Rare variant association analyses reveal the significant contribution of carbohydrate metabolic disturbance in severe adolescent idiopathic scoliosis.

BACKGROUND: Adolescent idiopathic scoliosis (AIS), the predominant genetic-influenced scoliosis, results in spinal deformities without vertebral malformations. However, the molecular aetiology of AIS remains unclear. METHODS: Using genome/exome sequencing, we studied 368 patients with severe AIS (Cobb angle >40°) and 3794 controls from a Han Chinese cohort. We performed gene-based and pathway-based weighted rare variant association tests to assess the mutational burden of genes and established biological pathways. Differential expression analysis of muscle tissues from 14 patients with AIS and 15 controls was served for validation. RESULTS: SLC16A8, a lactate transporter linked to retinal glucose metabolism, was identified as a novel severe AIS-associated gene (p=3.08E-06, false discovery rate=0.009). Most AIS cases with deleterious SLC16A8 variants demonstrated early onset high myopia preceding scoliosis. Pathway-based burden test also revealed a significant enrichment in multiple carbohydrate metabolism pathways, especially galactose metabolism. Patients with deleterious variants in these genes demonstrated a significantly larger spinal curve. Genes related to catabolic processes and nutrient response showed divergent expression between AIS cases and controls, reinforcing our genomic findings. CONCLUSION: This study uncovers the pivotal role of genetic variants in carbohydrate metabolism in the development of AIS, unveiling new insights into its aetiology and potential treatment.

The clinical utility and diagnostic implementation of human subject cell transdifferentiation followed by RNA sequencing.

RNA sequencing (RNA-seq) has recently been used in translational research settings to facilitate diagnoses of Mendelian disorders. A significant obstacle for clinical laboratories in adopting RNA-seq is the low or absent expression of a significant number of disease-associated genes/transcripts in clinically accessible samples. As this is especially problematic in neurological diseases, we developed a clinical diagnostic approach that enhanced the detection and evaluation of tissue-specific genes/transcripts through fibroblast-to-neuron cell transdifferentiation. The approach is designed specifically to suit clinical implementation, emphasizing simplicity, cost effectiveness, turnaround time, and reproducibility. For clinical validation, we generated induced neurons (iNeurons) from 71 individuals with primary neurological phenotypes recruited to the Undiagnosed Diseases Network. The overall diagnostic yield was 25.4%. Over a quarter of the diagnostic findings benefited from transdifferentiation and could not be achieved by fibroblast RNA-seq alone. This iNeuron transcriptomic approach can be effectively integrated into diagnostic whole-transcriptome evaluation of individuals with genetic disorders.

Core planar cell polarity genes VANGL1 and VANGL2 in predisposition to congenital vertebral malformations.

Congenital scoliosis (CS), affecting approximately 0.5 to 1 in 1,000 live births, is commonly caused by congenital vertebral malformations (CVMs) arising from aberrant somitogenesis or somite differentiation. While Wnt/ß-catenin signaling has been implicated in somite development, the function of Wnt/planar cell polarity (Wnt/PCP) signaling in this process remains unclear. Here, we investigated the role of Vangl1 and Vangl2 in vertebral development and found that their deletion causes vertebral anomalies resembling human CVMs. Analysis of exome sequencing data from multiethnic CS patients revealed a number of rare and deleterious variants in VANGL1 and VANGL2, many of which exhibited loss-of-function and dominant-negative effects. Zebrafish models confirmed the pathogenicity of these variants. Furthermore, we found that Vangl1 knock-in (p.R258H) mice exhibited vertebral malformations in a Vangl gene dose- and environment-dependent manner. Our findings highlight critical roles for PCP signaling in vertebral development and predisposition to CVMs in CS patients, providing insights into the molecular mechanisms underlying this disorder.

Effects of crystalline lens rise and anterior chamber parameters on vault after implantable collamer lens placement.

