MiR‐139‐5p inhibits the tumorigenesis and progression of oral squamous carcinoma cells by targeting HOXA9

Our study sought to clarify the effects of microRNA‐139‐5p (miR‐139‐5p) in the tumorigenesis and progression of oral squamous cell carcinoma (OSCC) by regulating HOXA9. MiR‐139‐5p and HOXA9 expression in OSCC tissues, tumour adjacent tissues, OSCC cells and normal cells were tested by qRT‐PCR. SAS and CAL‐27 cell lines were selected in among four OSCC cell lines and then transfected with miR‐139‐5p mimics, pEGFP‐HOXA9 and cotransfected with miR‐139‐5p mimics + pEGFP‐HOXA9. We used MTT, colony formation, transwell and wound healing assays to analyse cell viability, proliferation, invasion and migration. The target relationship between miR‐139‐5p and HOXA9 was verified by luciferase reporter assay and Western blot, respectively. MiR‐139‐5p was down‐regulated, whereas HOXA9 was up‐regulated in OSCC tissues and cells. The proliferation, invasion and migration ability of SAS and CAL‐27 cells in miR‐139‐5p mimics group were significantly weaker than those in the control group and the miR‐NC group (P < 0.01). MiR‐139‐5p can negatively regulate HOXA9. The proliferation, invasion and migration of SAS and CAL‐27 cells in the miR‐139‐5p mimics + pEGFP‐HOXA9 group were not significantly different from those in the blank control and negative control groups (P > 0.05). Our results indicated that miR‐139‐5p could directly inhibit HOXA9, which might be a potential mechanism in inhibiting the proliferation, invasiveness and migration of OSCC cells.     

Effect of microRNA-27a-5p on apoptosis and inflammatory response of pancreatic acinar cells in acute pancreatitis by targeting PTEN

To investigate the apoptosis and inflammatory response of microRNA-27a-5p (miR-27a-5p) in pancreatic acinar cells of acute pancreatitis (AP) and its related mechanisms. Rat pancreatic acinar cell line AR42J was treated with caerulein (10nmol/L) to construct an acute pancreatitis cell model. Quantitative real-time polymerase chain reaction was performed to measure the expression of miR-27a-5p; The miR-27a-5p mimic was transfected into cell, and the apoptosis rate of the cells was detected by flow cytometry; The levels of TNF-α, IL-1, and IL-6 in the culture supernatant were determined by enzyme-linked immunosorbent assay; TargetScans database predicted and dual luciferase reporter gene assay verified the relationship between miR-27a-5p and the phosphatase and tensin homolog deleted on chromosome 10 (PTEN); The recovery experiment explored the apoptosis and the effects of inflammatory responses. The expression of miR-27a-5p decreased gradually (P < 0.05) and the expression of PTEN increased gradually (P < 0.05) with the prolongation of acting time. Upregulation of miR-27a-5p significantly promoted cell apoptosis (P < 0.05) and inhibited inflammatory response (P < 0.05); The TargetScans database predicted that the 3'UTR of PTEN contains a base complementary to the miR-27a-5p seed region. Cotransfection of wild-type vector (PTEN-WT) with miR-27a-5p mimic or miR-27a-5p inhibitor significantly affected the relative activity of luciferase (P < 0.05), and no significant impact was observed in mutant PTEN-MUT. Compared with miR-27a-5p + pcDNA group, transfection of miR-27a-5p mimic and pcDNA-PTEN significantly increased the expression of PTEN (P < 0.05), decreased the apoptotic rate (P < 0.05), and increased the inflammatory response (P < 0.05). miR-27a-5p induced apoptosis and inhibited the inflammatory response of pancreatic acinar cells in AP by targeting PTEN.     

Analysis of factors of pulmonary fungal infection in mice in respiratory medicine department based on logistic regression analysis model and Progranulin

The objective of this research is to solve the current medical problems of a high incidence of fungal infections in the lungs, high misdiagnosis rate, and high mortality. In this study, firstly, the logistic regression model was used to conduct. Risk factors of pulmonary fungal infection in respiratory department were analyzed. Then a model of pulmonary fungal infection in mice was constructed, and the expression difference of Progranulin (PGRN) in serum was detected by enzyme-linked immuno sorbent assay (ELISA). The expression of PGRN in lung tissues of mice infected by pulmonary fungi was detected by Western bolt method and quantitative polymerase chain reaction (PCR). The PGRN protein and mRNA expression in the lung epithelial cells of mice were detected after the infection. Results logistic regression model was used to analyze the main risk factors affecting pulmonary infection in mice. The risk factors of pulmonary fungal infection were indent catheter, hypoproteinemia, long-term use of glucocorticoid and long-term use of antibiotics. The PGRN content in serum was obviously higher than that before pulmonary fungal infection (P < 0.01). The expression of PGRN mRNA and protein in lung tissue was obviously higher than that before infection (P < 0.01). The expression of PGRN mRNA and protein in lung tissues of the infected group was obviously higher than that of the non-infected group (P < 0.01). The expression of PGRN protein in the lung epithelial cells of mice was obviously higher at 24 h after infection than before infection (P < 0.01), and the expression of PGRN mRNA was obviously higher at 12 h after infection than before infection (P < 0.01), indicating that PGRN is highly expressed in fungal pulmonary infection and is involved in disease progression. Therefore, this study provides a new idea for the diagnosis and treatment of fungal pulmonary infection in the later stage and has a good guiding significance for the diagnosis and treatment of fungal pulmonary infection.     

