Protein Microarray Assay Research Articles

Estimating the force of infection of four dengue serotypes from serological studies in two regions of Vietnam.

Dengue is endemic in Vietnam with circulation of all four serotypes (DENV1-4) all year-round. It is hard to estimate the disease's true serotype-specific transmission patterns from cases due to its high asymptomatic rate, low reporting rate and complex immunity and transmission dynamics. Seroprevalence studies have been used to great effect for understanding patterns of dengue transmission. We tested 991 population serum samples (ages 1-30 years, collected 2013 to 2017), 531 from Ho Chi Minh City and 460 from Khanh Hoa in Vietnam, using a flavivirus protein microarray assay. By applying our previously developed inference framework to the antibody profiles from this assay, we can (1) determine proportions of a population that have not been infected or infected, once, or more than once, and (2) infer the infecting serotype in those infected once. With these data, we then use mathematical models to estimate the force of infection (FOI) for all four DENV serotypes in HCMC and KH over 35 years up to 2017. Models with time-varying or serotype-specific DENV FOI assumptions fit the data better than constant FOI. Annual dengue FOI ranged from 0.005 (95%CI: 0.003-0.008) to 0.201 (95%CI: 0.174-0.228). FOI varied across serotypes, higher for DENV1 (95%CI: 0.033-0.048) and DENV2 (95%CI: 0.018-0.039) than DENV3 (95%CI: 0.007-0.010) and DENV4 (95%CI: 0.010-0.016). The use of the PMA on serial age-stratified cross-sectional samples increases the amount of information on transmission and population immunity, and should be considered for future dengue serological surveys, particularly to understand population immunity given vaccines with differential efficacy against serotypes, however, there remains limits to what can be inferred even using this assay.

In Situ Protein Microarray for Identifying the Geminivirus-Arabidopsis Interactome.

The protein-protein interactions (PPI) by protein array technology complement other PPI assay technologies such as AP-MS and Y2H. The in situ protein array technology (NAPPA) enables low-cost, rapid, and comprehensive protein detection. It allows standardized and simultaneous assay of a wide range of proteins with a broad range of expression in cells. This technology facilitates the detection of protein-protein interactions within species and between heterologous species such as host-microbe. Here, we described the technique that identified a syntaxin-6 protein-mediated begomovirus infection using an array containing 4600 Arabidopsis genes. The protein microarray assay also identified several other viral protein-host protein interactions.

Emodin attenuates cardiomyocyte pyroptosis in doxorubicin-induced cardiotoxicity by directly binding to GSDMD

BackgroundDoxorubicin (Dox), which is an anticancer drug, has significant cardiac toxicity and side effects. Pyroptosis occurs during Dox-induced cardiotoxicity (DIC), and drug inhibition of this process is one therapeutic approach for treating DIC. Previous studies have indicated that emodin can reduce pyroptosis. However, the role of emodin in DIC and its molecular targets remain unknown. Hypothesis/PurposeWe aimed to clarify the protective role of emodin in mitigating DIC, as well as the mechanisms underlying this effect. MethodsThe model of DIC was established via the intraperitoneal administration of Dox at a dosage of 5 mg/kg per week for a span of 4 weeks. Emodin at two different doses (10 and 20 mg/kg) or a vehicle was intragastrically administered to the mice once per day throughout the Dox treatment period. Cardiac function, myocardial injury markers, pathological morphology of the heart, level of pyroptosis and mitochondrial function were assessed. Protein microarray, biolayer interferometry and pull-down assays were used to confirm the target of emodin. Moreover, GSDMD-overexpressing plasmids were transfected into GSDMD−/− mice and HL-1 cells to further verify whether emodin suppressed GSDMD activation. ResultsEmodin therapy markedly enhanced cardiac function and reduced cardiomyocyte pyroptosis in mice induced by Dox. Mechanistically, emodin binds to GSDMD and inhibits the activation of GSDMD by targeting the Trp415 and Leu290 residues. Moreover, emodin was able to mitigate Dox-induced cardiac dysfunction and myocardial injury in GSDMD−/− mice overexpressing GSDMD, as shown by increased EF and FS, decreased serum levels of CK-MB, LDH and IL-1β and mitigated cell death and cell morphological disorder. Additionally, emodin treatment significantly reduced GSDMD-N expression and plasma membrane disruption in HL-1 cells overexpressing GSDMD induced by Dox. In addition, emodin reduced mitochondrial damage by alleviating Dox-induced GSDMD perforation in the mitochondrial membrane. ConclusionEmodin has the potential to attenuate DIC by directly binding to GSDMD to inhibit pyroptosis. Emodin may become a promising drug for prevention and treatment of DIC.

