Cloning,expression and function of the extracellular fragment of human TRAIL gene

To obtain the recombinant soluble protein of the extracellular fragment of human TRAIL gene and to identify its function preliminarily,this gene fragment was amplified from peripheral blood mononu- clear cells(PBMC)by RT-PCR and cloned into vector pGEM-T-Easy for sequence analysis.The expres- sion vector pET-30a/TRAIL was then constructed by DNA recombination method with a His-tag gene at the front of the TRAIL fragment,and the recombinant protein was expressed in E.coli BL21(DE3). Meanwhile,the expressed target protein was purified with Ni-NTA chromatography column and identified by SDS-PAGE and Western blotting.The proliferation inhibition activity of TRAIL-His was detected by MTT assay.PI staining and Wright-Giemsa staining were used to detect the presence of the TRAIL-in- duced cell apoptosis.It was demonstrated that the target protein expressed in E.coli BL21 showed the same relative molecular mass as that the protein expected and could be recognized by both the anti-TRAIL polyclonal antibody and anti-His monoclonal antibody.In addition,this protein could also inhibit prolif- eration of human lymphoma cell line Jurkat cells or induce apoptosis of this cell line.It is apparent that a recombinant soluble TRAIL protein with biological activity is obtained and this prospective study can lay solid foundation for further research on the biological activity and application in the anti-tumor therapy.

ILF2 cooperates with E2F1 to maintain mitochondrial homeostasis and promote small cell lung cancer progression

Objective:Mitochondria play multifunctional roles in carcinogenesis.Deciphering uncertainties of molecular interactions within mitochondria will promote further understanding of cancer.Interleukin enhancer binding factor 2(ILF2)is upregulated in several malignancies,however,much remains unknown regarding ILF2 in small cell lung cancer(SCLC).In the current study,we explored ILF2's role in SCLC and demonstrated its importance in mitochondria quality control.Methods:Colony formation,cell proliferation,cell viability and xenograft studies were performed to examine ILF2's role on SCLC progression.Glucose uptake,lactate production,cellular oxygen consumption rate and extracellular acidification rate were measured to examine the effect of ILF2 on glucose metabolism.RNA-sequencing was utilized to explore genes regulated by ILF2.E2 F1 transcriptional activity was determined by dual luciferase reporter assay.Mitochondria quantification and mitochondrial membrane potential assays were performed to examine mitochondrial quality.Gene expression was determined by RT-qPCR,Western blot and IHC assay.Results:ILF2 promotes SCLC tumor growth in vitro and in vivo.ILF2 elevates oxidative phosphorylation expression and declines glucose intake and lactate production.Genome-wide analysis of ILF2 targets identified a cohort of genes regulated by E2 F1.In consistent with this,we found ILF2 interacts with E2 F1 in SCLC cells.Further studies demonstrated that suppression of E2 F1 expression could reverse ILF2-induced tumor growth and enhanced mitochondria function.Significantly,expression of ILF2 is progressively increased during SCLC progression and high ILF2 expression is correlated with higher histologic grades,which indicates ILF2's oncogenic role in SCLC.Conclusions:Our results demonstrate that ILF2 interacts with E2 F1 to maintain mitochondria quality and confers SCLC cells growth advantage in tumorigenesis.

Selection and characterization of the novel anti-human PD-1 FV78 antibody from a targeted epitope mammalian cell-displayed antibody library

Currently,display-based methods are well established and widely used in antibody engineering for affinity maturation and structural stability improvement.We obtained a novel anti-human programmed death 1(PD-1)antibody using computer-aided design and a mammalian cell display technology platform.We used computer-aided modeling and distance geometry methods to predict and assign the key residues that contributed to the binding of human PD-L1 to PD-1.Then,we analyzed the sequence of nivolumab(an anti-human PD-1 antibody,referred to as MIL75 in the article)to determine the template for antibody design and library construction.We identified a series of potential substitutions on the obtained template and constructed a virtual epitope-targeted antibody library based on the physicochemical properties and each possible location of the assigned key residues.The virtual antibody libraries were displayed on the surface of mammalian cells as the antigen-binding fragments of full-length immunoglobulin G.Then,we used flow cytometry and sequencing approaches to sort and screen the candidates.Finally,we obtained a novel anti-human PD-1 antibody named FV78.FV78 competitively recognized the PD-1 epitopes that interacted with MIL75 and possessed an affinity comparable to MIL75.Our results implied that FV78 possessed equivalent bioactivity in vitro and in vivo compared with MIL75,which highlighted the probability and prospect of FV78 becoming a new potential antibody therapy.

