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@#Among current novel druggable targets, protein–protein interactions (PPIs) are of considerable and growing interest. Diacylglycerol kinase α (DGKα) interacts with focal adhesion kinase (FAK) band 4.1-ezrin-radixin-moesin (FERM) domain to induce the phosphorylation of FAK Tyr397 site and promotes the malignant progression of esophageal squamous cell carcinoma (ESCC) cells. Chrysin is a multi-functional bioactive flavonoid, and possesses potential anticancer activity, whereas little is known about the anticancer activity and exact molecular mechanisms of chrysin in ESCC treatment. In this study, we found that chrysin significantly disrupted the DGKα/FAK signalosome to inhibit FAK-controlled signaling pathways and the malignant progression of ESCC cells both in vitro and in vivo, whereas produced no toxicity to the normal cells. Molecular validation specifically demonstrated that Asp435 site in the catalytic domain of DGKα contributed to chrysin-mediated inhibition of the assembly of DGKα/FAK complex. This study has illustrated DGKα/FAK complex as a target of chrysin for the first time, and provided a direction for the development of natural products-derived PPIs inhibitors in tumor treatment.
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Metastasis-associated drug resistance accounts for high mortality in ovarian cancer and remains to be a major barrier for effective treatment. In this study, SKOV3/T4, a metastatic subpopulation of ovarian cancer SKOV3 cells, was enriched to explore potential interventions against metastatic-associated drug resistance. Quantitative genomic and functional analyses were performed and found that slug was significantly increased in the SKOV3/T4 subpopulation and contributed to the high resistance of SKOV3/T4. Further studies showed that slug activated c-Met in a ligand-independent manner due to elevated levels of fibronectin and provoked integrin V function, which was confirmed by the significant correlation of slug and p-Met levels in 121 ovarian cancer patient samples. Intriguingly, c-Met inhibitor(s) exhibited greatly enhanced anti-cancer effects in slug-positive ovarian cancer models both and . Additionally, IHC analyses revealed that slug levels were highly correlated with reduced survival of ovarian cancer patients. Taken together, this study not only uncovers the critical roles of slug in drug resistance in ovarian cancer but also highlights a promising therapeutic strategy by targeting the noncanonical activation of c-Met in slug-positive ovarian cancer patients with poor prognosis.
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Background@#Enzyme-linked immunosorbent assay (ELISA) kit is used abundantly in order to measure the quantity of agents and their antibodies. Introducing the production of the kit in our own country for research and diagnostic uses will be beneficial for economy and time.@*Goal@#Producing enzyme-linked immunosorbent assay for the determination of IL-2 cytokine in biological fluids.@*Materials and Methods@#Elisa kits made by R&D systems in USA were used in our study. Anti-IL-2 monoclonal antibody was diluted with concentrations of 0.5-3μg/ml in polystyrene well, and then incubated with conjugate solution of pH 7.2-7.4 for 12 hours with the purpose of measuring the optimal starting concentration for the conjugate antibody. Later, the reaction was blocked with PBS blocking buffer with 1% BSA (Bovine serum albumin) for an hour. After washing the wells, 100ul of standard solutions with concentrations of 7.81-2000pg/ul were added and incubated for 60 or 120 minutes. Subsequently, secondary antibody and streptavidin-HRP were added as substrate, and the reaction was terminated with stop solution. Absorbance was measured immediately at 450 nm optical densities, and then regression equation was calculated using standard curve.@*Result@#The light absorbance was insufficient when then dilution of starting concentration for the conjugate antibody was 0.5μg/ml and the light absorbance decreased as the concentration of conjugate antibody reduced. The light absorbance increased when the concentration was 2-4μg/ml, but the standard curve was constructed nonlinear. When standard curve was constructed with 1 ug/ml, the regression equation had R2=0.96, which identifies the optimal concentration. The results were analogous when the incubation times were 60 and 120 minutes with the concentration of 4μg/ml.@*Conclusion@#The optimal condition for determining IL-2 cytokine was when ELISA conjugate concentration was 1μg/ml (sample and conjugate) and incubation time was 120 minutes, and the regression equation from the standard curve was y=0.0013x+0.1901, R<sup>2</sup>=0.96.
