OsVTC1-1 Gene Silencing Promotes a Defense Response in Rice and Enhances Resistance to Magnaporthe oryzae.

Rice blast disease is a serious disease in rice caused by Magnaporthe oryzae (M. oryzae). Ascorbic acid (AsA), or vitamin C, is a strong antioxidant that prevents oxidative damage to cellular components and plays an essential role in plant defense response. GDP-D-mannose pyrophosphorylase (GMP or VTC1) is an enzyme that generates GDP-D-mannose for AsA, cell wall, and glycoprotein synthesis. The OsVTC1 gene has three homologs in the rice genome: OsVTC1-1, OsVTC1-3, and OsVTC1-8. Using OsVTC1-1 RNAi lines, this study investigated the role of the OsVTC1-1 gene during rice blast fungus inoculation. The OsVTC1-1 RNAi inoculated with rice blast fungus induced changes to cell wall monosaccharides, photosynthetic efficiency, reactive oxygen species (ROS) accumulation, and malondialdehyde (MDA) content. Additionally, the OsVTC1-1 RNAi lines were shown to be more resistant to rice blast fungus than the wild type. Genes and proteins related to defense response, plant hormone synthesis, and signaling pathways, especially salicylic acid and jasmonic acid, were up-regulated in the OsVTC1-1 RNAi lines after rice blast inoculation. These results suggest that the OsVTC1-1 gene regulates rice blast resistance through several defense mechanisms, including hormone synthesis and signaling pathways.

Phosphorylation of CAD1, PLDdelta, NDT1, RPM1 Proteins Induce Resistance in Tomatoes Infected by Ralstonia solanacearum.

Ralstonia solanacaerum is one of the most devastating bacteria causing bacterial wilt disease in more than 200 species of plants, especially those belonging to the family Solanaceae. To cope with this pathogen, plants have evolved different resistance mechanisms depending on signal transduction after perception. Phosphorylation is the central regulatory component of the signal transduction pathway. We investigated a comparative phosphoproteomics analysis of the stems of resistant and susceptible tomatoes at 15 min and 30 min after inoculation with Ralstonia solanacearum to determine the phosphorylated proteins involved in induced resistance. Phosphoprotein profiling analyses led to the identification of 969 phosphoproteins classified into 10 functional categories. Among these, six phosphoproteins were uniquely identified in resistant plants including cinnamyl alcohol dehydrogenase 1 (CAD1), mitogen-activated protein kinase kinase kinase 18 (MAPKKK18), phospholipase D delta (PLDDELTA), nicotinamide adenine dinucleotide transporter 1 (NDT1), B3 domain-containing transcription factor VRN1, and disease resistance protein RPM1 (RPM1). These proteins are typically involved in defense mechanisms across different plant species. qRT-PCR analyses were performed to evaluate the level of expression of these genes in resistant and susceptible tomatoes. This study provides useful data, leading to an understanding of the early defense mechanisms of tomatoes against R. solanacearum.

Discovery of potential protein biomarkers associated with sugarcane white leaf disease susceptibility using a comparative proteomic approach.

Sugarcane white leaf disease (SCWLD) is caused by phytoplasma, a serious sugarcane phytoplasma pathogen, which causes significant decreases in crop yield and sugar quality. The identification of proteins involved in the defense mechanism against SCWLD phytoplasma may help towards the development of varieties resistant to SCWLD. We investigated the proteomes of four sugarcane varieties with different levels of susceptibility to SCWLD phytoplasma infection, namely K88-92 and K95-84 (high), KK3 (moderate), and UT1 (low) by quantitative label-free nano-liquid chromatography-tandem mass spectrometry (nano LC-MS/MS). A total of 248 proteins were identified and compared among the four sugarcane varieties. Two potential candidate protein biomarkers for reduced susceptibility to SCWLD phytoplasma were identified as proteins detected only in UT1. The functions of these proteins are associated with protein folding, metal ion binding, and oxidoreductase. The candidate biomarkers could be useful for further study of the sugarcane defense mechanism against SCWLD phytoplasma, and in molecular and conventional breeding strategies for variety improvement.