乳腺癌患者循环肿瘤细胞的检测及其临床应用(英文)

肿瘤转移是乳腺癌治疗失败的主要原因之一。肿瘤细胞进人外周血是发生远处转移的前提,早在1896年,Ashworth在1例因癌症死亡的患者外周血中发现了类似肿瘤的细胞,并首次提出了循环肿瘤细胞(circulating tumor cell.CTC)的概念。进人循环未被清除的肿瘤细胞可相互聚集形成微小癌栓,并在一定条件下发展为转移灶。越来越多的学者开始关注循环肿瘤细胞方面的研究,近年来,有关乳腺癌患者CTC检测及其临床应用的研究已取得了长足的进展。

Identification of p100 target promoters by chromatin immunoprecipitation-guided ligation and selection (ChIP-GLAS)

The multifunctional protein p100 is a vital transcriptional regulator that increases gene transcription by forming a physical bridge between promoter-specific transcription factors and the basal transcription machinery.To identify potential signal transduction pathways in which human p100 acts as a coregulator and to find target promoter regions that may interact with p100,we performed a promoter microarray assay called chromatin immunoprecipitation-guided ligation and selection(ChIP-GLAS).From this assay,we determined that a set of promoter fragments,including several factors in the transforming growth factor beta(TGF-β)signaling pathway,exhibited interaction with p100.The ChIP-GLAS data were validated by RT-PCR assessing the mRNA expression of various factors in the TGF-b signaling pathway in cell lines.

Phytochemistry,Metabolism, and Metabolomics of Ginseng

Ginseng, as a medicinal plant, has been used for thousands of years in China, Korea, and Japan, and the study on ginseng is a hotspot in the research field as evidenced by about 7000 scientific papers in PUBMED. In recent decades, many ginseng studies focused on the metabolism and metabolomics of ginseng or its active ingredients using modern bioanalytical technologies. To date, more than 200 ginsenosides and non-saponin constituents have been isolated and identified. In the past decades, rapid development of analytical technologies has facilitated the advancement of ginseng research in many ways. In this review, we focus on the advances of ginseng research in chemistry, pharmacology, and metabolomics. We also provide the comments on the significance as well as challenges of metabolomics-based ginseng studies.

Truth telling for patients with esophageal squamous cell carcinoma in Henan,China

Objective: This study aims to investigate the truth-telling status and the relevant factors of esophageal squamous cell carcinoma(ESCC) patients in Henan, China.Methods: A cross-sectional study from April to June 2015 using questionnaires was given to 301 family members of hospitalized ESCC patients based in three affiliated hospitals of Zhengzhou University(i.e., The First Hospital, The Second Hospital, and Tumor Hospital) and Anyang Tumor Hospital.Results: Among the 41.9%(126/301) hospitalized ESCC patients who knew of their true diagnoses, only 4.0% patients were informed by their corresponding responsible doctors, 39.7% by their family members, and 56.3% by themselves. Univariate analyses showed that disclosure of confirmed ESCC diagnosis to patients was correlated with gender, family history of cancer(FHC), education level, vocation, hospital administrative level, and attitudes of family members(P < 0.05). Furthermore,multivariate analysis indicated that attitude of family members was the most important and an independent factor for diagnosis disclosure. Those patients with a negative FHC, under-education, manual occupation, advanced stages, and hospitalized in municipal hospitals exhibited a low rate of truth telling.Conclusions: Truth telling for ESCC patients in Henan is not prevalent and may be improved through consultation with family members, particularly for patients with a negative FHC, poor education, manual occupation, and advanced stages.