BACKGROUND: To analyze vault effects of crystalline lens rise (CLR) and anterior chamber parameters (recorded by Pentacam) in highly myopic patients receiving implantable collamer lenses (ICLs), which may avoid subsequent complications such as glaucoma and cataract caused by the abnormal vault. METHODS: We collected clinical data of 137 patients with highly myopic vision, who were all subsequent recipients of V4c ICLs between June 2020 and January 2021. Horizontal ciliary sulcus-to-sulcus diameter (hSTS) and CLR were measured by ultrasonic biomicroscopy (UBM), and a Pentacam anterior segment analyzer was used to measure horizontal white-to-white diameter (hWTW), anterior chamber depth (ACD), anterior chamber angle (ACA), anterior chamber volume (ACV), CLR, and postoperative vault (Year 1 and Month 1). The lens thickness (LT) was determined by optical biometry (IOL Master instrument). The predictive model was generated through multiple linear regression analyses of influential factors, such as hSTS, CLR, hWTW, ACD, ACA, ACV, ICL size, and LT. The predictive performance of the multivariate model on vault after ICL was assessed using the receiver operating characteristic (ROC) curve with area under the curve (AUC) as well as the point of tangency. RESULTS: Average CLR assessed by UBM was lower than the average value obtained by Pentacam (0.561 vs. 0.683). Bland-Altman analysis showed a good consistency in the two measurement methods and substantial correlation (r = 0.316; P = 0.000). The ROC curve of Model 1 (postoperative Year 1) displayed an AUC of 0.847 (95% confidence interval [CI]: 74.19-95.27), with optimal threshold of 0.581 (sensitivity, 0.857; specificity, 0.724). In addition, respective values for Model 2 (postoperative Month 1) were 0.783 (95% CI: 64.94-91.64) and 0.522 (sensitivity, 0.917; specificity, 0.605). CONCLUSION: CLR and anterior chamber parameters are important determinants of postoperative vault after ICL placement. The multivariate regression model we constructed may serve in large part as a predictive gauge, effectively avoid postoperative complication.

Unraveling the genetic architecture of congenital vertebral malformation with reference to the developing spine.

Congenital vertebral malformation, affecting 0.13-0.50 per 1000 live births, has an immense locus heterogeneity and complex genetic architecture. In this study, we analyze exome/genome sequencing data from 873 probands with congenital vertebral malformation and 3794 control individuals. Clinical interpretation identifies Mendelian etiologies in 12.0% of the probands and reveals a muscle-related disease mechanism. Gene-based burden test of ultra-rare variants identifies risk genes with large effect sizes (ITPR2, TBX6, TPO, H6PD, and SEC24B). To further investigate the biological relevance of the genetic association signals, we perform single-nucleus RNAseq on human embryonic spines. The burden test signals are enriched in the notochord at early developmental stages and myoblast/myocytes at late stages, highlighting their critical roles in the developing spine. Our work provides insights into the developmental biology of the human spine and the pathogenesis of spine malformation.

[Analysis of CHIP-Related Mutation and Risk of Cardio-Cerebrovasculars Events in Patients with Myeloproliferative Neoplasms].

