SR9009 attenuates inflammation-related NPMSC pyroptosis and IVDD through NR1D1/NLRP3/IL-1ß pathway.

Intervertebral disc is a highly rhythmical tissue. As a key factor linking biorhythm and inflammatory response, the shielding effect of NR1D1 in the process of intervertebral disc degeneration remains unclear. Here, we first confirmed that NR1D1 in the nucleus pulposus tissue presents periodic rhythmic changes and decreases in expression with intervertebral disc degeneration. Second, when NR1D1 was activated by SR9009 in vitro, NLRP3 inflammasome assembly and IL-1ß production were inhibited, while ECM synthesis was increased. Finally, the vivo experiments further confirmed that the activation of NR1D1 can delay the process of disc degeneration to a certain extent. Mechanistically, we demonstrate that NR1D1 can bind to IL-1ß and NLRP3 promoters, and that the NR1D1/NLRP3/IL-1ß pathway is involved in this process. Our results demonstrate that the activation of NR1D1 can effectively reduce IL-1ß secretion, alleviate LPS-induced NPMSC pyroptosis, and protect ECM degeneration.

Vitamin C alleviates rheumatoid arthritis by modulating gut microbiota balance.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic and symmetric in-flammation. Our previous research revealed an imbalance in the gut flora of RA patients and showed that certain gut microbiota can accelerate RA progression by enhancing vitamin C degradation. However, it is unclear whether vitamin C supplementation could improve the gut microbiota to prevent the development of arthritis by interfering with the gut-joint axis. In this work, we aimed to evaluate the effects of vitamin C in regulating the gut microbiota and to elucidate its potential role in the onset and progression of RA in a mouse model, thus providing a basis for the development of new intervention strategies and treatments for RA. In this study, collagen-induced arthritis (CIA) mouse models, biochemical, histological and 16S rRNA microbiological methods were used to investigate the role and possible mechanism of vitamin C in rheumatoid arthritis. The results showed that treatment of CIA mice with vitamin C effectively rescued the gut mi-crobiota imbalance and suppressed the inflammatory response associated with RA, and effectively alleviated arthritis symptoms in mice in which levels of the pro-inflammatory cytokines IL-6 and TNF-α were specifi-cally reduced. In conclusion, our results demonstrate the potential of vitamin C as a potential therapeutic choice for RA.

Characterization of gene regulatory networks underlying key properties in human hematopoietic stem cell ontogeny.

Human hematopoiesis starts at early yolk sac and undergoes site- and stage-specific changes over development. The intrinsic mechanism underlying property changes in hematopoiesis ontogeny remains poorly understood. Here, we analyzed single-cell transcriptome of human primary hematopoietic stem/progenitor cells (HSPCs) at different developmental stages, including yolk-sac (YS), AGM, fetal liver (FL), umbilical cord blood (UCB) and adult peripheral blood (PB) mobilized HSPCs. These stage-specific HSPCs display differential intrinsic properties, such as metabolism, self-renewal, differentiating potentialities etc. We then generated highly co-related gene regulatory network (GRNs) modules underlying the differential HSC key properties. Particularly, we identified GRNs and key regulators controlling lymphoid potentiality, self-renewal as well as aerobic respiration in human HSCs. Introducing selected regulators promotes key HSC functions in HSPCs derived from human pluripotent stem cells. Therefore, GRNs underlying key intrinsic properties of human HSCs provide a valuable guide to generate fully functional HSCs in vitro.

Autophagy is essential for human myelopoiesis.

Emergency myelopoiesis (EM) is essential in immune defense against pathogens for rapid replenishing of mature myeloid cells. During the EM process, a rapid cell-cycle switch from the quiescent hematopoietic stem cells (HSCs) to highly proliferative myeloid progenitors (MPs) is critical. How the rapid proliferation of MPs during EM is regulated remains poorly understood. Here, we reveal that ATG7, a critical autophagy factor, is essential for the rapid proliferation of MPs during human myelopoiesis. Peripheral blood (PB)-mobilized hematopoietic stem/progenitor cells (HSPCs) with ATG7 knockdown or HSPCs derived from ATG7-/- human embryonic stem cells (hESCs) exhibit severe defect in proliferation during fate transition from HSPCs to MPs. Mechanistically, we show that ATG7 deficiency reduces p53 localization in lysosome for a potential autophagy-mediated degradation. Together, we reveal a previously unrecognized role of autophagy to regulate p53 for a rapid proliferation of MPs in human myelopoiesis.

