, 2023.

Prospective phase II trial of preoperative hypofractionated proton therapy for extremity and truncal soft tissue sarcoma: the PRONTO study rationale and design.

BACKGROUND: Oncologic surgical resection is the standard of care for extremity and truncal soft tissue sarcoma (STS), often accompanied by the addition of pre- or postoperative radiation therapy (RT). Preoperative RT may decrease the risk of joint stiffness and fibrosis at the cost of higher rates of wound complications. Hypofractionated, preoperative RT has been shown to provide acceptable outcomes in prospective trials. Proton beam therapy (PBT) provides the means to decrease dose to surrounding organs at risk, such as the skin, bone, soft tissues, and adjacent joint(s), and has not yet been studied in patients with extremity and truncal sarcoma. METHODS: Our study titled "PROspective phase II trial of preoperative hypofractionated protoN therapy for extremity and Truncal soft tissue sarcOma (PRONTO)" is a non-randomized, prospective phase II trial evaluating the safety and efficacy of preoperative, hypofractionated PBT for patients with STS of the extremity and trunk planned for surgical resection. Adult patients with Eastern Cooperative Group Performance Status ≤ 2 with resectable extremity and truncal STS will be included, with the aim to accrue 40 patients. Treatment will consist of 30 Gy radiobiological equivalent of PBT in 5 fractions delivered every other day, followed by surgical resection 2-12 weeks later. The primary outcome is rate of major wound complications as defined according to the National Cancer Institute of Canada Sarcoma2 (NCIC-SR2) Multicenter Trial. Secondary objectives include rate of late grade ≥ 2 toxicity, local recurrence-free survival and distant metastasis-free survival at 1- and 2-years, functional outcomes, quality of life, and pathologic response. DISCUSSION: PRONTO represents the first trial evaluating the use of hypofractionated PBT for STS. We aim to prove the safety and efficacy of this approach and to compare our results to historical outcomes established by previous trials. Given the low number of proton centers and limited availability, the short course of PBT may provide the opportunity to treat patients who would otherwise be limited when treating with daily RT over several weeks. We hope that this trial will lead to increased referral patterns, offer benefits towards patient convenience and clinic workflow efficiency, and provide evidence supporting the use of PBT in this setting. TRIAL REGISTRATION: NCT05917301 (registered 23/6/2023).

Effective treatment of MET exon 14 skipping mutation-positive non-small cell lung cancer using capmatinib following serious maculopapular rash caused by two MET inhibitors: a case report.

Background: Multi-gene panel testing and advancements in molecular targeted therapy have improved the overall survival of patients with driver mutation-positive non-small cell lung cancer (NSCLC). Mesenchymal-epithelial transition factor (MET) exon 14 skipping mutation-positive NSCLC, which remains untreated with MET inhibitors, shows a poorer prognosis than do cases of NSCLC without MET mutations. However, serious treatment-related adverse events (TRAEs) act as substantial treatment barriers. Case Description: Herein, we report a case of advanced NSCLC in a male in his 40s with MET exon 14 skipping mutation. A MET-inhibitory investigational drug was administered as first-line treatment; the development of grade 3 maculopapular rash necessitated dose reduction, which resulted in disease progression. Tepotinib was then administered with dexamethasone as a third-line treatment but was discontinued owing to the re-development of the grade 3 maculopapular rash. Finally, capmatinib administration as the fifth-line treatment appeared partially effective, with no serious adverse events. The patient could successfully resume work. Conclusions: This is the first report of MET exon 14 skipping mutation-positive NSCLC wherein partial response was achieved without severe TRAEs by alternating between two MET inhibitors. If no alternative treatments are available, cautious repeated re-administration of MET inhibitors after resolving serious rashes can be considered a potential approach.

Differences in the intestinal microbiota and association of host metabolism with hair coat status in cattle.