OBJECTIVE: To analyze the mutant spectrum of clonal hematopoiesis of indeterminate potential (CHIP) related mutations and clinical characteristics and to explore the correlation and the possible mechanism between CHIP-related mutations and cardio-cerebrovasculars events (CCEs) in patients with myeloproliferative neoplasms (MPNs). METHODS: The clinical data and next-generation sequencing results of 73 MPN patients in Beijing Anzhen Hospital from August 2019 to July 2022 were retrospectively analyzed. Statistical analyses were conducted by multivariate logistic regression for the effects of CHIP-related mutations and inflammatory cytokines on CCEs for MPNs patients. RESULTS: Fifty-five cases of MPN (75.3%) showed positive in CHIP-related genes. There was no significant difference in variant allele frequency of CHIP-related gene between essential thrombocythemia (ET) and polycythemia vera (PV). CHIP-related gene mutations were mainly single gene mutations, with mutation rate from high to low as JAK2V617F (63.0%, 46/73), ASXL1 (16.4%, 12/73), TET2 (11.0%, 8/73), DNMT3A (9.6%, 7/73), SRSF2 (6.9%, 5/73), SF3B1 (4.1%, 3/73), TP53(1.4%, 1/73) and PPM1D (1.4%, 1/73). The mutation rate of CHIP-related genes in MPN patients >60 years old was significantly higher than that in the patients ≤60 years old ï¼»91.7%(33/36) vs 59.5%(22/37)ï¼½. CCEs occurred in 27 MPNs patients (37.0%, MPNs/CCEs), and 5 had recurrent CCEs, all of which were arterial events. Age (62.8±12.8 years vs 53.9±15.8 years, P =0.015), IL-1ß level (17.7±26.0 vs 4.3±8.6, P =0.012), IL-8 level (360.7±598.6 vs 108.3±317.0, P =0.045), the proportion of the patients with thrombosis history (29.6% vs 2.2%, P =0.020), and the detection rate of CHIP-related mutations (88.9% vs 67.4%, P =0.040) in the group with CCEs were higher than those in the group without CCEs. Multivariate Logistic regression analysis showed that age（OR =0.917， 95%CI ：0.843-0.999, P =0.047）, thrombosis history (OR =34.148， 95%CI ：2.392-487.535, P =0.009), any CHIP-related mutations(OR =16.065， 95%CI ：1.217-212.024， P =0.035), and elevated level of IL-1ß (OR =0.929， 95%CI ：0.870-0.992， P =0.027) were independent risk factors for MPNs/CCEs. CHIP-related gene mutations were not associated with CCEs in MPN patients, but DNMT3A (OR =88.717， 95%CI ：2.690-292.482， P =0.012) and ASXL1 (OR =7.941， 95%CI ：1.045-60.353， P =0.045) were independent risk factors for CCEs in PV. CONCLUSION: There is a higher mutation rate of CHIP-related genes in MPN patients, especially those over 60 years old. Older age, thrombosis history, CHIP-related mutations and IL-1ß elevated levels are independent risk factors for CCEs in MPN. DNMT3A and ASXL1 mutations are independent risk factors for CCEs in PV patients. CHIP-related gene mutations and inflammatory cytokine IL-1 ß elevated levels may be the novel risk factors for CCEs in MPN.

Nontargeted metabolomic insights into the behavioral effects of 5-MeO-MiPT in zebrafish (Danio rerio).

5-Methoxy-N-methyl-N-isopropyltryptamine (5-MeO-MiPT) is a novel psychoactive substance exhibiting a tryptamine structure. Despite its increasing prevalence, the environmental impact of 5-MeO-MiPT remains unexplored. Our prior investigation revealed that 5-MeO-MiPT induced inhibited spontaneous movement and prompted anxiety-like behavior in adult zebrafish-a validated toxicological model. To elucidate this phenomenon and establish a correlation between metabolomics and behavioral changes induced by 5-MeO-MiPT, zebrafish were administered varying drug concentrations. Zebrafishes were subjected to injections of different 5-MeO-MiPT concentrations. Subsequent metabolomic analysis of endogenous metabolites affected by the drug unveiled substantial variations in metabolic levels between the control group and the drug-injected cohorts. A total of 22 distinct metabolites emerged as potential biomarkers. Further scrutiny identified seven pathways significantly influenced by 5-MeO-MiPT. A focused exploration into amino acid metabolism, lipid metabolism, and energy metabolism unveiled that the metabolic repercussions of 5-MeO-MiPT on zebrafish resulted in observable brain damage. Notably, the study identified a consequential disruption in the liver-brain pathway. The comprehensive metabolomic approach employed herein effectively discerned the impact of 5-MeO-MiPT on zebrafish metabolism. This approach also shed light on the mechanism underpinning the anxiety-like behavior observed in zebrafish post-drug injection. Specifically, our findings indicate that 5-MeO-MiPT induces brain damage, particularly within the liver-brain pathway.

Fluid-solid coupling numerical simulation of entire rat caudal vertebrae under dynamic loading.

