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IntroductionThe present study was conducted to explore the expression of serum inflammatory cytokines and oxidative stress markers in patients with coronary heart disease (CHD), with an attempt to analyze their relationship with the coronary artery calcium score (CACS) by coronary computed tomography angiography (CCTA).Material and methodsIt total 81 patients with coronary heart disease and 81 healthy adults were included as the observation group and the control group, respectively. The levels of serum interleukin (IL)-6 and IL-12 of the two groups were detected by ELISA, and serum superoxide dismutase (SOD) was detected by the hydroxylamine oxidation method. Micro-RNA-497-5p (miR-497-5p) was screened out as a possible new CHD biomarker and its serum level was measured by real-time fluorescence quantitative PCR. The CACS of patients in the observation group was calculated by the Agatston method to analyze the correlation between the abovementioned indexes and CACS.ResultsWith increase in the number of CHD lesions, the levels of IL-6, IL-12 and miR-497-5p rose gradually while the level of SOD decreased gradually. In the observation group, IL-6, IL-12 and miR-497-5p were positively correlated with CACS while SOD was negatively correlated with CACS.ConclusionsAbnormal expression levels of serum IL-6, IL-12, SOD and miR-497-5p may be able to reveal the severity of the disease, and the combination with CACS is of potential value in terms of evaluating the condition of patients harboring coronary heart disease.

Human aspartyl-(asparaginyl)-β-hydroxylase (HAAH) has recently been the subject of several studies, as it was previously observed to be overexpressed in numerous types of carcinoma cells and tissues in patient tumor samples. HAAH has been implicated in tumor invasion and metastasis, indicating that it may be an important target and biomarker for tumor diagnosis and treatment. However, the immunological tools currently available for the study of this protein, including monoclonal antibodies, are limited, as is the present knowledge regarding the role of HAAH in tumor therapy and diagnosis. In the present study, a recombinant C-terminal domain of HAAH was expressed in Pichia pastoris and a novel monoclonal antibody (mAb) targeting HAAH (HAAH-C) was constructed. Immunofluorescence and antibody-dependent cellular cytotoxicity (ADCC) assays were used to demonstrate the specificity and ADCC activity of this antibody. The results demonstrated that this anti-C-terminal HAAH mAB, in combination with an existing anti-N terminal HAAH mAb, exhibited a high response to native HAAH from carcinoma cell culture supernatant, as measured with a double antibody sandwich enzyme-linked immunosorbent assay. This validated novel mAB-HAAH-C may prompt further studies into the underlying mechanisms of HAAH, and the exploration of its potential in tumor diagnosis and therapy.

Lycopene is a nonprovitamin a carotenoid mainly found in fruits and vegetables, which has been reported to possess a variety of biological effects. The properties of lycopene on human natural killer (NK) cells after ex vivo expansion were assessed in the present study. Results showed that lycopene has a positive effect on NK cells viability and cytotoxicity. Aging and apoptosis started from the fourth week onwards in the cultured NK cells which were obtained from the peripheral blood mononuclear cells (PBMC). Supplemented with lycopene (5μM) can restore the decreased viability and cytotoxicity of NK cells and reduce NK cells apoptosis caused by aging during fourth-sixth week culture. Its anti-apoptosis effect on NK cells may be related to lycopene can down regulated the expression of caspase 3 and 9 genes. Furthermore, lycopene can enhance the IFN-γ expression in gene and protein level after 7d treatment. However, lycopene did not affect the functional receptor's (NKG2A, NKG2D, NKp30 and NKp44) expression on NK cells. These results indicated that lycopene has a positive effect on NK cells. As a health product, it may help to prolong the lifespan and enhance the cytotoxicity of NK cells after ex vivo expansion.

In recent years, paper-based point-of-care testing (POCT) has been widely used in medical diagnostics, food safety and environmental monitoring. However, a high-cost, time-consuming and equipment-dependent sample pretreatment technique is generally required for raw sample processing, which are impractical for low-resource and disease-endemic areas. Therefore, there is an escalating demand for a cost-effective, simple and portable pretreatment technique, to be coupled with the commonly used paper-based assay (e.g. lateral flow assay) in POCT. In this review, we focus on the importance of using paper as a platform for sample pretreatment. We firstly discuss the beneficial use of paper for sample pretreatment, including sample collection and storage, separation, extraction, and concentration. We highlight the working principle and fabrication of each sample pretreatment device, the existing challenges and the future perspectives for developing paper-based sample pretreatment technique.