Seasonal modulation of immunity by melatonin and gonadal steroids in a short day breeder goat Capra hircus

Role of melatonin in regulation of immunity and reproduction has never been studied in detail in goats. The aim of the present study was to explore hormonal regulation of immunity in goats with special reference to melatonin. Plasma of male and female goats (n = 18 per sex per season) was processed for hormonal (estrogen, testostrone, and melatonin) and cytokine (interleukin [IL-2], IL-6, and tumor necrosis factor α) measurements during three seasons, i.e., summer, monsoon, and winter. To assess cell-mediated immune response, percent stimulation ratio of thymocytes was recorded during three seasons. To support and establish the modulation by hormones, Western blot analysis for expressions of melatonin receptors (MT1, MT2), androgen receptor, and estrogen receptor α and estimations of marker enzymes, arylalkylamine N-acetyltransferase for melatonin and 3β-hydroxysteroid dehydrogenase activities for steroidogenesis were performed in thymus. All the hormones and cytokines were estimated by commercial kits. Biochemical, immunologic, and Western blot analyses were done by standardized protocols. We noted a significant increase in estrogen and testosterone levels (P < 0.05) in circulation during monsoon along with melatonin (P < 0.05) presenting a parallel relationship. Expressions of melatonin receptors (MT1 and MT2) in thymus of both the sexes were significantly high (P < 0.01) during winter. Estrogen receptor α expression in female thymus was significantly high during monsoon (P < 0.05). However, androgen receptor showed almost static expression pattern in male thymus during three seasons. Further, both arylalkylamineN-acetyltransferase and 3β-hydroxysteroid dehydrogenase enzyme activities were significantly high (P < 0.05; P < 0.01, respectively) during monsoon. These results suggest that there may be a functional parallelism between gonadal steroids and melatonin as melatonin is progonadotrophic in goats. Cell-mediated immune parameters (percent stimulation ratio of thymocytes) and circulatory levels of cytokines (IL-2, IL-6, and tumor necrosis factor α) were significantly high (P < 0.01) during monsoon. In vitro supplementation of gonadal steroids to T-cell culture suppressed immunity but cosupplementation with melatonin restored it. Further, we may also suggest that reproductive and immune seasonality are maintained by variations in circulatory hormones and local synthesis of melatonin and gonadal steroids. These functional interactions between melatonin and gonadal steroid might be of great importance in regulating the goat immunity by developing some hormonal microcircuit (gonadal steroid and melatonin) in lymphatic organs.     

上调OPN和TIP30表达对口腔鳞癌cal-27细胞的影响

目的:探讨上调骨桥蛋白(OPN)和TIP30表达对口腔鳞状细胞癌cal-27细胞增殖和迁移能力的影响。方法:采用过表达OPN载体病毒和过表达TIP30质粒分别感染或转染cal-27细胞株后,通过实时荧光定量PCR法和蛋白印迹western blot法检测OPN和TIP30的表达,CCK-8法检测细胞增殖能力,细胞划痕实验检测细胞的迁移能力。结果:过表达OPN载体病毒感染的cal-27-sh OPN细胞株OPN m RNA和蛋白的表达均明显高于cal-27-sh N细胞株(P0.001,P=0.002),且细胞的增殖能力和迁移能力强(P0.001,P=0.002)。过表达TIP30质粒转染的cal-27-p TIP30细胞株TIP30 m RNA和蛋白的表达均明显高于cal-27-pc3.0细胞株(P=0.001,P=0.003),且细胞的增殖能力和迁移能力弱(P0.001,P=0.005)。结论:OPN能促进cal-27细胞的增殖和迁移,TIP30抑制cal-27细胞的增殖和迁移。     

Association of interleukin‐27 gene polymorphisms with susceptibility to HIV infection and disease progression

