Identification of Seroreactive Proteins of Leptospira interrogans Serovar Copenhageni Using a High-Density Protein Microarray Approach

Background

Leptospirosis is a widespread zoonotic disease worldwide. The lack of an adequate laboratory test is a major barrier for diagnosis, especially during the early stages of illness, when antibiotic therapy is most effective. Therefore, there is a critical need for an efficient diagnostic test for this life threatening disease.

Methodology

In order to identify new targets that could be used as diagnostic makers for leptopirosis, we constructed a protein microarray chip comprising 61% of Leptospira interrogans proteome and investigated the IgG response from 274 individuals, including 80 acute-phase, 80 convalescent-phase patients and 114 healthy control subjects from regions with endemic, high endemic, and no endemic transmission of leptospirosis. A nitrocellulose line blot assay was performed to validate the accuracy of the protein microarray results.

Principal findings

We found 16 antigens that can discriminate between acute cases and healthy individuals from a region with high endemic transmission of leptospirosis, and 18 antigens that distinguish convalescent cases. Some of the antigens identified in this study, such as LipL32, the non-identical domains of the Lig proteins, GroEL, and Loa22 are already known to be recognized by sera from human patients, thus serving as proof-of-concept for the serodiagnostic antigen discovery approach. Several novel antigens were identified, including the hypothetical protein LIC10215 which showed good sensitivity and specificity rates for both acute- and convalescent-phase patients.

Conclusions

Our study is the first large-scale evaluation of immunodominant antigens associated with naturally acquired leptospiral infection, and novel as well as known serodiagnostic leptospiral antigens that are recognized by antibodies in the sera of leptospirosis cases were identified. The novel antigens identified here may have potential use in both the development of new tests and the improvement of currently available assays for diagnosing this neglected tropical disease. Further research is needed to assess the utility of these antigens in more deployable diagnostic platforms.     

Construction of dextrin and isomaltose-assimilating brewer’s yeasts for production of low-carbohydrate beer

Most Saccharomyces spp. cannot degrade or ferment dextrin, which is the second most abundant carbohydrate in wort for commercial beer production. Dextrin-degrading brewer’s bottom and top yeasts expressing the glucoamylase gene (GAM1) from Debaryomyces occidentalis were developed to produce low-carbohydrate (calorie) beers. GAM1 was constitutively expressed in brewer’s yeasts using a rDNA-integration system that contained yeast CUP1 gene coding for copper resistance as a selective marker. The recombinants secreted active glucoamylase, displaying both α-1,4- and α-1,6-debranching activities, that degraded dextrin and isomaltose and consequently grew using them as sole carbon source. One of the recombinant strains expressing GAM1 hydrolyzed 96 % of 2 % (w/v) dextrin and 98 % of 2 % (w/v) isomaltose within 5 days of growth. Growth, substrate assimilation, and enzyme activity of these strains were characterized.     

A cross-platform public domain PC image-analysis program for the comet assay

The single-cell gel electrophoresis, also known as the comet assay, has gained wide-spread popularity as a simple and reliable method to measure genotoxic and cytotoxic effects of physical and chemical agents as well as kinetics of DNA repair. Cells are generally stained with fluorescent dyes. The analysis of comets--damaged cells which form a typical comet-shaped pattern--is greatly facilitated by the use of a computer image-analysis program. Although several image-analysis programs are available commercially, they are expensive and their source codes are not provided. For Macintosh computers a cost-free public domain macro is available on the Internet. No ready for use, cost-free program exists for the PC platform. We have, therefore, developed such a public domain program under the GNU license for PC computers. The program is called CASP and can be run on a variety of hardware and software platforms. Its practical merit was tested on human lymphocytes exposed to gamma-rays and found to yield reproducible results. The binaries for Windows 95 and Linux, together with the source code can be obtained from: http://www.casp.of.pl.     

Functional expression of FIP-gts, a fungal immunomodulatory protein from Ganoderma tsugae in Sf21 insect cells

The mushrooms of diverse Lingzhi species have been traditionally consumed as luxurious functional food supplements in Chinese society. FIP-gts, a fungal immunomodulatory protein found in Song-Shan Lingzhi (Ganodera tsugae) has been proposed to possess therapeutic effects on cancer and autoimmune diseases. To produce active FIP-gts for evaluation of oral administration, a recombinant FIP-gts (rFIP-gts) fused with a 6His-tag at its C-terminus was expressed in Sf21 insect cells by the baculovirus expression system. High yield (about 70%) and purity (about 90%) of rFIP-gts was obtained by one-step nickel-affinity chromatography. The correctness of the harvested rFIP-gts was verified by Western blot and MALDI-MS analyses. Optimal expression of rFIP-gts was observed when the Sf21 cells were infected with multiplicity of infection of 10 for 72 h, and the yield was up to 47.2 microg/3 x 10(6) infected cells. The immunomodulatory activity of the purified rFIP-gts was detected as the induction of interleukin 2 released from murine splenocytes. Compared with the rFIP-gts produced in Escherichia coli cells, the rFIP-gts produced in Sf21 cells possessed evidently higher specific immunomodulatory activity.     