Trabeculae bone undergoes directional growth along the applied force under physiological loading. The growth of bone structure relies on the coordinated interplay among osteocytes, osteoblasts, and osteoclasts. Under normal circumstances, bone remodeling maintains a state of equilibrium. Excessive bone formation can lead to osteosclerosis, while excessive bone resorption can result in osteoporosis and osteonecrosis. The investigation of the structural characteristics of trabeculae and the mechanotransduction between bone cells plays a vital role in the treatment of bone-related diseases. In this study, a fluid-solid coupling model of the entire vertebral bone was established based on micro-CT images obtained from rat tail vertebrae subjected to tensile loading experiments. The flow characteristics of bone marrow and the mechanical response of osteocytes in different regions under physiological loading were investigated. The results revealed a U-shaped distribution of wall fluid shear stress (FSS) along the longitudinal axis in trabecular bone, with higher FSS regions exhibiting greater mechanical stimulation on osteocytes. These findings elucidate a positive correlation between the mechanical microenvironment among osteocytes, osteoblasts, and osteoclasts, providing potential strategies for the prevention and treatment of bone diseases.

SIGMA leverages protein structural information to predict the pathogenicity of missense variants.

Leveraging protein structural information to evaluate pathogenicity has been hindered by the scarcity of experimentally determined 3D protein. With the aid of AlphaFold2 predictions, we developed the structure-informed genetic missense mutation assessor (SIGMA) to predict missense variant pathogenicity. In comparison with existing predictors across labeled variant datasets and experimental datasets, SIGMA demonstrates superior performance in predicting missense variant pathogenicity (AUC = 0.933). We found that the relative solvent accessibility of the mutated residue contributed greatly to the predictive ability of SIGMA. We further explored combining SIGMA with other top-tier predictors to create SIGMA+, proving highly effective for variant pathogenicity prediction (AUC = 0.966). To facilitate the application of SIGMA, we pre-computed SIGMA scores for over 48 million possible missense variants across 3,454 disease-associated genes and developed an interactive online platform (https://www.sigma-pred.org/). Overall, by leveraging protein structure information, SIGMA offers an accurate structure-based approach to evaluating the pathogenicity of missense variants.

Impaired glycine neurotransmission causes adolescent idiopathic scoliosis.

Adolescent idiopathic scoliosis (AIS) is the most common form of spinal deformity, affecting millions of adolescents worldwide, but it lacks a defined theory of etiopathogenesis. Because of this, treatment of AIS is limited to bracing and/or invasive surgery after onset. Preonset diagnosis or preventive treatment remains unavailable. Here, we performed a genetic analysis of a large multicenter AIS cohort and identified disease-causing and predisposing variants of SLC6A9 in multigeneration families, trios, and sporadic patients. Variants of SLC6A9, which encodes glycine transporter 1 (GLYT1), reduced glycine-uptake activity in cells, leading to increased extracellular glycine levels and aberrant glycinergic neurotransmission. Slc6a9 mutant zebrafish exhibited discoordination of spinal neural activities and pronounced lateral spinal curvature, a phenotype resembling human patients. The penetrance and severity of curvature were sensitive to the dosage of functional glyt1. Administration of a glycine receptor antagonist or a clinically used glycine neutralizer (sodium benzoate) partially rescued the phenotype. Our results indicate a neuropathic origin for "idiopathic" scoliosis, involving the dysfunction of synaptic neurotransmission and central pattern generators (CPGs), potentially a common cause of AIS. Our work further suggests avenues for early diagnosis and intervention of AIS in preadolescents.

Acute pancreatitis associated with pleural effusion: MDCT manifestations and anatomical basis.