Immunological comparison of recombinant shrimp allergen Pen m 4, produced in Pichia pastoris and Escherichia coli.

Shellfish are a leading cause of allergies worldwide, affecting about one-tenth of the general population. The sarcoplasmic calcium-binding protein, also known as allergen Pen m 4, is an important factor in shrimp allergies. Our objective was to assess the most effective techniques for producing a recombinant Pen m 4 protein as a potential tool for diagnosing shrimp allergies. In this study, for the first time, we produced a functional recombinant Pen m 4 protein in a eukaryotic system, Pichia pastoris, and analyzed it against Escherichia coli-produced equivalents in enzyme-linked immunosorbent and reverse-phase protein microarray assays. A dual tag system based on the maltose-binding protein was successfully used to increase the yield of Pen m 4 by 1.3 to 2.3-fold in both bacteria and yeast, respectively. Immunological characterization showed that N-glycosylation is neither crucial for the folding of Pen m 4 nor its recognition by specific IgE. However, the Ca2+-depletion assay indicated a dependence on calcium ion presence in blood samples. Results demonstrate how a comparative analysis can elucidate essential allergen manufacturing points. In conclusion, E. coli-produced Pen m 4 protein fused with the maltose-binding protein should be the preferred option for further studies in Penaeus monodon allergy diagnostics.

The association between altered intestinal microbiome, impaired systemic and ocular surface immunity, and impaired wound healing response after corneal alkaline-chemical injury in diabetic mice.

We aim to investigate the effect of sustained hyperglycemia on corneal epithelial wound healing, ocular surface and systemic immune response, and microbiome indices in diabetic mice compared to controls after alkaline chemical injury of the eye. Corneal alkaline injury was induced in the right eye of Ins2Akita (Akita) mice and wild-type mice. The groups were observed at baseline and subsequently days 0, 3, and 7 after injury. Corneal re-epithelialization was observed under slit lamp with fluorescein staining using a cobalt blue light filter. Enucleated cornea specimens were compared at baseline and after injury for changes in cornea thickness under hematoxylin and eosin staining. Tear cytokine and growth factor levels were measured using protein microarray assay and compared between groups and time points. Flow cytometry was conducted on peripheral blood and ocular surface samples to determine CD3+CD4+ cell count. Fecal samples were collected, and gut microbiota composition and diversity pattern were measured using shotgun sequencing. Akita mice had significantly delayed corneal wound healing compared to controls. This was associated with a reduction in tear levels of vascular endothelial growth factor A, angiopoietin 2, and insulin growth factor 1 on days 0, 3, and 7 after injury. Furthermore, there was a distinct lack of upregulation of peripheral blood and ocular surface CD3+CD4+ cell counts in response to injury in Akita mice compared to controls. This was associated with a reduction in intestinal microbiome diversity indices in Akita mice compared to controls after injury. Specifically, there was a lower abundance of Firmicutes bacterium M10-2 in Akita mice compared to controls after injury. In diabetic mice, impaired cornea wound healing was associated with an inability to mount systemic and local immune response to ocular chemical injury. Baseline and post-injury differences in intestinal microbial diversity and abundance patterns between diabetic mice and controls may potentially play a role in this altered response.

DNA methylation-mediated silencing of Neuronatin promotes hepatocellular carcinoma proliferation through the PI3K-Akt signaling pathway