FBPAII and rpoBC, the Two Novel Secreted Proteins Identified by the Proteomic Approach from a Comparative Study between Antibiotic-Sensitive and Antibiotic-Resistant Helicobacter pylori-Associated Gastritis Strains.

Helicobacter pylori infection is the leading cause of chronic gastritis, which can develop into gastric cancer. Eliminating H. pylori infection with antibiotics achieves the prevention of gastric cancer. Currently, the prevalence of H. pylori resistance to clarithromycin and metronidazole, and the dual resistance to metronidazole and clarithromycin (C_R, M_R, and C/M_R, respectively), remains at a high level worldwide. As a means of exploring new candidate proteins for the management of H. pylori infection, secreted proteins from antibiotic-susceptible and antibiotic-resistant H. pylori-associated gastritis strains were obtained by in-solution tryptic digestion coupled with nano-liquid chromatography tandem mass spectrometry (nano-LC-MS/MS). A total of 583, 582, 590, and 578 differential expressed proteins were identified from C_R, M_R, C/M_R, and antibiotic-sensitive strain (S_S) samples, respectively. Of these, 23 overlapping proteins were found by Venn diagram analysis. Based on heat map analyses, the most and least differing protein expressions were observed from C/M_R strains and S_S strains, respectively. Of the proteins secreted by the S_S strain, only nine were found. After predicting the protein interaction with metronidazole and clarithromycin via the STITCH database, the two most interesting proteins were found to be rpoBC and FBPAII. After quantitative real-time reverse transcription PCR (qRT-PCR) analysis, a downregulation of rpoB from M_R strains was observed, suggesting a relationship of rpoB to metronidazole sensitivity. Inversely, an upregulation of fba from C_R, M_R, and C/M_R strains was noticed, suggesting the paradoxical expression of FBPAII and the fba gene. This report is the first to demonstrate the association of these two novel secreted proteins, namely, rpoBC and FBPAII, with antibiotic-sensitive H. pylori-associated gastritis strains.

In-gel digestion coupled with mass spectrometry (GeLC-MS/MS)-based salivary proteomic profiling of canine oral tumors.

BACKGROUND: Various types of oral tumors, either benign or malignant, are commonly found in dogs. Since saliva directly contacts the tumors and saliva collection is non-invasive, easily accessible and cost effective, salivary biomarkers are practical to be used for the diagnosis and/or prognosis of these diseases. However, there is limited knowledge of protein expression in saliva for canine oral tumors. The present study aimed to investigate novel biomarkers from the salivary proteome of dogs with early- and late-stage oral melanoma (EOM and LOM, respectively), oral squamous cell carcinoma (OSCC), benign oral tumors (BN), and periodontitis and healthy controls (CP), using an in-gel digestion coupled with mass spectrometry (GeLC-MS/MS). The relationships between protein candidates and chemotherapy drugs were explored and the expression of potential biomarkers in saliva and tissues was verified by western blot analysis. RESULTS: For saliva samples, increased expression of protein tyrosine phosphatase non-receptor type 5 (PTPN5) was shown in all tumor groups compared with the CP group. Marked expression of PTPN5 was also observed in LOM and OSCC compared with that in BN and EOM. In addition, tumor protein p53 (p53), which appeared in the PTPN5-drug interactions, was exhibited to be expressed in all tumor groups compared with that in the CP group. For tissue samples, increased expression of p53 was shown in LOM compared with the control group. CONCLUSION: PTPN5 and p53 were proposed to be potential salivary biomarkers of canine oral tumors.

Protein expression in female salivary glands of pyrethroid-susceptible and resistant strains of Aedes aegypti mosquitoes.

