Fluorescent monitoring osteogenic differentiation of osteosarcoma cells with an aggregation-induced emission probe.

Osteosarcoma is widely believed to be an osteogenic differentiation disorder. In recent years, to further understand this disease, a lot resources were poured into the potential link between differentiation defects and tumorigenesis. Long-term monitoring of the differentiation progress of osteosarcoma cells is of great importance. In order to better promote the research, we have developed a novel fluorescent probe called PTB-EDTA, which exhibits remarkable bio-compatibility and demonstrates high selectivity towards osteosarcoma cells. Not only is the PTB-EDTA is capable of live cell imaging while conventional histology requires to kill the cells, its fluorescence is also enhanced as the osteogenic differentiation proceeding. These properties make PTB-EDTA a promising tool for monitoring osteosarcoma cells.

Bioinformatics analysis and experimental verification of the cancer-promoting effect of DHODH in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is a malignant tumor of the urinary system. To explore the potential mechanisms of DHODH in ccRCC, we analyzed its molecular characteristics using public databases. TCGA pan-cancer dataset was used to analyze DHODH expression in different cancer types and TCGA ccRCC dataset was used to assess differential expression, prognosis correlation, immune infiltration, single-gene, and functional enrichment due to DHODH. The GSCALite and CellMiner databases were employed to explore drugs and perform molecular docking analysis with DHODH. Protein-protein interaction networks and ceRNA regulatory networks of DHODH were constructed using multiple databases. The effect of DHODH on ccRCC was confirmed in vitro. DHODH was highly expressed in ccRCC. Immune infiltration analysis revealed that DHODH may be involved in regulating the infiltration of immunosuppressive cells such as Tregs. Notably, DHODH influenced ccRCC progression by forming regulatory networks with molecules, such as hsa-miR-26b-5p and UMPS and significantly enhanced the malignant characteristics of ccRCC cells. Several drugs, such as lapatinib, silmitasertib, itraconazole, and dasatinib, were sensitive to DHODH expression and exhibited strong molecular binding with it. Thus, DHODH may promote ccRCC progression and is a candidate effective therapeutic target for ccRCC.

Stochastic distinguishability of Markovian trajectories.

The ability to distinguish between stochastic systems based on their trajectories is crucial in thermodynamics, chemistry, and biophysics. The Kullback-Leibler (KL) divergence, DKLAB(0,τ), quantifies the distinguishability between the two ensembles of length-τ trajectories from Markov processes A and B. However, evaluating DKLAB(0,τ) from histograms of trajectories faces sufficient sampling difficulties, and no theory explicitly reveals what dynamical features contribute to the distinguishability. This work provides a general formula that decomposes DKLAB(0,τ) in space and time for any Markov processes, arbitrarily far from equilibrium or steady state. It circumvents the sampling difficulty of evaluating DKLAB(0,τ). Furthermore, it explicitly connects trajectory KL divergence with individual transition events and their waiting time statistics. The results provide insights into understanding distinguishability between Markov processes, leading to new theoretical frameworks for designing biological sensors and optimizing signal transduction.

Nr4a1 enhances Wnt4 transcription to promote mesenchymal stem cell osteogenesis and alleviates inflammation-inhibited bone regeneration.

Intense inflammatory response impairs bone marrow mesenchymal stem cell (BMSC)-mediated bone regeneration, with transforming growth factor (TGF)-ß1 being the most highly expressed cytokine. However, how to find effective and safe means to improve bone formation impaired by excessive TGF-ß1 remains unclear. In this study, we found that the expression of orphan nuclear receptor Nr4a1, an endogenous repressor of TGF-ß1, was suppressed directly by TGF-ß1-induced Smad3 and indirectly by Hdac4, respectively. Importantly, Nr4a1 overexpression promoted BMSC osteogenesis and reversed TGF-ß1-mediated osteogenic inhibition and pro-fibrotic effects. Transcriptomic and histologic analyses confirmed that upregulation of Nr4a1 increased the transcription of Wnt family member 4 (Wnt4) and activated Wnt pathway. Mechanistically, Nr4a1 bound to the promoter of Wnt4 and regulated its expression, thereby enhancing the osteogenic capacity of BMSCs. Moreover, treatment with Nr4a1 gene therapy or Nr4a1 agonist Csn-B could promote ectopic bone formation, defect repair, and fracture healing. Finally, we demonstrated the correlation of NR4A1 with osteogenesis and the activation of the WNT4/ß-catenin pathway in human BMSCs and fracture samples. Taken together, these findings uncover the critical role of Nr4a1 in bone formation and alleviation of inflammation-induced bone regeneration disorders, and suggest that Nr4a1 has the potential to be a therapeutic target for accelerating bone healing.