AimsTo explore the methylation status, function, and underlying mechanism of the imprinted gene Neuronatin (NNAT) in hepatocellular carcinoma (HCC) progression. Main methodsImmunohistochemistry (IHC) was performed to evaluate the expression of NNAT in HCC samples. Bisulfite genomic sequencing PCR (BSP) was applied to examine the methylation status of the NNAT promoter. In addition, colony formation, 5-Ethynyl-20-deoxyuridine (EdU) assays and subcutaneous xenograft nude models were used to explore the roles of NNAT in HCC cell proliferation. Furthermore, RNA-seq and phospho-specific protein microarray assays were conducted to illustrate the underlying mechanism by which NNAT regulates HCC progression. Key findingsNNAT was obviously downregulated in HCC tissues, and its expression level was closely associated with tumor growth and patient prognosis. The downregulation of NNAT in HCC was induced by hypermethylation of CpG islands in the promoter region, and hypermethylation was correlated with overall survival of HCC. Moreover, the enforced expression of NNAT significantly inhibited HCC cell proliferation in vitro and in vivo. Transcriptome analysis showed that the alteration of NNAT expression was mainly related to dysregulation of the PI3K-Akt signaling pathway. Finally, phospho-specific antibody microarray detection further revealed that overexpressed NNAT can increase the phosphorylation levels of LKB1, Met, and elF4E and decrease the phosphorylation levels of PTEN, which are all involved in the PI3K-Akt signaling pathway. SignificanceOur research provides new insights into the epigenetic regulation of imprinted genes in tumorigenesis and implies that the imprinted gene NNAT may act as a prognostic biomarker and tumor suppressor in HCC.

Development and application of a novel triplex protein microarray method for rapid detection of antibodies against avian influenza virus, Newcastle disease virus, and avian infectious bronchitis virus.

Avian influenza virus (AIV), Newcastle disease virus (NDV), and avian infectious bronchitis virus (IBV) inflict immense damage on the global poultry industry annually. Serological diagnostic methods are fundamental for the effective control and prevention of outbreaks caused by these viruses. In this study, a novel triplex protein microarray assay was developed and validated for the rapid and simultaneous visualized detection of antibodies against AIV, NDV, and IBV in chicken sera. The AIV nuclear protein (NP), NDV phosphoprotein (P), and IBV nonstructural protein 5 (nsp5) were produced in a prokaryotic expression system, purified, and immobilized onto an initiator integrated poly(dimethylsiloxane) (iPDMS) film as probes to detect antibodies against these viruses in chicken sera. After optimization of the reaction conditions, no cross-reactivity was detected with infectious bursal disease virus, avian leukosis virus subgroup J and chicken anemia virus antisera. The lowest detectable antibody titers in this assay corresponded to hemagglutination inhibition (HI) titers of 24 and 21 for AIV and NDV, respectively, and to an IDEXX antibody titer of 103 for IBV, using the HI assay and IDEXX commercial ELISA kit as the reference methods. When156 serum samples were tested using the new assay, the HI test and the IBV IDEXX ELISA kit, the assay showed 96.8% (151/156), 97.4% (152/156) and 99.4% (155/156) diagnostic accuracy for detection of AIV, NDV and IBV antibody, respectively. The current study suggests that the newly developed triplex microarray is rapid, sensitive, and specific, providing a viable alternative assay for AIV, NDV, and IBV antibody screening in epidemiological investigations and vaccination evaluations.

Identification and validation of potential novel biomarkers for oral squamous cell carcinoma

ABSTRACT Our study aimed to explore potential new diagnostic biomarkers in patients with oral squamous cell carcinoma (OSCC) to find new target molecules involved in the progression of OSCC. Potential novel biomarkers of OSCC were identified using a protein microarray assay. Compared with the healthy control group, there were five proteins (I309, GDF15, AXL, MMP3, and CTACK) in the serum of in situ oral cancer group. However, there were four differentially expressed proteins (MCSF, I309, MMP3, and CTACK) in the serum of the OSCC group. Receiver operating characteristic (ROC) curve analysis results suggested that these six proteins (I309, GDF15, AXL, MMP3, CTACK, and MCSF) had diagnostic value for OSCC. Based on The Cancer Genome Atlas (TCGA) database, we found that only GDF15 expression was associated with the prognosis of OSCC. Subsequently, we verified the expression levels of six proteins in HSC-3 and HaCaT cells, and the results showed that the level of these six proteins was significantly higher in HSC-3 cells than in normal HaCaT cells. Similarly, in the OSCC nude mouse model, the expression levels of these proteins were significantly upregulated in OSCC tumor tissue compared to the normal tissue. GDF15, MMP3, AXL, MCSF, I309, and CTACK may be used as biomarkers for OSCC diagnosis and provide a novel study direction for the treatment of OSCC.