Introduction: The hair coat status of cattle serves as an easily observed indicator of economic value in livestock production; however, the underlying mechanism remains largely unknown. Therefore, the objective of the current study was to determine differences in the intestinal microbiota and metabolome of cattle based on a division of with either slick and shining (SHC) or rough and dull (MHC) hair coat in Simmental cows. Methods: Eight SHC and eight MHC late-pregnancy Simmental cows (with similar parities, body weights, and body conditions) were selected based on their hair coat status, and blood samples (plasma) from coccygeal venipuncture and fecal samples from the rectum were collected. The intestinal microbiota (in the fecal samples) was characterized by employing 16S rRNA gene sequencing targeting the V3-V4 hypervariable region on the Illumina MiSeq PE300 platform, and plasma samples were subjected to LC-MS/MS-based metabolomics with Progenesis QI 2.3. Plasma macromolecular metabolites were examined for differences in the metabolism of lipids, proteins, mineral elements, and hormones. Results: Notable differences between the SHC and MHC groups related to host hair coat status were observed in the host metabolome and intestinal microbiota (P < 0.05). The host metabolome was enriched in histidine metabolism, cysteine and methionine metabolism, and purine metabolism in the SHC group, and the intestinal microbiota were also enriched in histidine metabolism (P < 0.05). In the MHC group, the symbiotic relationship transitioned from cooperation to competition in the MHC group, and an uncoupling effect was present in the microbe-metabolite association of intestine microbiota-host interactions. The hubs mediating the relationships between intestinal microbiota and plasma metabolites were the intestinal bacterial genus g__norank_f__Eubacterium_coprostanoligenes_group, plasma inosine, triiodothyronine, and phosphorus, which could be used to differentiate cows' hair coat status (P < 0.05). Conclusion: Overall, the present study identified the relationships between the features of the intestinal microbiota and host hair coat status, thereby providing evidence and a new direction (intestine microbiota-host interplay) for future studies aimed at understanding the hair coat status of cattle.

Kaempferol efficacy in metabolic diseases: Molecular mechanisms of action in diabetes mellitus, obesity, non-alcoholic fatty liver disease, steatohepatitis, and atherosclerosis.

The incidence of metabolic diseases has progressively increased, which has a negative impact on human health and life safety globally. Due to the good efficacy and limited side effects, there is growing interest in developing effective drugs to treat metabolic diseases from natural compounds. Kaempferol (KMP), an important flavonoid, exists in many vegetables, fruits, and traditional medicinal plants. Recently, KMP has received widespread attention worldwide due to its good potential in the treatment of metabolic diseases. To promote the basic research and clinical application of KMP, this review provides a timely and comprehensive summary of the pharmacological advances of KMP in the treatment of four metabolic diseases and its potential molecular mechanisms of action, including diabetes mellitus, obesity, non-alcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH), and atherosclerosis. According to the research, KMP shows remarkable therapeutic effects on metabolic diseases by regulating multiple signaling transduction pathways such as NF-κB, Nrf2, AMPK, PI3K/AKT, TLR4, and ER stress. In addition, the most recent literature on KMP's natural source, pharmacokinetics studies, as well as toxicity and safety are also discussed in this review, thus providing a foundation and evidence for further studies to develop novel and effective drugs from natural compounds. Collectively, our manuscript strongly suggested that KMP could be a promising candidate for the treatment of metabolic diseases.

Enhancing astaxanthin accumulation through the expression of the plant-derived astaxanthin biosynthetic pathway in Dunaliella salina.