n-3 polyunsaturated fatty acids delay intervertebral disc degeneration by inhibiting nuclear receptor coactivator 4-mediated iron overload.

n-3 polyunsaturated fatty acids (PUFAs) are closely related to the progression of numerous chronic inflammatory diseases, but the role of n-3 PUFAs in the intervertebral disc degeneration (IVDD) remains unclear. In this study, male C57BL/6 wildtype mice (WT group, n = 30) and fat-1 transgenic mice (TG group, n = 30) were randomly selected to construct the IVDD model. The results demonstrated that the optimized composition of PUFAs in the TG mice had a significant impact on delaying IVDD and cellular senescence of intervertebral disc (IVD). Mechanismly, n-3 PUFAs inhibited IVD senescence by alleviating NCOA4-mediated iron overload. NCOA4 overexpression promoted iron overload and weakened the pro-proliferation and anti-senescence effect of DHA on the IVD cells. Furthermore, this study futher revealed n-3 PUFAs downregulated NCOA4 expression by inactiviting the LGR5/ß-catenin signaling pathway. This study provides an important theoretical basis for preventing and treating IVDD and low back pain.

Whitening of brown adipose tissue inhibits osteogenic differentiation via secretion of S100A8/A9.

The mechanism by which brown adipose tissue (BAT) regulates bone metabolism is unclear. Here, we reveal that BAT secretes S100A8/A9, a previously unidentified BAT adipokine (batokine), to impair bone formation. Brown adipocytes-specific knockout of Rheb (RhebBAD KO), the upstream activator of mTOR, causes BAT malfunction to inhibit osteogenesis. Rheb depletion induces NF-κB dependent S100A8/A9 secretion from brown adipocytes, but not from macrophages. In wild-type mice, age-related Rheb downregulation in BAT is associated with enhanced S100A8/A9 secretion. Either batokines from RhebBAD KO mice, or recombinant S100A8/A9, inhibits osteoblast differentiation of mesenchymal stem cells in vitro by targeting toll-like receptor 4 on their surfaces. Conversely, S100A8/A9 neutralization not only rescues the osteogenesis repressed in the RhebBAD KO mice, but also alleviates age-related osteoporosis in wild-type mice. Collectively, our data revealed an unexpected BAT-bone crosstalk driven by Rheb-S100A8/A9, uncovering S100A8/A9 as a promising target for the treatment, and potentially, prevention of osteoporosis.

Thrombospondin-1 promotes mechanical stress-mediated ligamentum flavum hypertrophy through the TGFß1/Smad3 signaling pathway.

Lumbar spinal canal stenosis is primarily caused by ligamentum flavum hypertrophy (LFH), which is a significant pathological factor. Nevertheless, the precise molecular basis for the development of LFH remains uncertain. The current investigation observed a notable increase in thrombospondin-1 (THBS1) expression in LFH through proteomics analysis and single-cell RNA-sequencing analysis of clinical ligamentum flavum specimens. In laboratory experiments, it was demonstrated that THBS1 triggered the activation of Smad3 signaling induced by transforming growth factor ß1 (TGFß1), leading to the subsequent enhancement of COL1A2 and α-SMA, which are fibrosis markers. Furthermore, experiments conducted on a bipedal standing mouse model revealed that THBS1 played a crucial role in the development of LFH. Sestrin2 (SESN2) acted as a stress-responsive protein that suppressed the expression of THBS1, thus averting the progression of fibrosis in ligamentum flavum (LF) cells. To summarize, these results indicate that mechanical overloading causes an increase in THBS1 production, which triggers the TGFß1/Smad3 signaling pathway and ultimately results in the development of LFH. Targeting the suppression of THBS1 expression may present a novel approach for the treatment of LFH.