We newly cloned the gene encoding the human aspartyl (asparaginyl) beta-hydroxylase (HAAH) from the surgical tissue of a patient with hepatocellular carcinoma. This study was designed to generate HAAH-specific monoclonal antibody (MAb) for further exploration of its structure and function. Mice were co-immunized with naked plasmid DNA containing N-terminal domain of encoding HAAH gene and recombinant HAAH polypeptide. Hybridomas were developed by the electrofusion of the splenocytes from mice immunized with plasmid DNA to Sp2/0 myeloma cells in vitro. Three hybridoma cell lines (designated G3, G9, and F11, respectively) stably secreting HAAH-specific MAbs were obtained. The specificity and sensitivity of MAbs were assessed by indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Results showed that the three MAbs belong to IgG1 kappa isotype, the titer of MAbs reached was 5 x 10(4) - 1 x 10(5), and the affinity constant (k(aff)) of MAbs ranged between 2.5 x 10(8) - 1.1 x 10(9). MAb G3 was preliminarily applied to detection expression of HAAH for seven tumor tissues, including hepatocellular carcinoma, lung cancer, kidney cancer, cholangiocarcinoma, prostate cancer, breast cancer, and glioblastoma by immunohistochemical stain. Our studies demonstrated that co-immunization of naked DNA containing encoding gene of target antigen and recombinant target protein, and combined with in vitro electrofusion, is an effective and simple method to raise MAbs.

This study was aimed to investigate the effects of carbon monoxide releasing molecule (CORM-2), a novel carbon monoxide carrier, on the reendothelialization of carotid artery in rat endothelial denudation model.Male rats subjected to carotid artery balloon injury were treated with CORM-2, inactive CORM-2 (iCORM-2) or dimethyl sulfoxide (DMSO). The reendothelialization capacity was evaluated by Evans Blue dye and the immunostaining with anti-CD31 antibody. The number of circulating endothelial progenitor cells (EPCs) was detected by flow cytometry. The proliferation, migration, and adhesion of human umbilical vein endothelial cells (HUVECs) were assessed by using [3H]thymidine, Boyden chamber and human fibronectin respectively. The expressions of protein were detected by using western blot analysis.CORM-2 remarkably accelerated the re-endothelialization 5 d later and inhibited neointima formation 28 d later. In addition, the number of peripheral EPCs significantly increased in CORM-2-treated rats than that in iCORM-2 or DMSO-treated rats after 5 d later. In vitro experiments, CORM-2 significantly enhanced the proliferation, migration and adhesion of HUVECs. The levels of Akt, eNOS phosphorylation, and NO generation in HUVECs were also much higher in CORM-2 treated group. Blocking of PI3K/Akt/eNOS signaling pathway markedly suppressed the enhanced migration and adhesion of HUVECs induced by CORM-2.CORM-2 could promote endothelial repair, and inhibit neointima formation after carotid artery balloon injury, which might be associated with the function changes of HUVECs regulated by PI3K/Akt/eNOS pathway.

To study the effect of autophagy in MTB's infection and the expression of its related gene.The formation of autophagy was induced by Rapamycin and observed by the transmission electron microscope. The cleaning role of autophagy to the MTB H37Rv virulent strain after its formation was detected by clone forming unit (CFU). Realtime PCR was used to detect the mRNA of the autophagy related gene was expressed.The RAW264.7 cell could form autophagosome under the induction of the Rapamycin, and it had the determinate cleaning role to the H37Rv strain in the cell after which formed. The mRNA of atg5, atg8 and atg12 which participated the formation of autophagy were expressed more, but the expression of atg7 had no change.Autophagy participated the process of immune response of anti-MTB. Atg5, atg8 and atg12 were the important molecule which control the formation of autophagy when MTB infected.

To obtain sufficient numbers of pure and activated human primary NK cells for potential clinical application.PBMC based ex vivo expansion of natural killer cells was set up. The expansion was initiated by co-culture of PBMC and stimulator cells (irradiate genetic modified K562 cells) in RPMI1640 medium with 1 000 U/mL IL-2. Genetic modified K562 cells 'K562D3' were prepared by expressing IL-15, 4-1BBL and IL-18 on K562 cell membrane.An average of 500 fold expansion of CD56(+)CD3(-) cells was observed after 3 weeks of co-culture. The NK cells population could reach 93% after expansion, comparing with 7% before expansion. The expanded NK cells lysed 95% of K562 targets in a 5:1 effector to target ratio. IL-15/4-1BBL bound K562 stimulatory cells could expand same fold NK cells as K562D3, but the cytotoxicity of NK cells expanded by 'K562D3' was 10% higher.The described method is a simple and efficient way for the expansion of human NK cells.