Dunaliella salina, a microalga that thrives under high-saline conditions, is notable for its high ß-carotene content and the absence of a polysaccharide cell wall. These unique characteristics render it a prime candidate as a cellular platform for astaxanthin production. In this study, our initial tests in an E. coli revealed that ß-ring-4-dehydrogenase (CBFD) and 4-hydroxy-ß-ring-4-dehydrogenase (HBFD) genes from Adonis aestivalis outperformed ß-carotene hydroxylase (BCH) and ß-carotene ketolase (BKT) from Haematococcus pluvialis counterparts by two-fold in terms of astaxanthin biosynthesis efficiency. Subsequently, we utilized electroporation to integrate either the BKT gene or the CBFD and HBFD genes into the genome of D. salina. In comparison to wild-type D. salina, strains transformed with BKT or CBFD and HBFD exhibited inhibited growth, underwent color changes to shades of red and yellow, and saw a nearly 50% decline in cell density. HPLC analysis confirmed astaxanthin synthesis in engineered D. salina strains, with CBFD + HBFD-D. salina yielding 134.88 ± 9.12 µg/g of dry cell weight (DCW), significantly higher than BKT-D. salina (83.58 ± 2.40 µg/g). This represents the largest amount of astaxanthin extracted from transgenic D. salina, as reported to date. These findings have significant implications, opening up new avenues for the development of specialized D. salina-based microcell factories for efficient astaxanthin production.

Christensenella strain resources, genomic/metabolomic profiling, and association with host at species level.

The gut commensal bacteria Christensenellaceae species are negatively associated with many metabolic diseases, and have been seen as promising next-generation probiotics. However, the cultured Christensenellaceae strain resources were limited, and their beneficial mechanisms for improving metabolic diseases have yet to be explored. In this study, we developed a method that enabled the enrichment and cultivation of Christensenellaceae strains from fecal samples. Using this method, a collection of Christensenellaceae Gut Microbial Biobank (ChrisGMB) was established, composed of 87 strains and genomes that represent 14 species of 8 genera. Seven species were first described and the cultured Christensenellaceae resources have been significantly expanded at species and strain levels. Christensenella strains exerted different abilities in utilization of various complex polysaccharides and other carbon sources, exhibited host-adaptation capabilities such as acid tolerance and bile tolerance, produced a wide range of volatile probiotic metabolites and secondary bile acids. Cohort analyses demonstrated that Christensenellaceae and Christensenella were prevalent in various cohorts and the abundances were significantly reduced in T2D and OB cohorts. At species level, Christensenellaceae showed different changes among healthy and disease cohorts. C. faecalis, F. tenuis, L. tenuis, and Guo. tenuis significantly reduced in all the metabolic disease cohorts. The relative abundances of C. minuta, C. hongkongensis and C. massiliensis showed no significant change in NAFLD and ACVD. and C. tenuis and C. acetigenes showed no significant change in ACVD, and Q. tenuis and Geh. tenuis showed no significant change in NAFLD, when compared with the HC cohort. So far as we know, this is the largest collection of cultured resource and first exploration of Christensenellaceae prevalences and abundances at species level.

The efficacy and safety of short-term and low-dose IL-2 combined with tocilizumab to treat rheumatoid arthritis.

Background: Immunotherapy targeting factors related to immune imbalance has been widely employed for RA treatment. This study aimed to evaluate the efficacy and safety of low-dose interleukin (IL)-2 combined with tocilizumab (TCZ), a biologics targeting IL-6, in RA patients. Methods: Fifty adults with active RA who met the criteria with complete clinical data were recruited, and divided into three groups: control group (n=15), IL-2 group (n=26), and IL-2+TCZ group (n=9). In addition to basic treatment, participants in the IL-2 group received IL-2 (0.5 MIU/day), while participants in the IL-2+TCZ group received IL-2 (0.5 MIU/day) along with one dose of TCZ (8 mg/kg, maximum dose: 800 mg). All subjects underwent condition assessment, laboratory indicators and safety indicators detection, and records before treatment and one week after treatment. Results: Compared with the baseline, all three groups showed significant improvement in disease conditions, as evidenced by significantly reduced disease activity indicators. The low-dose IL-2 and combination treatment groups demonstrated a violent proliferation of Tregs, while the absolute number of Th1, Th2, and Th17 cells in the latter group showed a decreasing trend. The decrease in the Th17/Treg ratio was more pronounced in the IL-2+TCZ groups. No significant adverse reactions were observed in any of the patients. Conclusion: Exogenous low doses of IL-2 combined TCZ were found to be safe and effective in reducing effector T cells and appropriately increasing Treg levels in RA patients with high effector T cell levels. This approach helps regulate immune homeostasis and contributes to the prevention of disease deterioration. Clinical trial registration: https://www.chictr.org.cn/showprojEN.html?proj=13909, identifier ChiCTR-INR-16009546.