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No advento dos antirretrovirais potentes, os indivíduos infectados pelo vírus da imunodeficiência humana (HIV) começaram a apresentar risco maior para o desenvolvimento de doença cardiovascular (DCV). Este aumento do risco cardiovascular pode ser associado tanto à infecção viral quanto ao tratamento antirretroviral (TARV), que provocam mudanças pró-aterogênicas como o aumento do colesterol total e da lipoproteína de baixa densidade (LDL), além da diminuição da lipoproteína de alta densidade (HDL). A ativação imune e as alterações lipídicas são mecanismos associados com a infecção pelo HIV e com o risco de DCV. Este trabalho utilizou ensaios imunoenzimáticos para a determinação plasmática de biomarcadores emergentes de risco cardiovascular relacionados com modificações da lipoproteína de baixa densidade, a saber: LDL eletronegativa [LDL(-)] e formas oxidadas da LDL, ou seja, LDL-oxi (resíduos lisina da apolipoproteína B100 modificados com malondialdeído), LDL-HNE (resíduos lisina da ApoB100 modificados com 4-hidroxinonenal) e LDL-CML (resíduos lisina da ApoB100 modificados por carboximetila), além de biomarcadores relacionados com a resposta imune-inflamatória, ou seja, autoanticorpos IgG e IgM anti-LDL(-), imunocomplexo de LDL(-) [IC-LDL(-)], proteína amiloide sérica A (SAA) e mieloperoxidase (MPO). Também foram determinadas as concentrações séricas dos biomarcadores de risco relacionados às apolipoproteínas: apolipoproteína A-I (ApoA-I), apolipoproteína B (ApoB) e apolipoproteína E (ApoE). A população estudada incluiu indivíduos com infecção pelo HIV, tratados (HIV-TARV) e não tratados (HIV-NT) com terapia antirretroviral e indivíduos sem infecção pelo HIV (controle). Não foram identificadas diferenças para as concentrações de LDL(-), IC-LDL(-), anti- LDL(-)-IgM, SAA, ApoA-I, ApoB e ApoE entre os grupos estudados (HIV-TARV, HIV-NT e controle). A ApoA-I correlacionou-se positivamente com ApoB e ApoE (rs= 0,418 e rs= 0,347, Spearman, p<0,01) e a ApoB com a ApoE (rs= 0,286, Spearman, p<0,01). Verificou-se correlação inversa entre as concentrações de LDL(-) e IC-LDL(-) (rs= -0,214, Spearman, p<0,05). Os níveis de anti-LDL(-)-IgG correlacionaram-se positivamente com IC-LDL(-) e anti-LDL(-)-IgM (rs= 0,240, Spearman, p<0,05 e rs= 0,348, Spearman, p<0,01). As concentrações de LDL-CML correlacionaram-se positivamente com LDL(-), LDL-oxi, LDL-HNE e IC-LDL(-) (rs= 0,212, Spearman, p<0,05; rs= 0,214, Spearman, p<0,05; rs= 0,573, Spearman, p<0,01 e rs= 0,219, Spearman, p<0,05). O grupo HIV-NT apresentou níveis mais elevados de anticorpos anti-LDL(-)-IgG comparado ao grupo controle (Kruskal-Wallis, p<0,01). Em contraste, observou-se no grupo HIV-NT diminuição das concentrações de MPO, LDL-HNE e LDL-CML em relação ao grupo controle (Kruskal-Wallis, p<0,01). A comparação dos grupos HIV-NT e HIV-TARV demonstrou que o TARV promoveu diminuição das concentrações dos anticorpos anti-LDL(-)-IgG e aumentou os níveis de LDL-oxi (Kruskal-Wallis, p<0,01). O grupo HIV-TARV apresentou aumento das concentrações de LDL-oxi e diminuição dos níveis de MPO, LDL-HNE e LDL-CML em relação ao controle (Kruskal-Wallis, p<0,01). Em conclusão, a infecção pelo HIV modificou o biomarcador de inflamação MPO e o perfil de biomarcadores relacionados às modificações da LDL (menor formação de LDL-HNE e LDL-CML), além aumentar a resposta imune-humoral à LDL eletronegativa [anti-LDL(-)-IgG], enquanto o tratamento com antirretrovirais inibiu esta resposta. Os outros biomarcadores estudados não foram modificados pela infecção viral ou pelo tratamento antirretroviral
In the advent of potent antiretroviral therapy, individuals infected with human immunodeficiency virus (HIV) have showed an increased risk for developing cardiovascular disease (DCV). Studies have discussed that the increased risk may be related to both the disease and antiretroviral treatment (TARV), that produced pro-atherogenic changes such as increased of total cholesterol and low density lipoprotein (LDL) and decreased high density lipoprotein. The immune activation and the lipid modifications are well known mechanisms related to HIV infection and the risk of DCV. This study used immunoassays for plasma quantification for emerging biomarkers of cardiovascular risk related to modification of low density lipoprotein: electronegative LDL [LDL(-)] and oxidized forms of LDL, LDL-oxi (lysine residues of apolipoprotein B100 modified