Controlling the immobilization process of an optically enhanced protein microarray for highly reproducible immunoassay

By virtue of its high throughput multiplex detection capability, superior read-out sensitivity, and tiny analyte consumption, an optically enhanced protein microarray assay has been developed as a promising diagnostic tool for various applications, ranging from the field of pharmacology to diagnostics. However, so far, the development of an optically enhanced protein microarray (OEPM) toward widespread commercial availability is mainly hampered by insufficient detection reproducibility. Here, we develop an OEPM platform with an order of magnitude optical enhancement induced by the interference effect. High assay reproducibility of the OEPM is achieved by optimizing the protein immobilization schemes, linking to the surface energy of the substrate, surfactant-tuned wetting ability, and the washing and drying dynamics. As a result, smearing-free and uniform spot arrays with a coefficient of variation less than 7% can be achieved. Furthermore, we demonstrate the assay performance of the OEPM by detecting five biomarkers, showing an order of magnitude higher sensitivity, many-fold higher throughput, and 10 times less analyte consumption than those of the commercial enzyme-linked immunosorbent assay kits. Our results provide new insight for improving the reproducibility of OEPMs toward practical and commercial diagnostic assays.

Adenosylhomocysteinase like 1 interacts with nonstructural 5A and regulates hepatitis C virus propagation.

Hepatitis C virus (HCV) life cycle is highly dependent on cellular proteins for viral propagation. In order to identify the cellular factors involved in HCV propagation, we previously performed a protein microarray assay using the HCV nonstructural 5A (NS5A) protein as a probe. Of ∼9,000 human cellular proteins immobilized in a microarray, adenosylhomocysteinase like 1 (AHCYL1) was among 90 proteins identified as NS5A interactors. Of these candidates, AHCYL1 was selected for further study. In the present study, we verified the physical interaction between NS5A and AHCYL1 by both in vitro pulldown and coimmunoprecipitation assays. Furthermore, HCV NS5A interacted with endogenous AHCYL1 in Jc1-infected cells. Both NS5A and AHCYL1 were colocalized in the cytoplasmic region in HCV-replicating cells. siRNAmediated knockdown of AHCYL1 abrogated HCV propagation. Exogenous expression of the siRNA-resistant AHCYL1 mutant, but not of the wild-type AHCYL1, restored HCV protein expression levels, indicating that AHCYL1 was required specifically for HCV propagation. Importantly, AHCYL1 was involved in the HCV internal ribosome entry site-mediated translation step of the HCV life cycle. Finally, we demonstrated that the proteasomal degradation pathway of AHCYL1 was modulated by persistent HCV infection. Collectively, these data suggest that HCV may modulate the AHCYL1 protein to promote viral propagation.

Cross-species reactivity of antibodies against Plasmodium vivax blood-stage antigens to Plasmodium knowlesi.

Malaria is caused by multiple different species of protozoan parasites, and interventions in the pre-elimination phase can lead to drastic changes in the proportion of each species causing malaria. In endemic areas, cross-reactivity may play an important role in the protection and blocking transmission. Thus, successful control of one species could lead to an increase in other parasite species. A few studies have reported cross-reactivity producing cross-immunity, but the extent of cross-reactive, particularly between closely related species, is poorly understood. P. vivax and P. knowlesi are particularly closely related species causing malaria infections in SE Asia, and whilst P. vivax cases are in decline, zoonotic P. knowlesi infections are rising in some areas. In this study, the cross-species reactivity and growth inhibition activity of P. vivax blood-stage antigen-specific antibodies against P. knowlesi parasites were investigated. Bioinformatics analysis, immunofluorescence assay, western blotting, protein microarray, and growth inhibition assay were performed to investigate the cross-reactivity. P. vivax blood-stage antigen-specific antibodies recognized the molecules located on the surface or released from apical organelles of P. knowlesi merozoites. Recombinant P. vivax and P. knowlesi proteins were also recognized by P. knowlesi- and P. vivax-infected patient antibodies, respectively. Immunoglobulin G against P. vivax antigens from both immune animals and human malaria patients inhibited the erythrocyte invasion by P. knowlesi. This study demonstrates that there is extensive cross-reactivity between antibodies against P. vivax to P. knowlesi in the blood stage, and these antibodies can potently inhibit in vitro invasion, highlighting the potential cross-protective immunity in endemic areas.

Nonstructural NS5A Protein Regulates LIM and SH3 Domain Protein 1 to Promote Hepatitis C Virus Propagation.

