Design of a polytopic construct of LACK,TSA and GP63 proteins for the diagnosis of cutaneous leishmaniasis: An in silico strategy

Cutaneous leishmaniasis (CL), which is caused by different species of the genus Leishmania, including L. major, is a significant parasitic disease worldwide. Commercial leishmaniasis diagnostic kits frequently target the entire parasite antigen, although this approach can reduce the specificity of the tests. The present study sought to increase the specificity of antigen detection tests by identifying the B-cell epitopes on the surface of the protozoa and developing an antigenic multiepitope construct to react with the system of B-cell epitopes unique to L. major. More specifically, the linear and conformational B-cell epitopes for three L. major proteins, namely, GP63, LACK, and TSA, were predicted using the ABCprd, IEDB, LBtope, CBTOPE, and DiscoTope servers. The three-dimensional structures of these proteins, as well as of the complete multiepitope, were predicted using the I-TASSER server. The multiepitope structure models were validated by means of Ramachandran plots and the Prosa and Verify3D servers. The multiepitope construct was evaluated using the ProtParam and SOLpro servers. The synthesized multiepitope was then successfully expressed into a pET28a plasmid. The expression was confirmed using SDS-PAGE and dot blot techniques. Moreover, the expressed protein was evaluated by means of Western blotting. A band of approximately 39 kDa was observed by SDS-PAGE. The rGLT-pET-28a showed a high response with the 1:1000 dilution of the anti-His-tag monoclonal antibody by Western blot. The results of this study indicated that the integrated GP63, LACK, and TSA multiepitope antigens of L. major represent a potential target for a novel diagnostic kit for CL.     

Rapid identification of adult whiteflies in plant consignments using monoclonal antibodies

A rapid and simple method for the identification of adult whiteflies (Homoptera: Aleyrodidae) would be valuable, both to facilitate pest management decision making and, more importantly, to prevent the introduction of new pests into non-infested areas. Considering the enormous economic losses that have resulted in the recent past from the accidental introduction of harmful species, there is a clear and urgent need for a reliable antibody-based test kit that would allow non-specialist growers, extension workers and customs authorities to identify whiteflies. As a first step in this process, this paper describes the development and characterisation of monoclonal antibodies capable of distinguishing between major pest species of whitefly, and application of these antibodies in an enzyme-linked immunosorbent assay (ELISA). A monoclonal antibody is described that reacts strongly with both male and female Trialeurodes vaporariorum (Westwood) but not with either Bemisia tabaci (Gennadius) or B. argentifolii Bellows & Perring. A second antibody is reported that gives a strong response to female Bemisia sp. that does not react with T. vaporariorum of either sex. The two antibodies used in combination provide a powerful means of distinguishing between adult whitefly, particularly at low levels of infestation in plant consignments, when pupae cannot easily be found.     

Commercially available luminometers and imaging devices for low-light level measurements and kits and reagents utilizing bioluminescence or chemiluminescence: Survey update 5

This survey was compiled in July 1997 and includes products not covered in the luminometer survey (Jan 1992: Stanley PE, J Biolumin Chemilumin 1992; 7:77–108 and 7:157–69), kits and reagent survey (Nov 1992; Stanley PE, J Biolumin Chemilumin 1993; 8:51–63), update 1 (June 1993: luminometers, kits and reagents, Stanley PE, J Biolumin Chemilumin 1993; 8:237–240) and update 2 (Dec 1993: luminometers, kits and reagents, Stanley PE, J Biolumin Chemilumin 1994; 9:51–3) and update 3 (Feb 1994: luminometers, kits and reagents, Stanley PE, J Biolumin Chemilumin 1994; 9:123–5) and update 4 (June 1996: Stanley PE, J Biolumin Chemilumin 1996; 11:175–91). Technical details are provided together with company address and contact information including email and website where known. Items include: Luminometers, radiometers, low-light imaging, CCD cameras, immunoassays, ATP rapid microbiology, hygiene monitoring, molecular probes, labels, nucleic acid hybridization, reporter genes. © 1997 John Wiley & Sons, Ltd.     