Lycopene is a nonprovitamin A carotenoid mainly found in fruits and vegetables, which has been reported to possess a variety of biological effects. The properties of lycopene on human natural killer (NK) cells after ex vivo expansion were assessed in the present study. Results showed that lycopene has a positive effect on NK cells viability and cytotoxicity. Aging and apoptosis started from the fourth week onwards in the cultured NK cells which were obtained from the peripheral blood mononuclear cells (PBMC). Supplemented with lycopene (5μM) can restore the decreased viability and cytotoxicity of NK cells and reduce NK cells apoptosis caused by aging during fourth-sixth week culture. Its anti-apoptosis effect in NK cells may be related to lycopene which can decrease the expression of caspase 3 and 9 genes. Furthermore, lycopene can enhance the IFN-γ expression in gene and protein level after 7d treatment. However, lycopene did not affect the functional receptor’s (NKG2A, NKG2D, NKp30 and NKp44) expression on NK cells. These results indicated that lycopene has a positive effect on NK cells. As a health product, it may help to prolong the lifespan and enhance the cytotoxicity of NK cells after ex vivo expansion.

To express nucleocapsid(N) protein of SARS coronavirus and produce monoclonal antibody(mAb) to N protein.N protein gene was amplified by RT-PCR. After being confirmed by DNA sequencing, the gene was subcloned into prokaryotic expression vector. N protein expressed in E.coli was recovered from SDS-PAGE gel and served as immunogen in the preparation of the mAb.DNA sequencing confirmed that the amplified fragment was N protein gene. SDS-PAGE analysis showed that the M(r) of the expressed protein was approximately 43 kd. This expressed protein could be further confirmed in Western blot by using serum from a convalescent SARS patient as primary antibody. Western blot analysis proved that three mAbs obtained could react specifically to the recombinant N protein.The prepared recombinant N protein and mAbs against N protein lay the foundation for further development of early diagnosis assays for SARS coronavirus infection.

To express Japanese encephalitis virus (JEV) E protein in methylotrophic yeast Pichia pastoris.The gene coding for JEV E protein was sub-cloned into vector pPIC9k. The constructed plasmid was transformed into yeast SMD1168 by electroporation. The recombinant transformants with a high copy number of the plasmid were selected by using MD plate and G418. The expression of E protein in yeast was induced by the addition of methanol and analyzed by SDS-PAGE and Western blot.The protein was produced at a yield of 50 mg per litre of culture. Owing to heterogeneous glycosylation, relative molecular mass (M(r)) of the expressed E protein was sized about 113 000 and 78 000.JEV E protein was expressed successfully in yeast Pichia pastoris, which should be useful for the production of diagnostic reagents and genetically engineered vaccine of JEV.

To probe the possibility of screening tPA with higher activity through DNA shuffling.Human, rat and rhesus monkey tPA cDNAs were used as the templates of "DNA family shuffling". The diverse tPA library was expressed in CHO cells, then the screening was carried out.Two shuffled clones, t9 and t17 were selected. The activity of tPA expressed by t9 clone was 4-fold higher than the activity of human tPA. tPA expressed by t17 clone was found having a 88-amino-acid deletion, but still showed the same activity as human tPA. DNA sequencing demonstrated that the sequences of t9 and t17 were mainly derived from human and monkey tPA cDNAs.Above work has laid the foundation for further experiments in the screening for higher activity of tPA.

To establish the role of vascular endothelial growth factor (VEGF) in the oncogenesis of human gastric carcinoma more directly.The expression of VEGF and its receptor kinase-domain insert containing receptor (KDR) in human gastric cancer tissue were observed by immunohistochemical staining. VEGF levels were manipulated in human gastric cancer cell using eukaryotic expression constructs designed to express the complete VEGF(165) complimentary DNA in either the sense or antisense orientation. The biological changes of the cells were observed in which VEGF was up-regulated or down-regulated.VEGF-positive rate was 50%, and VEGF was mainly localized in the cytoplasm and membrane of the tumor cells, while KDR was mainly located in the membrane of vascular endothelial cells in gastric cancer tissues and peri-cancerous tissue. In 2 cases of 50 specimens, the gastric cancer cells expressed KDR, localized in both the cytoplasm and membrane. Introduction of VEGF(165) antisense into human gastric cancer cells (SGC-7901, immunofluorescence intensity, 31.6%)) resulted in a significant reduction in VEGF-specific messenger RNA and total and cell surface VEGF protein (immunofluorescence intensity, 8.9%) (P0.05). Conversely, stable integration of VEGF(165) in the sense orientation resulted in an increase in cellular and cell surface VEGF (immunofluorescence intensity, 75.4%) (P0.05). Lowered VEGF levels were associated with a marked decrease in the growth of nude mouse xenografted tumor (at 33 days postimplantation, tumor volume: 345.40 +/- 136.31 mm3)(P0.05 vs control SGC-7901 group: 1534.40 +/- 362.88 mm3), whereas up-regulation of VEGF resulted in increased xenografted tumor size (at 33 days postimplantation, tumor volume: 2350.50 +/- 637.70 mm3) (P0.05 vs control SGC-7901 group).This study provides direct evidence that VEGF plays an important role in the oncogenesis of human gastric cancer.