BACKGROUND: A group of insecticides called pyrethroids has been used extensively worldwide and development of pyrethroid resistance within mosquito populations, especially in Aedes aegypti, has rapidly spread through populations. In this study, SDS-PAGE, 2-DE coupled with NanoLC-MS, and bioinformatics were used to analyze the female salivary gland proteins of pyrethroid-susceptible (PMD) and pyrethroid-resistant (PMD-R and UPK-R) strains of Ae. aegypti mosquitoes for the first time. RESULTS: SDS-PAGE analysis revealed that among the three strains at least nine major proteins were detected but one protein band (20 kDa) was found only in the PMD strain. Two-dimensional gel electrophoresis analysis revealed 19 similarly expressed proteins in the salivary glands of the three strains involved in blood-feeding process, stress response, immunogenic response, and metabolic process and five additional major protein spots differentially expressed in the susceptible and resistant strains. Comparative analysis of the expression volume of each protein spot between the PMD and the PMD-R strains showed three downregulated proteins of the PMD-R mosquitoes. For UPK-R strains, six major proteins were downregulated when compared to the PMD strain. Additionally, four downregulated proteins were found in the UPK-R when compared to the PMD-R strain. These results suggest that pyrethroids might induce alteration of salivary gland proteins in resistant mosquitoes. Network analysis by STITCH database 5.0 showed that SRPN23 interacted with sodium and calcium ions, suggesting that SRPN23 might be involved in insecticide resistance. CONCLUSIONS: Information obtained from this study will be useful for further studies on the roles of differentially expressed salivary gland proteins in resistance to insecticides and viral transmission.

NOTCH1 regulates the viability of cholangiocarcinoma cells via 14-3-3 theta.

Notch signaling has been reported to correlate with tumor progression and metastasis in several types of cancer. In cholangiocarcinoma (CCA), it has recently been shown that NOTCH1 is overexpressed in both nucleus and cytoplasm of CCA cells; however, the complete understanding of Notch signaling in CCA is still lacking. Here, we aimed to understand the functions of NOTCH1 in CCA cells and the molecular mechanisms that underlie those functions. We used retroviral vectors to overexpress active forms of NOTCH1, the NOTCH1 intracellular domain (N1ICD) and N1ICD that lacks the RBP-J-associated module (RAM), in human CCA cell lines RMCCA-1 and HuCCA-1. Our results showed that activation of Notch signaling by both N1ICD variants enhanced CCA cell proliferation and survival via upregulation of pro-survival protein Mcl-1 and Bcl-xL. Moreover, our LC-MS/MS proteomic studies demonstrated that NOTCH1 may cooperate with 14-3-3 theta to promote CCA cell survival. Knockdown of 14-3-3 theta in RMCCA-1 cells overexpressing N1ICD, diminished pro-survival effects of N1ICD under gemcitabine treatment. In conclusion, these data demonstrated that NOTCH1 plays a role in CCA cell proliferation and survival via the regulation of 14-3-3 theta in a RAM-independent fashion.

Comparative proteomic study of dog and human saliva.

Saliva contains many proteins that have an important role in biological process of the oral cavity and is closely associated with many diseases. Although the dog is a common companion animal, the composition of salivary proteome and its relationship with that of human are unclear. In this study, shotgun proteomics was used to compare the salivary proteomes of 7 Thai village dogs and 7 human subjects. Salivary proteomes revealed 2,532 differentially expressed proteins in dogs and humans, representing various functions including cellular component organization or biogenesis, cellular process, localization, biological regulation, response to stimulus, developmental process, multicellular organismal process, metabolic process, immune system process, apoptosis and biological adhesion. The oral proteomes of dogs and humans were appreciably different. Proteins related to apoptosis processes and biological adhesion were predominated in dog saliva. Drug-target network predictions by STITCH Version 5.0 showed that dog salivary proteins were found to have potential roles in tumorigenesis, anti-inflammation and antimicrobial processes. In addition, proteins related to regeneration and healing processes such as fibroblast growth factor and epidermal growth factor were also up-regulated in dogs. These findings provide new information on dog saliva composition and will be beneficial for the study of dog saliva in diseased and health conditions in the future.

Novel Potential Biomarkers for Opisthorchis viverrini Infection and Associated Cholangiocarcinoma.

