A Combination of M50I and V151I Polymorphic Mutations in HIV-1 Subtype B Integrase Results in Defects in Autoprocessing

We have recently reported that a recombinant HIV-1NL4.3 containing Met-to-Ile change at codon 50 of integrase (IN) (IN:M50I) exhibits suppression of the virus release below 0.5% of WT HIV, and the released viral particles are replication-incompetent due to defects in Gag/GagPol processing by inhibition of the initiation of autoprocessing of GagPol polyproteins in the virions and leads to replication-incompetent viruses. The coexisting Ser-to-Asn change at codon 17 of IN or Asn-to-Ser mutation at codon 79 of RNaseH (RH) compensated the defective IN:M50I phenotype, suggesting that both IN and RH regulate an HIV infectability. In the current study, to elucidate a distribution of the three mutations during anti-retroviral therapy among patients, we performed a population analysis using 529 plasma virus RNA sequences obtained through the MiSeq. The result demonstrated that 14 plasma HIVs contained IN:M50I without the compensatory mutations. Comparing the sequences of the 14 viruses with that of the defective virus illustrated that only Val-to-Ile change at codon 151 of IN (IN:V151I) existed in the recombinant virus. This IN:V151I is known as a polymorphic mutation and was derived from HIVNL4.3 backbone. A back-mutation at 151 from Ile-to-Val in the defective virus recovered HIV replication capability, and Western Blotting assay displayed that the back-mutation restored Gag/GagPol processing in viral particles. These results demonstrate that a combination of IN:M50I and IN:V151I mutations, but not IN:M50I alone, produces a defective virus.

Hydrogen Enhanced the Myogenic Differentiation of Adipose Mesenchymal Stem Cells Through P38 MAPK Signaling Pathway

Purpose: This study aims to clarify the systems underlying regulation and regulatory roles of hydrogen in the myogenic differentiation of adipose mesenchymal stem cells (ADSCs). Materials and methods: In this study, ADSCs acted as an in vitro myogenic differentiating mode. First, the Alamar blue Staining and mitochondrial tracer technique were used to verify whether hydrogen could promote cell proliferation. In addition, this study assessed myogenic differentiating markers (e.g., Myogenin, Mhc and Myod protein expressions) based on the Western blotting assay, analysis on cellular morphological characteristics (e.g., Myotube number, length, diameter and maturation index), RT-PCR (Mhc and Myod mRNA expression) and Immunofluorescence analysis (Desmin, Myosin and β-actin protein expression). Lastly, to verify the myogenic differentiating system of hydrogen, Western blotting assay was performed to detect p38 and p-p38 proteins expressions. Results: Hydrogen can remarkably enhance the proliferation of ADSCs in vitro by increasing the number of single-cell mitochondria and by up-regulating the expression of myogenic biomarkers (e.g., Myod, Mhc and myotube formation). The expressions of both p38 and p-p38 were up-regulated by hydrogen. The differentiating ability was suppressed when the cells were cultivated in combination with SB203580 (p38 MAPK signal pathway inhibitor). Conclusions: The present study initially indicated that hydrogen can promote myogenic differentiation via the p38 MAPK pathway. Thus, the mentioned results present insights into myogenic differentiation and are likely to generate one potential alternative strategy for skeletal muscle related diseases.

GALNT14 regulates ferroptosis and apoptosis of ovarian cancer through the EGFR/mTOR pathway

Background: Chemoresistance usually occurs in ovarian cancer. We aimed to explore the mechanisms of chemoresistance. Methods: Western blotting assay was used to detect the expression of GALNT14. Further cell function experiments were performed to investigate the effect of GALNT14 in ovarian cancer. Results: GALNT14 is significantly upregulated in ovarian cancer. Downregulation of GALNT14 significantly inhibits both apoptosis and ferroptosis of ovarian cancer cells. A further mechanism assay illustrated that downregulation of GALNT14 suppresses the activity of the mTOR pathway through modifying O-glycosylation of EGFR. Finally, an additive effect promoting cell death occurs with a combination of an mTOR inhibitor and cisplatin. Conclusion: Our study might provide a promising method to overcome cisplatin resistance for patients with ovarian cancer.