BACKGROUND: Acute pancreatitis (AP) is a severe condition with complications that can impact multiple organ systems throughout the body. Specifically, the diffusion of peripancreatic effusion to the pleural cavity is a significant phenomenon in AP. However, its pathways and implications for disease severity are not fully understood. OBJECTIVE: This study aims to investigate the anatomical routes of peripancreatic effusion diffusion into the pleural cavity in patients with AP and to analyze the correlation between the severity of pleural effusion (PE) and the computed tomography severity index (CTSI) and acute physiology and chronic health evaluation II (APACHE II) scoring system. METHODS: 119 patients with AP admitted to our institution were enrolled in this study (mean age 50 years, 74 male and 45 female). Abdominal CT was performed, and the CTSI and APACHE II index were used to evaluate the severity of the AP, Meanwhile, the prevalence and semiquantitative of PE were also mentioned. The anatomical pathways of peripancreatic effusion draining to pleural were analyzed. Finally, the correlation relationship between the severity of AP and the PE was analyzed. RESULTS: In 119 patients with AP, 74.8% of patients had PE on CT. The anatomic pathways of peripancreatic effusion draining to pleural included esophageal hiatus in 33.7% of patients, aortic hiatus in 6.7% of patients and inferior vena cava hiatus in 3.37% of patients. The rating of PE on CT was correlated with CTSI scores (r= 0.449, P= 0.000) and was slightly correlated with the APACHE II scores (r= 0.197, P= 0.016). CONCLUSION: PE is a common complication of AP, which can be caused by anatomic pathways such as diaphragmatic hiatus. Due to its correlation with the CTSI score, the PE may be a supplementary indicator in determining the severity of AP.

Efficacy and safety of corticosteroids, hyaluronic acid, and PRP and combination therapy for knee osteoarthritis: a systematic review and network meta-analysis.

OBJECTIVE: There are many injectable treatments for knee osteoarthritis with different characteristics and effects, the aim is to understand which one can lead to better and safer results. METHODS: The PRISMA principles were followed when doing the literature search. Web of Science databases, Embase, the Cochrane Library, PubMed, and the Wanfang database were searched to identified randomized controlled trials that assessed the efficacy of corticosteroids (CSC), platelet-rich plasma (PRP), hyaluronic acid (HA), and combination therapy in treating KOA. Risk of bias was assessed using the relevant Cochrane tools (version 1.0). The outcome measure included the visual analog scale (VAS) score, the Western Ontario and McMaster Universities Osteoarthritis (WOMAC) score, and treatment-related adverse events. The network meta-analysis was performed using STATA17 software and a Bayesian stratified random effects model. RESULTS: Network meta-analysis using the Bayesian random-effects model revealed 35 studies with 3104 participants. PRP showed the best WOMAC score at a 3-month follow-up, followed by PRP + HA, HA, placebo, and CSC; PRP + HA scored the highest VAS, followed by PRP, CSC, HA, and placebo. PRP, CSC, HA, and placebo had the highest WOMAC scores six months following treatment; PRP + HA showed the best VAS scores. PRP showed the best WOMAC score at 12 months, followed by PRP + HA, HA, placebo, and CSC; The best VAS score was obtained with PRP, followed by PRP + HA, HA, and CSC. No therapy demonstrated a rise in adverse events linked to the treatment in terms of safety. CONCLUSIONS: The current study found that PRP and PRP + HA were the most successful in improving function and alleviating pain after 3, 6, and 12 months of follow-up. CSC, HA, PRP, and combination therapy did not result in an increase in the incidence of treatment-related side events as compared to placebo.

Associations of dichlorophenol with metabolic syndrome based on multivariate-adjusted logistic regression: a U.S. nationwide population-based study 2003-2016.

