Ubiquitination of ASCL1 mediates CD47 transcriptional activation of the AKT signaling pathway, and glycolysis promotes osteogenic differentiation of hBMSCs.

Bones are extremely dynamic organs that continually develop and remodel. This process involves changes in numerous gene expressions. hBMSC cells can promote osteogenic differentiation. The purpose of this study was to elucidate the mechanism by which ASCL1 promotes osteogenic differentiation in hBMSC cells while decreasing glycolysis. hBMSCs were induced to differentiate into osteoblasts. The ASCL1 expression level during hBMSC osteogenic differentiation was measured by RT‒qPCR, Western blotting, and immunofluorescence. The differentiation level of osteoblasts was observed after staining with ALP and alizarin red. ChIP-qPCR were used to determine the relationship between ASCL1 and CD47, and the expression of glycolysis-related proteins was detected. Overexpression of ASCL1 was used to determine its impact on osteogenic differentiation. si-USP8 was used to verify the ubiquitination of ASCL1-mediated CD47/AKT pathway's impact on hBMSC glycolysis and osteogenic differentiation. The results showed that the expression of ASCL1 was upregulated after the induction of osteogenic differentiation in hBMSCs. From a functional perspective, knocking down USP8 can promote the ubiquitination of ASCL1, while the osteogenic differentiation ability of hBMSCs was improved after the overexpression of ASCL1, indicating that ASCL1 can promote the osteogenic differentiation of hBMSCs. In addition, USP8 regulates the ubiquitination level of ASCL1 and mediates CD47 transcriptional regulation of the AKT pathway to increase the glycolysis level of hBMSCs and cell osteogenic differentiation. USP8 ubiquitination regulates the level of ASCL1. In addition, ubiquitination of ASCL1 mediates CD47 transcription to activate the AKT signaling pathway and increase hBMSC glycolysis to promote osteogenic differentiation.

Nerve growth factor promotes osteogenic differentiation of MC3T3-E1 cells via BMP-2/Smads pathway.

BACKGROUND: Exogenous nerve growth factor (NGF) can induce osteogenic precursor cell differentiation and promote fracture healing. However, the molecular mechanism by which NGF induces osteogenesis is not well understood. BMP-2 has good osteogenic efficacy and is one of the most osteogenic-inducing growth factors known. Therefore, this study aimed to determine whether NGF induces osteogenic differentiation of mouse embryonic osteogenic precursor cell line MC3T3-E1 by BMP-2 and search further mechanisms of NGF on BMP-2. METHODS: MC3T3-E1 cells were treated with NGF at a concentration gradient for indicated times, after which the cell viability was measured by CCK-8 kit. Osteogenic differentiation was detected with quantification of alkaline phosphatase (ALP) activity also visualized with ALP staining. The transcription and expression of relevant genes were detected by qPCR and western blotting, respectively. NGF's effect on BMP2 was studied with qPCR and luciferase reporter assay. The phosphorylation of Smads was probed with specific antibodies by western blotting, and the location of Smads was observed through immunofluorescence. RESULTS: We found that NGF promoted proliferation and osteogenic differentiation of MC3T3-E1, increased the expression level of BMP-2, as well as the phosphorylation and nuclear translocation of Smad1/5/8. However, neutralization of BMP-2 with si-BMP-2 or BMP-2 signal inhibitors reversed NGF induced phosphorylation and nuclear translocation of Smad1/5/8, as well as the expression of Runx2, type I collagen, osteocalcin and osteopontin. In addition, si-BMP-2 abrogated NGF-induced ALP activity. CONCLUSION: NGF induced osteogenic differentiation of MC3T3-E1 cells through BMP-2/Smads pathway and induction of Runx2. Our study would provide a theoretical basis for clinical treatment of fractures using NGF.

Intra-articular Injection of Chitosan-Based Supramolecular Hydrogel for Osteoarthritis Treatment.