维生素E琥珀酸酯增强DR5抗体对Raji和K562细胞的凋亡作用(英文)

Objective: The aim of our study was to detect whether Vitamin E Succinic Acid (VES) could regulate the expression level of DR5 in the cells and further elucidate the potential mechanisms involving that VES enhances the effect of mDRA-6 to eradicate leukemia Raji and K562 cells. Methods: MTT method was used to detect the growth inhibition of VES and mDRA-6 to Raji and K562 cells. Annexin V-FITC/PI double staining assay was used to analysis the apoptosis of leukemia cell. Flow cytometry was used to detect the cell surface DR5 expression. Immunoblotting technique was used to detect the DR5 protein expression. Results: MTT detection showed that 10 μmol/L mDRA-6 on the cell death rates of Raji and K562 cells were 21.98% and 5.23%, respectively. While increasing concentration of VES (5 μmol/L, 10 μmol/L, 20 μmol/L) and mDRA-6 both on the cell viability of Raji or K562 cells, the mortality of Raji cells elevated to 24.67%, 35.65% (P<0.01) and 40.22% (P<0.01), respectively. Similarly, the mortality of K562 cells increase to 6%, 7.89% (P<0.01) and 8.67% (P<0.01), respectively. To further specify the increased cell death rate induced by mDRA-6 and VES, the treated cells were analyzed by Annexin-V/PI staining assay. As shown in Fig. 1, the apoptosis rates of Raji and K562 cells treated with 2 μg/mL mDRA-6 for 12 h were 20.79% and 7.74%. Compared with this, the proportion of apoptotic cells increased upon exposure to 2 μg/mL mDRA-6 combination with 10 μmol/L VES, the apoptosis rates of Raji and K562 cells were 43.18% and 16.99%, respectively. To examine the anticancer effects of a combination strategy based on mDRA-6 and VES. We analyzed whether VES could elevated the expression level of DR5 on Raji and K562 cytomembrane by FACS. Interestingly, after treated with 10 μmol/L VES for 12 h, the expression level of DR5 on Raji and K562 cell surface increased from 50.66% to 70.08%, and 15.02% to 16.38%, respectively. Immune imprinting technology test showed that, different concentrations of VES could increase Raji and K562 cell DR5 protein expression. Conclusion: VES enhances the effect of mDRA-6 to eradicate leukemia Raji and K562 cells. The proper mechanism is VES could enhance the Raji and K562 cell membrane expression of DR5, and VES can also enhance the DR5 protein expression of cells.

Inhibitory activity of Bifidobacterium adolescent combined with cisplatin on melanoma in mice and its mechanism

The aim of this study is to explore inhibitory activity of Bifidobacterium adolescent combined with cis-platin on the growth of melanoma(B16)in mice and the underlying mechanism.C57 mice were inoculated with B16 cancer cells to construct mouse model of melanoma and treated with bifidobacterium adolescent combined with cisplatin.Ratios of inhibitory activity on the growth of melanoma(B16)were calculated.Pathology changes of the tumor were observed by HE staining.B16 cell cycles were examined on a flow cytometer.Lymphocyte prolif-eration was measured with MTT assay and the T-cell sub-set was measured by double marked fluorescence.When bifidobacterium of 1010 cfu/L was injected,the ratio of inhibitory activity on the growth of melanoma(B16)reached 54%,which was similar to that of cisplatin group.The ratio of inhibitory activity reached 74.45%when the mice were treated by bifidobacterium combined with cis-platin.HE staining shows that bifidobacterium inhibited B16 cell proliferation and enhanced the cisplatin`s killing activity on B16 cells.The results of flow cytometry demonstrated that B16 cell proliferation was arrested at G1 stage after treatment with bifidobacterium.The B16 cell proliferation was arrested at S stage after treatment with cisplatin.The CD4+percentage increased and the difference was significant compared with the normal group after treatment with bifidobacterium,indicating that T-cell immune activity was enhanced.Treatment with bifidobacterium combined with cisplatin can enhance the inhibitory activity on the growth of melanoma(B16)of cisplatin.The mechanism of the inhibitory activity on B16 cell proliferation is correlated with the enhanced immune activity in mice.