Astragalin flavonoid inhibits proliferation in human lung carcinoma cells mediated via induction of caspase-dependent intrinsic pathway, ROS production, cell migration and invasion inhibition and targeting JAK/STAT signalling pathway

The aim of the current study was to investigate the anti-lung cancer effects of astragalin. Studies were also undertaken to evaluate its effects on apoptosis induction, ROS production, cellular migration and invasion and JAK/STAT3 signalling pathway. MTT assay was used to evaluate cell viability in NSCLC A549 cells after exposure to astragalin molecule. Apoptosis was investigated using AO/EB staining, comet assay and western blotting assay. Fluorescence microscopy was implemented to estimate ROS production. Cell migration and invasion were measured using transwell chambers assay. Effects of astragalin on JAK/STAT pathway were investigated using western blotting assay. Results showed astragalin molecule induced inhibition of proliferation in A549 cells in a dose-dependent fashion. Further, the antiproliferative effects were found to mediate via apoptosis as suggested by AO/EB staining and western blotting assay. Astragalin modulated the expressions of caspase-3, caspase-9, Bax, Bak, Cyt-c Bcl-2, XIAP and Bcl-xL. Astragalin induced DNA damage in A549 cells which too indicated apoptotic cell death. Astragalin molecule enhanced the production of ROS by A549 cells. It inhibited both cell migration and invasion of A549 cells in a concentration-dependent manner. Finally, astragalin drug was observed with remarkable potential of targeting JAK/STAT pathway in A549 NSCLC cells. These results indicated that astragalin drug could prove helpful in lung cancer treatment and research provided more in-vivo studies are performed.

Recombinant human IL-37 inhibited endometriosis development in a mouse model through increasing Th1/Th2 ratio by inducing the maturation of dendritic cells

Background Endometriosis is a serious reproductive and general health consequences. Recombinant human IL-37 (rhIL-37) is an inhibitor of inflammation. Methods ELISA assay was performed to detect the concentration of cytokines. Flow cytometry was used to analyze cell proportion. Besides, qRT-PCR and western blotting assay were used to detect the level of gene and protein, respectively. Transwell co-culture system was used for the co-culture of dendritic cells (DCs) and CD4+T cells. Results Our data showed that rhIL-37 inhibited the development of ectopic lesions in the mice with endometriosis, increased Th1/Th2 ratio and induced DCs maturation. The co-culture system of DCs and CD4+T cells demonstrated that rhIL-37 increased Th1/Th2 cell ratio through promoting DCs maturation. Moreover, the expression of IL-4 in the DCs derived from healthy mice was inhibited by rhIL-37 treatment. rhIL-37 increased Th1/Th2 cell ratio through inhibiting IL-4 in DCs. Subsequently, our results proved that rhIL-37 promoted the maturation of DCs via inhibiting phosphorylation of STAT3. Activation of STAT3 could reverse rhIL-37-induced maturation of DCs. Conclusion Overall, rhIL-37 could protect against endometriosis through increasing the ratio of Th1/Th2 cells via inducing DCs maturation and inhibiting IL-4 expression in the DCs. Furthermore, rhIL-37 induced DCs maturation by inhibiting STAT3 phosphorylation. Our data confirmed the protective effect of rhIL-37 in endometriosis. These data may provide a novel idea for the treatment of the disease. Graphical abstract

The Effect and Mechanism of lncRNA NR2F1-As1/miR-493-5p/MAP3K2 Axis in the Progression of Gastric Cancer

Background. LncRNA NR2F1-AS1 has been identified as an oncogene in some human tumors, such as breast cancer, nonsmall cell lung cancer, and esophageal squamous cell carcinoma. Nonetheless, whether NR2F1-AS1 is involved in the progression of gastric cancer (GC) remains unknown. Methods. The expression patterns of NR2F1-AS1, MAP3K2, and miR-493-5p in GC tissues and cells were detected by RT-qPCR. The protein expression of MAP3K2 was assessed by the Western blotting assay. The MTT assay and flow cytometry were performed to measure cell proliferation and cell apoptosis in GC cells. The transwell assay was adopted to assess cell migration in GC cells. The relationship between NR2F1-AS1, MAP3K2, and miR-493-5p was verified by a dual-luciferase reporter assay. Results. The increased NR2F1-AS1 and MAP3K2 expressions were discovered in GC tissues and cells compared with control groups. Knockdown of NR2F1-AS1 and MAP3K2 dramatically suppressed cell proliferation and migration, while it enhanced cell apoptosis in GC cells. In addition, NR2F1-AS1 was found to be a sponge of miR-493-5p, and MAP3K2 was a downstream gene of miR-493-5p. Moreover, the expression of MAP3K2 was notably reduced by miR-493-5p, and NR2F1-AS1 counteracted the inhibition of miR-493-5p. Conclusion. Thus, NR2F1-AS1 was verified to regulate GC cell progression by sponging miR-493-5p to upregulate MAP3K2 expression.