BACKGROUND: Pain and cartilage destruction caused by osteoarthritis (OA) is a major challenge in clinical treatment. Traditional intra-articular injection of hyaluronic acid (HA) can relieve the disease, but limited by the difficulty of long-term maintenance of efficacy. METHODS: In this study, an injectable and self-healing hydrogel was synthesized by in situ crosslinking of N-carboxyethyl chitosan (N-chitosan), adipic acid dihydrazide (ADH), and hyaluronic acid-aldehyde (HA-ALD). RESULTS: This supramolecular hydrogel sustains good biocompatibility for chondrocytes. Intra-articular injection of this novel hydrogel can significantly alleviate the local inflammation microenvironment in knee joints, through inhibiting the inflammatory cytokines (such as TNF-α, IL-1ß, IL-6 and IL-17) in the synovial fluid and cartilage at 2- and even 12-weeks post-injection. Histological and behavioral test indicated that hydrogel injection protected cartilage destruction and relieved pain in OA rats, in comparison to HA injection. CONCLUSION: This kind of novel hydrogel, which is superior to the traditional HA injection, reveals a great potential for the treatment of OA.

Chemical profiling of Dioscorea septemloba by High Performance Liquid Chromatography Coupled to Electrospray Ionization and Quadrupole Time-of-Flight Mass Spectrometry.

The rhizomes of Dioscorea septemloba (DS) is one of traditional Chinese medicine, which can be used for treatments of urethral and renal infections, as well as rheumatism. Nevertheless, detailed information on DS chemical compositions is still scarce. Therefore, the present work is aimed at investigating the phytochemical components of DS using high performance liquid chromatography coupled to electrospray ionization and quadrupole time-of-flight mass spectrometry in negative ion mode. The proposed method provided tentative identification of 32 components, 6 of which were determined to be new. The present study highlighted the importance of DS as a promising source of medicinal ingredients and provides valuable information on the new potential components in DS.

Photocatalytic reduction for graphene oxide by PbTiO3 with high polarizability and its electrocatalytic application in pyrrole detection.

Quick recombination of photogenerated electrons and holes in photocatalytic process remains a huge challenge. And the routine efforts are concentrated on heterojunction, metal decoration and surface defect strategies. PbTiO3 as a typical perovskite ferroelectrics is with a strong built-inelectric field as self-junction caused by internal spontaneous polarization, facilitating the charge separation in the photocatalytic process. Here, under UV irradiation, L-shaped PbTiO3 with active (1 1 0) facet as a photocatalyst was applied to photo-reduce graphene oxide (GO), where a specific reduced graphene oxide (rGO)/PbTiO3 composite was synthesized in presence of isopropanol, a hole-trapping agent. According to the linear optical properties, the polarizability of PbTiO3 is calculated to 1.01 × 10-23 cm3 (2.68 times that of P25 (TiO2)), inducing the photo-excited charge separation by PbTiO3. Based on XPS characterization, a TiOC chemical bond is identified on the interface between rGO and PbTiO3. The response peak current for an electrochemical sensor based on rGO/PbTiO3 was proportional to the concentration of pyrrole (6.6 × 10-9-3.1 × 10-7 M, R2 = 0.999), and an extremely low limit reaches to 2.38 × 10-9 M. In addition, polypyrrole during the pyrrole detection was realized by the multi-cycle oxidation process. And also, the electrochemical detection has been successfully applied for the pyrrole quantification in real samples.

Solalyratins A and B, new anti-inflammatory metabolites from Solanum lyratum.

A new coumestan (solalyratin A, 1) and a novel cyclic eight-membered α,ß-unsaturated ketone (solalyratin B, 3), together with three known compounds, puerariafuran (2), coumestrol (4) and 9-hydroxy-2',2'-dimethylpyrano[5',6':2,3]-coumestan (5), were isolated from the whole plant of Solanum lyratum. Their structures were elucidated on the basis of spectroscopic analyses. In vitro, compounds 1-5 showed anti-inflammatory activities, with IC(50) values in the range 6.3-9.1 µM.

Two new cytotoxic ent-clerodane diterpenoids from Scutellaria barbata.

Two new ent-clerodane diterpenoids have been isolated from Scutellaria barbata, and their structures were established by detailed spectroscopic analyses as (13R)-6α,7ß-dihydroxy-8ß,13-epoxy-11ß-nicotinyloxy-ent-clerodan-3-en-15,16-olide (scutelinquanine D, 1) and (11E)-6α-acetoxy-7ß,8ß-dihydroxy-ent-clerodan-3,11,13-trien-15,16-olide (6-acetoxybarbatin C, 2). In vitro, the isolated two new compounds showed significant cytotoxic activities against three human cancer cell lines (HONE-1 nasopharyngeal, KB oral epidermoid carcinoma, and HT29 colorectal carcinoma cells), and gave IC(50) values in the range of 2.5-6.6 µM.

New anti-inflammatory 4-hydroxyisoflavans from Solanum lyratum.

