Circulating proteomic biomarkers for diagnosing sporadic amyotrophic lateral sclerosis: a cross-sectional study.

JOURNAL/nrgr/04.03/01300535-202408000-00039/figure1/v/2023-12-16T180322Z/r/image-tiff Biomarkers are required for the early detection, prognosis prediction, and monitoring of amyotrophic lateral sclerosis, a progressive disease. Proteomics is an unbiased and quantitative method that can be used to detect neurochemical signatures to aid in the identification of candidate biomarkers. In this study, we used a label-free quantitative proteomics approach to screen for substantially differentially regulated proteins in ten patients with sporadic amyotrophic lateral sclerosis compared with five healthy controls. Substantial upregulation of serum proteins related to multiple functional clusters was observed in patients with sporadic amyotrophic lateral sclerosis. Potential biomarkers were selected based on functionality and expression specificity. To validate the proteomics profiles, blood samples from an additional cohort comprising 100 patients with sporadic amyotrophic lateral sclerosis and 100 healthy controls were subjected to enzyme-linked immunosorbent assay. Eight substantially upregulated serum proteins in patients with sporadic amyotrophic lateral sclerosis were selected, of which the cathelicidin-related antimicrobial peptide demonstrated the best discriminative ability between patients with sporadic amyotrophic lateral sclerosis and healthy controls (area under the curve [AUC] = 0.713, P < 0.0001). To further enhance diagnostic accuracy, a multi-protein combined discriminant algorithm was developed incorporating five proteins (hemoglobin beta, cathelicidin-related antimicrobial peptide, talin-1, zyxin, and translationally-controlled tumor protein). The algorithm achieved an AUC of 0.811 and a P-value of < 0.0001, resulting in 79% sensitivity and 71% specificity for the diagnosis of sporadic amyotrophic lateral sclerosis. Subsequently, the ability of candidate biomarkers to discriminate between early-stage amyotrophic lateral sclerosis patients and controls, as well as patients with different disease severities, was examined. A two-protein panel comprising talin-1 and translationally-controlled tumor protein effectively distinguished early-stage amyotrophic lateral sclerosis patients from controls (AUC = 0.766, P < 0.0001). Moreover, the expression of three proteins (FK506 binding protein 1A, cathelicidin-related antimicrobial peptide, and hemoglobin beta-1) was found to increase with disease progression. The proteomic signatures developed in this study may help facilitate early diagnosis and monitor the progression of sporadic amyotrophic lateral sclerosis when used in combination with current clinical-based parameters.

Synovial fluid exosome-derived miR-182-5p alleviates osteoarthritis by downregulating TNFAIP8 and promoting autophagy through LC3 signaling.

OBJECTIVE: To investigate the role of exosomal miRNAs from synovial fluid (SF) in osteoarthritis (OA) patients and investigate the underlying molecular mechanism. METHODS: Degenerated knee tissues were collected from male and female OA patients. Enzyme-linked immunosorbent assay (ELISA) was used to detect the differences in the expression of inflammatory indicators, including TNF-α, IL-6, and IL-10, between the degenerative and injury groups. Exosomes were isolated from SF using the Exoquick kit, and a microarray was used to identify differentially expressed miRNAs (DEmiRNAs), which were analyzed using bioinformatics. The predicted relationship between DEmiRNAs and target genes was verified using a luciferase reporter gene assay. CCK-8 and transwell assays were used to assess cell viability and migration. Immunofluorescence and TUNEL assay were used to detect cell autophagy and apoptosis. The interaction between proteins was detected by immunoprecipitation and verified by Mab rescue assay. RESULTS: The relative expression of TNF-α/IL6 was significantly higher in the degeneration group than in the injury group. The OA degeneration group released significantly more and smaller exosomes than the injury group. The expression of miR-182-5p was markedly reduced in OA patients and had a higher correlation with inflammatory indicators. Tumor necrosis factor α-induced protein 8 (TNFAIP8) was a target of miR-182-5p, and its overexpression promoted chondrocyte proliferation, migration, and invasion and enhanced the wound healing efficiency. We also found a direct interaction of TNFAIP8 with autophagy-related gene 3 (ATG3). TNFAIP8 triggered ATG3 LC3-mediated autophagy. CONCLUSION: The downregulation of exosomal miR-182-5p inhibits OA degeneration by targeting TNFAIP8 via the ATG/LC3 pathway.

METTL3 promotes pancreatic cancer proliferation and stemness by increasing stability of ID2 mRNA in a m6A-dependent manner.