中国海桑属小志

本文描述中国海桑属一新种:拟海桑(Sonneratia paracaseolaris Ko, E. Y. Chen et W. Y.Chen).重新发表海桑属7种检索表,并将原隶属海桑组的海南海桑S.hainanensis Ko, E. Y.Chenet W.Y.Chen改隶无瓣海桑组,因其过去被认为是花瓣的部分实为退化雄蕊.     

Mutagenicity and polycyclic aromatic hydrocarbon content of fumes from heated cooking oils produced in Taiwan

According to epidemiologic studies, exposure of women to fumes from cooking oils appears to be an important risk factor for lung cancer. Fume samples from three different commercial cooking oils frequently used in Taiwan were collected and analyzed for mutagenicity in the Salmonella/microsome assay. Polycyclic aromatic hydrocarbons were extracted from the samples and identified by HPLC chromatography. Extracts from three cooking oil fumes were found to be mutagenic in the presence of S9 mix. All samples contained dibenz[a,h]anthracene (DB[a,h]A) and benz[a]anthracene (B[a]A). Concentration of DB[a,h]A and B[a]A were 1.9 and 2.2 μg/m³ in fumes from lard oil, 2.1 and 2.3 μg/m³ in soybean oil, 1.8 and 1.3 μg/m³ in peanut oil, respectively. Benzo[a]pyrene (B[a]P) was identified in fume samples of soybean and peanut oil, in concentrations of 19.6 and 18.3 μg/m³, in this order. These results provide experimental evidence and support the findings of epidemiologic observations, in which women exposed to the emitted fumes of cooking oils are at increased risk of contracting lung cancer.     

Seasonal changes of free amino acids and thermal hysteresis in overwintering heteropteran insect, Pyrrhocoris apterus

Overwintering adults of Pyrrhocoris apterus do not tolerate freezing of their body fluids and rely on a supercooling strategy and seasonal accumulation of polyols to survive at subzero body temperatures. We sampled the adults monthly in the field during the cold season 2008-2009 and found active thermal hysteresis factors (THFs) in hemolymph of winter-sampled adults. The hysteresis between the equilibrium melting and freezing points ranged from 0.18°C to 0.30°C. No signs of THFs activity were found in the autumn- and spring-sampled insects. The total free amino acid pool almost doubled during winter time. The sum concentrations of 27 free amino acids ranged between 35 and 40mM in whole body water and 40-45mM in hemolymph during December-February. Two amino acids, Pro and α-Ala most significantly contributed to the seasonal increase, while Gln showed the most dramatic seasonal decrease. Moderate levels of amino acid accumulation in overwintering P. apterus suggest that they are by-products of protein degradation and pentose pathway activity during the state of metabolic suppression imposed by diapause and low body temperature. Potential colligative effects of accumulated amino acids, extending the supercooling capacity of overwintering P. apterus, are negligible. Non-colligative effects require further study.     

Stress-induced expression and structure of the putative gene hyp-1 for hypericin biosynthesis

The phenolic oxidative coupling protein (Hyp-1) with proposed activity in the biosynthesis of hypericin in Hypericum perforatum shares about 50 % sequence similarity with Bet.v.1-like/PR-10 proteins. In our previous study, we showed that this protein is not a limiting factor in hypericin biosynthesis. To ascertain the role of Hyp-1 in defense mechanisms, we have analyzed some structural features of the hyp-1 gene in 14 Hypericum species with different abilities to synthesise hypericin. We show that the hyp-1 gene possesses characteristics typical for genes encoding plant PR-10 proteins. The coding sequence of the hyp-1 gene is interrupted by a single 86- to 125-bp intron localised strictly in codon 62, which is a typical feature of the dicot PR-10 subfamily. The localisation of the intron is conserved in all 14 tested Hypericum species indicating a common evolutionary history with genes encoding PR-10 proteins. In addition, we report that the hyp-1 gene exhibits a similar response to stress conditions as the PR-10 proteins encoding genes. Following either wounding or infection by Agrobacterium tumefaciens, all analysed Hypericum species exhibited rapid and significant upregulation of hyp-1 gene expression; this was particularly observed in hypericin-producing species. On the other hand, in the presence of high levels of abscisic acid, different levels of gene expression were observed.