Use of random amplified polymorphic DNA to identify several novel markers for sex identification in the crested serpent eagle and crested goshawk

The crested serpent eagle (Spilornis cheela hoya) has no distinct sexual dimorphic traits. In the current study, we report the results of an EE0.6 (EcoRI 0.6-kb fragment) sequence applied to S. cheela hoya and a novel random amplified polymorphic DNA (RAPD) marker that can be used to sex individuals within the species S. cheela hoya and Accipiter trivigatus formosae (crested goshawk). We used sex-specific primers for the avian CHD1 (chromo-helicase-DNA-binding 1) gene and the EE0.6 sequence in PCR assays to determine sex. In addition, 120 random primers were used for RAPD fingerprinting to search for novel sex-specific fragments of S. cheela hoya. The OPBB08 random primer generated a 1241-bp sex-specific fragment in all female S. cheela hoya. From the nucleotide sequence, PCR primers were designed to amplify 553-, 895-, and 194-bp sex-specific fragments present in all female S. cheela hoya. One of these primer pairs (ScBB08-7F/R) also amplified a male/female common fragment that can be used as an internal control (543 bp). Moreover, one of the primer pairs (ScBB08-5aF/5bR) could be used to identify genders of A. trivigatus formosae. In conclusion, we identified novel sex-specific DNA markers of S. cheela hoya and A. trivigatus formosae that can be used for rapid and accurate sex identification.     

Non-specificity of a colorimetric method for the estimation of N-acetyl-d-glucosamine

The colorimetric method of Reissig et al. for the estimation of N-acetylamino sugars, is often used as a specific method for the quantification of the N-acetyl-d-glucosamine. Although this assay is more sensitive to the monomer, it recognizes all soluble N-acetyl-d-glucosamine oligomers. This result is very important because this method is extensively used in biology for the estimation of chitinolytic activity.     

Roosting Ecology of the Tent‐Roosting Bat Artibeus watsoni (Chiroptera: Phyllostomidae) in Southwestern Costa Rica1

We described the plants used as roost resources by Artibeus watsoni in southwestern Costa Rica, assessed roost fidelity, and compared roosting ecology between two sites, Golfito and Corcovado, which vary in the degree of human influence. A total of 349 tents from 25 different plant species were used by A. watsoni as roosts; some plant species (e.g., Carludovica palmata, Asplundia alata, Heliconia imbricata and Calathea lutea) were modified into tents with significantly higher frequency than others. The highest tents above the ground were observed in Philodendron popenoei and Rhodospatha wendlandii, whereas tents in Philodendron grandipes and A. alata were significantly lower than any other species. Asplundia alata and R. wendlandii also had the highest frequency of leaves modified per plant. Fidelity of bats to tents was low, although bats used several tents intermittently within a restricted area. Males generally were more faithful to tents than females, although not significantly so. This observation, along with indirect evidence of leaf modification, suggests that males are primarily responsible for tent construction. The two study sites differed in the plants used for roosting and in tent fidelity. Bats in Corcovado used a greater variety of plant species for tent roosting, whereas bats in Golfito were more faithful, suggesting that roosting resources were scarcer at the latter site.     

Key to the eggs of the equid stomach bot flies Gasterophilus Leach 1817 (Diptera: Gasterophilidae) utilizing scanning electron microscopy