Melatonin alleviates oxidative stress-induced injury to nucleus pulposus-derived mesenchymal stem cells through activating PI3K/Akt pathway.

Background: The changes in the microenvironment of degenerative intervertebral discs cause oxidative stress injury and excessive apoptosis of intervertebral disc endogenous stem cells. The purpose of this study was to explore the possible mechanism of the protective effect of melatonin on oxidative stress injury in NPMSCs induced by H2O2. Methods: The Cell Counting Kit-8 assay was used to evaluate the cytotoxicity of hydrogen peroxide and the protective effects of melatonin. ROS content was detected by 2'7'-dichlorofluorescin diacetate (DCFH-DA). Mitochondrial membrane potential (MMP) was detected by the JC-1assay. Transferase mediated d-UTP Nick end labeling (TUNEL) and Annexin V/PI double staining were used to determine the apoptosis rate. Additionally, apoptosis-associated proteins and PI3K/Akt signaling pathway-related proteins were evaluated by immunofluorescence, immunoblotting and PCR. ECMs were evaluated by RT‒PCR and immunofluorescence. In vivo, X-ray, Magnetic resonance imaging (MRI) and Histological analyses were used to evaluate the protective effect of melatonin. Results: Melatonin had an obvious protective effect on NPMSCs treated with 0-10 µM melatonin for 24 h. In addition, melatonin also had obvious protective effects on mitochondrial dysfunction, decreased membrane potential and cell senescence induced by H2O2. More importantly, melatonin could significantly reduce the apoptosis of nucleus pulposus mesenchymal stem cells induced by H2O2 by regulating the expression of apoptosis-related proteins and decreasing the rate of apoptosis. After treatment with melatonin, the PI3K/Akt pathway was significantly activated in nucleus pulposus mesenchymal stem cells, while the protective effect was significantly weakened after PI3K-IN-1 treatment. In vivo, the results of X-ray, MRI and histological analyses showed that therapy with melatonin could partially reduce the degree of intervertebral disc degeneration. Conclusion: Our research demonstrated that melatonin can effectively alleviate the excessive apoptosis and mitochondrial dysfunction of nucleus pulposus mesenchymal stem cells induced by oxidative stress via the PI3K/Akt pathway, which provides a novel idea for the therapy of intervertebral disc degeneration. The translational potential of this article: This study indicates that melatonin can effectively alleviate the excessive apoptosis and mitochondrial dysfunction of NPMSCs through activating the PI3K/Akt pathway. Melatonin might serve as a promising candidate for the prevention and treatment of Intervertebral disc degeneration disease (IVDD) in the future.

Significance of cuproptosis-related lncRNA signature in LUAD prognosis and immunotherapy: A machine learning approach.

OBJECTIVE: Cuproptosis-related genes are closely related to lung adenocarcinoma (LUAD), which can be analyzed via the analysis of long noncoding RNA (lncRNA). To date, the clinical significance and function of cuproptosis-related lncRNAs are still not well elucidated. Further analysis of cuproptosis-related prognostic lncRNAs is of great significance for the treatment, diagnosis, and prognosis of LUAD. METHODS: In this study, a multiple machine learning (ML)-based computational approach was proposed for the identification of the cuproptosis-related lncRNAs signature (CRlncSig) via comprehensive analysis of cuproptosis, lncRNAs, and clinical characteristics. The proposed approach integrated multiple ML algorithms (least absolute shrinkage and selection operator regression analysis, univariate and multivariate Cox regression) to effectively identify the CRlncSig. RESULTS: Based on the proposed approach, the CRlncSig was identified from the 3450 cuproptosis-related lncRNAs, which consist of 13 lncRNAs (CDKN2A-DT, FAM66C, FAM83A-AS1, AL359232.1, FRMD6-AS1, AC027237.4, AC023090.1, AL157888.1, AL627443.3, AC026355.2, AC008957.1, AP000346.1, and GLIS2-AS1). CONCLUSIONS: The CRlncSig could well predict the prognosis of different LUAD patients, which is different from other clinical features. Moreover, the CRlncSig was proved to be an effective indicator of patient survival via functional characterization analysis, which is relevant to cancer progression and immune infiltration. Furthermore, the results of RT-PCR assay indicated that the expression level of FAM83A-AS1 and AC026355.2 in A549 and H1975 cells (LUAD) was significantly higher than that in BEAS-2B cells (normal lung epithelial).

