Utilization of ELISA Using Thioredoxin Peroxidase-1 and Tandem Repeat Proteins for Diagnosis of Schistosoma japonicum Infection among Water Buffaloes

Background

The presence of animal reservoirs in Schistosoma japonicum infection has been a major obstacle in the control of schistosomiasis. Previous studies have proven that the inclusion of control measures on animal reservoir hosts for schistosomiasis contributed to the decrease of human cases. Animal surveillance should therefore be included to strengthen and improve the capabilities of current serological tests.

Methodology/Principal Findings

Thioredoxin peroxidase-1 (SjTPx-1) and four tandem repeat proteins (Sj1TR, Sj2TR, Sj4TR, Sj7TR) were initially evaluated against human sera. The previous test showed high sensitivity and specificity for antibody detection against SjTPx-1 and Sj7TR. In this study, the immunodiagnostic potential of these recombinant proteins was evaluated using enzyme-linked immunoassay on 50 water buffalo serum samples collected in Cagayan, the Philippines as compared with the soluble egg antigen (SEA). For specificity, 3 goat serum samples positive with Fasciola hepatica were used and among the antigens used, only SEA showed cross-reaction. Stool PCR targeting the S. japonicum 82 bp mitochondrial NAD 1 gene was done to confirm the true positives and served as the standard test. Twenty three samples were positive for stool PCR. SjTPx-1 and Sj1TR gave the highest sensitivity among the recombinant proteins tested for water buffalo samples with 82.61% and 78.26% respectively which were higher than that of SEA (69.57%).

Conclusions/Significance

These results prove that SjTPx-1 works both for humans and water buffaloes making it a good candidate antigen for zoonotic diagnosis. Sj1TR showed good results for water buffaloes and therefore can also be used as a possible candidate for detecting animal schistosome infection.     

Nucleotide-induced conformational changes of PMP70, an ATP binding cassette transporter on rat liver peroxisomal membranes

Nucleotide-induced conformational changes of the 70-kDa peroxisomal membrane protein (PMP70) were investigated by means of limited-trypsin digestion. Rat liver peroxisomes preincubated with various nucleotides were subsequently digested by trypsin. The digestion products were subjected to immunoblot analysis with an anti-PMP70 antibody that recognizes the carboxyl-terminal 15 amino acids of the protein. PMP70 was initially cleaved in the boundary region between the transmembrane and nucleotide-binding domains and a carboxyl-terminal 30-kDa fragment resulted. The fragment in turn was progressively digested at the helical domain between the Walker A and B motifs. The fragment, however, could be stabilized with MgATP or MgADP. In contrast to MgATP, MgATP-gammaS protected whole PMP70 as well as the fragment. The 30-kDa fragment processed by trypsin was recovered in the post-peroxisomal fraction as a complex with a molecular mass of about 60 kDa irrespective of the presence of MgATP. These results suggest that PMP70 exists as a dimer on the peroxisomal membranes and the binding and hydrolysis of ATP induce conformational changes in PMP70 close to the boundary between the transmembrane and nucleotide binding domains and the helical domain between the Walker A and B motifs.     

Hsp105α suppresses Adriamycin-induced cell death via nuclear localization signal-dependent nuclear accumulation

Heat shock protein 105 (Hsp105) is a molecular chaperone, and the isoforms Hsp105α and Hsp105β exhibit distinct functions with different subcellular localizations. Hsp105β localizes in the nucleus and induces the expression of the major heat shock protein Hsp70, whereas cytoplasmic Hsp105α is less effective in inducing Hsp70 expression. Hsp105 shuttles between the cytoplasm and the nucleus; the subcellular localization is governed by the relative activities of the nuclear localization signal (NLS) and nuclear export signal (NES). Here, we show that nuclear accumulation of Hsp105α but not Hsp105β is involved in Adriamycin (ADR) sensitivity. Knockdown of Hsp105α induces cell death at low ADR concentration, at which ADR is less effective in inducing cell death in the presence of Hsp105α. Of note, Hsp105 is localized in the nucleus under these conditions, even though Hsp105β is not expressed, indicating that Hsp105α accumulates in the nucleus in response to ADR treatment. The exogenously expressed Hsp105α but not its NLS mutant localizes in the nucleus of ADR-treated cells. In addition, the expression level of the nuclear export protein chromosomal maintenance 1 (CRM1) was decreased by ADR treatment of cells, and CRM1 knockdown caused nuclear accumulation of Hsp105α both in the presence and absence of ADR. These results indicating that Hsp105α accumulates in the nucleus in a manner dependent on the NLS activity via the suppression of nuclear export. Our findings suggest a role of nuclear Hsp105α in the sensitivity against DNA-damaging agents in tumor cells.     

