[Analysis of genetic variants and molecular pathogenesis in a Chinese pedigree affected with Multiple epiphyseal dysplasia].

OBJECTIVE: To analyze the genetic variant and molecular pathogenesis in a Chinese pedigree affected with Multiple epiphyseal dysplasia (MED). METHODS: A MED pedigree which had presented at the Beijing Jishuitan Hospital Affiliated to Capital Medical University on September 13, 2020 was selected as the study subject. Clinical data of the pedigree were collected. Peripheral blood samples were drawn from pedigree members for the extraction of genomic DNA. Whole exome sequencing (WES) was carried out for the pedigree. Candidate variant was verified by Sanger sequencing. Wild type and mutant SLC26A2 expression plasmids were constructed and transfected into human primary chondrocytes. The effect of the variants on the protein localization and cell proliferation was determined by immunofluorescence and CCK8 assays. RESULTS: WES and Sanger sequencing revealed that the proband has harbored compound heterozygous variants of the SLC26A2 gene, including a paternally derived c.484G>T (p.Val162Leu) missense variant and a maternally derived c.485_486delTG (p.Val162Glyfs*12) frameshifting variant. The SLC26A2WT and its mutant SLC26A2Val162Leu and SLC26A2Val162Glyfs*12 expression plasmids were distributed in the nuclei and cytoplasm of human primary chondrocytes. Compared with SLC26A2WT, the expressions of SLC26A2Val162Leu and SLC26A2Val162Glyfs*12 were decreased, along with reduced proliferation of human primary chondrocytes. CONCLUSION: The c.484G>T and c.485_486delTG compound heterozygous variants of the SLC26A2 gene may affect the proliferation of human primary chondrocytes and underlay the pathogenesis of MED in this pedigree.

BMSC-Derived Exosomes Attenuate Rat Osteoarthritis by Regulating Macrophage Polarization through PINK1/Parkin Signaling Pathway.

OBJECTIVE: Exosomes derived from bone marrow mesenchymal stem cells (BMSC-Exos) may modulate the M1/M2 polarization of macrophages during osteoarthritis (OA). However, the underlying mechanisms of BMSC-Exos in this process still need to be elucidated. In this study, we explored the role of BMSC-Exos in the polarization of macrophages in vitro and the OA rats in vivo. METHODS: The effects of BMSC-Exos on RAW264.7 cells were determined, including the production of reactive oxygen species (ROS) and the protein expression of Akt, PINK1, and Parkin. We prepared an OA model by resecting the anterior cruciate ligament and medial meniscus of Sprague-Dawley (SD) rats. Hematoxylin-eosin (H&E) and safranin O-fast green staining, immunohistochemistry and immunofluorescence analyses, and the examination of interleukin 6 (IL-6), interleukin 1ß (IL-1ß), tumor necrosis factor alpha (TNF-α), and interleukin 10 (IL-10) were performed to assess changes in cartilage and synovium. RESULTS: BMSC-Exos inhibited mitochondrial membrane damage, ROS production, and the protein expression of PINK1 and Parkin. Akt phosphorylation was downregulated under lipopolysaccharide (LPS) induction but significantly recovered after treatment with BMSC-Exos. BMSC-Exos alleviated cartilage damage, inhibited M1 polarization, and promoted M2 polarization in the synovium in OA rats. The expression of PINK1 and Parkin in the synovium and the levels of IL-6, IL-1ß, and TNF-α in the serum decreased, but the level of IL-10 increased when BMSC-Exos were used in OA rats. CONCLUSION: BMSC-Exos ameliorate OA development by regulating synovial macrophage polarization, and one of the underlying mechanisms may be through inhibiting PINK1/Parkin signaling.

ERK-estrogen receptor α signaling plays a role in the process of bone marrow mesenchymal stem cell-derived exosomes protecting against ovariectomy-induced bone loss.