Discovery of Taurocholic Acid Sodium Hydrate as a Novel Repurposing Drug for Intervertebral Disc Degeneration by Targeting MAPK3.

OBJECTIVE: Nowadays, more than 90% of people over 50 years suffer from intervertebral disc degeneration (IDD), but there are exist no ideal drugs. The aim of this study is to identify a new drug for IDD. METHODS: An approved small molecular drug library including 2040 small molecular compounds was used here. We found that taurocholic acid sodium hydrate (NAT) could induce chondrogenesis and osteogenesis in mesenchymal stem cells (MSCs). Then, an in vivo mouse model of IDD was established and the coccygeal discs transcriptome analysis and surface plasmon resonance analysis (SPR) integrated with liquid chromatography-tandem mass spectrometry assay (LC-MS) were performed in this study to study the therapy effect and target proteins of NAT for IDD. Micro-CT was used to evaluate the cancellous bone. The expression of osteogenic (OCN, RNX2), chondrogenic (COL2A1, SOX9), and the target related (ERK1/2, p-ERK1/2) proteins were detected. The alkaline phosphatase staining was performed to estimate osteogenic differentiation. Blood routine and blood biochemistry indexes were analyzed for the safety of NAT. RESULTS: The results showed that NAT could induce chondrogenesis and osteogenesis in MSCs. Further experiments confirmed NAT could ameliorate the secondary osteoporosis and delay the development of IDD in mice. Transcriptome analysis identified 128 common genes and eight Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for NAT. SPR-LC-MS assay detected 57 target proteins for NAT, including MAPK3 (mitogen-activated protein kinase 3), also known as ERK1 (extracellular regulated protein kinase 1). Further verification experiment confirmed that NAT significantly reduced the expression of ERK1/2 phosphorylation. CONCLUSION: NAT would induce chondrogenesis and osteogenesis of MSCs, ameliorate the secondary osteoporosis and delay the progression of IDD in mice by targeting MAPK3.Furthermore, MAPK3, especially the phosphorylation of MAPK3, would be a potential therapeutic target for IDD treatment.

Epidemiological and clinical features, treatment status, and economic burden of traumatic spinal cord injury in China: a hospital-based retrospective study.

Traumatic spinal cord injury is potentially catastrophic and can lead to permanent disability or even death. China has the largest population of patients with traumatic spinal cord injury. Previous studies of traumatic spinal cord injury in China have mostly been regional in scope; national-level studies have been rare. To the best of our knowledge, no national-level study of treatment status and economic burden has been performed. This retrospective study aimed to examine the epidemiological and clinical features, treatment status, and economic burden of traumatic spinal cord injury in China at the national level. We included 13,465 traumatic spinal cord injury patients who were injured between January 2013 and December 2018 and treated in 30 hospitals in 11 provinces/municipalities representing all geographical divisions of China. Patient epidemiological and clinical features, treatment status, and total and daily costs were recorded. Trends in the percentage of traumatic spinal cord injuries among all hospitalized patients and among patients hospitalized in the orthopedic department and cost of care were assessed by annual percentage change using the Joinpoint Regression Program. The percentage of traumatic spinal cord injuries among all hospitalized patients and among patients hospitalized in the orthopedic department did not significantly change overall (annual percentage change, -0.5% and 2.1%, respectively). A total of 10,053 (74.7%) patients underwent surgery. Only 2.8% of patients who underwent surgery did so within 24 hours of injury. A total of 2005 (14.9%) patients were treated with high-dose (≥ 500 mg) methylprednisolone sodium succinate/methylprednisolone (MPSS/MP); 615 (4.6%) received it within 8 hours. The total cost for acute traumatic spinal cord injury decreased over the study period (-4.7%), while daily cost did not significantly change (1.0% increase). Our findings indicate that public health initiatives should aim at improving hospitals' ability to complete early surgery within 24 hours, which is associated with improved sensorimotor recovery, increasing the awareness rate of clinical guidelines related to high-dose MPSS/MP to reduce the use of the treatment with insufficient evidence.

Probability and predictors of long-term smoking relapse among Chinese adult smokers: A longitudinal study.