Abstract. A key to the eggs of the equid stomach bot flies is presented. Scanning electron photomicrographs of eggs are used to illustrate differences among the eight Gasterophilus species. The eggs include those of G.haemorrhoidalis (Linnaeus, 1758), G.inermis (Brauer, 1858), G.intestinalis (De Geer, 1776), G.meridionalis (Piller and Evans, 1926), G.nasalis (Linnaeus, 1758), G.nigricornis (Loew, 1863), G.pecorum (Fabricius, 1794), and G.ternicinctus Gedoelst, 1912. The eggs of G.meridionalis and G.ternicinctus are shown for the first time. Egg profile is the same for a particular species and is used as a key character for egg identification. Colour of eggs is used in some couplets but only as a supplemental character. Absence or presence of striae on the eggs is used as a primary contrasting character to separate G.pecorum from the other seven species. Shape of the striae varies on eggs of the same species, even those dissected from the same specimen, and is therefore deemed an unreliable taxonomic character for further separation of the Gasterophilus species. Eggs of the same species taken from specimens throughout the world appear the same in profile. Two sets of eggs require close inspection for adequate identification: G.inermis and G.nigricornis separated primarily by the shape of the microphylar region; and G.intestinalis and G.ternicinctus separated by the shape of the egg ventrum. All other eggs have very unique and distinctive profiles. Only G.pecorum was found to possess the Type-II egg attachment organ (AO) used for adherence of the egg to plants or flat surfaces. The eggs of the remaining seven species possess a Type-I AO used to attach the eggs to hair shafts. The type of AO and the degree that the Type-I AO is extended posteriorly were used as key characters in the first and second couplet respectively.     

Diagnostic evaluation of a nanobody with picomolar affinity toward the protease RgpB from Porphyromonas gingivalis

Porphyromonas gingivalis is one of the major periodontitis-causing pathogens. P. gingivalis secretes a group of proteases termed gingipains, and in this study we have used the RgpB gingipain as a biomarker for P. gingivalis. We constructed a naive camel nanobody library and used phage display to select one nanobody toward RgpB with picomolar affinity. The nanobody was used in an inhibition assay for detection of RgpB in buffer as well as in saliva. The nanobody was highly specific for RgpB given that it did not bind to the homologous gingipain HRgpA. This indicated the presence of a binding epitope within the immunoglobulin-like domain of RgpB. A subtractive inhibition assay was used to demonstrate that the nanobody could bind native RgpB in the context of intact cells. The nanobody bound exclusively to the P. gingivalis membrane-bound RgpB isoform (mt-RgpB) and to secreted soluble RgpB. Further cross-reactivity studies with P. gingivalis gingipain deletion mutants showed that the nanobody could discriminate between native RgpB and native Kgp and RgpA in complex bacterial samples. This study demonstrates that RgpB can be used as a specific biomarker for P. gingivalis detection and that the presented nanobody-based assay could supplement existing methods for P. gingivalis detection.     

Mapping of genes affecting linolenic acid content in Brassica rapa ssp. oleifera

F₂ progeny segregating for linolenic acid content were used to identify genes and develop markers for linolenic acid in spring turnip rape (Brassica rapa ssp. oleifera). A candidate gene approach applying the rapeseed fad3 gene and bulked segregant analysis with RAPD markers was used. A total of 27 markers were distributed in three linkage groups which each exhibited a QTL for linolenic acid. Jointly the three QTLs accounted for 73.5% of the variation in linolenic acid level in this population. The fad3 gene was mapped near one QTL controlling 23.5% of the variation. Allele-specific markers were developed for fad3 and can be used for marker-assisted selection in future spring turnip rape breeding programmes.     

Alkanes of four related moth species,Helicoverpa and Heliothis

Comparison of the presence and quantities of cuticular hydrocarbons has been used successfully for identifying sibling species and races of several groups of insects. This approach has been extended to four species of moths previously regarded as belonging to the same genus, Heliothis. Gas chromatography was used to quantify the numerous high-molecular weight alkanes found on the cuticle of two pairs of closely related species: Helicoverpa zea and Helicoverpa armigera, and Heliothis virescens and Heliothis subflexa. Both sexes of H. zea and H. armigera contained different quantities of several alkanes that could be used for unambiguous identification. Similar comparisons of H. subflexa and H. virescens showed four peak ratios that were different for each species. Sexual dimorphism was minor in H. subflexa and H. virescens.     

Impact of Aspergillus oryzae genomics on industrial production of metabolites

Aspergillus oryzae is used extensively for the production of the traditional Japanese fermented foods sake (rice wine), shoyu (soy sauce), and miso (soybean paste). In recent years, recombinant DNA technology has been used to enhance industrial enzyme production by A. oryzae. Recently completed genomic studies using expressed sequence tag (EST) analyses and whole-genome sequencing are quickly expanding the industrial potential of the fungus in biotechnology. Genes that have been newly discovered through genome research can be used for the production of novel valuable enzymes and chemicals, and are important for designing new industrial processes. This article describes recent progress of A . oryzae genomics and its impact on industrial production of enzymes, metabolites, and bioprocesses.     