BACKGROUND: Exosomes derived from bone marrow mesenchymal stem cells (BMSC-Exos) are considered as candidates for osteoporosis (OP) therapy. Estrogen is critical in the maintenance of bone homeostasis. However, the role of estrogen and/or its receptor in BMSC-Exos treatment of OP, as well as its methods of regulation during this process remain unclear. METHODS: BMSCs were cultured and characterized. Ultracentrifugation was performed to collect BMSC-Exos. Transmission electron microscopy, nanoparticle tracking analysis, and western blotting were used to identify BMSC-Exos. We examined the effects of BMSC-Exos on the proliferation, osteogenic differentiation, mineralization, and cell cycle distribution of MG-63 cells. The protein expression of estrogen receptor α (ERα) and the phosphorylation of ERK were investigated through western blotting. We determined the effects of BMSC-Exos on the prevention of bone loss in female rats. The female Sprague-Dawley rats were divided into three groups: the sham group, ovariectomized (OVX) group, and the OVX + BMSC-Exos group. Bilateral ovariectomy was performed in the OVX and OVX + BMSC-Exos groups, while a similar volume of adipose tissue around the ovary was removed in the sham group. The rats in OVX group and OVX + BMSC-Exos group were given PBS or BMSC-Exos after 2 weeks of surgery. Micro-CT scanning and histological staining were used to evaluate the in vivo effects of BMSC-Exos. RESULTS: BMSC-Exos significantly enhanced the proliferation, alkaline phosphatase activity, and the Alizarin red S staining in MG-63 cells. The results of cell cycle distribution demonstrated that BMSC-Exos increased the proportion of cells in the G2 + S phase and decreased the proportion of cells in the G1 phase. Moreover, PD98059, an inhibitor of ERK, inhibited both the activation of ERK and the expression of ERα, which were promoted by administration of BMSC-Exos. Micro-CT scan showed that in the OVX + BMSC-Exos group, bone mineral density, bone volume/tissue volume fraction, trabecular number were significantly upregulated. Additionally, the microstructure of the trabecular bone was preserved in the OVX + BMSC-Exos group compared to that in the OVX group. CONCLUSION: BMSC-Exos showed an osteogenic-promoting effect both in vitro and in vivo, in which ERK-ERα signaling might play an important role.

The molecular mechanism of a novel derivative of BTO-956 induced apoptosis in human myelomonocytic lymphoma cells.

Acute myeloid leukemia (AML) is a malignant cancer of the hematopoietic system. Although the effectiveness of arsenic compounds has been recognized and applied clinically, some patients are still found resistant to this chemotherapy. In this study, we investigated that a synthetic thyroid hormone analog (TA), 2-iodo-4-nitro-1-(o-tolyloxy) benzene, had a strong apoptosis effect on U937 cells. U937 cells were treated with TA, and examinted the generation of reactive oxygen species (ROS), dysfunction of mitochondria, expression of pro-apoptosis and anti-apoptosis, and cleavage of caspase-3 and Poly (ADP-ribose) polymerase (PARP). Further, it is also evaluated that insight molecular mechanism and signaling pathways involved in the study. It is found that TA significantly induced apoptosis in U937 cells through production of ROS, dysfunction of mitochondria, and activation of caspase cascade. It was also observed that MAPK signaling pathway including ERK, JNK, and P38 signals are involved in the induction of apoptosis. Moreover, marked activation of autophagy and ER stress markers such as LC3, P62, Beclin1 and GRP78, CHOP were observed, respectively. Pretreatment with ER stress inhibitor tauroursodeoxycholic acid (TUDCA) and autophagy inhibitor 3-Methyladenine (3-MA) have successfully attenuated and aggravated TA-induced apoptosis, respectively. We further confirmed the active involvement of ER stress and autophagy signals. In conclusion, TA induced apoptosis through ER stress and activation of autophagy, and the latter is not conducive to TA-induced cell death. Our results may provide a new insight into the strategic development of novel therapy for the treatment of AML.

Alpha-enolase (ENO1), identified as an antigen to monoclonal antibody 12C7, promotes the self-renewal and malignant phenotype of lung cancer stem cells by AMPK/mTOR pathway.