BACKGROUND/AIM: Early detection of disease is a pivotal factor for determining prognosis and clinical outcome of patients with cancer. As cholangiocarcinoma (CCA) is currently difficult to detect and most cases of such cancer present with late-stage disease at the time of initial diagnosis, we employed proteomic analysis of the bile to identify potential candidate biomarkers for Opisthorchis viverrini (OV)-associated CCA. MATERIALS AND METHODS: Proteins in pooled bile samples from patients with CCA and OV infection, with CCA without OV infection, with OV infection but no CCA, and with neither OV infection nor CCA were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-gel trypsin digestion and analyzed by liquid chromatography-tandem mass spectrometry. RESULTS: According to our analysis, three proteins, namely aristaless-like homeobox1 isoform X1 (ALX1), major histocompatibility complex polypeptide-related sequence A (MICA), and uncharacterized protein C14orf105 isoform X12 were found to be potential markers for OV infection, as they were predominantly found in all OV-infected groups. Although these proteins were detected in both OV-infected patients with and without CCA, their abundance was 2.90-, 7.06-and 3.65-fold higher, respectively, in those with CCA. In patients with CCA, potential novel biomarkers wre immunoglobulin heavy chain, translocated in liposarcoma (TLS), visual system homeobox 2 (VSX2) and an unnamed protein product. CONCLUSION: We provided novel information regarding potential biomarkers for OV infection and CCA. These two protein profiles could benefit diagnosis as well as monitoring of CCA.

Culture degeneration in conidia of Beauveria bassiana and virulence determinants by proteomics.

The quality of Beauveria bassiana conidia directly affects the virulence against insects. In this study, continuous subculturing of B. bassiana on both rice grains and potato dextrose agar (PDA) resulted in 55 and 49 % conidial yield reduction after 12 passages and 68 and 60 % virulence reduction after 20 and 12 passages at four d post-inoculation, respectively. The passage through Tenebrio molitor and Spodoptera exigua restored the virulence of rice and PDA subcultures, respectively. To explore the molecular mechanisms underlying the conidial quality and the decline of virulence after multiple subculturing, we investigated the conidial proteomic changes. Successive subculturing markedly increased the protein levels in oxidative stress response, autophagy, amino acid homeostasis, and apoptosis, but decreased the protein levels in DNA repair, ribosome biogenesis, energy metabolism, and virulence. The nitro blue tetrazolium assay verified that the late subculture's colony and conidia had a higher oxidative stress level than the early subculture. A 2A-type protein phosphatase and a Pleckstrin homology domain protein Slm1, effector proteins of the target of rapamycin (TOR) complex 1 and 2, respectively, were dramatically increased in the late subculture. These results suggest that TOR signalling might be associated with ageing in B. bassiana late subculture, in turn affecting its physiological characteristics and virulence.

Nevirapine induced mitochondrial dysfunction in HepG2 cells.

Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor frequently used in combination with other antiretroviral agents for highly active antiretroviral therapy (HAART) of patients infected with the human immunodeficiency virus type 1 (HIV-1). However NVP can cause serious, life-threatening complications. Hepatotoxicity is one of the most severe adverse effects, particularly in HIV patients with chronic hepatitis C virus co-infection as these patients can develop liver toxicity after a relatively short course of treatment. However, the mechanism of NVP-associated hepatotoxicity remains unclear. This study sought to investigate the effect of NVP on protein expression in liver cells using a proteomic approach. HepG2 cells were treated or not treated with NVP and proteins were subsequently resolved by two-dimensional gel electrophoresis. A total of 33 differentially regulated proteins were identified, of which nearly 40% (13/33) were mitochondrial proteins. While no obvious differences were observed between NVP treated and untreated cells after staining mitochondria with mitotracker, RT-PCR expression analysis of three mitochondrially encoded genes showed all were significantly up-regulated in NVP treated cells. Mitochondrial dysfunction was observed in response to treatment even with slightly sub-optimal therapeutic treatment concentrations of NVP. This study shows that NVP induces mitochondrial dysregulation in HepG2 cells.

Characterization of cellulolytic enzyme system of Schizophyllum commune mutant and evaluation of its efficiency on biomass hydrolysis.