Interleukin‐27 (IL‐27) gene polymorphisms are linked to infectious disease susceptibility and IL‐27 plasma level is associated with HIV infection. Therefore, we aimed to investigate the association between IL‐27 polymorphisms and susceptibility to HIV infection and disease progression. A total of 300 patients with HIV infection (48 long‐term nonprogressors and 252 typical progressors) and 300 healthy controls were genotyped for three IL‐27 polymorphisms, rs17855750, rs181206, rs40837 which were performed by using multiple single nucleotide primer extension technique. Significant association was found between IL‐27 rs40837 polymorphisms with susceptibility to HIV infection (AG vs AA: adjusted OR = 1.60, 95% CI, 1.11‐2.30, P = 0.012; AG+GG vs AA: adjusted OR = 1.44, 95% CI, 1.02‐2.03, P = 0.038) and disease progression (LTNP: AG vs AA: adjusted OR = 2.33, 95% CI, 1.13‐4.80, P = 0.021; TP: AG vs AA: adjusted OR = 1.50, 95% CI, 1.04‐2.24, P = 0.030). Serum IL‐27 levels were significantly lower in cases compared to controls (P < 0.001). There were lower serum IL‐27 levels in TPs than in LTNPs (P < 0.001). We further found that LTNPs with rs40837 AG or GG genotype had lower serum IL‐27 levels than with AA genotype (P < 0.05). The CD4⁺T counts in cases were significantly lower than controls (P < 0.001). In contrast, individuals with rs40837 AG genotype had lower CD4⁺T counts than with AA genotype in cases (P < 0.05). In addition, CD4⁺T counts in TPs were significantly lower than LTNPs (P < 0.001). IL‐27 rs40837 polymorphism might influence the susceptibility to HIV infection and disease progression probably by regulating the level of serum IL‐27 or the quantity of CD4⁺T.     

Nanoscale characterization coupled to multi-parametric optimization of Hi5 cell transient gene expression

Polyethylenimine (PEI)-based transient gene expression (TGE) is nowadays a well-established methodology for rapid protein production in mammalian cells, but it has been used to a much lower extent in insect cell lines. A fast and robust TGE methodology for suspension Hi5 (Trichoplusia ni) cells is presented. Significant differences in size and morphology of DNA:PEI polyplexes were observed in the different incubation solutions tested. Moreover, minimal complexing time (&lt; 1 min) between DNA and PEI in 150 mM NaCl solution provided the highest transfection efficiency. Nanoscopic characterization by means of cryo-EM revealed that DNA:PEI polyplexes up to 300–400 nm were the most efficient for transfection. TGE optimization was performed using eGFP as model protein by means of the combination of advanced statistical designs. A global optimal condition of 1.5 × 10⁶ cell/mL, 2.1 μg/mL of DNA, and 9.3 μg/mL PEI was achieved through weighted-based optimization of transfection, production, and viability responses. Under these conditions, a 60% transfection and 0.8 μg/10⁶ transfected cell·day specific productivity were achieved. The TGE protocol developed for Hi5 cells provides a promising baculovirus-free and worthwhile approach to produce a wide variety of recombinant proteins in a short period of time.

     

DNA methylation and histone deacetylation regulating insulin sensitivity due to chronic cold exposure

In this study, we investigated the causal relationship between chronic cold exposure and insulin resistance and the mechanisms of how DNA methylation and histone deacetylation regulate cold-reduced insulin resistance. 46 adult male mice from postnatal day 90–180 were randomly assigned to control group and cold-exposure group. Mice in cold-exposure group were placed at temperature from -1 to 4 °C for 30 days to mimic chronic cold environment. Then, fasting blood glucose, blood insulin level and insulin resistance index were measured with enzymatic methods. Immunofluorescent labeling was carried out to visualize the insulin receptor substrate 2 (IRS2), Obese receptor (Ob-R, a leptin receptor), voltage-dependent anion channel protein 1 (VDAC1), cytochrome C (cytC), 5-methylcytosine (5-mC) positive cells in hippocampal CA1 area. Furthermore, the expressions of some proteins mentioned above were detected with Western blot. The results showed: ① Chronic cold exposure could reduce the insulin resistance index (P < 0.01) and increase the number of IRS2 positive cells and Ob-R positive cells in hippocampus (P < 0.01). ② The expressions of mitochondrial energy-relative proteins, VDAC1 and cytC, were higher in cold-exposure group than in control group with both immunohistochemical staining and Western blot (P < 0.01). ③ Chronic cold exposure increased DNA methylation and histone deacetylation in the pyramidal cells of CA1 area and led to an increase in the expression of histone deacetylase 1 (HDAC1) and DNA methylation relative enzymes (P < 0.01). In conclusion, chronic cold exposure can improve insulin sensitivity, with the involvement of DNA methylation, histone deacetylation and the regulation of mitochondrial energy metabolism. These epigenetic modifications probably form the basic mechanism of cold-reduced insulin resistance.     