A regionally adapted HRM-based technique to screen MMACHC carriers for methylmalonic acidemia with homocystinuria in Shandong Province, China.

Methylmalonic acidemia with homocystinuria (MMA-cblC) is an autosomal recessive genetic disorder of organic acid metabolism. Shandong, a northern province of China, has a significantly high incidence of about 1/4,000, suggesting a high carrying rate among the local population. The current study established a PCR technique involving high-resolution melting (HRM) to screen for carriers based on hotspot mutation analysis to further develop a preventive strategy to reduce the local incidence of this rare disease. Whole-exome sequencing of 22 families with MMA-cblC and a comprehensive literature review were used to identify MMACHC hotspot mutations in Shandong Province. Subsequently, a PCR-HRM assay based on the selected mutations was established and optimized for large-scale hotspot mutation screening. The accuracy and efficiency of the screening technique was validated using samples from 69 individuals with MMA-cblC and 1,000 healthy volunteers. Six hotspot mutations in the MMACHC gene (c.609G>A, c.658_660delAAG, c.80A>G, c.217C>T, c.567dupT and c.482G>A), which account for 74% of the alleles associated with MMA-cblC, were used to establish a screening technique. The established PCR-HRM assay detected 88 MMACHC mutation alleles in a validation study with 100% accuracy. In the general population in Shandong, the carrying rate of 6 MMACHC hotspot mutations was 3.4%. In conclusion, the 6 hotspots identified cover the majority of the MMACHC mutation spectrum, and the Shandong population has a particularly high carrying rate of MMACHC mutations. The PCR-HRM assay is highly accurate, cost-effective, and easy to use, making it an ideal choice for mass carrier screening.

Metabolic disorder and intestinal microflora dysbiosis in chronic inflammatory demyelinating polyradiculoneuropathy.

OBJECTIVE: Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a rare acquired immune-mediated neuropathy. Although microbial infection is potentially a contributing factor, a causative link between CIDP and microbial infection remains unclear. There is also no definitive biomarker for CIDP diagnostics and therapies. The present study aimed to characterize the serum metabolic profile and gut microbiome structure in CIDP. METHODS: Targeted metabolomics profiling of serum, using liquid chromatography-mass spectrometry, and metagenomics sequencing of stool samples from a cohort of CIDP and non-CIDP subjects were performed to evaluate serum metabolic profiles and gut microbiome structure in CIDP subjects relative to healthy controls. RESULTS: Metabolome data revealed that the bile acids profile was perturbed in CIDP with bile acids and arachidonic acid enriched significantly in CIDP versus non-CIDP controls. Metagenome data revealed that opportunistic pathogens, such as Klebsiella pneumonia and Megamonas funiformis, and genes involved in bacterial infection were notably more abundant in CIDP subjects, while gut microbes related to biotransformation of secondary bile acids were abnormal in CIDP versus non-CIDP subjects. Correlation analysis revealed that changes in secondary bile acids were associated with altered gut microbes, including Bacteroides ovatus, Bacteroides caccae, and Ruminococcus gnavus. CONCLUSION: Bile acids and arachidonic acid metabolism were disturbed in CIDP subjects and might be affected by the dysbiosis of gut microbial flora. These findings suggest that the combination of bile acids and arachidonic acid could be used as a CIDP biomarker and that modulation of gut microbiota might impact the clinical course of CIDP.

Association of maternal TSH and neonatal metabolism: A large prospective cohort study in China.

Aims: Neonatal metabolites are very important in neonatal disease screening, and maternal thyroid hormones play an important role in fetal and neonatal health. Our study aimed to explore the association of maternal thyroid hormones with neonatal metabolites and identify an important time windows. Methods: Pregnant women were recruited in Jinan Maternity and Child Care Hospital and followed up until delivery. Multivariate generalized linear regression models (GLMs) and restricted cubic spline (RCS) regression analysis models were used to investigate the associations of maternal TSH and FT4 with neonatal metabolites. Results: In total, 6,653 pairs of mothers and newborns were enrolled in our study. We identified 5 neonatal metabolites, including arginine/ornithine (Arg/Orn), C14:1/C2, C18:1, C3DC+C4OH and C8:1, that were significantly associated with maternal serum TSH during the whole pregnancy (P < 0.05), especially in the first trimester. Moreover, 10 neonatal metabolites were significantly associated with maternal serum FT4 (P < 0.05), most of which had positive correlations with maternal FT4 in the first trimester (P < 0.05). Some neonatal metabolites also had linear or nonlinear dose-effect relationships with maternal serum TSH and FT4 during the whole pregnancy, particularly in the first trimester. Conclusions: Our study, for the first time, provides epidemiological evidence that maternal serum TSH and FT4, especially during the first trimester, are associated with linear or nonlinear variations in neonatal metabolites. Efforts to identify newborn metabolism levels should carefully consider the effects of maternal thyroid function.