Use of copper-cysteamine nanoparticles to simultaneously enable radiotherapy,oxidative therapy and immunotherapy for melanoma treatment

Dear Editor,Melanoma,squamous cell carcinoma(SCC),and basal cell carcinoma(BCC)are three major types of skin cancer.Among them,melanoma is the most severe form and accounts for~4%of all newly diagnosed cancers annually in the United States.It is estimated that approximately 9500 people are diagnosed with skin cancer every day,and more than 1 million Americans are living with melanoma.Melanoma treatment is still a major challenge in the clinic.Photodynamic therapy(PDT)is composed of targeted ablation and immune activation,is less invasive than other therapies and has been widely used in the treatment of various cancers.

Respective IL-17A production by γδ T and Th17 cells and its implication in host defense against chlamydial lung infection

The role of IL-17A is important in protection against lung infection with Chlamydiae,an obligate intracellular bacterial pathogen.In this study,we explored the producers of IL-17A in chlamydial lung infection and specifically tested the role of major IL-17A producers in protective immunity.We found thatγδT cells and Th17 cells are the major producers of IL-17A at the early and later stages of chlamydial infection,respectively.Depletion ofγδT cells in vivo at the early postinfection(p.i.)stage,when mostγδT cells produce IL-17A,failed to alter Th1 responses and bacterial clearance.In contrast,the blockade of IL-17A at the time when IL-17A was mainly produced by Th17(day 7 p.i.)markedly reduced the Th1 response and increased chlamydial growth.The data suggest that theγδT cell is the highest producer of IL-17A in the very early stages of infection,but the protection conferred by IL-17A is mainly mediated by Th17 cells.In addition,we found that depletion ofγδT cells reduced IL-1αproduction by dendritic cells,which was associated with a reduced Th17 response.This finding is helpful to understand the variable role of IL-17A in different infections and to develop preventive and therapeutic approaches against infectious diseases by targeting IL-17A.

Characterization of a Novel Anti-DR5 Monoclonal Antibody WD1 with the Potential to Induce Tumor Cell Apoptosis

TNF-related apoptosis-inducing ligand （TRAIL） is a TNF family member capable of inducing apoptosis. Death receptor 5 （DR 5） is a key receptor of TRAIL and plays an important role in TRAIL-induced apoptosis. To prepare monoclonal antibodies （mAbs） against DR5, cDNA encoding soluble DR5 （sDR5） was firstly amplified by reverse transcriptase-polymerase chain reaction （RT-PCR） with specific primers, and then inserted into a prokaryotic expression vector pET-30a. The recombinant plasmid was expressed in Escherichia coli strain BL21 （DE3）, and sDR5 was purified by nickel affinity chromatography. As an antigen, sDR5 was used to immunize mice. Hybridomas secreting antibodies against sDR5 were identified. One positive done was selected to produce antibody, WD1. ELISA and immunofluorescence demonstrated that WD1 could bind recombinant sDR5 and membranebound DR5 （mDR5） on Jurkat and Molt-4 cells. ATPLite assays showed that Jurkat and Molt-4 cells were sensitive to the antibody in a dose dependent manner. The Annexin V/PI assays and Giemsa＇s staining both showed that WD1 could induce Jurkat cell apoptosis efficiently. Transient transfection of 293T cells and indirect immunofluorescence assay demonstrated that mAb （WD1） couldn＇t cross-react with DR4. Our findings indicated that the novel antibody, WD1 could act as a direct agonist, bind DR5 characteristically, and initiate efficient apoptotic signaling and tumor regression. Thus, WD1 would be a leading candidate for potential cancer therapeutics. Cellular ＆ Molecular Immunology.

Inefficiency of C3H/HeN Mice to Control Chlamydial Lung Infection Correlates with Downregulation of Neutrophil Activation during the Late Stage of Infection

We previously reported that massive infiltration of neutrophils in C3H/HeN （C3H） mice could not efficiently control Chlamydia muridarum （Cm） infection and might contribute to the high susceptibility of these mice to lung infection. To further define the nature of neutrophil responses in C3H mice during chlamydial infection, we examine the expression of adhesion molecules and CDllb related to neutrophils infiltration and activation, respectively, following intranasal Cm infection. The results showed that the expression of selectins （E-selectin, P-selectin and L-selectin）, and intercellular cell adhesion molecule-1 （ICAM-1） in the lung of C3H mice increased more significantly than in C57BL/6 （B6） mice, the more resistant strain. These results correlated well with the massive neutrophils infiltration in C3H mice. In contrast, CDllb expression on peripheral blood and lung neutrophils in C3H mice exhibited a significant reduction compared with B6 mice during the late phage of infection （day 14）. These findings suggest that the high-level expression of adhesion molecules in C3H mice may enhance neutrophils recruitment to the lung, but the decline of CDllb expression on neutrophils may attenuate neutrophil function. Therefore, CDllb down-regulation on neutrophils may contribute to the failure of C3H mice to control chlamydial lung infection.