Numerous smokers attempt to quit smoking, but most cessation efforts prove unsuccessful. Scarce evidence exists regarding predictors of long-term relapse in China. This study aims to evaluate the probability of relapse and examine factors may contribute to relapse among Chinese adults. A dynamic cohort of 6,036 observations on 2,378 adult quitters was constructed from the China Family Panel Studies in 2010, 2012, 2014, 2016 and 2018. The life table method was employed to calculate the probability of relapse for long-term smoking abstinence. Multivariate complementary log-log survival models were developed to examine the predictors of smoking relapse. We found that the probability of relapse decreased as the duration of abstinence increased, with rates of 49.07 %, 20.05 %, 10.29 %, and 6.63 % at 2, 4, 6, and 8 years of abstinence, respectively. The cumulative probability of relapse within 8 years was 65.89 %. Age ≥65 years, higher educational attainment, respiratory disease, and a satisfying lifestyle were associated with a reduced likelihood of relapse. Conversely, higher occupational prestige, alcohol drinking, cohabitant smoking, and greater future confidence were associated with an increased risk of relapse. These findings demonstrated that the probability of relapse decreased progressively over time, with most relapses occurring in the initial two years following quit attempts. Predictors of Chinese quitters' relapse behavior in our study were similar to those in previous studies. Drinking and cohabitant smoking were identified as strong predictors of relapse in this population.

Therapeutic Effect of Human Bone Marrow- and Adipose-Derived Stem Cells on Intervertebral Disc Degeneration: A Comparative Study on Rats.

BACKGROUND: Intervertebral disc degeneration (IVD) is a pain-inflicting disorder, posing a serious threat to the elderly, and new therapies are urgently needed. In this study, we examined the potential therapeutic effect of mesenchymal stem cells (MSCs) transplantation on IVD. METHODS: Both human adipose-derived stem cells (hADSCs) and human bone marrow mesenchymal stem cells (hBMSCs) provided by a volunteer were non-contact co-cultured with the human nucleus pulposus cells (hNPCs) to determine the efficacy of hNPCs-oriented differentiation. Flow cytometry was used to characterize the purity of hADSCs/hBMSCs. We determined the expression of surface antigen molecules, such as CD73, CD105, CD90, CD31, HLA-DR, CD34 and CD45, using flow cytometry. Osteogenic and lipogenic differentiations demonstrated by the cells were identified with Alizarin red and Oil red O staining, respectively, and changes in type II collagen and proteoglycan levels were detected by immunofluorescence. Myeloid cell-related mRNA and protein expression levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. The therapeutic effect of hADSCs and hBMSCs on IVD was evaluated in experimental rats, in which degeneration was induced by needling the annulus fibrosus of the caudal intervertebral disc. RESULTS: As evidenced by the presence of hNPCs-like morphology, both hBMSCs and hADSC could effectively differentiate into hNPCs. Using flow cytometry assays, we found high expression of type II collagen (COL2) and aggrecan (ACAN) protein in the hNPCs-like tissue. Treatment with hADSCs and hBMSCs attenuated IVD progression in the rats, and most importantly, there was no significant difference between the therapeutic effects of both types of cells on IVD, on the basis of the COL2 and SRY-Box Transcription Factor 9 (SOX9) protein expression and the histological results. Findings from the animal studies also suggested that both hADSCs and hBMSCs transplantation could be applied in IVD treatment. CONCLUSIONS: In summary, both hADSCs and hBMSCs can attenuate the progression of IVD by delaying, rather than completely reversing the deterioration of disc degeneration, and there is no significant difference between hADSCs and hBMSCs on the therapeutic effects.

Nonequilibrium Theoretical Framework and Universal Design Principles of Oscillation-Driven Catalysis.

At stationary environmental conditions, a catalyst's reaction kinetics may be restricted by its available designs and thermodynamic laws. Thus, its stationary performances may experience practical or theoretical restraints (e.g., catalysts cannot invert the spontaneous direction of a chemical reaction). However, many works have reported that if environments change rapidly, catalysts can be driven away from stationary states and exhibit anomalous performance. We present a general geometric nonequilibrium theory to explain anomalous catalytic behaviors driven by rapidly oscillating environments where stationary-environment restraints are broken. It leads to a universal design principle of novel catalysts with oscillation-pumped performances. Even though a single free energy landscape cannot describe catalytic kinetics at various environmental conditions, we propose a novel control-conjugate landscape to encode the reaction kinetics over a range of control parameters λ, inspired by the Arrhenius form. The control-conjugate landscape significantly simplifies the design principle applicable to large-amplitude environmental oscillations.