VEGF165 mediates formation of complexes containing VEGFR-2 and neuropilin-1 that enhance VEGF165-receptor binding

Co-expression of NRP1 and (VEGFR-2) KDR on the surface of endothelial cells (EC) enhances VEGF165 binding to KDR and EC chemotaxis in response to VEGF165. Overexpression of NRP1 by prostate tumor cells in vivo results in increased tumor angiogenesis and growth. We investigated the molecular mechanisms underlying NRP1-mediated angiogenesis by analyzing the association of NRP1 and KDR. An intracellular complex containing NRP1 and KDR was immunoprecipitated from EC by anti-NRP1 antibodies only in the presence of VEGF165. In contrast, VEGF121, which does not bind to NRP1, did not support complex formation. Complexes containing VEGF165, NRP1, and KDR were also formed in an intercellular fashion by co-culture of EC expressing KDR only, with cells expressing NRP1 only, for example, breast carcinoma cells. VEGF165 also mediated the binding of a soluble NRP1 dimer to cells expressing KDR only, confirming the formation of such complexes. Furthermore, the formation of complexes containing KDR and NRP1 markedly increased 125I-VEGF165 binding to KDR. Our results suggest that formation of a ternary complex of VEGF165, KDR, and NRP1 potentiates VEGF165 binding to KDR. These complexes are formed on the surface of EC and in a juxtacrine manner via association of tumor cell NRP1 and EC KDR.     

NTH201, a novel class II KNOTTED1-like protein, facilitates the cell-to-cell movement of Tobacco mosaic virus in tobacco

NTH201, a novel class II KNOTTED1-like protein gene, was cloned from tobacco (Nicotiana tabacum cv. Xanthi) and its role in Tobacco mosaic virus (TMV) infection was analyzed. Virus-induced gene silencing of NTH201 caused a delay in viral RNA accumulation as well as virus spread in infected tobacco plants. Overexpression of the gene in a transgenic tobacco plant (N. tabacum cv. Xanthi nc) infected by TMV showed larger local lesions than those of the nontransgenic plant. NTH201 exhibited no intercellular trafficking ability but did exhibit colocalization with movement protein (MP) at the plasmodesmata. When NTH201-overexpressing tobacco BY-2 cultured cells were infected with TMV, the accumulation of MP but not of viral genomic and subgenomic RNA clearly was accelerated compared with those in nontransgenic cells at an early infection period. The formation of virus replication complexes (VRC) also was accelerated in these transgenic cells. Conversely, NTH201-silenced cells showed less MP accumulations and fewer VRC formations than did nontransgenic cells. These results suggested that NTH201 might indirectly facilitate MP accumulation and VRC formation in TMV-infected cells, leading to rapid viral cell-to-cell movement in plants at an early infection stage.     

Assessment of border control measures and community containment measures used in Japan during the early stages of Pandemic (H1N1) 2009

Background

In the early stages of Pandemic (H1N1) 2009, border control measures were taken by quarantine stations to block the entry of infected individuals into Japan and community containment measures were implemented to prevent the spreading. The objectives of this study were to describe these measures and the characteristics of infected individuals, and to assess the measures'' effectiveness.

Methodology/Principal Findings

Border control and community containment measures implemented from April to June (Period I: April 28–May 21, Period II: May 22–June 18) 2009 were described. Number of individuals identified and disease characteristics were analyzed. For entry screening, a health declaration form and an infrared thermoscanner were used to detect symptomatic passengers. Passengers indicated for the rapid influenza test underwent the test followed by RT-PCR. Patients positive for H1N1 were isolated, and close contacts were quarantined. Entry cards were handed out to all asymptomatic passengers informing them about how to contact a health center in case they developed symptoms. Nine individuals were identified by entry screening and 1 during quarantine to have Pandemic (H1N1) 2009. Health monitoring by health centers was performed in period I for passengers arriving from affected countries and in period II for those who had come into contact with the individuals identified by entry screening. Health monitoring identified 3 infected individuals among 129,546 in Period I and 5 among 746 in Period II. Enhanced surveillance, which included mandatory reporting of details of the infected individuals, identified 812 individuals, 141 (18%) of whom had a history of international travel. Twenty-four of these 141 passengers picked up by enhanced surveillance had been developing symptoms on entry and were missed at screening.

Conclusion/Significance

Symptomatic passengers were detected by the various entry screening measures put in place. Enhanced surveillance provided data for the improvement of public health measures in future pandemics.     

A series of novel indazole derivatives of Sirt 1 activator as osteogenic regulators

A series of indazole derivatives were identified as Sirt 1 activators though high-throughput screening. Optimization of each substituent on the indazole ring led to the identification of compound 13. Compound 13 appeared to give the best Sirt 1 activity of the compounds tested and also showed osteogenesis activity in a cell assay. Sirt 1 activators are therefore potential candidates for the treatment of osteoporosis.