Therapeutic effect of autologous bone marrow stem cell mobilization combined with anti-infective therapy on moyamoya disease

ObjectiveThe purpose of this study is to explore the therapeutic effect of autologous bone marrow stem cell (ABMSC) mobilization combined anti-infection therapy on patients with moyamoya disease (MMD), and to provide reference for the clinical treatment of MMD and cerebrovascular disease.Methods54 adult patients with MMD diagnosed in Henan Provincial People’s Hospital from March 2017 to March 2019 were chosen as research objects. All patients were randomly divided into study group (SG) and control group (CG), with 27 patients in each group. Patients in both groups received conventional drug treatment after diagnosis of MMD, and received dura turnover of brain - temporal muscle - superficial temporal artery application surgery during indirect vascular reconstruction. On the basis of surgical treatment, patients in the SG were given ABMSC mobilization combined with low-dose dexamethasone for anti-inflammatory and anti-infection treatment. ABMSCs were mobilized by recombinant human granulocyte colony stimulating factor (rhG-csF) and recombinant human granulocyte - macrophage colony stimulating factor (rhoM-esF). The therapeutic effects of the patients were evaluated BF, one month after treatment (AF), three months AF, and six months AF. The number of hematopoietic stem cells (HpCs) and inflammatory indicators were compared between the two groups before and 4 weeks AF.ResultsFirstly, the Barthcl index of patients in the two groups showed a gradual increase trend at the 3rd and 6th months AF, and the ascensional range in the research group was higher than that in the CG (P < 0.05). Secondly, at the 3rd and 6th month AF, national institute of heath stroke scale (NIHSS) scores of patients in the CG were lower than those before treatment (BF), and there was an important change in NIHSS scores between the two groups at the same period (P < 0.05). Thirdly, after 1 month of treatment and 3 months of treatment, Chinese stroke scale (CSS) scores of patients in both groups decreased obviously compared with those BF, and the SG was lower than the CG, with statistical changes (P < 0.05). Fourthly, after 4 weeks of treatment, the hematopoietic stem cell counts in both groups were higher than those BF, and the hematopoietic stem cell counts in the SG were obviously higher than those in the CG (P < 0.05). All three inflammatory indicators were improved compared with those BF, and the SG was better than the CG (P < 0.05).ConclusionAutogenous bone marrow stem cell mobilization combined with dexamethasone anti-inflammation and anti-infection treatment after revascularization in patients with MMD can accelerate the recovery of nerve function and promote the formation of new blood vessels. At the same time, it can reduce inflammation and improve patients' quality of life, which is worthy of clinical reference.     

Inhibition of the long non-coding RNA ZFAS1 attenuates ferroptosis by sponging miR-150-5p and activates CCND2 against diabetic cardiomyopathy

Diabetic cardiomyopathy (DbCM) is responsible for increased morbidity and mortality in patients with diabetes and heart failure. However, the pathogenesis of DbCM has not yet been identified. Here, we investigated the important role of lncRNA-ZFAS1 in the pathological process of DbCM, which is associated with ferroptosis. Microarray data analysis of DbCM in patients or mouse models from GEO revealed the significance of ZFAS1 and the significant downregulation of miR-150-5p and CCND2. Briefly, DbCM was established in high glucose (HG)–treated cardiomyocytes and db/db mice to form in vitro and in vivo models. Ad-ZFAS1, Ad-sh-ZFAS1, mimic miR-150-5p, Ad-CCND2 and Ad-sh-CCND2 were intracoronarily administered to the mouse model or transfected into HG-treated cardiomyocytes to determine whether ZFAS1 regulates miR-150-5p and CCND2 in ferroptosis. The effect of ZFAS1 on the left ventricular myocardial tissues of db/db mice and HG-treated cardiomyocytes, ferroptosis and apoptosis was determined by Masson staining, immunohistochemical staining, Western blotting, monobromobimane staining, immunofluorescence staining and JC-1 staining. The relationships among ZFAS1, miR-150-5p and CCND2 were evaluated using dual-luciferase reporter assays and RNA pull-down assays. Inhibition of ZFAS1 led to reduced collagen deposition, decreased cardiomyocyte apoptosis and ferroptosis, and attenuated DbCM progression. ZFAS1 sponges miR-150-5p to downregulate CCND2 expression. Ad-sh-ZFAS1, miR-150-5p mimic, and Ad-CCND2 transfection attenuated ferroptosis and DbCM development both in vitro and in vivo. However, transfection with Ad-ZFAS1 could reverse the positive effects of miR-150-5p mimic and Ad-CCND2 in vitro and in vivo. lncRNA-ZFAS1 acted as a ceRNA to sponge miR-150-5p and downregulate CCND2 to promote cardiomyocyte ferroptosis and DbCM development. Thus, ZFAS1 inhibition could be a promising therapeutic target for the treatment and prevention of DbCM.     