Impact of One-Lung Ventilation on Oxygenation and Ventilation Time in Thoracoscopic Heart Surgery: A Comparative Analysis with Median Thoracotomy.

BACKGROUND One-lung ventilation is the separation of the lungs by mechanical methods to allow ventilation of only one lung, particularly when there is pathology in the other lung. This retrospective study from a single center aimed to compare 49 patients undergoing thoracoscopic cardiac surgery using one-lung ventilation with 48 patients undergoing thoracoscopic cardiac surgery with median thoracotomy. MATERIAL AND METHODS This single-center retrospective study analyzed patients who underwent thoracoscopic cardiac surgery based on one-lung ventilation (experimental group, n=49). Other patients undergoing a median thoracotomy cardiac operation were defined as the comparison group (n=48). The oxygenation index and the mechanical ventilation time were also recorded. RESULTS There was no significant difference in the immediate oxygenation index between the experimental group and comparison group (P>0.05). There was no significant difference for the oxygenation index between men and women in both groups (P>0.05). The cardiopulmonary bypass time significantly affected the oxygenation index (F=7.200, P=0.009). Operation methods (one-lung ventilation thoracoscopy or median thoracotomy) affected postoperative ventilator use time (F=8.337, P=0.005). Cardiopulmonary bypass time (F=16.002, P<0.001) and age (F=4.384, P=0.039) had significant effects on ventilator use time. There was no significant effect of sex (F=0.75, P=0.389) on ventilator use time. CONCLUSIONS Our results indicated that one-lung ventilation thoracoscopic cardiac surgery did not affect the immediate postoperative oxygenation index; however, cardiopulmonary bypass time did significantly affect the immediate postoperative oxygenation index. Also, one-lung ventilation thoracoscopic cardiac surgery had a shorter postoperative mechanical ventilation use time than did traditional median thoracotomy cardiac surgery.

Comprehensive genetic analysis of facioscapulohumeral muscular dystrophy by Nanopore long-read whole-genome sequencing.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is a high-prevalence autosomal dominant neuromuscular disease characterized by significant clinical and genetic heterogeneity. Genetic diagnosis of FSHD remains a challenge because it cannot be detected by standard sequencing methods and requires a complex diagnosis workflow. METHODS: We developed a comprehensive genetic FSHD detection method based on Oxford Nanopore Technologies (ONT) whole-genome sequencing. Using a case-control design, we applied this procedure to 29 samples and compared the results with those from optical genome mapping (OGM), bisulfite sequencing (BSS), and whole-exome sequencing (WES). RESULTS: Using our ONT-based method, we identified 59 haplotypes (35 4qA and 24 4qB) among the 29 samples (including a mosaic sample), as well as the number of D4Z4 repeat units (RUs). The pathogenetic D4Z4 RU contraction identified by our ONT-based method showed 100% concordance with OGM results. The methylation levels of the most distal D4Z4 RU and the double homeobox 4 gene (DUX4) detected by ONT sequencing are highly consistent with the BSS results and showed excellent diagnostic efficiency. Additionally, our ONT-based method provided an independent methylation profile analysis of two permissive 4qA alleles, reflecting a more accurate scenario than traditional BSS. The ONT-based method detected 17 variations in three FSHD2-related genes from nine samples, showing 100% concordance with WES. CONCLUSIONS: Our ONT-based FSHD detection method is a comprehensive method for identifying pathogenetic D4Z4 RU contractions, methylation level alterations, allele-specific methylation of two 4qA haplotypes, and variations in FSHD2-related genes, which will all greatly improve genetic testing for FSHD.

Investigating shared genetic architecture between inflammatory bowel diseases and primary biliary cholangitis.

