IS6 family insertion sequences promote optrA dissemination between plasmids varying in transfer abilities.

Plasmids are the primary vectors for intercellular transfer of the oxazolidinone and phenicol cross-resistance gene optrA, while insertion sequences (ISs) are mobile genetic elements that can mobilize plasmid-borne optrA intracellularly. However, little is known about how the IS-mediated intracellular mobility facilitates the dissemination of the optrA gene between plasmid categories that vary in transfer abilities, including non-mobilizable, mobilizable, and conjugative plasmids. Here, we performed a holistic genomic study of 52 optrA-carrying plasmids obtained from searches guided by the Comprehensive Antibiotic Resistance Database. Among the 132 ISs identified within 10 kbp from the optrA gene in the plasmids, IS6 family genes were the most prevalent (86/132). Homologous gene arrays containing IS6 family genes were shared between different plasmids, especially between mobilizable and conjugative plasmids. All these indicated the central role of IS6 family genes in disseminating plasmid-borne optrA. Thirty-three of the 52 plasmids were harbored by Enterococcus faecalis found mainly in humans and animals. By Nanopore sequencing and inverse PCR, the potential of the enterococcal optrA to be transmitted from a mobilizable plasmid to a conjugative plasmid mediated by IS6 family genes was further confirmed in Enterococcus faecalis strains recovered from the effluents of anaerobic digestion systems for treating chicken manure. Our findings highlight the increased intercellular transfer abilities and dissemination risk of plasmid-borne optrA gene caused by IS-mediated intracellular mobility, and underscore the importance of routinely monitoring the dynamic genetic contexts of clinically important antibiotic resistance genes to effectively control this critical public health threat. KEY POINTS: • IS6 was prevalent in optrA-plasmids varying in intercellular transfer abilities. • Enterococcal optrA-plasmids were widespread among human, animal, and the environment. • IS6 elevated the dissemination risk of enterococcal optrA-plasmids.

N-Acetyl-Glucosamine Sensitizes Non-Small Cell Lung Cancer Cells to TRAIL-Induced Apoptosis by Activating Death Receptor 5.

BACKGROUND/AIMS: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential anti-cancer agent due to its selective toxicity. However, many human non-small cell lung cancer (NSCLC) cells are partially resistant to TRAIL, thereby limiting its clinical application. Therefore, there is a need for the development of novel adjuvant therapeutic agents to be used in combination with TRAIL. METHODS: In this study, the effect of N-acetyl-glucosamine (GlcNAc), a type of monosaccharide derived from chitosan, combined with TRAIL was evaluated in vitro and in vivo. Thirty NSCLC clinical samples were used to detect the expression of death receptor (DR) 4 and 5. After GlcNAc and TRAIL co-treatment, DR expression was determined by real-time PCR and western blotting. Cycloheximide was used to detect the protein half-life to further understand the correlation between GlcNAc and the metabolic rate of DR. Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to detect receptor clustering, and the localization of DR was visualized by immunofluorescence under a confocal microscope. Furthermore, a co-immunoprecipitation assay was performed to analyze the formation of death-inducing signaling complex (DISC). O-linked glycan expression levels were evaluated following DR5 overexpression and RNA interference mediated knockdown. RESULTS: We found that the clinical samples expressed higher levels of DR5 than DR4, and GlcNAc co-treatment improved the effect of TRAIL-induced apoptosis by activating DR5 accumulation and clustering, which in turn recruited the apoptosis-initiating protease caspase-8 to form DISC, and initiated apoptosis. Furthermore, GlcNAc promoted DR5 clustering by improving its O-glycosylation. CONCLUSION: These results uncovered the molecular mechanism by which GlcNAc sensitizes cancer cells to TRAIL-induced apoptosis, thereby highlighting a novel effective agent for TRAIL-mediated NSCLC-targeted therapy.

Anticancer effect of fufang yiliu yin on human hepatocellular carcinoma SMMC-7721 cells.

Chinese herbal medicine utilizes clinically effective adjuvants that can potentiate the effects of hepatectomy and molecule-targeted drugs for the treatment of hepatocellular carcinoma (HCC). The aim of this study was to investigate the possible molecular mechanisms underlying the antitumor effect of fufang yiliu yin (FYY) on HCC cells. We investigated the effects of FYY on the proliferation, migration, invasion, and apoptosis of SMMC-7721 cells in vitro and in mouse subcutaneous xenograft models in vivo. FYY significantly inhibited the proliferation of SMMC-7721 cells compared to that of normal hepatocytes; cell proliferation was blocked at the G2/M phase in accordance with reduced expression of proliferating cell nuclear antigen. FYY treatment resulted in the activation of caspase-8, caspase-3 and poly (ADP-ribose) polymerase, with reduced protein levels of tumor necrosis factor receptor-associated factor 2, indicating an induction of cell apoptosis. In addition, we observed decreases in the protein expression of matrix metalloproteinase-2 and -9 along with an inhibition of cell migration and invasion after FYY treatment. Furthermore, FYY treatment significantly inhibited the growth of tumors in vivo. These data demonstrate the strong inhibitory effects of FYY on SMMC-7721 cells, and we propose FYY as a novel potential anticancer adjuvant.