BACKGROUND: Para-dichlorobenzene (p-DCB) exposure associated with oxidative stress has indeed raised public concerns. However, whether p-DCB is linked with metabolic syndrome (MetS) remains unclear. We hypothesized that higher exposure to p-DCB would be linked with a higher risk of MetS in the U.S population. This study aimed to examine the associations of exposure to p-DCB with MetS prevalence. METHODS: We included 10,428 participants (5,084 men and 5,344 women), aged ≥ 20 years, from the National Health and Nutrition Examination Survey (2003-2016). The cases of MetS were diagnosed by NCEP/ATPIII. Logistic regression models were conducted to calculate the odds ratios (ORs) and 95% confidence intervals (CIs) of MetS prevalence. Moreover, the mix associations of p-DCB metabolites were assessed using quantile sum (WQS) regression and quantile g-computation (qgcomp) methods. RESULTS: We documented 2,861 (27.1%) MetS cases. After adjustment for the potential risk factors, the ORs (95% CI) of MetS prevalence across the quartile of urinary 2,5-dichlorophenol (2,5-DCP) were 1.09 (0.93-1.28), 1.22 (1.00-1.49), and 1.34 (1.04-1.73). Moreover, 2,5 DCP is significantly associated with a higher prevalence of abdominal obesity [ORQ4vsQ1 (95% CI): 1.23 (1.03-1.48)]. The WQS and qgcomp index also showed significant associations between p-DCB metabolites and MetS. Moreover, we further examined that 2,5 DCP was correlated with higher systolic blood pressure (r = 0.022, P = 0.027), waist circumference (r = 0.099, P < 0.001), and glycohemoglobin (r = 0.027, P = 0.008) and a lower high density cholesterol (r = -0.059, P < 0.001). In addition, the significant positive associations between 2,5 DCP and MetS were robust in the subgroup and sensitivity analyses. CONCLUSION: These findings indicated that increased urinary p-DCB concentration, especially 2,5 DCP, had a higher MetS prevalence. These results should be interpreted cautiously and further research is warranted to validate our findings.

Effect of Mechanical Stimulations on the Cytoskeleton of Mouse Oocytes During Maturation.

In the reproductive system of female mammals, the early embryos grow and develop in the fallopian tube, where they are stimulated by fluid flow and ciliary vibration. The mechanical environment of the fallopian tube affects the development of embryos. This study is focused on the role of mechanical stimulation on the cytoskeleton of oocytes during oocyte maturation in vitro. The 3 Hz microvibration and tilting stimulations were applied to mouse immature oocytes. The oocyte maturation rate and area of the first polar body under dynamic stimulation were compared with those of the static culture group. A fluorescence assay was used to study the localization of the cytoskeleton during oocyte maturation. The results indicated that mechanical stimulation can change the localization of F-actin and spindle, and oocytes discharged smaller areas of the first polar body.

TET1 Participates in Complete Freund's Adjuvant-induced Trigeminal Inflammatory Pain by Regulating Kv7.2 in a Mouse Model.

Trigeminal inflammatory pain is one of the most severe pain-related disorders in humans; however, the underlying mechanisms remain largely unknown. In this study, we investigated the possible contribution of interaction between ten-eleven translocation methylcytosine dioxygenase 1 (TET1) and the voltage-gated K+ channel Kv7.2 (encoded by Kcnq2) to orofacial inflammatory pain in mice. We found that complete Freund's adjuvant (CFA) injection reduced the expression of Kcnq2/Kv7.2 in the trigeminal ganglion (TG) and induced orofacial inflammatory pain. The involvement of Kv7.2 in CFA-induced orofacial pain was further confirmed by Kv7.2 knockdown or overexpression. Moreover, TET1 knockdown in Tet1flox/flox mice significantly reduced the expression of Kv7.2 and M currents in the TG and led to pain-like behaviors. Conversely, TET1 overexpression by lentivirus rescued the CFA-induced decreases of Kcnq2 and M currents and alleviated mechanical allodynia. Our data suggest that TET1 is implicated in CFA-induced trigeminal inflammatory pain by positively regulating Kv7.2 in TG neurons.

METTL3 Promotes the Differentiation of Goat Skeletal Muscle Satellite Cells by Regulating MEF2C mRNA Stability in a m6A-Dependent Manner.

The development of mammalian skeletal muscle is a highly complex process involving multiple molecular interactions. As a prevalent RNA modification, N6-methyladenosine (m6A) regulates the expression of target genes to affect mammalian development. Nevertheless, it remains unclear how m6A participates in the development of goat muscle. In this study, methyltransferase 3 (METTL3) was significantly enriched in goat longissimus dorsi (LD) tissue. In addition, the global m6A modification level and differentiation of skeletal muscle satellite cells (MuSCs) were regulated by METTL3. By performing mRNA-seq analysis, 8050 candidate genes exhibited significant changes in expression level after the knockdown of METTL3 in MuSCs. Additionally, methylated RNA immunoprecipitation sequencing (MeRIP-seq) illustrated that myocyte enhancer factor 2c (MEF2C) mRNA contained m6A modification. Further experiments demonstrated that METTL3 enhanced the differentiation of MuSCs by upregulating m6A levels and expression of MEF2C. Moreover, the m6A reader YTH N6-methyladenosine RNA binding protein C1 (YTHDC1) was bound and stabilized to MEF2C mRNA. The present study reveals that METTL3 enhances myogenic differentiation in MuSCs by regulating MEF2C and provides evidence of a post-transcriptional mechanism in the development of goat skeletal muscle.