by malondialdehyde), LDL-HNE (lysine residues of ApoB100 modified by 4-hydroxynonenal) and LDL-CML (lysine residues of ApoB100 modified by carboxymethyl) and biomarkers associated to immune and inflammatory responses, IgG and IgM autoantibodies anti-LDL(-) and immunecomplexe of LDL(-) [IC-LDL(-)], serum amyloid A protein (SAA) and myeloperoxidase (MPO). Also, were determined serum concentrations of risk biomarkers related to apolipoproteins: apolipoprotein A-I (ApoA-I), apolipoprotein B (ApoB) and apolipoprotein E (ApoE). The studied population included patients with HIV infection, treated (HIV-TARV) and untreated (HIV-NT) with antiretroviral therapy and individuals without HIV infection (controle). No differences were identified for concentrations of LDL(-), ICLDL(-), anti-LDL(-)-IgM, SAA, ApoA-I, ApoB and ApoE between studied groups (HIV-TARV, HIV-NT and controle). The ApoA-I was positively correlated to ApoB and ApoE (rs= 0,418 e rs= 0,347, Spearman, p<0,01) and ApoB to ApoE (rs= 0,286, Spearman, p<0,01). There was an inverted correlation between LDL(-) and IC-LDL(-) (rs= -0.214, Spearman, p<0,05). The levels of anti-LDL(-)-IgG were positively correlated to IC-LDL(-) and antibodies anti-LDL(-)-IgM (rs= 0.240; Spearman; p <0.05 and rs= 0.348; Spearman; p <0.01). The concentrations of LDL-CML were positively correlated to LDL(-), LDL-oxi, LDL-HNE e IC-LDL(-) (rs= 0,212, Spearman, p<0,05; rs= 0,214, Spearman, p<0,05; rs= 0,573, Spearman, p<0,01 e rs= 0,219, Spearman, p<0,05). The HIV-NT group showed higher levels of anti-LDL(-)-IgG compared to Control group (Kruskal-Wallis, p<0,01). In contrast, was observed lower levels for HIV-NT group to MPO, LDL-HNE and LDL-CML when compared to Control group (Kruskal-Wallis, p<0,01). The comparison of HIV-NT and HIV-TARV groups demonstrated that TARV caused a decrease of concentrations of anti-LDL(-)-IgG antibodies and an increased of LDL-oxi levels (Kruskal-Wallis, p <0.01). The HIV-TARV group showed increased LDL-oxi concentrations and decreased at levels of MPO, LDL-HNE e LDL-CML when compared to Control (Kruskal-Wallis, p<0,01). In conclusion, the HIV infection changed the biomarker of inflammation MPO and the profile of biomarkers related to modifications of LDL (lower concentrations of LDL-HNE and LDL-CML), as well as increased the humoral-immune response to electronegative LDL [anti-LDL(-)-IgG], while treatment with antiretroviral therapy inhibited this response. The other studied biomarkers were not modified either by viral infection or antiretroviral treatment
Subject(s)
Biomarkers/analysis , Cardiovascular System , Immunoenzyme Techniques/methods , HIV/metabolism , Antiretroviral Therapy, Highly Active/classification , Low Density Lipoprotein Receptor-Related Protein-1 , Atherosclerosis/complicationsABSTRACT
Introduction: Scrub typhus is a rickettsial infection which is caused by Orientia tsutsugamushi and transmitted by the bite of the chigger of a mite. Delay in diagnosis can be fatal otherwise the treatment is simple, doxycycline being the drug of choice. Indirect immunofl urescence is considered gold standard but it is not used in India as it is costly and also not available. There is need for rapid, economic and simple test for the diagnosis of scrub typhus. This study was taken up to study the seroprevalence of scrub typhus in Andhra Pradesh and to compare two commonly used serological methods; rapid test and IgM ELISA. Materials and methods: This is a prospective study in which 100 serum samples from clinically suspected cases collected over a period of 3 months were processed for the detection of IgM antibodies for scrub typhus by ELISA and Rapid test. Samples were also tested for leptospirosis and dengue fever which the other common causes of fever prevalent in this region. Results: Total number of samples processed was 100 of which 52 were males and 48 females. Among the hundred samples 39 were seropositive. Positivity was higher in the age group of patients between 16 and 30 yrs of age. There was 97% correlation between ELISA and rapid method. Of the 100 samples only three samples positive by ELISA were negative by rapid method. Fever was the most common manifestation and there was no eschar and no mortality reported. Conclusion: Scrub typhus should be included in the differential diagnosis of fever of unknown origin along with dengue, malaria and leptospirosis which are the other common endemic infections in this part of the country.