miR-495 andmiR-5688 are down-regulated in non-small cell lung cancer under hypoxia to maintain interleukin-11 expression

Background:Hypoxia is a hallmark of cancer and is associated with poor prognosis.However,the molecular mechanism by which hypoxia promotes tumor progression remains unclear.MicroRNAs dysregulation has been shown to play a critical role in the tumor and tumor microenvironment.Here,we investigated the roles ofmiR-495 and miR-5688 in human non-small cell lung cancer(NSCLC)and their underlying mechanism.Methods:The expression levels of miR-495 and miR-5688 in human NSCLC tissue specimens were measured by quantitative real-time polymerase chain reaction(qRT-PCR).Deferoxamine(DFO)was used to determine whether the regulation of miR-495 and miR-5688 under hypoxia was dependent on hypoxia-inducible factor 1-alpha(HIF-1α).Furthermore,the functions of miR-495 and miR-5688 in tumor progression were evaluated using colony formation,3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium(MTS),wound healing,transwell assays,and xenograft model.Two algorithms,PicTAR and Targetscan,were used to predict the target gene of these two miRNAs,and dual-luciferase reporter assay was conducted to confirm the target.The unpaired two-tailed t test,Pearson correlation analysis,and Fisher’s exact probability test were performed for statistical analyses.Results:Two miRNAs,miR-495 and miR-5688,were found to participate in NSCLC progression under hypoxia.They were down-regulated in NSCLC tissues compared with normal tissues.We determined that hypoxia led to the down-regulation of miR-495 and miR-5688 in NSCLC cells,which was independent of HIF-1αand cellular metabolic energy.In addition,miR-495 and miR-5688 suppressed cell proliferation,migration,and invasion in vitro.The NSCLC xenograft model showed that miR-495 and miR-5688 inhibited tumor formation in vivo.Interestingly,we found that miR-495 and miR-5688 had the same target,interleukin-11(IL-11).Recombinant human IL-11 counteracted the effects of miR-495 and miR-5688 on NSCLC cells,suggesting that miR-495 and miR-5688 executed their tumor suppressive role by repressing IL-11 expression.Conclusion:We found that hypoxia down-regulated the expression levels of miR-495 and miR-5688 in NSCLCto enhance IL-11 expression and tumor progression,indicating that the miR-495/miR-5688/IL-11 axismay serve as a therapeutic target and potential biomarker for NSCLC.

The S 1P, receptor-selective agonist CYM-5442 reduces the severity of acute GVHD byinhibiting macrophage recruitment

FTY720, an agonist for four of the five known sphingosine- 1-phosphate （SIP） receptors, has been reported to inhibit acute graft-versus-host disease （aGVHD）. Because FTY720 functions through multiple SIP receptors, the mechanism of action through one or more of these receptors may account for its side effects. Thus, more selective SIP receptor modulators are needed to evaluate the roles of different S1P receptors and their therapeutic efficacies. In this study, we investigated the effect of an SIPl-selective agonist, CYM-5442, on the progression of aGVHD. We showed that CYM-5442 significantly inhibited but did not prevent aGVHD. CYM-5442 did not affect the infiltration of the donor T cells into the target organs, while the number of macrophages in GVHD organs was significantly reduced by CYM-5442 treatment. In vivo proliferation assays showed that the proliferation of macrophages was not suppressed by CYM-5442. Further studies using human endothelial cells demonstrated that CYM-5442 treatment downregulated CCL2 and CCL7 expression in endothelial cells, therefore reducing the migration of monocytes, from which tissue macrophages originate. Our data demonstrate the therapeutic efficacy of an SIPl-selective agonist in aGVHD and its possible mechanism of action. The results suggest that further investigations are needed regarding CYM-5442 as a potential therapeutic regimen for aGVHD.