The effect of Arbas Cashmere goat bone marrow stromal cells on production of transgenic cloned embryos

The aim of this study was to develop a method for the in vitro separation and culture of Arbas Cashmere goat bone marrow stromal cells (gBMSCs). Arbas Cashmere gBMSCs were isolated and cultured in vitro, and cell surface markers were identified immunohistochemically. The gBMSCs were differentiated into neurocytes and osteoblasts, and the expression of neuron-specific enolase and osteocalcin was identified by immunohistochemistry. The gBMSCs and goat fetal fibroblast cells (gFFCs) were compared for transient transfection efficiency and fluorescent colony-forming efficiency with Arbas Cashmere gFFCs as a control. pDsRed2-1 encodes DsRed2, a variant of the Discosoma sp. red fluorescent protein (DsRed). In addition, the coding sequence for DsRed2 contains a series of silent base-pair changes for higher expression in mammalian cells. Of the gBMSCs-pDsRed2-1, one fraction was tested for pluripotency, whereas the other fraction was manipulated using somatic cell nuclear transfer, and the in vitro growth status of transgenic embryos derived from gBMSCs-pDsRed2-1 and gFFCs-pDsRed2-1 was compared. The findings showed that gBMSCs were isolated and amplified to express CD29, CD44, and CD90 through adherent culture, with no marked signs of aging after multiple passages. Expression of neuron-specific enolase and osteocalcin by gBMSCs and gBMSCs-pDsRed2-1 was strongly induced by neuronal and osteogenic differentiation, whereas the integrated exogenous genes did not influence pluripotency (P > 0.05). The transient transfection efficiencies of gBMSCs and gFFCs after 48 hours were not significantly different; however, the fluorescent colony-forming efficiency of gBMSCs-pDsRed2-1 after G418 screening was approximately 13% higher than that of gFFCs-pDsRed2-1. The convergence and cleavage rates of cloned embryos derived from gBMSCs-pDsRed2-1 were higher than those derived from gFFCs-pDsRed2-1, whereas their eight-cell and blastocyst rates were similar. The red fluorescent protein expression levels were higher in transgenic embryos derived from gBMSCs-pDsRed2-1 compared with those derived from gFFCs-pDsRed2-1 (48.8% vs. 31.1%, respectively) (P < 0.01). Real-time quantitative Polymerase Chain Reaction analysis showed that DsRed2-1 messenger RNA expression of cloned embryos derived from gBMSCs was 2.24 greater than that of embryos derived from gFFCs-pDsRed2-1 (P < 0.01). Similarly, Western blot analysis showed that DsRed2 protein expression of cloned embryos derived from gBMSCs-pDsRed2-1 was 1.29 greater than that of embryos derived from gFFCs-pDsRed2-1 (P < 0.01). In this study, gBMSCs were also used for somatic cell nuclear transfer and shown to provide effective nuclear donor cells for breeding new genetically modified varieties of livestock.     

Hyaluronate decorated polyethylene glycol linked poly(lactide-co-glycolide) nanoparticles encapsulating MUC-1 peptide augmented mucosal immune response in Balb/c mice through inhalation route

Background and objectivesNSCLC (Non-Small Cell Lung Cancer) clutches highest mortality rate in man and women globally. The present study was conducted to target MUC-1 peptide (M-1) into antigen presenting cells by cargo the peptide into hyaluronic acid decorated polyethylene glycol linked poly (D, l-lactide-co-glycolide) nanoparticles (M-1-PL-co-GA-PEG-sHA-NPs) for generating mucosal immunity through inhalation (i.h.) route.Methodology and resultsThe mean particle size and surface charge of M-1-PL-co-GA-PEG-sHA-NPs was measured to be 136.2 ± 18.38-nm and ? 28.34 ± 6.77-mV, respectively, prepared by non-aggregated emulsion-diffusion evaporation method. The 28.42% percentage release of M-1 peptide from M-1-PL-co-GA-PEG-NPs was observed to be at 2 h and 95.29% at 8 h while the percentage release of M-1 peptide from M-1-PL-co-GA-PEG-sHA-NPs was observed to be 26.02% at 4 h and 97.95% at 24 h that proved the prolonged release of antigen. M-1-PL-co-GA-PEG-sHA-NPs demonstrated higher (P < 0.05) cellular uptake of 86.2% in RAW 264.7 cells in comparison to 27.6% of M-1-PL-co-GA-PEG-NPs. In addition, M-1-PL-co-GA-PEG-sHA-NPs induced remarkably (P < 0.05) elevated release of 80.6-pg/ml of TNF-α in comparison to 5-pg/ml by culture medium and 57.9-pg/ml of TNF-α by M-1-PL-co-GA-PEG-NPs. Similarly, M-1-PL-co-GA-PEG-sHA-NPs persuade remarkably (P < 0.05) elevated release of 225-pg/ml of IL-1β in comparison to 47-pg/ml by culture medium and 161.9-pg/ml of IL-1β by M-1-PL-co-GA-PEG-NPs. M-1-PL-co-GA-PEG-sHA-NPs might have been endocytosed through receptor mediated pathway owing to presence of sHA. Mice immunized through i.h. route with M-1-PL-co-GA-PEG-sHA-NPs induced strong (P < 0.05) IgA antibody titre as compared to M-1-PL-co-GA-PEG-NPs and M-1 peptide in dose-dosage regimen.ConclusionM-1-PL-co-GA-PEG-sHA-NPs nanovaccine warrants further analysis in xenograft model of NSCLC to showcase its antitumor capability.     