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Background & objectives: The challenge of malaria and efforts targeted at developing malaria vaccines triggered this study on the reactivity of IgG and its subclasses in the test serum specific to CSP. This work was directed at assessing the influence of age and gender on host humoral antibody against Plasmodium falciparum recombinant circumsporozoite antigen in Nigerian children. Methods: In all, 67 serum samples (>10,000 parasites/μl of blood) collected from malaria-infected children at the University College Hospital, Ibadan during the transmission season were analyzed by ELISA. Results: The mean absorbance values of IgG subclasses reactive against P. falciparum CSP appeared to be agedependent and ranged from 0.01 for IgG4 in younger children to 0.95 for IgG3 in older children. The sixty-seven subjects investigated in this study had significantly higher mean IgG1 and IgG3 than the uninfected controls (p <0.01). This follows the order IgG3 >IgG1>IgG2>IgG4 which confirmed the prevalence of the cytophilic antibodies (IgG1 and IgG3) in 65% of the malaria infected children over the non-cytophilic subclasses (IgG2 and IgG4). Similarly, there was low production of IgG4 and IgG2 levels in 35% of the subjects compared with control. IgG was detected in the serum of North American Subjects (NAS) which served as negative control for CSP-specific IgG subclasses. Although the NAS titre was lower than that of the malaria subjects in Nigeria, its IgG2 was, however, higher (0.16) than that of other subclasses. The mean absorbance values of total serum IgG subclass were higher than those of IgG subclasses specific to P. falciparum circumsporozoite antigen. The mean absorbance values of the total serum IgG subclass follows the order IgG2>IgG1>IgG4>IgG3. Interpretation & conclusion: Age and gender-dependent correlations of results suggest that acquired immunity could play a significant role in protection from malaria. Antibody levels are higher in male than female children of the same age group. Antibody levels also increase with age in both the male and female children.
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Objective To optimize the conditions of the expression of fusion protein GST-SjMP10 and to evaluate the value of fusion protein GST-SjMP10 for diagnosis of schistosomiasis.Methods The optimal concentration of IPTG for the expression of fusion protein GST-SjMP10 was chosen in inducing the expression of GST-SjMP10 with different concentration of IPTG,and the soluble fusion protein GST-SjMP10 was identified by SDS-PAGE.The fusion protein GST-SjMP10 was purified by chromatographic affinity with glutathione Sepharose 4B gel.The sensitivity and specificity of purified fusion protein GST-SjMP10 for diagnosis of schistosomiasis were determined by enzyme linked immunosorbent assay(ELISA)to detect the IgG antibody in sera from the patients with acute schistosomiasis,advanced schistosomiasis and clonorchiasis as well as healthy subjects.Results Most of the expressed fusion protein GST-SjMP10 was in soluble status when the concentration of IPTG was reduced to 0.1 mmol/L and the fusion protein GST-SjMP10 could be purified by chromatographic affinity.The positive rate of anti-GST-SjMP10 antibody in the sera from the patients with acute and chronic schistosomiasis japonica was 97.5% and 96.7% respectively.No cross reactivity of the fusion protein GST-SjMP10 was found in the detection for the sera from clonorchiasis patients,and no false positive was found in the detection for the sera of healthy subjects.Conclusion The fusion protein GST-SjMP10 was expressed successfully and showed high sensitivity and specificity for the diagnosis of schistosomiasis japonicum.
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Objective To explore changes of myelin basic pr otein(MBP)and S-100B protein in serum and cerebrospinal fluid(CSF)after acute spinal cord injury(ASCI )and after tetramethylpyrazine trea tment.Methods Dog models with ASCI at T13were made in th e methods of Allens and an epidural t ube was set in the subarachnoid space,with one opening out of the skin and blocked in order to gather CSF.The dogs were divided randomly into 3groups.Grou p A(n =6)was the control group which received only laminectomy of T13without a str iking to the spinal cord.Group B(n =6),acute spinal cord injury group and Group C(n =6),tetramethylpyrazine treatment group,all received a striking to the spinal cord with the device of Allens after the dura was exposed.Blood an d CSF were taken out of the dogs respec tively at the time intervals of 2h,4h,6h,8h,10h,24h,48h,72h and 96h after the operation.The concentration of MBP and S-100B in the serum and CSF was measur ed by the methods of ELISA and feature s of the change were studied.Results After acute spinal cord injury,concentrations of MBP and S-100B in the se rum and CSF increased significantly in Group B and Group C,and the levels changed dynamically.But the levels in tetrame thylpyrazine treatment group were l ower than those in spinal cord injury group.Conclusions MBP and S-100B protein change dynami cally after acute spinal cord injury.Tetramethylpyrazine has a t herapeutic effect on acute spinal cord injury.Whether MBP and S-100B pro tein can be served as the marker for ASCI needs fu rther research to evaluate their correlation to the severity and prognosis of ASCI.[