Functions of PARylation in DNA Damage Repair Pathways

Protein poly ADP-ribosylation（PARylation） is a widespread post-translational modification at DNA lesions,which is catalyzed by poly（ADP-ribose） polymerases（PARPs）.This modification regulates a number of biological processes including chromatin reorganization,DNA damage response（DDR）,transcriptional regulation,apoptosis,and mitosis.PARP1,functioning as a DNA damage sensor,can be activated by DNA lesions,forming PAR chains that serve as a docking platform for DNA repair factors with high biochemical complexity.Here,we highlight molecular insights into PARylation recognition,the expanding role of PARylation in DDR pathways,and the functional interaction between PARylation and ubiquitination,which will offer us a better understanding of the biological roles of this unique post-translational modification.

Therapeutic effects of a novel BAFF blocker on arthritis

B-cell targeted therapy is effective for autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis(RA),although there are setbacks in RA clinical trials.In this study,we designed a novel B-cell activating factor(BAFF)antagonist:BAFF-Trap,a recombinant glycoprotein with BAFF-binding domains of two BAFF receptors(TACI and Br3)linked to Fc domain of human IgG1.Unlike TACI-Fc,BAFF-Trap bound BAFF but not APRIL(a proliferation-inducing ligand),and significantly suppressed the development of collagen-induced arthritis and adjuvant-induced arthritis.Furthermore,BAFF-Trap inhibited proinflammatory cytokine expression,ameliorated joint damage and suppressed B-and T-cell activation.BAFF-Trap reduced dendritic cells in joints,and increased regulatory T cell,regulatory B-cell,and M2 macrophage.The function of BAFF-Trap was related to inhibition of canonical and noncanonical NF-κB activation.Thus,BAFF-Trap may be a valuable agent for the effective treatment of RA.

Anti-CD44 mAb remodels biological behaviors of spheroid cells with stemness from human ovarian cancer cell line SKOV-3

There is accumulating evidence that cancer stem cells (CSCs) play an important role in tumor progression. Novel strategies targeting CSCs have been widely researched. In the present study, we explored whether such CSCs existed in human ovarian cancer (OVCA) cell line and whether anti-CD44 antibody had effects on such subpopulation. We isolated and identified spheroid cells from SKOV-3. Then we used A3D8, an anti-CD44 mAb to treat spheroid cells with so-called "stemness". Effects of A3D8 on spheroid cells' biological behaviors were examined. Our findings showed that there was a small subpopulation that had so-called "stemness" in SKOV-3 cell line. Against spheroid cells, A3D8 can (1) inhibit cell proliferation; (2) change cell cycle distribution and expression of p21, CDK2 and cyclinA; (3) enhance cisplatin (DDP)-induced apoptosis; (4) promote cell differentiation; (5) inhibit clone formation efficiency; (6) reduce invasive efficacy; (7) inhibit tumorigenicity. Thus, to sum up points which we have just showed, spheroid cells isolated from SKOV-3 can be used as an appropriate in vitro model for relevant study of human ovarian CSCs. And our results reasoned that anti-CD44 therapy may become a potential promising strategy for OVCA treatment.

Functional Identification of the Stable Transfection C5aR Cell Line Molt-4

The complement C5 anaphylatoxin receptor is a member of the seven transmembrane-spanning G protein-coupled receptor superfamily that signals through Gcxi and Gtz16. C5aR is mostly expressed on neutrophils, macrophages and endothelial cells. C5a and C5aR interaction plays an important role in numerous biological effects such as in vivo cytokine storm which results in inflammatory damage. Considering the limitation of collection of human peripheral blood neutrophils and their short half life, the stably transfected cell line for studying the biological effects of C5aR is needed. In this study, we transfected C5aR gene into Molt-4 cell line and examined the function of ectopic C5aR. Our results showed stable expression of the C5aR in Molt-4 cell line and their interaction with human C5a induced ERKI/2 phosphorylation, Ca＋＋ influx. This stable transfected cell line may provide a useful tool for studying signal pathways related to C5a and C5aR interplay and antibody development specific for C5aR. Cellular ＆ Molecular Immunology.