Heterologous expression of cholesterol oxidase in<Emphasis Type="Italic"> Bifidobacterium longum</Emphasis> under the control of 16S rRNA gene promoter of bifidobacteria

We have constructed a constitutive high-level-expression vector for the genus Bifidobacterium and used it to express cholesterol oxidase from Streptomyces coelicola. The promoter region of the 16S rRNA gene was amplified by inverse PCR and used for the construction of pBES16PR. The optimal ribosome-binding site (RBS) for Bifidobacterium was incorporated in pBES16PR. In order to test the efficacy of this expression vector, we constructed pBES16PR-CHOL with the structural gene for cholesterol oxidase under the control of the 16S rRNA promoter, and used it to transform Bifidobacterium longum. The gene was successfully expressed and high level of cholesterol oxidase activity was obtained in B. longum. This is the first report of an expression vector for the genus Bifidobacterium using a 16S rRNA gene promoter and successful expression of cholesterol oxidase. Myeong Soo Park and Bin Kwon contributed equally.     

Retrofitting ampicillin resistant vectors by recombination for use in generating C. elegans transgenic animals by bombardment

Caenorhabditis elegans is an important model organism for modern biologic research. An essential aspect of C. elegans research is the production of transgenic animals for study. These are often generated via microinjection, but biolistic bombardment has become increasingly popular. However, many of the plasmids previously generated for use in microinjection are not readily used for bombardment due to the lack of a convenient marker. The unc-119 gene is often used as a marker since unc-119 rescue can be observed at low magnification, allowing rescued animals to be easily distinguished from the larger number of non-rescued animals. Here we report the use of homologous recombination in Escherichia coli as a method to insert a cassette containing the unc-119 gene into commonly used plasmids at the site of the ampicillin resistance gene which is simpler than other methods like subcloning. These cassettes are flanked by regions homologous to the 5′ and 3′ ends of the ampicillin resistance gene and contain either the unc-119 gene and the kanamycin resistance gene or a unc-119:mCherry fusion gene and the kanamycin resistance gene. The resulting plasmids may be used for biolistic bombardment to yield animals that display unc-119 rescue, and also express the recipient plasmid transgene.     

牡丹组植物的药用民族植物学研究与考证

牡丹干燥根皮自古以来就有入药的传统,尤其在中药和民族药中被广泛使用。为阐明牡丹组植物在古籍中的记载情况和民族药中的利用现状,该文对中国八部经典医学古籍、37部地方志和民族药传统知识进行整理,采用民族植物学编目方法,对牡丹组植物在古籍和民族药中的入药种类、地理分布、入药部位、炮制方法和功效等相关传统知识进行考证和分析研究。结果表明:古籍中记载的牡丹组植物种类被考证为2种,分别为牡丹(Paeonia suffruticosa)和滇牡丹(P.delavayi),有14种炮制方法和18类功效;现有9个民族药使用4种牡丹组植物入药,为牡丹(P.suffruticosa)、滇牡丹(P.delavayi)、紫斑牡丹(P.rockii)和四川牡丹(P.decomposita);在古籍和民族药中,牡丹(P.suffruticosa)入药的频率高于其他品种;古籍以根、丹皮和花入药与民族药记载相一致,入药部位以根和丹皮的使用频率最高。芍药属牡丹组植物有治疗糖尿病、高血压、肺炎、急性高烧、乌头中毒、急性阑尾炎、中风、癫疾、炭疽,以及安神、润泽肌肤等多种药用、保健和护肤功效,为该类植物资源的研发提供了知识原型和应用基础研究。     

High efficiency electrotransformation of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> spp. <Emphasis Type="Italic">lactis</Emphasis>cells pretreated with lithium acetate and dithiothreitol