Angiopoietin-2 Promotes Mechanical Stress-induced Extracellular Matrix Degradation in Annulus Fibrosus Via the HIF-1α/NF-κB Signaling Pathway.

OBJECTIVE: Mechanical stress is an important risk factor for intervertebral disc degeneration (IVDD). Angiopoietin-2 (ANG-2) is regulated by mechanical stress and is widely involved in the regulation of extracellular matrix metabolism. In addition, the signaling cascade between HIF-1α and NF-κB is critical in matrix degradation. This study aims to investigate the role and molecular mechanism of ANG-2 in regulating the degeneration of annulus fibrosus (AF) through the HIF-1α/NF-κB signaling pathway. METHODS: The bipedal standing mice IVDD model was constructed, and histological experiments were used to evaluate the degree of IVDD and the expression of ANG-2 in the AF. Mouse primary AF cells were extracted in vitro and subjected to mechanical stretching experiments. Western blot assay was used to detect the effect of mechanical stress on ANG-2, and the role of the ANG-2-mediated HIF-1α/NF-κB pathway in matrix degradation. In addition, the effect of inhibiting ANG-2 expression by siRNA or monoclonal antibody on delaying IVDD was investigated at in vitro and in vivo levels. One-way ANOVA with the least significant difference method was used for pairwise comparison of the groups with homogeneous variance, and Dunnett's method was used to compare the groups with heterogeneous variance. RESULTS: In IVDD, the expressions of catabolic biomarkers (mmp-13, ADAMTS-4) and ANG-2 were significantly increased in AF. In addition, p65 expression was increased while HIF-1α expression was significantly decreased. The results of western blot assay showed mechanical stress significantly up-regulated the expression of ANG-2 in AF cells, and promoted matrix degradation by regulating the activity of HIF-1α/NF-κB pathway. Exogenous addition of Bay117082 and CoCl2 inhibited matrix degradation caused by mechanical stress. Moreover, injection of neutralizing antibody or treatment with siRNA to inhibit the expression of ANG-2 improved the matrix metabolism of AF and inhibited IVDD progression by regulating the HIF-1α/NF-κB signaling pathway. CONCLUSION: In IVDD, mechanical stress could regulate the HIF-1α/NF-κB signaling pathway and matrix degradation by mediating ANG-2 expression in AF degeneration.

Hesperetin ameliorates spinal cord injury by inhibiting NLRP3 inflammasome activation and pyroptosis through enhancing Nrf2 signaling.

Neuroinflammation is a prominent feature of traumatic spinal cord injuries (SCIs). Hesperetin exhibits anti-inflammatory effects in neurological disorders; however, the potential neuroprotective effects of hesperetin in cases of SCI remain unclear. Sprague-Dawley rats with C5 hemi-contusion injuries were used as an SCI model. Hesperetin was administered to the experimental rats in order to investigate its neuroprotective effects after SCI, and BV2 cells were pretreated with hesperetin or silencing of nuclear factor erythroid 2-related factor 2 (siNrf2), and then stimulated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP). The therapeutic impact and molecular mechanism of hesperetin were elucidated in a series of in vivo and in vitro investigations conducted using a combination of experiments. The results of the present in vivo experiment indicated that hesperetin improved functional recovery and protected spinal cord tissue after SCI. Hesperetin attenuated oxidative stress and microglial activation, lowered malondialdehyde (MDA) levels, and elevated catalase (CAT), glutathione (GSH)-Px, and superoxide dismutase (SOD) levels. Moreover, hesperetin downregulated the expression of advanced oxygenation protein products (AOPPs), ionized calcium-binding adapter molecule 1 (Iba-1), NOD-like receptor protein 3 (NLRP3), and interleukin-1 beta (IL-1ß), but increased the expression of Nrf2. In vitro studies have shown that hesperetin inhibits the generation of reactive oxygen species (ROS), as well as the neuroinflammation associated with the upregulation of Nrf2 and heme oxygenase-1 (HO-1) in BV2 cells. The results of the present study indicated that hesperetin inhibited BV2 cell pyroptosis and significantly blocked the expression of NLRP3 inflammasome proteins (NLRP3 Caspase-1 p10 apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain [ASC]) and pro-inflammatory mediators (IL-18, IL-1ß). Furthermore, the silencing of Nrf2 by small interfering ribonucleic acid (siRNA) partially abolished its antioxidant effect in the aforementioned cell experiments. Collectively, these findings illustrate that through an increase in Nrf2 signaling hesperetin reduces oxidative stress and neuroinflammation by suppressing NLRP3 inflammasome activation and pyroptosis.