Gut morphology of diploid and triploid Atlantic cod,Gadus morhua

Little is known about the functional consequences of triploidy in Atlantic cod. This study compared the gut morphology of diploid and triploid offspring of wild and selected broodstock. Three‐year‐old triploid offspring of wild cod (mean weight 1695 ± 346 g) had approximately 18% fewer pyloric caeca (125 ± 9 vs 172 ± 14, P < 0.001) and a 23% shorter intestine [Relative Gut Length, (RGL); 1.40 ± 0.17 vs 1.81 ± 0.17, P < 0.05] than their diploid siblings (mean weight: 1820 ± 262 g). Two‐year‐old triploid offspring of selected broodstock (mean weight: 640 ± 64 g) had 20% fewer pyloric caeca (309 ± 17 vs 387 ± 27, P < 0.001) but similar RGL to their diploid siblings (mean weight: 820 ± 69 g). The average number of mucus cells in the columnar epithelium of pyloric caeca was significantly higher in triploid than in diploid cod (54 ± 9 vs 25 ± 5, P < 0.001). There was no correlation between pyloric caeca number and RGL, or between mucus cells and pyloric caeca number, and no significant differences between sexes for any of the measured variables. Overall, the observations highlight some differences in the digestive system of these two ploidy groups that could have an influence on nutrient utilization and performance capacity in triploids compared to diploids.     

Effect of polymorphisms in the CAMKMT gene on growth traits in Ujumqin sheep

Our previous genome‐wide association study in sheep revealed that OAR3‐84073899.1 (SNP31) in intron 8 of the CAMKMT gene was significantly associated with post‐weaning gain at the genomic level. Herein, we performed a replication study to investigate single nucleotide polymorphisms (SNPs) within the CAMKMT gene exons, and 1000 bp of the 5′‐ and 3′‐intranslated regions (UTRs) and their associations with growth traits in Ujumqin sheep. Five SNPs were identified through DNA pool sequencing technology: SNP26 in the 5′‐UTR, SNP06 in exon 5, SNP07 in exon 8 and SNP27 and SNP28 in the 3′‐UTR. Six SNPs, including SNP31 in intron 8, were genotyped in the validation group of 343 Ujumqin sheep, and each SNP was classified into three genotypes. The chi‐square test suggested that all the variations were in Hardy–Weinberg equilibrium (P > 0.05) except for SNP28 and SNP31. Linkage disequilibrium analysis showed that SNP07 and SNP31 were strongly linked. An association analysis suggested that SNP06 was significantly associated with chest girth at 6 months of age (P < 0.05). SNP07 exhibited significant correlation with body weight and chest girth at 4 months of age and with body weight, chest girth and chest width at 6 months of age (P < 0.05). SNP27 was highly associated with body weight and chest girth at 4 months of age (P < 0.05), and SNP28 was extremely significantly associated with body weight and chest girth at 4 months of age and with chest girth at 6 months of age (P < 0.01). SNP31 was significantly associated with body weight and shin circumference at 4 months of age and with post‐weaning gain (P < 0.05). Association analysis of the combined effect of SNP07 and SNP31 showed significant correlation with body weight and chest girth at four of months of age (P < 0.05) and with body weight and chest girth at 6 months of age (P < 0.05). These results indicate that the SNPs could be used as meritorious and available genetic markers in growth traits breeding and that the CAMKMT gene may be one of the key candidate genes that affect Ujumqin economic traits.     