Hepatitis C virus (HCV) propagation is highly dependent on cellular proteins. To identify the host factors involved in HCV propagation, we previously performed protein microarray assays and identified the LIM and SH3 domain protein 1 (LASP-1) as an HCV NS5A-interacting partner. LASP-1 plays an important role in the regulation of cell proliferation, migration, and protein-protein interactions. Alteration of LASP-1 expression has been implicated in hepatocellular carcinoma. However, the functional involvement of LASP1 in HCV propagation and HCV-induced pathogenesis has not been elucidated. Here, we first verified the protein interaction of NS5A and LASP-1 by both in vitro pulldown and coimmunoprecipitation assays. We further showed that NS5A and LASP-1 were colocalized in the cytoplasm of HCV infected cells. NS5A interacted with LASP-1 through the proline motif in domain I of NS5A and the tryptophan residue in the SH3 domain of LASP-1. Knockdown of LASP-1 increased HCV replication in both HCV-infected cells and HCV subgenomic replicon cells. LASP-1 negatively regulated viral propagation and thereby overexpression of LASP-1 decreased HCV replication. Moreover, HCV propagation was decreased by wild-type LASP-1 but not by an NS5A binding-defective mutant of LASP-1. We further demonstrated that LASP-1 was involved in the replication stage of the HCV life cycle. Importantly, LASP-1 expression levels were increased in persistently infected cells with HCV. These data suggest that HCV modulates LASP-1 via NS5A in order to regulate virion levels and maintain a persistent infection.

Use of meat juice and blood serum with a miniaturised protein microarray assay to develop a multi-parameter IgG screening test with high sample throughput potential for slaughtering pigs

BackgroundSerological screening of pig herds at the abattoir is considered a potential tool to improve meat inspection procedures and herd health management. Therefore, we previously reported the feasibility of a miniaturised protein microarray as a new serological IgG screening test for zoonotic agents and production diseases in pigs. The present study investigates whether the protein microarray-based assay is applicable for high sample throughput using either blood serum or meat juice.Material and methodsMicroarrays with 12 different antigens were produced by Abbott (formerly Alere Technologies GmbH) Jena, Germany in a previously offered ‘ArrayTube’ platform and in an ‘ArrayStrip’ platform for large-scale use. A test protocol for the use of meat juice on both microarray platforms was developed. Agreement between serum and meat juice was analysed with 88 paired samples from three German abattoirs. Serum was diluted 1:50 and meat juice 1:2. ELISA results for all tested antigens from a preceding study were used as reference test to perform Receiver Operating Characteristic analysis for both test specimens on both microarray platforms.ResultsHigh area under curve values (AUC > 0.7) were calculated for the analysis of T. gondii (0.87), Y. enterocolitica (0.97), Mycoplasma hyopneumoniae (0.84) and Actinobacillus pleuropneumoniae (0.71) with serum as the test specimen and for T. gondii (0.99), Y. enterocolitica (0.94), PRRSV (0.88), A. pleuropneumoniae (0.78) and Salmonella spp. (0.72) with meat juice as the test specimen on the ArrayStrip platform. Cohens kappa values of 0.92 for T. gondii and 0.82 for Y. enterocolitica were obtained for the comparison between serum and meat juice. When applying the new method in two further laboratories, kappa values between 0.63 and 0.94 were achieved between the laboratories for these two pathogens.ConclusionFurther development of a miniaturised pig-specific IgG protein microarray assay showed that meat juice can be used on microarray platforms. Two out of twelve tested antigens (T. gondii, Y. enterocolitica) showed high test accuracy on the ArrayTube and the ArrayStrip platform with both sample materials.

Human adipose-derived stem cells support lymphangiogenesis in vitro by secretion of lymphangiogenic factors

Lymphedema is a chronic progressive disease ultimately resulting in severe, disfiguring swelling and permanent changes of the affected tissues. Presently, there is no causal treatment approach of lymphedema. Therefore, most therapies are purely symptomatic. However, the recent use of stem cell-based therapies has offered new prospects for alternative treatment options. The present study was performed to investigate the effects of human adipose-derived stem cells (ADSCs) on human dermal lymphatic endothelial cells (HDLECs) in terms of basic in vitro lymphangiogenic assays (WST-8 assay, scratch assay, transmigration assay, sprouting assay, tube formation assay).The influence of ADSC-conditioned medium (ADSC-CM) on HDLECs was compared to recombinant VEGF-C, bFGF and HGF. Further ADSC-CM was characterized by protein microarray and enzyme-linked immunosorbent assay (ELISA). Although key-lymphangiogenic growth factors - like VEGF-C - could only be detected in low concentrations within the conditioned medium (CM), HDLECs were potently stimulated to proliferate, migrate and to form tube like structures by ADSC-CM. Despite concentrations more than hundredfold higher than those found in the conditioned medium, stimulation with recombinant VEGF-C, bFGF and HGF was still weaker compared to ADSC-CM. These results highlight the effectiveness of growth factors secreted by ADSC to stimulate HDLEC, potentially providing a promising new therapeutic approach for the treatment of lymphedema.