Correlated responses on litter size traits and survival traits after two-stage selection for ovulation rate and litter size in rabbits

Farmer profit depends on the number of slaughter rabbits. The improvement of litter size (LS) at birth by two-stage selection for ovulation rate (OR) and LS could modify survival rate from birth to slaughter. This study was aiming to estimate direct and correlated response on LS traits and peri- and postnatal survival traits in the OR_LS rabbit line selected first only for OR (first period) and then for OR and LS using independent culling levels (second period). The studied traits were OR, LS measured as number of total born, number of kits born alive (NBA) and dead (NBD), and number of kits at weaning (NW) and young rabbits at slaughter (NS). Prenatal survival (LS/OR) and survival at birth (NBA/LS), at weaning (NW/NBA) and at slaughter (NS/NW) were also studied. Data were analysed using Bayesian inference methods. Heritability for LS traits were low, 0.07 for NBA, NW and NS. Survival traits had low values of heritability 0.07, 0.03 and 0.03 for NBA/LS, NW/NBA and NS/NW, respectively. After six generations of selection by OR (first period), a small increase in NBD and a slight decrease in NBA/LS were found. However, no correlated responses on NW/NBA and NS/NW were observed. After 11 generations of two-stage selection for OR and LS (second period), correlated responses on NBA, NW and NS were 0.12, 0.12 and 0.11 kits per generation, respectively, whereas no substantial modifications on NBA/LS, NW/NBA and NS/NW were found. In conclusion, two-stage selection improves the number of young rabbits at slaughter without modifying survival from birth to slaughter.     

Early solid additional feeding of suckling rabbits from 3 to 15 days of age

Studies have shown that nutrient requirement of suckling kits is not satisfied, but they can be fed a double quantity of milk (double nursing) resulting in improved BW and weight gain. The aim of our trials was to give additional solid feed during the early suckling period (3 to 15 days of age) when rabbit kits drink exclusively milk. Two experiments were conducted with animals from Pannon Rabbit Breeding Program. In experiment 1 (n=77 does, 734 kits) the does received commercial feed (C) or C pellet supplemented with 0.2 g powdered thyme/kg (CT). Within both dietary groups of the does three groups of litters were formed: no additional solid creep feeding (N); soya bean-based pellet (S); S pellet with 1% added powdered thyme (ST). In group S and ST, cylinder-shaped solid pellets were made. At the beginning (3 days of age) two pieces of pellets were placed daily into the nestbox after nursing. Later on it was increased to six pellets till 15 days of age. The kits consumed the additional solid feed (S and ST), however, it did not affect the BW, weight gain or survival. In experiment 2 (n=30 does, 240 kits) all does consumed commercial feed. The additional feed for kits was based on commercial piglet feed. Three groups were formed: the litters in control group were fed no additional solid feed (K), kits were fed additionally with pellets (8 mm of diameter) based on piglet feed powder, pellet adhesive and water (PI), and extra glycerin powder was added to the mixture of piglet feed powder and water (PG). The experiment lasted from the age of 3 days till 21 days. At the beginning six pellets were placed on the nest material. Later on the amount was gradually increased to 24 pellets till age of 15 days. The kits consumed the pellets. The BW of PI group differed from group PG at age of 5, 9, 12 and 21 days by +7.3%, +6.5%, +5.9%, +4.8%, respectively (P<0.05) and from group K at age of 12 days by +5.9% (P< 0.05). The differences were more expressed at age of 16 and 19 days in favour of group PI (from K by +7.1%, +6.9% and from PG by +5.9%, +5 8%, respectively, P<0.01) and at 21 days of age (from K by +6.2%, P<0.01). To find appropriate composition of creep feed for kits further studies are needed.     