BACKGROUND: Tumor-associated antigens (TAAs) can be targeted in cancer therapy. We previously identified a monoclonal antibody (mAb) 12C7, which presented anti-tumor activity in lung cancer stem cells (LCSCs). Here, we aimed to identify the target antigen for 12C7 and confirm its role in LCSCs. METHODS: Immunofluorescence was used for antigen localization. After targeted antigen purification by electrophoresis and immunoblot, the antigen was identified by LC-MALDI-TOF/TOF mass spectrometry, immunofluorescence, and immunoprecipitation. The overexpression or silence of ENO1 was induced by lentiviral transduction. Self-renewal, growth, and invasion of LCSCs were evaluated by sphere formation, colony formation, and invasion assay, respectively. High-throughput transcriptome sequencing (RNA-seq) and bioinformatics analysis were performed to analyze downstream targets and pathways of targeted antigen. RESULTS: Targeted antigen showed a surface antigen expression pattern, and the 43-55 kDa protein band was identified as α-enolase (ENO1). Self-renewal, growth, and invasion abilities of LCSCs were remarkably inhibited by ENO1 downregulation, while enhanced by ENO1 upregulation. RNA-seq and bioinformatics analysis eventually screened 4 self-renewal-related and 6 invasion-related differentially expressed genes. GSEA analysis and qRT-PCR verified that ENO1 regulated self-renewal, invasion-related genes, and pathways. KEGG pathway analysis and immunoblot demonstrated that ENO1 inactivated AMPK pathway and activated mTOR pathway in LCSCs. CONCLUSIONS: ENO1 is identified as a targeted antigen of mAb 12C7 and plays a pivotal role in facilitating self-renewal, growth, and invasion of LCSCs. These findings provide a potent therapeutic target for the stem cell therapy for lung cancer and have potential to improve the anti-tumor activity of 12C7.

Preoperative imaging differences of patients with cervical spondylosis with cervical vertigo indicate the prognosis after cervical total disc replacement.

OBJECTIVE: To evaluate the relationship between the preoperative imaging differences and prognosis in patients with cervical spondylosis with cervical vertigo who underwent total disc replacement (TDR). METHODS: This was a retrospective study of patients with cervical spondylosis with cervical vertigo treated with single-segment TDR. The severity of pre- and postoperative cervical vertigo was evaluated separately. Paired samples t-tests were used to compare the severity of the symptoms before and after surgery. Characteristics of plain films, computed tomography myelography and magnetic resonance imaging were compared between patients with different outcomes by analysis of variance and Fisher's exact tests. RESULTS: The severity of cervical vertigo was significantly different after single-segment TDR. Three groups with different treatment outcomes were not significantly different with regard to gender, age, type of the cervical spondylosis, follow-up time, segment of surgery, cervical curve, range of motion, T2WI high signal in the spinal cord, and location of compression. The type of compression was significantly different between the three groups. CONCLUSIONS: Cervical vertigo was improved in patients with cervical spondylosis through the TDR procedure. Those in whom a herniated disc was the main source of compression may have a better prognosis following TDR.

Analgesic Efficacy of Adductor Canal Block in Total Knee Arthroplasty: A Meta-analysis and Systematic Review.

The aim of this meta-analysis and systematic review of randomized controlled trials (RCTs) was to evaluate the efficacy and safety of adductor canal block (ACB) for early postoperative pain management in patients undergoing total knee arthroplasty (TKA). Relevant manuscripts comparing ACB with saline or femoral nerve block (FNB) in TKA patients were searched for in the databases of PubMed, EMBASE, and Cochrane library. The outcomes assessed included cumulative analgesic consumption, pain at rest or during movement, ability to ambulate, quadriceps strength, and complications (nausea, vomiting or sedation). For continuous outcomes, pooled effects were measured using weighted mean difference (WMD) or standard mean difference (SMD), together with 95% confidence intervals (CIs). For outcomes without sufficient data for synthesis, qualitative interpretation of individual studies was summarized. Finally, 11 RCTs involving 675 patients met the inclusion criteria. The pooled results showed that ACB resulted in less postoperative analgesic consumption than saline (WMD, -12.84 mg; 95% CI, -19.40 mg to -6.27 mg; P < 0.001) and less pain at rest or during activity. No conclusions could be drawn regarding ability to ambulate and quadriceps strength, because only one study reported these variables. Most studies comparing ACB and FNB reported similar effects on postoperative analgesic consumption (WMD, -0.56 mg; 95% CI, -8.05 mg to 6.93 mg; P = 0.884) and pain; however, ability to ambulate and quadriceps strength were significantly better with ACB (SMD, 0.99; 95% CI, 0.04-1.94; P = 0.041). Additionally, ACB did not increase the rate of complications. Our results suggest that, compared with saline, ACB decreases analgesic consumption and offers short-term advantages in terms of pain relief. Compared with FNB, ACB was associated with better ability to ambulate and quadriceps strength.

Cordycepin prevents hyperlipidemia in hamsters fed a high-fat diet via activation of AMP-activated protein kinase.