Variants in the SOX9 transactivation middle domain induce axial skeleton dysplasia and scoliosis.

SOX9 is an essential transcriptional regulator of cartilage development and homeostasis. In humans, dysregulation of SOX9 is associated with a wide spectrum of skeletal disorders, including campomelic and acampomelic dysplasia, and scoliosis. The mechanism of how SOX9 variants contribute to the spectrum of axial skeletal disorders is not well understood. Here, we report four novel pathogenic variants of SOX9 identified in a large cohort of patients with congenital vertebral malformations. Three of these heterozygous variants are in the HMG and DIM domains, and for the first time, we report a pathogenic variant within the transactivation middle (TAM) domain of SOX9 . Probands with these variants exhibit variable skeletal dysplasia, ranging from isolated vertebral malformation to acampomelic dysplasia. We also generated a Sox9 hypomorphic mutant mouse model bearing a microdeletion within the TAM domain ( Sox9 Asp272del ). We demonstrated that disturbance of the TAM domain with missense mutation or microdeletion results in reduced protein stability but does not affect the transcriptional activity of SOX9. Homozygous Sox9 Asp272del mice exhibited axial skeletal dysplasia including kinked tails, ribcage anomalies, and scoliosis, recapitulating phenotypes observed in human, while heterozygous mutants display a milder phenotype. Analysis of primary chondrocytes and the intervertebral discs in Sox9 Asp272del mutant mice revealed dysregulation of a panel of genes with major contributions of the extracellular matrix, angiogenesis, and ossification-related processes. In summary, our work identified the first pathologic variant of SOX9 within the TAM domain and demonstrated that this variant is associated with reduced SOX9 protein stability. Our finding suggests that reduced SOX9 stability caused by variants in the TAM domain may be responsible for the milder forms of axial skeleton dysplasia in humans.

COL11A2 as a candidate gene for vertebral malformations and congenital scoliosis.

Human vertebral malformations (VMs) have an estimated incidence of 1/2000 and are associated with significant health problems including congenital scoliosis (CS) and recurrent organ system malformation syndromes such as VACTERL (vertebral anomalies; anal abnormalities; cardiac abnormalities; tracheo-esophageal fistula; renal anomalies; limb anomalies). The genetic cause for the vast majority of VMs are unknown. In a CS/VM patient cohort, three COL11A2 variants (R130W, R1407L and R1413H) were identified in two patients with cervical VM. A third patient with a T9 hemivertebra and the R130W variant was identified from a separate study. These substitutions are predicted to be damaging to protein function, and R130 and R1407 residues are conserved in zebrafish Col11a2. To determine the role for COL11A2 in vertebral development, CRISPR/Cas9 was used to create a nonsense mutation (col11a2L642*) as well as a full gene locus deletion (col11a2del) in zebrafish. Both col11a2L642*/L642* and col11a2del/del mutant zebrafish exhibit vertebral fusions in the caudal spine, which form due to mineralization across intervertebral segments. To determine the functional consequence of VM-associated variants, we assayed their ability to suppress col11a2del VM phenotypes following transgenic expression within the developing spine. While wildtype col11a2 expression suppresses fusions in col11a2del/+ and col11a2del/del backgrounds, patient missense variant-bearing col11a2 failed to rescue the loss-of-function phenotype in these animals. These results highlight an essential role for COL11A2 in vertebral development and support a pathogenic role for two missense variants in CS.

