A novel peptide ELISA for universal detection of antibodies to human H5N1 influenza viruses

Background

Active serologic surveillance of H5N1 highly pathogenic avian influenza (HPAI) virus in humans and poultry is critical to control this disease. However, the need for a robust, sensitive and specific serologic test for the rapid detection of antibodies to H5N1 viruses has not been met.

Methodology/Principal Findings

Previously, we reported a universal epitope (CNTKCQTP) in H5 hemagglutinin (HA) that is 100% conserved in H5N1 human isolates and 96.9% in avian isolates. Here, we describe a peptide ELISA to detect antibodies to H5N1 virus by using synthetic peptide that comprises the amino acid sequence of this highly conserved and antigenic epitope as the capture antigen. The sensitivity and specificity of the peptide ELISA were evaluated using experimental chicken antisera to H5N1 viruses from divergent clades and other subtype influenza viruses, as well as human serum samples from patients infected with H5N1 or seasonal influenza viruses. The peptide ELISA results were compared with hemagglutinin inhibition (HI), and immunofluorescence assay and immunodot blot that utilize recombinant HA1 as the capture antigen. The peptide ELISA detected antibodies to H5N1 in immunized animals or convalescent human sera whereas some degree of cross-reactivity was observed in HI, immunofluorescence assay and immunodot blot. Antibodies to other influenza subtypes tested negative in the peptide-ELISA.

Conclusion/Significance

The peptide-ELISA based on the highly conserved and antigenic H5 epitope (CNTKCQTP) provides sensitive and highly specific detection of antibodies to H5N1 influenza viruses. This study highlighted the use of synthetic peptide as a capture antigen in rapid detection of antibodies to H5N1 in human and animal sera that is robust, simple and cost effective and is particularly beneficial for developing countries and rural areas.     

Mechanism of Infrared Detection and Transduction by Beetle Melanophila Acuminata In memory of Jerry Wolken

The Melanophila acuminata beetle is attracted to forest fires via a pair of infrared sensory organs composed of sensilla. Our histological work showed that each sensillum contains lipid layers surrounding a protein layer and a unique polysaccharide base that is associated with a neuron to each sensillum. Infrared microscopy showed that the protein region maximally absorbs infrared radiation at 3 μm wavelength and at 10 μm, which corresponds to the known radiation produced by forest fires at 3 μm.Mathematical calculations showed that the physical properties of the sensilla are such that the expected temperature rise is insufficient for transduction of the infrared signal through mechanical means or as a thermal receptor as previously thought;hence the protein plays the pivotal role in perception of single photons and transmission of the signal within the sensilla.     

Aptness of Beauveria bassiana and enhanced diatomaceous earth (DEBBM) for control of Rhyzopertha dominica F.

A diatomaceous earth formulation enhanced with bitterbarkomycin (DEBBM) combined with Beauveria bassiana (Balsamo) Vuillemin was evaluated against lesser grain borer Rhyzopertha dominica F. (Coleoptera: Bostrychidae) under laboratory conditions. DEBBM was applied at the rates of 15 and 30 ppm alone as well as in combination with 6.69 × 10⁶, 6.69 × 10⁸ and 6.69 × 10¹⁰ conidia/kg of wheat. Mortality of treated adults was recorded after 5, 10 and 15 days of exposure. Bioassays were carried out at 20, 25 and 30°C with 55 and 75% relative humidity. The emergence of progeny was also assessed 60 days post exposure. The combined use of DEBBM and B. bassiana considerably increased adult mortality especially at increasing temperatures and longer exposure intervals compared with DEBBM and B. bassiana alone. Progeny production was less in wheat treated with high dose rates of DEBBM +B. bassiana. The per cent mycosis in the cadavers was maximum where B. bassiana was applied at low dose rates. The results of the present study indicated that a combination of DEBBM and B. bassiana may provide effective control of R. dominica.     

Effect of modified meridic diet on the development and growth of tomato fruitworm Helicoverpa armigera (Lepidoptera: Noctuidae)

The efficacy of one new modified and two old meridic diets on Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) for rearing six successive generations was studied. Duration of larval development for insects fed on the modified diet was considerably shortened as most of them went through only five stadia before pupation, while the per cent pupation and per cent eclosion were relatively higher than on other diets. The lowest pupal mortality (6.33 ± 0.13%) was recorded in the F₁ generation reared on the modified diet, whereas the highest pupal mortality (19.49 ± 0.15%) was observed in insects reared on a natural diet in the F₆ generation. Blending of chickpea Cicer arietinum L. and red kidney bean Phaseolus vulgaris L. flours with tomato paste proved highly favorable for adult reproduction. These results suggest that the vitality of the tomato fruitworm did not decline obviously after rearing on a modified diet for several generations.     