Energy landscape design principle for optimal energy harnessing by catalytic molecular machines.

Under temperature oscillation, cyclic molecular machines such as catalysts and enzymes could harness energy from the oscillatory bath and use it to drive other processes. Using an alternative geometrical approach, under fast temperature oscillation, we derive a general design principle for obtaining the optimal catalytic energy landscape that can harness energy from a temperature-oscillatory bath and use it to invert a spontaneous reaction. By driving the reaction against the spontaneous direction, the catalysts convert low free-energy product molecules to high free-energy reactant molecules. The design principle, derived for arbitrary cyclic catalysts, is expressed as a simple quadratic objective function that only depends on the reaction activation energies, and is independent of the temperature protocol. Since the reaction activation energies are directly accessible by experimental measurements, the objective function can be directly used to guide the search for optimal energy-harvesting catalysts.

Reprogramming of Mitochondrial Respiratory Chain Complex by Targeting SIRT3-COX4I2 Axis Attenuates Osteoarthritis Progression.

Mitochondrial homeostasis is of great importance for cartilage integrity and associated with the progression of osteoarthritis (OA); however, the underlying mechanisms are unknown. This study aims to investigate the role of mitochondrial deacetylation reaction and investigate the mechanistic relationship OA development. Silent mating type information regulation 2 homolog 3 (SIRT3) expression has a negative correlation with the severity of OA in both human arthritic cartilage and mice inflammatory chondrocytes. Global SIRT3 deletion accelerates pathological phenotype in post-traumatic OA mice, as evidenced by cartilage extracellular matrix collapse, osteophyte formation, and synovial macrophage M1 polarization. Mechanistically, SIRT3 prevents OA progression by targeting and deacetylating cytochrome c oxidase subunit 4 isoform 2 (COX4I2) to maintain mitochondrial homeostasis at the post-translational level. The activation of SIRT3 by honokiol restores cartilage metabolic equilibrium and protects mice from the development of post-traumatic OA. Collectively, the loss of mitochondrial SIRT3 is essential for the development of OA, whereas SIRT3-mediated proteins deacetylation of COX4I2 rescues OA-impaired mitochondrial respiratory chain functions to improve the OA phenotype. Herein, the induction of SIRT3 provides a novel therapeutic candidate for OA treatment.

Identifying Candidate Genes Associated with Sporadic Amyotrophic Lateral Sclerosis via Integrative Analysis of Transcriptome-Wide Association Study and Messenger RNA Expression Profile.

Amyotrophic lateral sclerosis, a fatal neurodegeneration disease affecting motor neurons in the brain and spinal cord, is difficult to diagnose and treat. The objective of this study is to identify novel candidate genes related to ALS. Transcriptome-wide association study of ALS was conducted by integrating the genome-wide association study summary data (including 1234 ALS patients and 2850 controls) and pre-computed gene expression weights of different tissues. The ALS-associated genes identified by TWAS were further compared with the differentially expressed genes detected by the mRNA expression profiles of the sporadic ALS. Functional enrichment and annotation analysis of identified genes were performed by an R package and the functional mapping and annotation software. TWAS identified 761 significant genes (PTWAS < 0.05), 627 Gene ontology terms, and 8 Kyoto Encyclopedia of Genes and Genomes pathways for ALS, such as C9orf72, with three expression quantitative trait loci were found significantly: rs2453554 (PTWAS CBRS = 4.68 × 10-10, PTWAS CBRS = 2.54 × 10-9), rs10967976 (PTWAS CBRS = 7.85 × 10-10, PTWAS CBRS = 8.91 × 10-9, PTWAS CBRS = 1.49 × 10-7, PTWAS CBRS = 5.59 × 10-7), rs3849946 (PTWAS CBRS = 7.69 × 10-4, PTWAS YBL = 4.02 × 10-2), Mitochondrion (Padj = 4.22 × 10-16), and Cell cycle (Padj = 2.04 × 10-3). Moreover, 107 common genes, 4 KEGG pathways and 41 GO terms were detected by integrating mRNA expression profiles of sALS, such as CPVL (FC = 2.06, PmRNA = 6.99 × 10-6, PTWAS CBR = 2.88 × 10-2, PTWAS CBR = 4.37 × 10-2), Pyrimidine Metabolism (Padj = 2.43 × 10-2), and Cell Activation (Padj = 5.54 × 10-3). Multiple candidate genes and pathways were detected for ALS. Our findings may provide novel clues for understanding the genetic mechanism of ALS.

