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Context: Rhizoma coptidis is a broadly used traditional Chinese medicine (TCM). The investigation of the influence of species and geographical origins on the phytochemicals of R. coptidis is crucial for its reasonable application and quality control., Objective: Development of an effective method to systematically study the phytochemical variations of the rhizomes of three Coptis species (Ranunculaceae) (Coptis chinensis Franch, Coptis deltoidea C.Y. Cheng et Hsiao and Coptis teeta Wall.) and a species (i.e., C. chinensis) obtained from both Daodi and non-Daodi production regions., Results: The three species had significant differences in their phytochemicals. The rhizome of C. chinensis contained more epiberberine (13.52 ± 2.65 mg/g), palmatine (18.20 ± 2.89 mg/g), coptisine (23.32 ± 4.27 mg/g) and columbamine (4.89 ± 1.16 mg/g), whereas the rhizomes of C. deltoidea and C. teeta showed the highest level of jatrorrhizine (8.52 ± 1.36 mg/g) and berberine (81.06 ± 4.83 mg/g), respectively. Moreover, the rhizome of C. chinensis from three Daodi production regions (Shizhu, Lichuan and Emeishan) contained more alkaloids than those from three non-Daodi production regions (Mianyang, Shifang and Zhenping)., Discussion and Conclusion: It is necessary to use the three R. coptidis species differentially in TCM clinical practice. Daodi C. chinensis medicinal materials have better quality than most non-Daodi ones, and so they should be preferred for TCM prescription. The combination of HPLC-based fingerprint analysis and quantification of multi-ingredients with statistical analysis provided an effective approach for species discrimination and quality evaluation of R. coptidis.

Culture-based identification methods have been the gold standard for the diagnosis of fungal infection. Currently, molecular technologies such as real-time PCR assays with short turnaround time can provide desirable alternatives for the rapid detection of Candida microbes. However, most of the published PCR primer sets are not Candida specific and likely to amplify DNA from common environmental contaminants, such as Aspergillus microbes. In this study, we designed pan-Candida primer sets based on the ribosomal DNA-coding regions conserved within Candida but distinct from those of Aspergillus and Penicillium. We demonstrate that the final two selected pan-Candida primer sets would not amplify Aspergillus DNA and could be used to differentiate eight medically important Candida pathogens in real-time PCR assays based on their melting profiles, with a sensitivity of detection as low as 10 fg of Candida genomic DNA. Moreover, we further evaluated and selected species-specific primer sets covering Candida albicans, Candida glabrata, Candida tropicalis, and Candida dubliniensis and show that they had high sensitivity and specificity. These real-time PCR primer sets could potentially be assembled into a single PCR array for the rapid detection of Candida species in various clinical settings, such as corneal transplantation.

Morphometric data from spores of ten myxosporean species were statistically analysed to explore myxosporean intraspecific variation in measurements when obtained from a sample from: (1) the same plasmodium, (2) different plasmodia from the same host and (3) different host individuals and localities. In some cases, significant differences in spore dimensions were found between samples from the same plasmodium, highlighting the difficulty of obtaining representative measurements of myxosporean spore. In addition, significant differences in spore dimensions were found when plasmodia from the same site of infection were compared, suggesting that measurements of spores should come from several different plasmodia of the sampling to increase the reliability of the morphology data. Moreover, significant differences in spore dimensions were observed for most spore dimensions when data were compared between localities. In all cases, there was clear overlap in ranges of dimensions even when means differed significantly. The present statistical analysis shows that intraspecific morphometric variation of myxosporean species commonly occurs, highlighting the importance of reporting ranges of measurements for a species, not just the mean dimensions, and taking into account all evidence when assigning or describing myxosporean species.

Allergies are an increasing issue in human health and can, eventually, cause severe anaphylactic shock. Aspergillus fumigatus and Candida albicans are leading causes of life-threatening invasive fungal infections in immunocompromised patients, but can also cause severe allergic responses in otherwise healthy individuals. The glycolytic enzyme enolase is known as a major allergen despite its function in intracellular metabolism. Therefore, its presentation on surfaces of different fungal species was investigated by using antibodies raised against recombinant enolases from A. fumigatus and C. albicans. Examination of antibody specificity revealed cross-reactivity to cell-free extracts from Aspergillus terreus, Aspergillus flavus, Aspergillus nidulans and Candida glabrata, but not against any of the three human enolases. Antibody specificity was further confirmed by hybridization with other recombinant fungal enolases, where the antibodies recognized different subsets of fungal enolases. When surface presentation of enolase was tested on intact fungal cells, a positive staining was obtained with those antibodies that also recognized the enzyme from the respective cell-free extract. This implies a general surface presentation of this glycolytic enzyme among fungal species and provides hints for its predominant recognition as an allergen. Additionally, A. fumigatus and C. albicans enolase bound to human plasminogen, which remained accessible for the plasminogen activator uPA. This implies a potential role of enolase in the invasion and dissemination process during fungal infections., (Crown Copyright © 2015. Published by Elsevier GmbH. All rights reserved.)