2930例应用两种实验方法诊断细菌性阴道炎的比较

目的探讨细菌性阴道炎（BV）实验方法在临床的应用。方法对2009年3月至2009年8月在我院门诊就诊的患者2930例,随机分为2组,采用生理盐水直接涂片镜检法与细菌性阴道病联合测定试剂盒检测法进行比较。结果直接涂片镜检法阳性率为22.03%;应用细菌性阴道病联合测定试剂盒检测法,阳性率为45.81%,其中单患BV组占22.20%,合并白细胞酯酶2个加号占7.70%,合并白细胞酯酶3个加号占16.08%。结论细菌性阴道炎合并感染时,运用细菌性阴道病联合测定试剂盒检测法进行阴道分泌物检查,检出率明显高于涂片镜检法。     

Commercially available luminometers and imaging devices for low-light level measurements and kits and reagents utilizing bioluminescence or chemiluminescence: Survey update 3

This survey was compiled in February 1994 and includes products not covered in the luminometer survey (Jan 1992: Stanley PE, J Biolumin Chemilumin 1992; 7:77–108 and 7:157–69), kits and reagent survey (Nov 1992: Stanley PE, J Biolumin Chemilumin 1993; 8:51–63), update 1 (June 1993 luminometers, kits and reagents, Stanley PE, J Biolumin Chemilumin 1993; 8:237–40) and update 2 (Dec 1993: luminometers, kits and reagents, Stanley PE, J Biolumin Chemilumin 1994; 9:51–3). Technical details are provided together with company addresses and contact information.     

Comparison of five commercial extraction kits for subsequent membrane protein profiling

Membrane proteins account for 70–80% of all pharmaceutical targets emphasizing their clinical relevance. Identification of new, differentially expressed membrane proteins reflecting distinct disease properties is thus of high importance. Unfortunately, isolation and analysis of membrane-bound proteins is hampered by their relative low abundance in total cell lysates, their frequently large size and their hydrophobic properties. We thus aimed to identify protocols that allow for highly efficient isolation and purification of membrane-bound proteins for subsequent protein profiling. We present a comparative study of different membrane protein extraction methods that vary in total protein yield between 0.02 and 4.8 mg using constant cell pellets of the colorectal carcinoma cell line SW620. We also demonstrate by means of polyacrylamide gel electrophoresis (SDS–PAGE) and Western blot analysis that the majority of commercial membrane extraction kits harbor a substantial cytosolic contamination of their membranous fraction. Based on purity of membranous fraction, protein yield, time and costs, we show superiority of two commercial extraction kits for downstream proteome analyses of membrane proteins.     

Development of molecular tools for the detection of freshwater diatoms

This study was undertaken to develop a reliable and reproducible procedure for the detection and quantitative determination of diatoms in environmental samples. A comparative study of seven different DNA extraction kits was carried out to establish conditions for analysis of diatom containing samples. The best performers were identified using both standard and real-time PCR. We show that the yield of diatom DNA is generally quite low when using commercially available extraction kits; in addition, a new protocol was devised to obtain samples suitable for DNA amplification without the need to perform all the steps required for DNA extraction. This method was tested on environmental samples spiked, in a wide range of total cell mass, with the rarely occurring diatom Neidium affine together with a highly species-specific oligonucleotide designed on the small subunit (SSU) rRNA gene. Thus, we propose a fast and effective procedure that, combined with the use of species-specific oligonucleotide probes can detect minute amounts of a spiked diatom within a complex diatom community. This study provides experimental conditions for a fast and accurate detection of diatoms, and demonstrates the feasibility of the use of molecular tools in the evaluation of water quality.     

Commercially available luminometers and imaging devices for low-light measurements and kits and reagents utilizing bioluminescence or chemiluminescence: Survey update I

This survey update was compiled in June 1993 and includes products not covered in the luminometer survey (Jan 1992; Stanley, PE. J Biolumin Chemilumin 1992; 7:77–108 and 7:157–69) and kit and reagent survey (Nov 1992; Stanley, PE. J Biolumin Chemilumin 1993; 8:51–63). Some products are new whilst for others details have only recently become available to the author. Technical details are provided together with company address and contact information.