Schizophyllum commune is a basidiomycete equipped with an efficient cellulolytic enzyme system capable of growth on decaying woods. In this study, production of lignocellulose-degrading enzymes from S. commune mutant G-135 (SC-Cel) on various cellulosic substrates was examined. The highest cellulase activities including CMCase, FPase, and ß-glucosidase were obtained on Avicel-PH101 while a wider range of enzymes attacking non-cellulosic polysaccharides and lignin were found when grown on alkaline-pretreated biomass. Proteomic analysis of SC-Cel also revealed a complex enzyme system comprising seven glycosyl hydrolase families with an accessory carbohydrate esterase, polysaccharide lyase, and auxiliary redox enzymes. SC-Cel obtained on Avicel-PH101 effectively hydrolyzed all agricultural residues with the maximum glucan conversion of 98.0% using corn cobs with an enzyme dosage of 5 FPU/g-biomass. The work showed potential of SC-Cel on hydrolysis of various herbaceous biomass with enhanced efficiency by addition external ß-xylosidase.

Protein Profiles Associated with Anoikis Resistance of Metastatic MDA-MB-231 Breast Cancer Cells.

Resistance to anoikis, a cell-detachment induced apoptosis, is one of the malignant phenotypes which support tumor metastasis. Molecular mechanisms underlying the establishment of this phenotype require further investigation. This study aimed at exploring protein expression profiles associated with anoikis resistance of a metastatic breast cancer cell. Cell survival of suspension cultures of non-metastatic MCF-7 and metastatic MDA-MB-231 cells were compared with their adherent cultures. Trypan blue exclusion assays demonstrated a significantly higher percentage of viable cells in MDA-MB-231 than MCF-7 cell cultures, consistent with analysis of annexin V-7-AAD stained cells indicating that MDA-MB-231 possess anti-apoptotic ability 1.7 fold higher than MCF-7 cells. GeLC-MS/MS analysis of protein lysates of MDA-MB-231 and MCF-7 cells grown under both culture conditions identified 925 proteins which are differentially expressed, 54 of which were expressed only in suspended and adherent MDA-MB-231 but not in MCF-7 cells. These proteins have been implicated in various cellular processes, including DNA replication and repair, transcription, translation, protein modification, cytoskeleton, transport and cell signaling. Analysis based on the STITCH database predicted the interaction of phospholipases, PLC and PLD, and 14-3-3 beta/alpha, YWHAB, with the intrinsic and extrinsic apoptotic signaling network, suggesting putative roles in controlling anti-anoikis ability. MDA-MB-231 cells grown in the presence of inhibitors of phospholipase C, U73122, and phospholipase D, FIPI, demonstrated reduced ability to survive in suspension culture, indicating functional roles of PLC and PLD in the process of anti-anoikis. Our study identified intracellular mediators potentially associated with establishment of anoikis resistance of metastatic cells. These proteins require further clarification as prognostic and therapeutic targets for advanced breast cancer.

mRNA Expression of Bax, Bcl-2, p53, Cathepsin B, Caspase-3 and Caspase-9 in the HepG2 Cell Line Following Induction by a Novel Monoclonal Ab Hep88 mAb: Cross-Talk for Paraptosis and Apoptosis.

Monoclonal antibodies with specific antigens have been widely used as targeted therapy for cancer. Hep88 mAb is a monoclonal antibody which shows specific binding with anti-cancer effects against the HepG2 cell line. However, its mechanisms of action are still not completely understood. We examined cell cycling and apoptosis by flow cytometry and mRNA expression of factors involved in apoptosis and paraptosis in Hep88 mAb-treated HepG2 cells by real-time PCR. The cell-cycle analysis demonstrated that growth-inhibitory activity was associated with G2/M cell cycle arrest. Hep88 mAb induced a significant increase in apoptotic cell populations in a dose- and time-dependent manner. The mRNA expression results also suggested that the process triggered by Hep88 mAb involved up-regulation of tumor suppressor p53, pro-apoptotic Bax, Cathepsin B, Caspase-3 and Caspase-9, with a decrease of anti-apoptotic Bcl-2 - thus confirming paraptosis and apoptosis programmed cell death. These findings represent new insights into the molecular mechanisms underlying the anti-cancer properties of Hep88 mAb in liver cancer cells.

Plasmodium vivax inhibits erythroid cell growth through altered phosphorylation of the cytoskeletal protein ezrin.