Stage-specific roles of microbial dysbiosis and metabolic disorders in rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is a progressive disease including four stages, where gut microbiome is associated with pathogenesis. We aimed to investigate stage-specific roles of microbial dysbiosis and metabolic disorders in RA. METHODS: We investigated stage-based profiles of faecal metagenome and plasma metabolome of 76 individuals with RA grouped into four stages (stages I-IV) according to 2010 RA classification criteria, 19 individuals with osteroarthritis and 27 healthy individuals. To verify bacterial invasion of joint synovial fluid, 16S rRNA gene sequencing, bacterial isolation and scanning electron microscopy were conducted on another validation cohort of 271 patients from four RA stages. RESULTS: First, depletion of Bacteroides uniformis and Bacteroides plebeius weakened glycosaminoglycan metabolism (p<0.001), continuously hurting articular cartilage across four stages. Second, elevation of Escherichia coli enhanced arginine succinyltransferase pathway in the stage II and stage III (p<0.001), which was correlated with the increase of the rheumatoid factor (p=1.35×10-3) and could induce bone loss. Third, abnormally high levels of methoxyacetic acid (p=1.28×10-8) and cysteine-S-sulfate (p=4.66×10-12) inhibited osteoblasts in the stage II and enhanced osteoclasts in the stage III, respectively, promoting bone erosion. Fourth, continuous increase of gut permeability may induce gut microbial invasion of the joint synovial fluid in the stage IV. CONCLUSIONS: Clinical microbial intervention should consider the RA stage, where microbial dysbiosis and metabolic disorders present distinct patterns and played stage-specific roles. Our work provides a new insight in understanding gut-joint axis from a perspective of stages, which opens up new avenues for RA prognosis and therapy.

Development of a human iPSC line (SMBCi017-A) from a healthy donor.

The human induced pluripotent stem cell (iPSC) line SMBCi017-A was derived from urine cells of a 20 year old male healthy control donor. The generated iPSCs had normal karyotype, expressed chromosomal stability pluripotency hallmarks and differentiated into the three primary germ layers.

Generation of a non-integrated induced pluripotent stem cell line from urine cells of a Chinese osteogenesis imperfecta type I patient.

Osteogenesis imperfecta (OI) is a group of genetic disorders characterized mainly by fractures and bone deformities. It has been established that gene mutations, particularly those in COL1A1 and COL1A2, account for most phenotypes. Here, we generated an induced pluripotent stem cells (iPSCs) line named SMBCi014-A using urine cells (UCs) derived from a 15-year-old female OI type I patient who carried the frame-shift mutation of the COL1A1 gene (exon35:c.2450delC:p.P817fs). The patient had a family history of mild fractures and a blue sclera. Therefore, our study established a patient-derived site-specific cellular model of OI to better understand the osteogenic mechanism.

Generation of induced pluripotent stem cells named SMBCi019-A from a methylmalonic acidemia patient carrying the MMACHC mutations.

Methylmalonic acidemia(MMA) is an autosomal recessive hereditary disease caused by methylmalonyl-CoA mutase defect or its coenzyme cobalamin metabolism defect. The mutation of the MMACHC gene leads to metabolic disorder of coenzyme cobalamin, resulting in abnormal accumulation of methylmalonic acid, and finally leads to impairment of multiple organs' functions. Here we generated an induced pluripotent stem cells (iPSCs) line named SMBCi019-A, using urine cells (UCs) derived from a 10-year-old male MMA patient who carried two heterozygous gene mutations in MMACHC c.438G > A (p.w146x) and c.609G > A (p.w203x). The generated iPSCs retained the mutations can function as a cellular model of MMA.

Establishment of a control induced pluripotent stem cell line SMBCi018-A from a patient with congenital talipes equinovarus.