High cell density transient transfection of CHO cells for TGF‐β1 expression

High cell densities for transient transfection with polyethyleneimine (PEI) can be used for rapid and maximal production of recombinant proteins. High cell densities can be obtained by different cultivation systems, such as batch or perfusion systems. Herein, densities up to 18 million cells/mL were obtained by centrifugation for transfection evaluation. PEI transfection efficiency was easily determined by transfected enhanced green fluorescence protein (EGFP) reporter plasmid DNA (pDNA). A linear correlation between fluorescence intensity and transfection efficiency was improved. The transfection efficiency of PEI was highly dependent on the transfection conditions and directly related to the level of recombinant protein. Several factors were required to optimize the transient transfection process; these factors included the media type (which is compatible with low or high cell density transfection), the preculture CHO‐K1 suspension cell density, and the pDNA to PEI level. Based on design of experiment (DoE) analyses, the optimal transfection conditions for 10 × 10⁶ cells/mL in the CHOMACS CD medium achieved 73% transfection efficiency and a cell viability of over 80%. These results were confirmed for the production of transforming growth factor‐beta 1 (TGF‐β1) in a shake flask. The purified TGF‐β1 protein concentration from 60 mL supernatant was 27 µg/mL, and the protein was biologically active.     

A protective effect of curcumin on cardiovascular oxidative stress indicators in systemic inflammation induced by lipopolysaccharide in rats

ObjectiveInflammation has been considered as an important factor in cardiovascular diseases (CVD). Curcumin has been well known for its anti-inflammatory effects. In current research, protective effect of curcumin on cardiovascular oxidative stress indicators in systemic inflammation induced by lipopolysaccharide (LPS) was investigated in rats.Material and methodsThe animals were divided into five groups and received the treatments during two weeks [1]: Control in which vehicle was administered instead of curcumin and saline was injected instead of LPS [2], LPS group in which vehicle of curcumin plus LPS (1 mg/kg) was administered [3-5], curcumin groups in them three doses of curcumin (5, 10 and 15 mg/kg) before LPS were administered.ResultsAdministration of LPS was followed by an inflammation status presented by an increased level of white blood cells (WBC) (p < 0.001). An oxidative stress status was also occurred after LPS injection which was presented by an increased level of malondialdehyde (MDA) while, a decrease in thiols, superoxide dismutase (SOD) and catalase(CAT) in all heart, aorta and serum (p < 0.001). The results also showed that curcumin decreased WBC (doses: 10 and 15 mg/kg) (p < 0.001) accompanying with a decrease in MDA (P < 0.01 and P < 0.001). Curcumin also improved the thiols and the activities of SOD and catalase (P < 0.05, P < 0.01 and P < 0.001).ConclusionBased on our findings, curcumin can ameliorates oxidative stress and inflammation induced by LPS in rats to protect the cardiovascular system.     

Protective role of scutellarin on LPS induced – Acute lung injury and regulation of apoptosis,oxidative stress and reduction of mitochondrial dysfunction

Lung fluid accumulation was determined using wet/dry lung mass ratio. Rats subjected to LPS-induced acute lung injury (2.8 ± 0.33, P < 0.05) presented with a significantly higher wet to dry lung weight ration ratio than sham rats (1.6 ± 0.23, P < 0.05). These results demonstrate that acutely inured rats' lungs were oedematous. On the other hand, treatment with scutellarin alone and in combination with a JNK inhibitor, SP600125, both significantly attenuated pulmonary edema as shown via reduced wet/dry lung mass ratios (1.7 ± 0.09 and 1.8 ± 0.23; P < 0.05, respectively). These results showed that the interventions were effective against LPS-induced edema of the lungs. However, the difference between treatment groups' weight ratios was not statistically significant (P > 0.05). In the sham control rats, the levels of ROS and SOD production were maintained at a low and at a high concentration, respectively (P < 0.05). However, following LPS infusion, the ROS levels skyrocketed while that of SOD decreased significantly relative to the control rats (P < 0.05). Furthermore, we noted that pre-treatment with scutellarin reduced the ROS levels in LPS-injured rats while the SOD was increased to near control levels (P < 0.05). Moreover, the combined effect of scutellarin and JNK inhibitor SP600125 on the levels of ROS and the SOD activity followed a similar trend to that of scutellarin alone albeit with a lower magnitude of change. Our results also showed that the combinatorial treatment was not significantly different from scutellarin alone in terms of influence on the levels of ROS production and SOD activity (P > 0.05). The effect of Scutellarin on broncho-alveolar lavage fluid (BALF) cytokine secretion The expression of interleukins-1β, ?18 and ?6 in the broncho-alveolar lavage fluid were significantly upregulated by LPS infusion (P < 0.05). The rise was, however, attenuated via pre-treatment with scutellarin only or in conjunction with SP600125, a JNK inhibitor (all P < 0.05). On the contrary, we observed that LPS injection caused a reduction of interlekins ?4 and ?10 secreted in the BALF. Pre-treatment with scutellarin alone (P < 0.05) and not in combination with SP600125 or SP600125 was able to significantly reverse this noted down-regulation (all P > 0.05).     