Lentiviral vector-mediated doxycycline-inducible USP39 shRNA or cDNA expression in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC), characterized by distinct biological and clinicopathological features, has a poor prognosis due to lack of effective therapeutic targets. Our previous data revealed that high levels of USP39 were selectively present in TNBC samples compared with their normal breast tissue samples and USP39 was also expressed at different levels in cultured TNBC cells and normal breast cells. Yet, the underlying cellular and molecular mechanisms of USP39 remain unclear. In the present study, we describe a doxycycline (DOX)-regulated lentiviral vector system expressing shRNA or cDNA of the USP39 gene in the TNBC cell line MDA-MB-231. USP39 expression was knocked down by the miR-30-based inducible lentiviral short hairpin RNA (shRNA) delivery system or overexpressed by the inducible cDNA system. The inducible shRNA-mediated downregulation of USP39 expression markedly reduced the proliferation and colony-forming ability of MDA-MB-231 cells, while overexpression of USP39 by the inducible system did not promote cancer cell proliferation. The lentiviral vector-mediated Tet-on system demonstrated efficient and inducible knockdown of USP39 or overexpression of USP39 in TNBC cells, facilitating a wide variety of applications for gene knockdown and overexpression experiments in gene functional studies in vitro and in vivo.

[Effect of platelet lysate on chondrogenic differentiation of human umbilical cord derived mesenchymal stem cells in vitro].

OBJECTIVE: To study the effect of platelet lysate (PL) on chondrogenic differentiation of human umbilical cord derived mesenchymal stem cells (hUCMSCs) in vitro. METHODS: Umbilical cords were voluntarily donated by healthy mothers. The hUCMSCs were isolated by collagenase digestion and cultured in vitro. The surface markers of the cells were detected by flow cytometer. According to different components of inductive medium, the cultured hUCMSCs were divided into 3 groups: group A [H-DMEM medium, 10% fetal bovine serum (FBS), and 10%PL]; group B [H-DMEM medium, 10%FBS, 10 ng/mL transforming growth factor beta1 (TGF-beta1), 1 x 10(-7) mol/L dexamethasone, 50 microg/mL Vitamin C, and 1% insulin-transferrin-selenium (ITS)]; and group C (H-DMEM medium, 10%FBS, 10 ng/mL TGF-beta1, 1 x 10(-7) mol/L dexamethasone, 50 microg/mL vitamin C, 1%ITS, and 10%PL). The hUCMSCs were induced in the mediums for 2 weeks. Toluidine blue staining was used to detect the secretion of chondrocyte matrix. Immunofluorescence method was used to identify the existence of collagen type II. The expressions of Aggrecan and collagen type II were detected by semiquantitative RT-PCR. RESULTS: Flow cytometer results showed that the hUCMSCs did not express the surface markers of hematopoietic cell CD34, CD45, and human leukocyte antigen DR, but expressed the surface markers of adhesion molecule and mesenchymal stem cells CD44, CD105, and CD146. Toluidine blue staining and immunofluorescence showed positive results in group C, weak positive results in group B, and negative results in group A. Semiquantitative RT-PCR showed the expressions of Aggrecan and collagen type II at mRNA level in groups B and C, but no expression in group A. The mRNA expressions of Aggrecan and collagen type II were higher in group C than in group B (P < 0.05). CONCLUSION: Only 10%PL can not induce differentiation of hUCMSCs into chondrocytes, but it can be a supplement to the induced mediums. PL can improve hUCMSCs differentiating into chondrocytes obviously in vitro. This study provides new available conditions for constructing tissue engineered cartilage.

[Nutrient cycling in Castanea mollissima B1 forest at the Miyun reservoir watershed, Beijing].

Studies on the nutrient cycling in Castanea mollissima B1 forest at the Miyun reservoir watershed, Beijing, showed that the total biomass of the Castanea mollissima B1 stands at age 22 was 38,638 kg.hm-2, and the biomass of their stem, branch, leaf, blossom, chestnut, seed capsule and root was 20,160, 8,430, 1429, 873, 1024, 800 and 5,922 kg.hm-2, occupying 52.18%, 21.82%, 3.70%, 2.26%, 2.65%, 2.07%, 15.33% of the total biomass, respectively. The annual average growth amount of stem, branch, and root was 916, 383, and 269 kg.hm-2, respectively, and the total annual average growth amount was 5,694 kg.hm-2. The nutrient contents in different organs of Castanea mollissima B1 stands showed that the N content sequence was leaf > blossom > chestnut > seed capsule > branch > stem, P content sequence was leaf > blossom > branch > stem > seed capsule > chestnut, K content sequence was chestnut > blossom > leaf > chestnut > branch > stem, Ca content sequence leaf > seed capsule > branch > stem > blossom > chestnut, and Mg content sequence was leaf > blossom > branch > chestnut > seed capsule > stem. The storage of N, P, K, Ca and Mg in Castanea mollissima B1 forest was 89.47, 17.34, 74.68, 105.49 and 28.40 kg.hm-2, respectively. The nutrient annual assimilation was 79.17 kg.hm-2, the total annual returning amount 106.55 kg.hm-2, and the annual retention amount was 11.25 kg.hm-2. Among of the total returning, atmospheric dry and wet deposition was 38.36 kg.hm-2, and the litter returning was 58.08 kg.hm-2. The nutrient input was a little more than the output. The storage of the five nutrient elements in 0(-)-30 cm soil layer was 206,427.59 kg.hm-2, and their storage amount in stands only occupied about 0.15% of the total storage in soil. The absorption coefficient of the stands was N > P > K > Ca > Mg, the utilization coefficient was K > N > Mg > P > Ca, and the cycling coefficient was K > N > P > Mg > Ca. The turnover period of the N, P, K, Ca and Mg was 4.34, 7.51, 3.31, 12.90 and 6.45 yr, respectively.