In eukaryotes, N6-methyladenosine (m6A) is the most prevalent epigenetic alteration. Methyltransferase-like 3 (METTL3) is a key player in the control of m6A, although its function in pancreatic cancer is incompletely understood. In this study, we examined the role that METTL3 plays in pancreatic cancer cell proliferation and stemness. We discovered that in pancreatic cancer cells, METTL3-mediated m6A alterations regulate ID2 as a downstream target. The stability of ID2 mRNA was decreased and m6A modification was effectively eliminated by METTL3 knockdown in pancreatic cancer cells. We also demonstrate that m6a-YTHDF2 is necessary for the METTL3-mediated stabilization of ID2 mRNA. Additionally, we show that ID2 controls the stemness molecules NANOG and SOX2 via the PI3K-AKT pathway to support pancreatic cancer growth and stemness maintenance. Our data suggest that METTL3 may post-transcriptionally upregulate ID2 expression in an m6A-YTHDF2-dependent manner to further promote the stabilization of ID2 mRNA, which may be a new target for pancreatic cancer treatment.

Magnesium promotes osteogenesis via increasing OPN expression and activating CaM/CaMKIV/CREB1 pathway.

Magnesium (Mg) based alloy has been used as a biodegradable implant for fracture repair with considerable efficacy, and it has been proved that magnesium ion (Mg2+ ), one of the degradation products, could stimulate osteogenesis. Here, we investigated the osteogenesis property of magnesium both in vitro and in vivo, and to identify the cellular and molecular mechanisms that mediate these effects. Results showed that magnesium exerts a dose-dependent increase in the proliferation of MC3T3 and MG63 cells, and in the expression of osteopontin (OPN), a promising biomarker of osteogenesis. Subsequently, the protein-protein interaction (PPI) network analysis showed the interactions between calmodulin (CaM) and calmodulin-dependent protein kinase (CaMK) and CREB1. The ratio of p-CaMKIV/CaMKIV and p-CREB1/CREB were increased at protein level in MC3T3 and MG63 cells after treatment with Mg2+ . Dual-luciferase reporter gene assay showed that p-CREB1 could directly bind to OPN promoter and up-regulate the transcription of OPN after nuclear entry. Meanwhile, the expression of OPN and p-CREB1, which increased after Mg2+ treatment, was down-regulated by sh-CaMKIV or sh-CREB1. Moreover, the mineralized deposit and expression of OPN were reduced after treatment with an inhibitor of CaMKIV, KN93. In addition, massive cavities in the cortical bone around the Mg screw were showed in vivo after injection of KN93. These data indicated that the osteogenic effect of Mg is related to the activation OPN through CaM/CaMKIV/CREB1 signaling pathway.

Upregulating CXCR7 accelerates endothelial progenitor cell-mediated endothelial repair by activating Akt/Keap-1/Nrf2 signaling in diabetes mellitus.

BACKGROUND: Endothelial progenitor cell (EPC) dysfunction contributes to vascular disease in diabetes mellitus. However, the molecular mechanism underlying EPC dysfunction and its contribution to delayed reendothelialization in diabetes mellitus remain unclear. Our study aimed to illustrate the potential molecular mechanism underlying diabetic EPC dysfunction in vivo and in vitro. Furthermore, we assessed the effect of EPC transplantation on endothelial regeneration in diabetic rats. METHODS: Late outgrowth EPCs were isolated from the bone marrow of rats for in vivo and in vitro studies. In vitro functional assays and Western blotting were conducted to reveal the association between C-X-C chemokine receptor type 7 (CXCR7) expression and diabetic EPC dysfunction. To confirm the association between cellular CXCR7 levels and EPC function, CXCR7 expression in EPCs was upregulated and downregulated via lentiviral transduction and RNA interference, respectively. Western blotting was used to reveal the potential molecular mechanism by which the Stromal-Derived Factor-1 (SDF-1)/CXCR7 axis regulates EPC function. To elucidate the role of the SDF-1/CXCR7 axis in EPC-mediated endothelial regeneration, a carotid artery injury model was established in diabetic rats. After the model was established, saline-treated, diabetic, normal, or CXCR7-primed EPCs were injected via the tail vein. RESULTS: Diabetic EPC dysfunction was associated with decreased CXCR7 expression. Furthermore, EPC dysfunction was mimicked by knockdown of CXCR7 in normal EPCs. However, upregulating CXCR7 expression reversed the dysfunction of diabetic EPCs. The SDF-1/CXCR7 axis positively regulated EPC function by activating the AKT-associated Kelch-like ECH-associated protein 1 (keap-1)/nuclear factor erythroid 2-related factor 2 (Nrf2) axis, which was reversed by blockade of AKT and Nrf2. Transplantation of CXCR7-EPCs accelerated endothelial repair and attenuated neointimal hyperplasia in diabetes mellitus more significantly than transplantation of diabetic or normal EPCs. However, the therapeutic effect of CXCR7-EPC transplantation on endothelial regeneration was reversed by knockdown of Nrf2 expression. CONCLUSIONS: Dysfunction of diabetic EPCs is associated with decreased CXCR7 expression. Furthermore, the SDF-1/CXCR7 axis positively regulates EPC function by activating the AKT/keap-1/Nrf2 axis. CXCR7-primed EPCs might be useful for endothelial regeneration in diabetes-associated vascular disease.