Congenital talipes equinovarus (CTEV) is a congenital malformation affecting approximately 1/700-1/1000 of live borns. To date extensive epidemiological and biological studies have been operated to solve this issue, the most meaningful findings in clubfoot genetics involve PITX1 variants, which were associated with clubfoot phenotype in mice and humans. According to recent studies, the PITX1-TBX4 transcriptional pathway regulatory for early limb development has identified a key developmental pathway in clubfoot etiology by the common disease-rare hypothesis. However, the precise mechanisms causing this disease remain elusive. The pluripotent stem cell line SMBCi018-A will enable proper in vitro disease modeling of CTEV.

Generation of a transgene-free induced pluripotent stem cell line (SMBCi011-A) from a patient with Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder caused by deficiency of paternal gene expression in the 15q11.2-q13 chromosome imprinted region. Hyperphagia and dysgnosia are typical clinical features in the early-childhood of patient. We generated an induced pluripotent stem cell (iPSC) line SMBCi011-A from a 6 years old male PWS patient, the line expressed pluripotent signs and had ability to differentiate into three germ layers in vivo.

Penfluridol targets acid sphingomyelinase to inhibit TNF signaling and is therapeutic against inflammatory autoimmune diseases.

BACKGROUND: Penfluridol, isolated from an FDA-approved small-molecule drug library as an inhibitor of tumor necrosis factor α (TNFα)-stimulated NF-κB activation, is clinically used to treat chronic schizophrenia and related disorders. This study is aimed to investigate the therapeutic effect of penfluridol on TNFα-stimulated inflammatory autoimmune diseases, particularly inflammatory arthritis. METHODS: Various in vitro studies to confirm the inhibitory effect of penfluridol on TNFα-induced NF-κB activity in bone marrow-derived macrophages or Raw 264.7 macrophage cell line. In vivo studies assessed the therapeutic effects of penfluridol in various disease models, including TNFα transgenic mice, collagen-induced arthritis, DSS-induced colitis, and TNBS-induced colitis. Identification and characterization of the binding of penfluridol to acid sphingomyelinase using bioinformatics and drug affinity responsive target stability assay. Acid sphingomyelinase activity assays to reveal penfluridol-mediated inhibition of acid sphingomyelinase activity. siRNA knockdown experiments to illustrate the dependence of penfluridol's anti-TNF activity on acid sphingomyelinase. RESULTS: Penfluridol effectively inhibited TNFα-induced NF-κB activation in vitro and alleviated the severity of arthritis and colitis in vivo. Mechanistic studies revealed that penfluridol bound to acid sphingomyelinase and inhibited its activation. In addition, knockdown of acid sphingomyelinase largely abolished the inhibitory effects of penfluridol on TNFα-induced inflammatory cytokine production. Furthermore, penfluridol suppressed the differentiation of spleen naive CD4+T cells to TH1 and TH17 and inhibited M1 macrophage polarization. CONCLUSION: This study provides the rationale for the possible innovative use of penfluridol as a newly identified small-molecule drug for TNFα-driven diseases, such as inflammatory arthritis and colitis.

Generation of a non-integrated induced pluripotent stem cell line (SMBCi009-A) from urine-derived cells of a Chinese Familial hypercholesterolemia patient.

Familial hypercholesterolemia (FH; OMIM: # 143890) is a common inherited autosomal dominant disease, characterized by high-level low-density lipoprotein cholesterol (LDL-C) in plasma. Elevated LDL-C levels is closely related with atherosclerotic plaques and premature cardiovascular disease if not treated in time. Here we generated an induced pluripotent stem cell (iPSC) line using urine cells (UCs) derived from an 8-year-old male FH patient who carrying two coding and pathogenic mutations of low-density lipoprotein receptor (LDLR) gene (exon12:c.C1747T and exon13: c. 1948 del G). This induced pluripotent stem cell line named SMBCi009-A can be used to understand more cellular details about FH.

Development and identification of a induced pluripotent stem cells line (SMBCi013-A) derived from urine cells of a patient with Wilson's disease.

The human induced pluripotent stem cell (iPSC) line SMBCi013-A was derived from urine cells of a 29 year old male affected by Wilson's disease. Two heterozygous mutations in the ATP7B gene were detected by whole exon sequencing (c.2303C > A and c.3334C > T). The reprogramming factors (OCT-4, SOX-2, KLF4, miR-302-367) were delivered using non-integrating episomal plasmids. The resulting iPSCs were pluripotent, have chromosomal stability, and the ability to differentiate into the three germ layers.