Background  

A goal for the food industry has always been to improve strains of Lactococcus lactis and stabilize beneficial traits. Genetic engineering is used extensively for manipulating this lactic acid bacterium, while electropolation is the most widely used technique for introducing foreign DNA into cells. The efficiency of electrotransformation depends on the level of electropermealization and pretreatment with chemicals which alter cell wall permeability, resulting in improved transformation efficiencies is rather common practice in bacteria as in yeasts and fungi. In the present study, treatment with lithium acetate (LiAc) and dithiothreitol (DTT) in various combinations was applied to L. lactis spp. lactis cells of the early-log phase prior to electroporation with plasmid pTRKH3 (a 7.8 kb shuttle vector, suitable for cloning into L. lactis). Two strains of L. lactis spp. lactis were used, L. lactis spp. lactis LM0230 and ATCC 11454. To the best of our knowledge these agents have never been used before with L. lactis or other bacteria.     

<Emphasis Type="Italic">In situ</Emphasis> Quantification of Phytoplankton in Reservoirs Using a Submersible Spectrofluorometer

A submersible in situ spectrofluorometer, which permits the differentiation of four algal groups (green algae, diatoms, cryptophytes and cyanobacteria), was used for phytoplankton monitoring in five reservoirs with varying levels of eutrophication and composition of their phytoplankton communities. Data obtained in situ were compared to standard laboratory methods for phytoplankton quantification; concentration of chlorophyll a and microscopy analysis. A high correlation (r = 0.95, n = 96) between chlorophyll a levels using different methods was found in all types of phytoplankton community. Taxonomic analyses and cell counts were closely related to the ratio of algal classes measured by the in situ spectrofluorometer. The submersible device used in the study measures in a continuous mode, which is advantageous in comparison with discrete sampling. This method appears to be a good tool for water quality management and can be used in the detection of natural horizontal and vertical variability in phytoplankton communities or for the early detection of cyanobacterial blooms. The device used in this study is recommended as a screening tool that enables more effective sampling that can be focused on the localities and depths where changes in phytoplankton composition occur.     

Molecular ecology ofFrankia: Advantages and disadvantages of the use of DNA probes

Ecological studies on the actinomyceteFrankia are often influenced by the difficulty to isolate and identify this microorganism. The application of molecular biological techniques offers possibilities to detect microbes without isolation and cultivation.Nif genes or whole plasmids can serve as targets for the design of specific probes. Alternatively, ribosomal RNA (rRNA), commonly used in modern phylogenetic studies, can be used as a target molecule in ecological studies. This paper gives an overview of new developments on the use of 16S rRNA as a target molecule for oligonucleotide probes. Group-specific sequences in the 16S rRNA ofFrankia can be used as targets for oligonucleotide probes that a) recognize ineffectiveFrankia strains onAlnus, b) recognize effective strains onAlnus, c) recognize allFrankia strains tested so far. The present paper summarizes the essential steps needed for the use of these probes for the detection ofFrankia strains in soil without isolation and cultivation.     

Influence of artificial accelerated ageing on the colour stability of paints used for ocular prosthesis iris painting

doi: 10.1111/j.1741‐2358.2010.00473.x
Influence of artificial accelerated ageing on the colour stability of paints used for ocular prosthesis iris painting Objectives: To evaluate the colour stability of paints used for ocular prosthesis iris painting submitted for accelerated artificial ageing (AAA). Materials and methods: Forty specimens of acrylic resin for sclera (16 × 2 mm) were made and separated into eight groups (n = 10) according to the type of paint (gouache, GP; oil, OP; acrylic AP; and composite resin for characterisation, CR) and the colours used (blue/brown). After drying (72 h), a new layer of colourless acrylic resin was applied and the initial colour readout was performed (Spectrophotometer PCB 6807). New colour readouts were performed after AAA, and ΔE was calculated. Results: Statistical analysis (two‐way anova –Bonferroni, p < 0.05) demonstrated that the brown colour showed lower ΔE means in comparison with the blue colour, with statistically significant difference for AP only. Blue colour showed no statistically significant difference with regard to the type of paint used. Brown AP showed lower ΔE than the other groups, with significant difference for OP and GP. GP showed greater alteration in ΔE for the brown colour, being statistically similar only to OP. Conclusions: Only the AP group for brown pigment shows clinically acceptable values for colour stability after AAA.     