Three new 4-hydroxyisoflavans, named lyratin A (1), lyratin B (2) and lyratin C (3), along with a known compound, 4,7,2'trihydroxy-4'-methoxyisoflavan (4), were isolated from the whole plant of Solanum lyratum. Their structures were established by means of detailed physical data analyses. In vitro, four compounds showed anti-inflammatory activities with inhibitory ratios of release of beta-glucuronidase from polymorphonuclear leukocytes of rats in the range of 30.3-38.6% at 10 microM.

New neo-clerodane diterpenoids from Scutellaria barbata with cytotoxic activities.

Two new neo-clerodane diterpenoids have been isolated from the whole plant of Scutellaria barbata D. Don, and their structures were established by detailed spectral analyses as scutehenanine H (1) and 6-(2,3-epoxy-2-isopropyl-n-propoxyl)barbatin C (2). In vitro, the isolated two new compounds showed significant cytotoxic activities against three human cancer lines, and gave IC(50) values in the range OF 2.0-4.2 µΜ.

[A specific PCR assay and a nested PCR assay for the screening of ovine pulmonary adenomatosis].

Ovine pulmonary adenomatosis (OPA) is a naturally occurring contagious lung tumor of sheep which was caused by an exogenous retrovirus of sheep, jaagsiekte retrovirus (JSRV). Although no specific circulating antibodies against the virus coud be detected in infected sheep, exogenous JSRV proviral DNA sequences (exJSRV) and JSRV RNA transcripts could be detected in lung tumors, lymphoreticular system and peripheral blood mononuclear cells (PBMC) from sheep affected by OPA. The sheep genome carried 15 to 20 copies of endogenous retrovirus loci (enJSRV) that were similar to JSRV in structural genes but the divergene in U3. Therefore, primers specific for the U3 sequences of exJSRV were designed for the specific PCR and nested PCR (n-PCR). Sensitivity between specific PCR assay and n-PCR assay was compared by using serial dilutions of positive plasmid pJSRV-LTR in a background of 700ng sheep genome DNA. Sensitivity of n-PCR was ten-fold higher than specific PCR. The n-PCR was only available in blood test for detection of JSRV infected sheep and might be useful in epidemiological studies and disease control of OPA.

[Contents of different soil fluorine forms in North Anhui and their affecting factors].

By the method of consecutive extraction, this paper studied the contents and vertical distribution of soil fluorine (F) forms in North Anhui, with their relations to the soil physical and chemical properties analyzed. The results showed that the soil total F (T-F) content in North Anhui was ranged from 265.8 mg . kg(-1) to 612.8 mg . kg(-1), with an average of 423.7 mg . kg(-1), and decreased in the sequence of vegetable soil > fluvo-aquic soil > paddy soil > shajiang black soil > yellow brown soil. Among the T-F, residual F (Res-F) was the main form, occupying > 95% of total F, followed by water soluble F (Ws-F), being about 1.5% of the total, and organic-F (Or-F), Fe and Mn oxide-F (Fe/Mn-F) and exchangeable-F (Ex-F) only had very small amount. The Ws-F content in test soils ranged from 1.35 mg . kg(-1) to 17.98 mg . kg(-1), with a mean value of 6.62 mg . kg(-1). Vegetable soil, fluvo-aquic soil and shajiang black soil had a relatively higher content of Ws-F, while yellow brown soil was in adverse. Soil pH and the contents of soil organic matter, total and available phosphorus, and physical clay were the main factors affecting the contents of various F forms. Soil Ws-F was significantly positively correlated with soil pH and soil total and available phosphorus, soil Ex-F was significantly positively correlated with soil clay ( < 0.01 mm and <0.001 mm), soil Fe/Mn-F was significantly positively correlated with soil total phosphorus, and soil Or-F had a significant positive correlation with soil organic matter. Soil Ws-F content also had a close connection to the parent material. The soil developed from shallow lacustrine and marsh sediments usually had the highest Ws-F content, followed by those developed from Huang River alluvial deposit, Q3 loess, Huaihe River alluvial deposit, and light-texture yellow brown soil, with the mean Ws-F content being 9.05, 8.12, 2.97, 2.05 and 1.91 mg . kg(-1), respectively. The contents of soil Or-F and Fe/Mn-F decreased with increasing soil depth, and those of T-F and Ws-F in vegetable soil were higher in upper than in deeper soil layers.