Identification of B cell marker genes based on single-cell sequencing to establish a prognostic model and identify immune infiltration in osteosarcoma.

Background: Tumor-infiltrating B cells play a crucial role in the promotion or inhibition of tumor development. However, the role of B cells in osteosarcoma remains largely unknown. The aim of this study was to investigate the effect of B cells on the prognosis and immunity infiltration of osteosarcoma. Methods: Marker genes of B cells were identified based on the single-cell sequencing results of osteosarcoma in the GEO database. The prognostic model was established by the TCGA database and verified by the GEO data. The divergence in immune infiltration between the low-risk and high-risk groups was then compared according to the established prognostic model. Finally, the differential genes in the low-risk and high-risk groups were enriched and analyzed. Results: A total of 261 B cell marker genes was obtained by single-cell sequencing and a prognostic model of 4 B cell marker genes was established based on TCGA data. The model was found to have a good prediction performance in the TCGA and GEO data. A remarkable difference in immune infiltration between the low-risk and high-risk groups was also observed. The obtained results were verified by enrichment analysis. Conclusion: In summary, a prognostic model with good predictive performance was established that revealed the indispensable role of B cells in the development of osteosarcoma. This model also provides a predictive index and a novel therapeutic target for immunotherapy for clinical patients.

Revealing the novel autophagy-related genes for ligamentum flavum hypertrophy in patients and mice model.

Background: Fibrosis is a core pathological factor of ligamentum flavum hypertrophy (LFH) resulting in degenerative lumbar spinal stenosis. Autophagy plays a vital role in multi-organ fibrosis. However, autophagy has not been reported to be involved in the pathogenesis of LFH. Methods: The LFH microarray data set GSE113212, derived from Gene Expression Omnibus, was analyzed to obtain differentially expressed genes (DEGs). Potential autophagy-related genes (ARGs) were obtained with the human autophagy regulator database. Functional analyses including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, Gene Set Enrichment Analysis (GSEA), and Gene Set Variation Analysis (GSVA) were conducted to elucidate the underlying biological pathways of autophagy regulating LFH. Protein-protein interaction (PPI) network analyses was used to obtain hub ARGs. Using transmission electron microscopy, quantitative RT-PCR, Western blotting, and immunohistochemistry, we identified six hub ARGs in clinical specimens and bipedal standing (BS) mouse model. Results: A total of 70 potential differentially expressed ARGs were screened, including 50 up-regulated and 20 down-regulated genes. According to GO enrichment and KEGG analyses, differentially expressed ARGs were mainly enriched in autophagy-related enrichment terms and signaling pathways related to autophagy. GSEA and GSVA results revealed the potential mechanisms by demonstrating the signaling pathways and biological processes closely related to LFH. Based on PPI network analysis, 14 hub ARGs were identified. Using transmission electron microscopy, we observed the autophagy process in LF tissues for the first time. Quantitative RT-PCR, Western blotting, and immunohistochemistry results indicated that the mRNA and protein expression levels of FN1, TGFß1, NGF, and HMOX1 significantly higher both in human and mouse with LFH, while the mRNA and protein expression levels of CAT and SIRT1 were significantly decreased. Conclusion: Based on bioinformatics analysis and further experimental validation in clinical specimens and the BS mouse model, six potential ARGs including FN1, TGFß1, NGF, HMOX1, CAT, and SIRT1 were found to participate in the fibrosis process of LFH through autophagy and play an essential role in its molecular mechanism. These potential genes may serve as specific therapeutic molecular targets in the treatment of LFH.