Background & Aims: Inflammatory bowel disease (IBD) is commonly associated with extraintestinal complications, including autoimmune liver disease. The co-occurrence of IBD and primary biliary cholangitis (PBC) has been increasingly observed, but the underlying relationship between these conditions remains unclear. Methods: Using summary statistics from genome-wide association studies (GWAS), we investigated the causal effects between PBC and IBD, including Crohn's disease (CD) and ulcerative colitis (UC). We also analyzed the shared genetic architecture between IBD and PBC using data from GWAS, bulk-tissue RNA sequencing, and single cell RNA sequencing, and explored potential functional genes. Result: There was a strong positive genetic correlation between PBC and IBD (linkage disequilibrium score regression: rg = 0.2249, p = 3.38 × 10-5). Cross-trait analysis yielded 10 shared-risk single nucleotide polymorphisms (SNPs), as well as nine novel SNPs, which were associated with both traits. Using Mendelian randomization, a stable causal effect was established of PBC on IBD. Genetically predicted PBC was found to have a risk effect on IBD (1.105; 95% CI: 1.058-1.15; p = 1.16 × 10-10), but not vice versa. Shared tissue-specific heritability enrichment was identified for PBC and IBD (including CD and UC) in lung, spleen, and whole-blood samples. Furthermore, shared enrichment was observed of specific cell types (T cells, B cells, and natural killer cells) and their subtypes. Nine functional genes were identified based on summary statistics-based Mendelian randomization. Conclusions: This study detected shared genetic architecture between IBD and PBC and demonstrated a stable causal relationship of genetically predicted PBC on the risk of IBD. These findings shed light on the biological basis of comorbidity between IBD and PBC, and have important implications for intervention and treatment targets of these two diseases simultaneously. Impact and Implications: The discovery of novel shared single nucleotide polymorphisms (SNPs) and functional genes provides insights into the common targets between inflammatory bowel disease (IBD) and primary biliary cholangitis (PBC), serving as a basis for new drug development and contributing to the study of disease pathogenesis. Additionally, the established significant causality and genetic correlation underscore the importance of clinical intervention in preventing the comorbidity of IBD and PBC. The enrichment of SNP heritability in specific tissues and cell types reveals the role of immune factors in the potential disease mechanisms shared between IBD and PBC. This stimulates further research on potential interventions and could lead to the development of new targets for immune-based therapies.

TCERG1L hypermethylation is a risk factor of diabetic retinopathy in Chinese children with type 1 diabetes.

AIM: To identify the differential methylation sites (DMS) and their according genes associated with diabetic retinopathy (DR) development in type 1 diabetes (T1DM) children. METHODS: This study consists of two surveys. A total of 40 T1DM children was included in the first survey. Because no participant has DR, retina thinning was used as a surrogate indicator for DR. The lowest 25% participants with the thinnest macular retinal thickness were included into the case group, and the others were controls. The DNA methylation status was assessed by the Illumina methylation 850K array BeadChip assay, and compared between the case and control groups. Four DMS with a potential role in diabetes were identified. The second survey included 27 T1DM children, among which four had DR. The methylation patterns of the four DMS identified by 850K were compared between participants with and without DR by pyrosequencing. RESULTS: In the first survey, the 850K array revealed 751 sites significantly and differentially methylated in the case group comparing with the controls (|Δß|>0.1 and Adj.P<0.05), and 328 of these were identified with a significance of Adj.P<0.01. Among these, 319 CpG sites were hypermethylated and 432 were hypomethylated in the case group relative to the controls. Pyrosequencing revealed that the transcription elongation regulator 1 like (TCERG1L, cg07684215) gene was hypermethylated in the four T1DM children with DR (P=0.018), which was consistent with the result from the first survey. The methylation status of the other three DMS (cg26389052, cg25192647, and cg05413694) showed no difference (all P>0.05) between participants with and without DR. CONCLUSION: The hypermethylation of the TCERG1L gene is a risk factor for DR development in Chinese children with T1DM.