Proteasome inhibitor MG132 induces apoptosis in human osteosarcoma U2OS cells

MG132 is a potent, reversible, and cell-permeable 20S proteasome inhibitor and it is derived from a Chinese medicinal plant. The purpose of this study is to investigate the anticancer effects of MG132 against human osteosarcoma U2OS cells. We first performed MTT and colony formation assays to investigate the anti-proliferative effects of MG132. The results demonstrated that MG132 suppressed the proliferation of U2OS cells. Furthermore, we found that treatment with MG132 increased apoptosis and induced DNA damage in U2OS cells. Additionally, zymography, wound healing, and invasion assays showed that MG132 suppressed the enzymatic activity of matrix metalloproteinases, cell migration, and invasion, respectively of U2OS cells. Furthermore, western blotting assay was performed to investigate the apoptotic signaling pathways in MG132-treated U2OS cells. Our results showed that MG132 downregulated the expression of antiapoptotic proteins, including CDK2, CDK4, Bcl-xL, and Bcl-2, whereas it upregulated the expression of proapoptotic proteins, including p21, p27, p53, p-p53 (ser15, ser20, and ser46), cleaved forms of caspase-3, caspase-7, caspase-9, and PARP, and FOXO3 in U2OS cells. These results demonstrated that MG132 activated apoptotic signaling pathways in U2OS cells. Interestingly, MG132 downregulated the phosphorylation of Akt and Erk. Taken together, our results suggest that MG132 has anticancer effects in U2OS cells. Therefore, MG132 may be a potential therapeutic agent for the treatment of osteosarcoma.

Exosomes from adipose-derived mesenchymal stem cells can promote fibroblasts proliferation, migration, and collagen synthesis via activating Wnt/β-catenin signaling pathway during wound healing

Background Differentiation, migration, proliferation of skin fibroblasts are identified as the key factors during the cutaneous wound healing. Adipose-derived mesenchymal stem cells (ADMSCs) have been recorded as possible candidates for wound treatment because of their positive effect on the regeneration of many tissues. Exosomes derived from ADMSCs (ADMSC-Exos), an important signal transduction substance secreted by ADMSCs, have a similar role to ADMSCs in wound healing. However, the effects of ADMSC‐Exos on cutaneous wound healing remain to be unclear. In this study, we tried to explore the role and mechaninsm of ADMSC‐Exos during cutaneous wound healing. Methods Human skin fibroblasts (HSF) and ADMSCs were isolated from skin and adipose tissues of healthy person. ADMSC-Exos were purified from human ADMSCs culture medium by differential ultracentrifugation and identified by Electron microscopy, Nanoparticle tracking, and Western blotting assay. Fibroblasts were treated with different concentrations of ADMSC‐Exos. The proliferation and migration abilities of fibroblasts were analyzed by CCK-8 assay and scratch method. The synthesis of collagen type I (Col-I), collagen type III (Col-III), and α-smooth muscle actin (α-SMA) in fibroblasts was assessed by real-time quantitative polymerase chain reaction and Western blotting assay. A tensional wound model on rat back was used to evaluate the effect of ADMSC-Exos on wound healing. The expression levels of Wntb2 and β-catenin were analyzed by Western blotting and immunohistochemical assay. Results ADMSC-Exos were successfully obtained. ADMSC-Exos could significantly promote the migration and proliferation ability of fibroblasts in a dose-dependent manner in vitro. Compared with the treatment without ADMSC-Exos, the expression levels of Col-I and Col-III in fibroblasts treated with ADMSC-Exos were significantly increased, while the expression level of α-SMA is decreased. Besides, the enhanced expression of Wnt2b and β-catenin proteins confirmed the activation of the Wnt/β-catenin signaling pathway. Conclusions ADMSC-Exos can promote fibroblasts proliferation, migration, and collagen synthesis in a dose-dependent manner and may play a positive role in skin wound healing through Wnt/β-catenin signaling pathway. So our study elucidates part of the mechanism of ADMSC-Exos in wound healing, which illustrates the therapeutic potential of ADMSC-Exos as a new therapeutic approach to promote skin wound healing.

Neutrophil Extracellular Traps and Macrophage Polarization Formation In The Inner And Outer Area of Carotid Plaque Arteries

Introduction: It is noteworthy that vast data exists which links NETs to arterial and venous thrombosis in both animal models and humans. In the current study, the level of extracellular neutrophil networks and macrophage polarization in the area outside and inside the carotid plaque of patients with carotid stenosis were assessed.Material and Methods: Ten patients were included in this pilot study. Confirmed cases of carotid stenosis were selected to participate in the study using the simple sampling method. Samples of carotid plaques of each patient were divided into two halves with a transverse incision; the terms inner part and outer part were used for the plaque’s inner part and the adjacent area, respectively. Carotid plaque was excised, and half of them were sorted in 10% formalin for CD163,CD11c ,MPO and histone H3 immunohistochemistry assessment while the other halves were stored at -80 ° C for western blotting assay with PDA4 marker. For statistical analysis, we used independent samples T-test or its non-parametric equivalents.Results: Results of this study showed that the extracellular neutrophil chicks in the inner part of the carotid plaque were significantly increased (P <0.0001), while the number of M1 and M2 macrophages was higher in the inner part compared with the outer part of the carotid plaque (P <0.0001).Conclusion: NETs and Macrophages have great potential for further investigation to find a better treatment for carotid plaque.