Botanicals for the prevention and treatment of cutaneous melanoma

Cutaneous melanoma, a cancer of melanocytes, when detected at later stages is arguably one of the most lethal cancers and the cause of more years of lost life than any other cancer among young adults. There is no standard therapy for advanced-stage melanoma and the median survival time for patients with metastatic melanoma is <1 yr. An urgent need for novel strategies against melanoma has directed research towards the development of new chemotherapeutic and biologic agents that can target the tumor by several different mechanisms. Recently, several dietary agents are being investigated for their role in the prevention and treatment of various forms of cancer and may represent the future modality of the treatment. Here, we have reviewed emerging data on botanicals that are showing promise for their potential inhibitory effect against cutaneous melanoma.     

Clinical proteomics of the neglected human malarial parasite Plasmodium vivax

Recent reports highlight the severity and the morbidity of disease caused by the long neglected malaria parasite Plasmodium vivax. Due to inherent difficulties in the laboratory-propagation of P. vivax, the biology of this parasite has not been adequately explored. While the proteome of P. falciparum, the causative agent of cerebral malaria, has been extensively explored from several sources, there is limited information on the proteome of P. vivax. We have, for the first time, examined the proteome of P. vivax isolated directly from patients without adaptation to laboratory conditions. We have identified 153 proteins from clinical P. vivax, majority of which do not show homology to any previously known gene products. We also report 29 new proteins that were found to be expressed in P. vivax for the first time. In addition, several proteins previously implicated as anti-malarial targets, were also found in our analysis. Most importantly, we found several unique proteins expressed by P. vivax.This study is an important step in providing insight into physiology of the parasite under clinical settings.     

Non-invasive imaging of cysteine cathepsin activity in solid tumors using a 64Cu-labeled activity-based probe

The papain family of cysteine cathepsins are actively involved in multiple stages of tumorigenesis. Because elevated cathepsin activity can be found in many types of human cancers, they are promising biomarkers that can be used to target radiological contrast agents for tumor detection. However, currently there are no radiological imaging agents available for these important molecular targets. We report here the development of positron emission tomography (PET) radionuclide-labeled probes that target the cysteine cathepsins by formation of an enzyme activity-dependent bond with the active site cysteine. These probes contain an acyloxymethyl ketone (AOMK) functional group that irreversibly labels the active site cysteine of papain family proteases attached to a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) tag for labeling with (64)Cu for PET imaging studies. We performed biodistribution and microPET imaging studies in nude mice bearing subcutaneous tumors expressing various levels of cysteine cathepsin activity and found that the extent of probe uptake by tumors correlated with overall protease activity as measured by biochemical methods. Furthermore, probe signals could be reduced by pre-treatment with a general cathepsin inhibitor. We also found that inclusion of a Cy5 tag on the probe increased tumor uptake relative to probes lacking this fluorogenic dye. Overall, these results demonstrate that small molecule activity-based probes carrying radio-tracers can be used to image protease activity in living subjects.     

Glucose and fatty acids synergize to promote B-cell apoptosis through activation of glycogen synthase kinase 3β independent of JNK activation

Background

The combination of elevated glucose and free-fatty acids (FFA), prevalent in diabetes, has been suggested to be a major contributor to pancreatic β-cell death. This study examines the synergistic effects of glucose and FFA on β-cell apoptosis and the molecular mechanisms involved. Mouse insulinoma cells and primary islets were treated with palmitate at increasing glucose and effects on apoptosis, endoplasmic reticulum (ER) stress and insulin receptor substrate (IRS) signaling were examined.

Principal Findings

Increasing glucose (5–25 mM) with palmitate (400 µM) had synergistic effects on apoptosis. Jun NH2-terminal kinase (JNK) activation peaked at the lowest glucose concentration, in contrast to a progressive reduction in IRS2 protein and impairment of insulin receptor substrate signaling. A synergistic effect was observed on activation of ER stress markers, along with recruitment of SREBP1 to the nucleus. These findings were confirmed in primary islets. The above effects associated with an increase in glycogen synthase kinase 3β (Gsk3β) activity and were reversed along with apoptosis by an adenovirus expressing a kinase dead Gsk3β.

Conclusions/Significance

Glucose in the presence of FFA results in synergistic effects on ER stress, impaired insulin receptor substrate signaling and Gsk3β activation. The data support the importance of controlling both hyperglycemia and hyperlipidemia in the management of Type 2 diabetes, and identify pancreatic islet β-cell Gsk3β as a potential therapeutic target.