Expression and localization of progesterone receptor membrane component 1 and 2 and serpine mRNA binding protein 1 in the bovine corpus luteum during the estrous cycle and the first trimester of pregnancy

The aim of this study was to evaluate the mRNA and protein expression and the localization of progesterone receptor membrane component 1 (PGRMC1), PGRMC2, and the PGRMC1 partner serpine mRNA binding protein 1 (SERBP1) in the bovine CL on Days 2 to 5, 6 to 10, 11 to 16, and 17 to 20 of the estrous cycle as well as during Weeks 3 to 5, 6 to 8, and 9 to 12 of pregnancy (n = 5–6 per each period). The highest levels of PGRMC1 and PGRMC2 mRNA expression were found on Days 6 to 16 (P < 0.05) and 11 to 16, respectively, of the estrous cycle and during pregnancy (P < 0.001). The level of PGRMC1 protein was the highest (P < 0.05) on Days 11 to 16 of the estrous cycle compared with the other stages of the estrous cycle and pregnancy, whereas PGRMC2 protein expression (P < 0.001) was the highest on Days 17 to 20 and also during pregnancy. The mRNA expression of SERBP1 was increased (P < 0.05) on Days 11 to 16, whereas the level of its protein product was decreased (P < 0.05) on Days 6 to 10 of the estrous cycle and was at its lowest (P < 0.001) on Days 17 to 20. In pregnant cows, the patterns of SERBP1 mRNA and protein expression remained constant and were comparable with those observed during the estrous cycle. Progesterone receptor membrane component 1 and PGRMC2 localized to both large and small luteal cells, whereas SERBP1 was observed mainly in small luteal cells and much less frequently in large luteal cells. All proteins were also localized in the endothelial cells of blood vessels. The data obtained indicate the variable expression of PGRMC1, PGRMC2, and SERBP1 mRNA and protein in the bovine CL and suggest that progesterone may regulate CL function via its membrane receptors during both the estrous cycle and pregnancy.     

Repression of CRNDE enhances the anti-tumour activity of CD8 + T cells against oral squamous cell carcinoma through regulating miR-545-5p and TIM-3

Immunotherapy has been identified a promising treatment of cancers, including Oral squamous cell carcinoma (OSCC). CRNDE is highly overexpressed in various cancers. Many lncRNAs have been reported in CD8 T lymphocytes. Little is investigated about their effects in the functions of CD8 + T cells in OSCC. Currently, the influence of lncRNA CRNDE on the function of CD8 + T cells in OSCC progression was investigated. Here, CRNDE was obviously elevated and negatively correlated with IFN-γ production in tumour-infiltrating CD8 + T cells isolated from OSCC patients. CRNDE can exhibit a crucial role in activating CD8 + T-cell exhaustion. Mechanistically, CRNDE specifically sponged miR-545-5p to induce T-cell immunoglobulin and mucin domain-3 (TIM-3), thus contributing to CD8 + T-cell exhaustion. The function of miR-545-5p on T-cell function remains poorly known. TIM-3 is a significant immune checkpoint, and it inhibits cancer immunity. TIM-3 can demonstrate an important role in CD8 + T-cell exhaustion. In summary, loss of CRNDE could induce miR-545-5p and inhibit TIM3 expression, thus significantly activated the anti-tumour effect of CD8 + T cells.     

MiR-210-3p-EphrinA3-PI3K/AKT axis regulates the progression of oral cancer

This study aimed to explore new therapeutic targets to improve the survival rate of patients with oral squamous cell carcinoma (OSCC).MiR-210-3p, EphrinA3 and EMT related indices were evaluated in OSCC tissues and cell lines. In addition, the relationship between differential EphrinA3 expression and tumour progression was explored through molecular biology techniques, in vitro functional experiments and tumour xenotransplantation models. The expression of EphrinA3 (r_s = −0.719, P < .05) and E-cadherin (r_s = −0.856, P < .05) was negatively correlated with the pathological grading in OSCC tissues. Protein clustering shows EphrinA3 may be associated with tumour progression. EphrinA3 also can regulate the biological behaviour of oral cancer cells. And it regulates the EMT by the PI3K/AKT signalling pathway. MiR-210-3p targeted the gen EFNA3. Up-regulation of miR-210-3p expression can decrease the expression of EphrinA3 and further to influence the biological behaviour of OSCC. The miR-210-3p-EphrinA3-PI3K/AKT signalling axis plays an important role in the progress of OSCC. EphrinA3 may serve as a novel target for oral cancer treatment.