BACKGROUND: The underlying causes of severe malarial anaemia are multifactorial. In previously reports, Plasmodium vivax was found to be able to directly inhibited erythroid cell proliferation and differentiation. The molecular mechanisms underlying the suppression of erythropoiesis by P. vivax are remarkably complex and remain unclear. In this study, a phosphoproteomic approach was performed to dissect the molecular mechanism of phosphoprotein regulation, which is involved in the inhibitory effect of parasites on erythroid cell development. METHODS: This study describes the first comparative phosphoproteome analysis of growing erythroid cells (gECs), derived from human haematopoietic stem cells, exposed to lysates of infected erythrocytes (IE)/uninfected erythrocytes (UE) for 24, 48 and 72 h. This study utilized IMAC phosphoprotein isolation directly coupled with LC MS/MS analysis. RESULTS: Lysed IE significantly inhibited gEC growth at 48 and 72 h and cell division resulting in the accumulation of cells in G0 phase. The relative levels of forty four phosphoproteins were determined from gECs exposed to IE/UE for 24-72 h and compared with the media control using the label-free quantitation technique. Interestingly, the levels of three phosphoproteins: ezrin, alpha actinin-1, and Rho kinase were significantly (p < 0.05) altered. These proteins display interactions and are involved in the regulation of the cellular cytoskeleton. Particularly affected was ezrin (phosphorylated at Thr567), which is normally localized to gEC cell extension peripheral processes. Following exposure to IE, for 48-72 h, the ezrin signal intensity was weak or absent. This result suggests that phospho-ezrin is important for actin cytoskeleton regulation during erythroid cell growth and division. CONCLUSIONS: These findings suggest that parasite proteins are able to inhibit erythroid cell growth by down-regulation of ezrin phosphorylation, leading to ineffective erythropoiesis ultimately resulting in severe malarial anaemia. A better understanding of the mechanisms of ineffective erythropoiesis may be beneficial in the development of therapeutic strategies to prevent severe malarial anaemia.

Hep88 mAb-mediated paraptosis-like apoptosis in HepG2 cells via downstream upregulation and activation of caspase-3, caspase-8 and caspase-9.

Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide. Presently, targeted therapy via monoclonal antibodies to specific tumor-associated antigens is being continuously developed. Hep88 mAb has proven to exert tumoricidal effects on the HepG2 cell via a paraptosis-like morphology. To verify the pathway, we then demonstrated downstream up-regulation of caspase-3, caspase-8 and caspase-9, assessingmRNA expression by real-time PCR and associated enzyme activity by colorimetric assay. Active caspase-3 determination was also accomplished by flow cytometry. Active caspase-3 expression was increased by Hep88 mAb treatment in a dose-and time-dependent manner. All of the results indicated that Hep88 mAb induced programmed cell death in the HepG2 cell line from paraptosis-like to apoptosis by downstream induction of caspases. These conclusions imply that Hep88mAb might be a promising tool for the effective treatment of HCC in the future.

Comprehensive proteomic analysis of white blood cells from chikungunya fever patients of different severities.

BACKGROUND: Chikungunya fever (CHIKF) is a recently re-emerged mosquito transmitted viral disease caused by the chikungunya virus (CHIKV), an Alphavirus belonging to the family Togaviridae. Infection of humans with CHIKV can result in CHIKF of variable severity, although the factors mediating disease severity remain poorly defined. METHODS: White blood cells were isolated from blood samples collected during the 2009-2010 CHIKF outbreak in Thailand. Clinical presentation and viral load data were used to classify samples into three groups, namely non chikungunya fever (non-CHIKF), mild CHIKF, and severe CHIKF. Five samples from each group were analyzed for protein expression by GeLC-MS/MS. RESULTS: CHIKV proteins (structural and non-structural) were found only in CHIKF samples. A total of 3505 human proteins were identified, with 68 proteins only present in non-CHIKF samples. A total of 240 proteins were found only in CHIKF samples, of which 65 and 46 were found only in mild and severe CHIKF samples respectively. Proteins with altered expression mapped predominantly to cellular signaling pathways (including toll-like receptor and PI3K-Akt signaling) although many other processes showed altered expression as a result of CHIKV infection. Expression of proteins consistent with the activation of the inflammasome was detected, and quantitation of (pro)-caspase 1 at the protein and RNA levels showed an association with disease severity. CONCLUSIONS: This study confirms the infection of at least a component of white blood cells by CHIKV, and shows that CHIKV infection results in activation of the inflammasome in a manner that is associated with disease severity.