Hyperlipidemia is a major risk factor for cardiovascular diseases. In this study, we investigated the potential effects of cordycepin (3'-deoxyadenosine), a bioactive component of the fungus Cordyceps militaris, on hyperlipidemia. We found that in male Syrian golden hamsters fed a high-fat diet (HFD), daily administration of cordycepin effectively reduced the accumulation of serum total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-c) and suppressed HFD-associated increases in relative retroperitoneal fat. It also increased the levels of phospho-AMP-activated protein kinase (AMPK) and phospho-acetyl-CoA carboxylase (phospho-ACC) in liver and retroperitoneal adipose tissues. In HepG2 cells, cordycepin stimulated robust concentration- and time-dependent AMPK activation that correlated with the activation of ACC and the suppression of lipid biosynthesis. However, pretreatment with compound C, a specific inhibitor of AMPK, substantially abolished the effects of cordycepin on AMPK activation and lipid biosynthesis inhibition. These results indicate that cordycepin prevents hyperlipidemia via activation of AMPK. Experiments on abnormal metabolic mice indicated that cordycepin can also improve insulin sensitivity effectively.

[Intermittent low-dose administration of recombinant human parathyroid hormone (1-34) promotes the expression of Runx2 during early stage of fracture healing].

OBJECTIVE: To study the correlation of intermittent low-dose administration of recombinant human parathyroid hormone (1-34) [rhPTH (1-34)] and the expression of Runx2 during early stage of fracture healing. METHOD: Sixty 2-month old male Sprague-Dawley rats underwent close unilateral femoral fracture and intramedullary nail fixation, and then were randomly divided into 2 equal groups: treatment group undergoing subcutaneous injection of rhPTH (1-34) 10 microgxkg(-1)xd(-1) immediately after the operation, and control group undergoing subcutaneous injection of normal saline of the same dose. Six rats from each group were killed with their bilateral femurs taken out on days 2, 4, 7, 14, and 21 after the operation to undergo X-ray photography Tissue RNA and protein were extracted from the bone tissues and the levels of Runx2 mRNA and protein expression were evaluated through real time quantitative PCR and Western-blotting. Blood samples were collected from the abdominal aorta before the rats were killed to undergo detection of serum calcium, phosphorus, and alkaline phosphatase (AKP). RESULTS: The Runx2 mRNA levels in the fractured femurs of the rhPTH (1-34) group on days 14 and 21 after the operation were 2.6 and 3.8 times as those of the control group respectively (both P<0.05). The Runx2 protein levels in the fractured femurs of the rhPTH (1-34) group on days 14 and 21 after the operation were significantly higher than those of the control group respectively too. Since day 14 fracture healing was seen, and the status of fracture healing was better in the rhPTH (1-34) group than in the control group on days 14 and 21. The levels of serum calcium ion on days 14 and 21 of the rhPTH (1-34) group were both significantly higher than those of the control group (both P<0.05). There were not significant differences in the serum levels of AKP, osteocalcin and collagen type Iat any time point between the 2 groups. CONCLUSION: Intermittent low-dose administration of rhPTH (1-34) up-regulates the levels of osteogenesis-specific Runx2 mRNA and protein expression to accelerate the early stage fracture healing during the early stage.

On the hepatic mechanism of HDL assembly by the ABCA1/apoA-I pathway.

The mechanism for the assembly of HDL with cellular lipid by ABCA1 and helical apolipoprotein was investigated in hepatocytes. Both HepG2 cells and mouse primary culture hepatocytes produced HDL with apolipoprotein A-I (apoA-I) whether endogenously synthesized or exogenously provided. Probucol, an ABCA1 inactivator, inhibited these reactions, as well as the reversible binding of apoA-I to HepG2. Primary cultured hepatocytes of ABCA1-deficient mice also lacked HDL production regardless of the presence of exogenous apoA-I. HepG2 cells secreted apoA-I into the medium even when ABCA1 was inactivated by probucol, but it was all in a free form as HDL production was inhibited. When a lipid-free apoA-I-specific monoclonal antibody, 725-1E2, was present in the culture medium, production of HDL was suppressed, whether with endogenous or exogenously added apoA-I, and the antibody did not influence HDL already produced by HepG2 cells. We conclude that the main mechanism for HDL assembly by endogenous apoA-I in HepG2 cells is an autocrine-like reaction in which apoA-I is secreted and then interacts with cellular ABCA1 to generate HDL.