A novel fasting regimen revealed protein reservation and complement C3 down-regulation after 14-day's continual dietary deprivation.

Objectives: The aim is to evaluate the effect of a novel 14-day fasting regimen on the balance between skeletal muscle and adipose tissue composition which might associate with inflammatory factors. Our analysis includes basic physical examinations, clinical laboratory analysis, bioelectrical impedance and biochemical analytic assessments of healthy volunteers. Methods: Eight healthy subjects were randomly selected from a pool of volunteers to undergo a continual dietary deprivation (CDD) regimen. Individuals were assigned to take Flexible Abrosia (FA, prebiotic combination) plus appropriate mineral supplement of potassium and magnesium at 3 mealtime every day to prevent potential injury from starved intestinal flora and avoid spasms of smooth muscle due to hunger. Physical and medical examinations were conducted and blood samples were collected at following timepoints: before CDD as self-control (0D), day 7 and day 14 during fasting, and 7-21days and/or 2~3mo after refeeding. Results: The combination of FA and mineral supplements significantly decreased self-reported physical response of starvation, with tolerable hunger-mediated sensations experienced during CDD. Bioelectrical and biochemical results indicated significant reduction in both muscle lean and fat mass on day 7. Meanwhile, markers related to fat composition consistently decreased during and after CDD. In addition, most biochemical marker levels, including serum proteins, reached their inflection points at the 7th day of CDD as compared to the control measurements. Levels of these factors started to show a relative plateau, or reversed direction upon the 14th day of CDD. The exceptions of above factors were myostatin and complement protein C3, which remained at lower concentrations in the blood throughout CDD, and were unable to fully recover toward baseline levels even after 3 months' refeeding. Conclusion: Our results indicated that human subjects undergoing prolonged dietary restriction were well protected by FA and mineral ions from gut injury or physical discomfort of starvation. Most factors showed a relative plateau response at the end of 14D-CDD. The muscle tissues were well preserved during prolonged fasting, and an improved protein/lipid ratio was observed. Upon refeeding, constant lower levels of myostatin and complement C3 were maintained after CDD implies a long-term beneficial effect in dealing with anti-aging and inflammation.

Structured diagnostic scheme clinical experience sharing: a prospective study of 320 cases of fever of unknown origin in a tertiary hospital in North China.

BACKGROUND: There has been little research on the long-term clinical outcomes of patients discharged due to undiagnosed fevers of unknown origin (FUO). The purpose of this study was to determine how fever of unknown origin (FUO) evolves over time and to determine the prognosis of patients in order to guide clinical diagnosis and treatment decisions. METHODS: Based on FUO structured diagnosis scheme, prospectively included 320 patients who hospitalized at the Department of Infectious Diseases of the Second Hospital of Hebei Medical University from March 15, 2016 to December 31,2019 with FUO, to analysis the cause of FUO, pathogenetic distribution and prognosis, and to compare the etiological distribution of FUO between different years, genders, ages, and duration of fever. RESULTS: Among the 320 patients, 279 were finally diagnosed through various types of examination or diagnostic methods, and the diagnosis rate was 87.2%. Among all the causes of FUO, 69.3% were infectious diseases, of which Urinary tract infection 12.8% and lung infection 9.7% were the most common. The majority of pathogens are bacteria. Among contagious diseases, brucellosis is the most common. Non-infectious inflammatory diseases were responsible for 6.3% of cases, of which systemic lupus erythematosus(SLE) 1.9% was the most common; 5% were neoplastic diseases; 5.3% were other diseases; and in 12.8% of cases, the cause was unclear. In 2018-2019, the proportion of infectious diseases in FUO was higher than 2016-2017 (P < 0.05). The proportion of infectious diseases was higher in men and older FUO than in women and young and middle-aged (P < 0.05). According to follow-up, the mortality rate of FUO patients during hospitalization was low at 1.9%. CONCLUSIONS: Infectious diseases are the principal cause of FUO. There are temporal differences in the etiological distribution of FUO, and the etiology of FUO is closely related to the prognosis. It is important to identify the etiology of patients with worsening or unrelieved disease.