Fractionated irradiation of right thorax induces abscopal damage on testes leading to decline in fertility

Radiation-induced abscopal effect (RIAE) may influence radiotherapy efficiency. However, it is unknown whether RIAE triggers abnormal genetic consequence. We present a novel evidence that, when mice were given fractionated irradiation on right thorax, the ultrastructure of blood-testis barrier was damaged in company with apoptosis induction in testes, and the sperm number and vitality were drastically decreased so that both the fertility and the survival of their offspring were reduced. Protein microarray assay and hormone detection showed that some cytokines especially TNF-α, TGF-β and estradiol in the serum of irradiated mice increased to higher levels in consistent with abscopal damage, and this conditioned serum had toxic effect on TM4 cells in vitro. When the mice were fed with cimetidine, the above abscopal responses were significantly attenuated. This study demonstrates in the first time that the thoracic irradiation (Th-IR) induces structural and functional damage in the distal testes and further cause fertility decline of irradiated male mice, which may have important implications in the strategy development of radiotherapy in avoiding abnormal genetic consequence.

Middle East Respiratory Syndrome: Its Occurrence, Aetiology, Epidemiology, Associated Clinicopathological Findings, Diagnosis, Prevention and Control in Humans and Animals: An Overview

Middle East respiratory syndrome (MERS) is a serious zoonotic disease. It is characterized by severe infection of the lower respiratory pathway in humans. Dromedary camels are considered to be the likeliest source of the very pathogenic aetiologic agent, Middle East respiratory syndrome coronavirus (MERS-CoV). Although, the first case of MERS-CoV was initially identified in Kingdom of Saudi Arabia (KSA) in September 2012, most of the reported cases have been detected in Arabian Peninsula but worthy of note is that travel-associated MERS cases have been reported around the world especially in Europe, Asia, Africa and North-America. This virus is very endemic in camel populations of East Africa and the Arabian Peninsula but worryingly, the zoonotic transmission of MERS-CoV are now well reported. It is diagnosed in camels using IgG immunofluorescence assay, protein microarray and virus neutralisation assay Diagnosis in humans is based on chest radiographs and computed tomographic scans but confirmatory diagnosis of suspected MERS patients could be done by taking a intratracheal aspirate for detection test of MERS-CoV RNA positive real-time reverse transcriptase polymerase chain reaction assays. The major control measures of MERS-CoV spread include strict regulation of camel movement, regular herd screening and culling of infected camels.

Cigarette smoking differentially affects immunoglobulin class levels in serum and saliva: An investigation and review.

The aim of the present study was to compare concentrations of IgG, IgA, IgM and IgD in both serum and saliva samples from smoking and non-smoking individuals using a protein microarray assay. The findings were also compared to previous studies. Serum and saliva were collected from 48 smoking male individuals and 48 age-matched never-smoker male individuals. The protein microarray assays for detection of human IgG, IgM, IgA and IgD were established and optimized using Ig class-specific affinity-purified goat anti-human Ig-Fc capture antibodies and horseradish peroxidase (HRP)-conjugated goat anti-human Ig-Fc detection antibodies. The Ig class specificity of the microarray assays was verified, and the optimal dilutions of serum and saliva samples were determined for quantification of Ig levels against standard curves. We found that smoking is associated with reduced IgG concentrations and enhanced IgA concentrations in both serum and saliva. By contrast, smoking differentially affected IgM concentrations-causing increased concentrations in serum, but decreased concentrations in saliva. Smoking was associated with decreased IgD concentrations in serum and did not have a significant effect on the very low IgD concentrations in saliva. Thus, cigarette smoking differentially affects the levels of Ig classes systemically and in the oral mucosa. Although there is variation between the results of different published studies, there is a consensus that smokers have significantly reduced levels of IgG in both serum and saliva. A functional antibody deficiency associated with smoking may compromise the body's response to infection and result in a predisposition to the development of autoimmunity.