Hep88 mAb-initiated paraptosis-like PCD pathway in hepatocellular carcinoma cell line through the binding of mortalin (HSPA9) and alpha-enolase.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most prevalent hepatic cancer worldwide. Currently, a targeted therapy via monoclonal antibodies (mAbs) specific to tumor-associated antigen is undergoing continual development in HCC treatment. METHODS: In this regard, after establishing and consequently exploring Hep88 mAb's tumoricidal effect on hepatocellular carcinoma cell line (HepG2 cell line), the Hep88 mAb's specific antigens from both membrane and cytoplasmic fractions of HepG2 cell line were identified by 2-D gel electrophoresis and western blot analysis. After in-gel digestion and subsequent analysis by liquid chromatography-mass spectrometry (LC-MS), mortalin (HSPA9) and alpha-enolase were identified. The recombinant proteins specific to Hep88 mAb were cloned and expressed in E. coli BL21(DE3). Moreover, alteration of HepG2 and Chang liver cell line after being induced by Hep88 mAb for 1-3 days was investigated using a transmission electron microscope. RESULTS: The result demonstrated that Hep88 mAb can bind to the recombinant mortalin (HSPA9) and alpha-enolase. In addition, the gradual appearing of mitochondria vacuolization and endoplasmic reticulum dilatation were observed. Those characteristics might be explained by the paraptosis-like program cell death (PCD), which is induced by the binding of Hep88 mAb to mortalin (HSPA9). Mortalin depletion resulting from the formation of Hep88 mAb-mortalin (HSPA9) complex might initiate transcription-independence of p53-mediated apoptosis. Additionally, Hep88mAb-alpha-enolase complex might initiate HepG2 cells energy exhaustion by glycolysis pathway obstruction. CONCLUSION: These fascinating results imply that Hep88 mAb might be a promising tool for the development of an effective treatment of HCC in the next decade.

Identification of prohibitin as a Chikungunya virus receptor protein.

Chikungunya virus (CHIKV) has recently re-emerged causing millions of infections in countries around the Indian Ocean. While CHIKV has a broad host cell range and productively infects a number of different cell types, macrophages have been identified as a potential viral reservoir serving to increase the duration of symptoms. To date no CHIKV interacting protein has been characterized and this study sought to identify CHIKV binding proteins expressed on target cell membranes. Two-dimensional virus overlay identified prohibitin (PHB) as a microglial cell expressed CHIKV binding protein. Co-localization, co-immunoprecipitation as well as antibody and siRNA mediated infection inhibition studies all confirmed a role for PHB in mediating internalization of CHIKV into microglial cells. PHB is the first identified CHIKV receptor protein, and this study is evidence that PHB may play a role in the internalization of multiple viruses.

Proteomic analysis of chikungunya virus infected microgial cells.

Chikungunya virus (CHIKV) is a recently re-emerged public health problem in many countries bordering the Indian Ocean and elsewhere. Chikungunya fever is a relatively self limiting febrile disease, but the consequences of chikungunya fever can include a long lasting, debilitating arthralgia, and occasional neurological involvement has been reported. Macrophages have been implicated as an important cell target of CHIKV with regards to both their role as an immune mediator, as well evidence pointing to long term viral persistence in these cells. Microglial cells are the resident brain macrophages, and so this study sought to define the proteomic changes in a human microglial cell line (CHME-5) in response to CHIKV infection. GeLC-MS/MS analysis of CHIKV infected and mock infected cells identified some 1455 individual proteins, of which 90 proteins, belonging to diverse cellular pathways, were significantly down regulated at a significance level of p<0.01. Analysis of the protein profile in response to infection did not support a global inhibition of either normal or IRES-mediated translation, but was consistent with the targeting of specific cellular pathways including those regulating innate antiviral mechanisms.