大型综合医院科研绩效评价指标体系的构建研究

目的 为大型综合医院构建较为科学合理的科研绩效评价指标体系。方法 采用德尔菲法获得大型综合医院科研绩效评价的指标体系,运用层次分析法对各级指标进行权重的确定。结果 建立了大型综合医院科研绩效评价指标体系,其中一级指标2个,二级指标12个,三级指标40个。结论 构建的医院科研评价指标体系具有科学性、可操作性,可以作为评估医院科研绩效的工具。     

Development of a CAPS (cleaved amplified polymorphics sequence) assay for sex identification of the emu (Dromaius novaehollandiae)

Polymerase chain reaction (PCR) based procedures that have been used to identify the sex of most birds cannot be used in ratites. This paper describes the identification of a female (W‐chromosome) specific randomly amplified polymorphic (RAPD) 1.3 kb DNA fragment (ESEXW) in the emu (Dromaius novaehollandiae). Southern blot experiments and sequence analysis revealed that a related (96% similarity) sequence exists on the emu Z‐chromosome (ESEXZ). The sequences of ESEXW and ESEXZ have been used for the development of a two‐primer CAPS (cleaved amplified polymorphic sequence) assay for reliable sex identification of the emu.     

Comparative symbiotic performance of native rhizobia of the Flooding Pampa and strains currently used for inoculating Lotus tenuis in this region

The Flooding Pampa (FP) is the most important area for cattle breeding in Argentina. In this region, persistence and yield of typical forage legumes are strongly limited by soil salinity and alkalinity, which affect around 30% of the total area. Instead, naturalized Lotus tenuis is the main forage legume in this region. Rhizobial strains currently used for inoculating L. tenuis in the FP are exotic or native from non-saline soils of this region, their taxonomic identity being unknown. Assuming that rhizobia native from the most restrictive environments are well adapted to adverse conditions, the use of such isolates could improve the productivity of L. tenuis in the FP. Hence, the goal of this study was to evaluate the symbiotic efficiency of selected L. tenuis rhizobia native from the FP, as compared with strains currently used for field inoculation of this legume. Under non-stressing conditions, the symbiotic performance of native strains of FP exceeded those ones currently used for L. tenuis. Moreover, the symbiotic performance of the native strain ML103 was considerably high under salt stress, compared with strains currently used as inoculants. Analysis of 16S rRNA gene sequencing revealed that unclassified rhizobia currently used for field inoculation of L. tenuis and native strains grouped with the genus Mesorhizobium. As a whole, results obtained demonstrate that soils of the FP are a source of efficient and diverse rhizobia that could be used as a sustainable agronomic tool to formulate inoculants that improve forage yield of L. tenuis in this region.     

Agrobacterium-mediated sorghum transformation

Agrobacterium tumefaciens was used to genetically transform sorghum. Immature embryos of a public (P898012) and a commercial line (PHI391) of sorghum were used as the target explants. The Agrobacterium strain used was LBA4404 carrying a `Super-binary' vector with a bar gene as a selectable marker for herbicide resistance in the plant cells. A series of parameter tests was used to establish a baseline for conditions to be used in stable transformation experiments. A number of different transformation conditions were tested and a total of 131 stably transformed events were produced from 6175 embryos in these two sorghum lines. Statistical analysis showed that the source of the embryos had a very significant impact on transformation efficiency, with field-grown embryos producing a higher transformation frequency than greenhouse-grown embryos. Southern blot analysis of DNA from leaf tissues of T₀ plants confirmed the integration of the T-DNA into the sorghum genome. Mendelian segregation in the T₁ generation was confirmed by herbicide resistance screening. This is the first report of successful use of Agrobacterium for production of stably transformed sorghum plants. The Agrobacterium method we used yields a higher frequency of stable transformation that other methods reported previously.