Development and validation of different indirect ELISAs for MERS-CoV serological testing

Since 2012, MERS-CoV has caused up to 2220 cases and 790 deaths in 27 countries with Saudi Arabia being the most affected country with ~83.1% of the cases and ~38.8% local death rate. Current serological assays such as microneutralization (MN), plaque reduction neutralization, immunofluorescence, protein microarray or pseudoparticle neutralization assays rely on handling of live MERS-CoV in high containment laboratories or need for expensive and special equipment and reagents and highly trained personnel which represent a technical hurdle for most laboratories in resource-limited MERS-CoV endemic countries. Here, we developed, compared and evaluated three different indirect ELISAs based on MERS-CoV nucleocapsid protein (N), spike (S) ectodomain (amino acids 1–1297) and S1 subunit (amino acids 1–725) and compared them with MN assay. The developed ELISAs were evaluated using large number of confirmed seropositive (79 samples) and seronegative (274 samples) MERS-CoV human serum samples. Both rS1- and rS-ELISAs maintained high sensitivity and specificity (≥90%) across a wider range of OD values compared to rN-ELISA. Moreover, rS1- and rS-based ELISAs showed better agreement and correlation with MN assay in contrast to rN-ELISA. Collectively, our data demonstrate that rS1-ELISA and rS-ELISA are more reliable than rN-ELISA and represent a suitable choice for seroepidemiological testing and surveillance in MERS-CoV endemic regions.

A novel HBx genotype serves as a preoperative predictor and fails to activate the JAK1/STATs pathway in hepatocellular carcinoma

Genetic variability in the hepatitis B virus X gene (HBx) is frequently observed and is associated with hepatocellular carcinoma (HCC) progression. However, a genotype classification based on the full-length HBx sequence and the impact of genotypes on hepatitis B virus (HBV)-related HCC prognosis remain unclear. We therefore aimed to perform this genotype classification and assess its clinical impact. We classified the genotypes of the full-length HBx gene through sequencing and a cluster analysis of HBx DNA from a cohort of patients with HBV-related HCC, which served as the primary cohort (n = 284). Two independent HBV-related HCC cohorts, a validation cohort (n = 171) and a serum cohort (n = 168), were used to verify the results. Protein microarray assay analysis was performed to explore the underlying mechanism. In the primary cohort, the HBx DNA was classified into 3 genotypes: HBx-EHBH1, HBx-EHBH2, and HBx-EHBH3. HBx-EHBH2 (HBx-E2) indicated better recurrence-free survival and overall survival for patients with HCC. HBx-E2 was significantly correlated with the absence of liver cirrhosis, a small tumor size, a solitary tumor, complete encapsulation and Barcelona Clinic Liver Cancer (BCLC) stage A-0 tumors. Additionally, HBx-E2 served as a significant prognostic factor for patients with BCLC stage B HCC after hepatectomy. Mechanistically, HBx-E2 is unable to promote proliferation in HCC cells and normal hepatocytes. It also fails to activate the Janus kinase 1 (JAK1)/signal transducer and activator of transcription 3 (STAT3)/STAT5 pathway. Our study identifies a novel HBx genotype that is unable to promote the proliferation of HCC cells and suggests a potential marker to preoperatively predict the prognosis of patients with BCLC stage B, HBV-associated, HCC. We classified a novel genotype of the full-length hepatitis B virus X gene (HBx), HBx-E2. This genotype was identified in tumor and nontumor tissues from patients with hepatitis B virus-related hepatocellular carcinoma. HBx-E2 could preoperatively predict the prognosis of patients with intermediate stage hepatocellular carcinoma, after resection.

Protein Microarrays and Liposome: A Method for Studying Lipid-Protein Interactions.

Interactions between specific lipids and proteins can provide important information regarding the functions of proteins and lipids. One of the novel and powerful methods to identify lipid-protein interactions is using protein microarrays and liposomes. Liposomes are spherical vesicles that are surrounded by phospholipid bilayers in which the lipid of interest can be incorporated. Thus, liposomes can be used to detect lipid-protein interactions and to analyze interactions between thousands of proteins and a small number of lipids with a single experiment. This chapter presents the methods and procedures for using protein microarray assays and liposome fabrication to analyze protein-lipid interactions. Up-to-date research reports are also reviewed briefly.