An increasing number of studies of hybridization in recent years have revealed that complete reproductive isolation between species is frequently not finalized in more or less closely related organisms. Most of these species do, however, seem to retain their phenotypical characteristics despite the implication of gene flow, highlighting the remaining gap in our knowledge of how much of an organism's genome is permeable to gene flow, and which factors promote or prevent hybridization. We used AFLP markers to investigate the genetic composition of three populations involving two interfertile Rhododendron species: two sympatric populations, of which only one contained hybrids, and a further hybrid-dominated population. No fixed differences between the species were found, and only 5.8% of the markers showed some degree of species differentiation. Additionally, 45.5% of highly species-differentiating markers experienced significant transmission distortion in the hybrids, which was most pronounced in F1 hybrids, suggesting that factors conveying incompatibilities are still segregating within the species. Furthermore, the two hybrid populations showed stark contrasting composition of hybrids; one was an asymmetrically backcrossing hybrid swarm, while in the other, backcrosses were absent, thus preventing gene flow.

Morphological differences in the otoliths of Macrodon atricauda from Rio Grande do Sul State (southern Brazil) and of M. ancylodon from Pará State (northern Brazil) were examined. Until recently, these were considered two populations of the same species. Although relatively similar in their general shape, those of large M. atricauda (>300 mm total length) were shorter and thinner and their outer surface was convex (those of the smallest specimens had a more lobullated antero-dorsal margin) whereas those of similarly sized M. ancylodon were larger, thicker and less lobullated. These features of the otoliths allow these morphologically similar species to be discriminated. The pattern of translucent and opaque bands in sectioned otoliths also differed. The otoliths of M. atricauda showed clear annual banding which allowed ageing, while those of M. ancylodon had no distinct alternate bands. [ABSTRACT FROM AUTHOR]

We propose a real-time decoding sequencing strategy in which a template is determined without directly measuring base sequence but by decoding two sets of encodings obtained from two parallel sequencing runs. This strategy relies on adding a mixture of different two-base pair, A+G, C+T, A+C, G+T, A+T or C+G (abbreviated as AG, CT, AC, GT, AT, or CG), into the reaction each time. When a template is cyclically interrogated twice with any two kinds of dual mononucleotide addition (AG/CT, AC/GT, and AT/CG), two sets of encodings are obtained sequentially. The two sets of encodings allow for the bases to be sequentially decoded, moving from first to last, in a deterministic manner. This strategy applies fewer cycles to obtain longer read length compared to the traditional real-time sequencing strategy. Partial rnpB gene was applied to verify the applicability of the decoding strategy via pyrosequencing. The results indicated that the sequence could be reconstructed by decoding two sets of encodings. Moreover, streptococcal strains could be differentiated by comparing signal intensity in each cycle and encoding size of each template. This strategy is likely to be applied to differentiate nucleic acid sequence as encoding size and signal intensity in each cycle vary with the base size and composition. Furthermore, it has the potential in building a promising strategy that could be utilized as an alternative to conventional sequencing systems., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Understanding the drivers of species adaptations to changing environments on the one hand and the limits for hybridization on the other hand is among the hottest questions in evolutionary biology. Parasites represent one of the major selective forces driving host evolution and at least those with free-living stages are at the same time dependent on the ecological conditions of their host's habitat. Local immunological adaptations of host species to varying parasite pressure are therefore expected and might represent the genetic basis for ecological speciation and the maintenance of recently diverged species. Madagascar provides one of the rare examples where two partially sympatric primate species (Microcebus griseorufus, M. murinus) and their hybrids, as well as an allopatric species (M. cf rufus) live in close proximity along a very steep environmental gradient ranging from southern dry spiny bush to gallery forest to evergreen eastern humid rain forest, thus mimicking the situation encountered during extensions and retreats of vegetation formations under changing climatic conditions. This system was used to study parasite infection and immune gene (MHC) adaptations to varying parasite pressure that might provide selective advantages to pure species over hybrids. Parasite burdens increased with increasing humidity. M. griseorufus, M. murinus, and their hybrids but not M. rufus shared the same MHC alleles, indicating either retention of ancestral polymorphism or recent gene flow. The hybrids had much higher prevalence of intestinal parasites than either of the parent species living under identical environmental conditions. The different representation of parasites can indicate a handicap for hybrids that maintains species identities.

Species members of the genus Parapoxvirus (PPV) within the family Poxviridae cause contagious pustular dermatitis in small ruminants (Orf virus, ORFV) and mostly mild localized inflammation in cattle (bovine papular stomatitis virus, BPSV and pseudocowpox virus, PCPV). All PPVs are known to be zoonotic, leading to circumscribed skin lesions in humans, historically known as milker's nodules. Human PPV isolates are often ill defined concerning their allocation to an animal origin. Here we present a comparative molecular analysis of a unique collection of 21 historic and recent human and animal PPV cell culture isolates (and two PPV DNA samples). Cell culture PPV propagation was restricted to primary ruminant fibroblasts and was strictly kept at low passages to avoid genomic changes by in vitro influences. For molecular arrangement of the isolate DNAs and their attribution to established PPV species DNA fragments of the PPVs were generated by two different discriminating PCR protocols, targeting the major part of the open reading frame (ORF) 011 (B2L gene) and the complete ORF 032. Multiple sequence alignments and phylogenetic analysis of both genes resulted in affiliation to the known PPV species. The sequences from the ORF 032 allowed discrimination of the isolate DNAs at a higher resolution. Human PPV isolates could be clearly assigned to the PPV species belonging to the reported or assumed animal host of transmission. For the first time, a whole PPV genome sequence comparison of a human biopsy derived virus (B029) and its ovine counterpart (B015) originating from a defined Orf outbreak in Germany is provided, revealing their well conserved relationship. Thus human PPVs can be molecularly retraced to the PPV species indicating the animal of transmission. After transmission to the human host, molecular conservation of the animal's virus peculiarities indicative for a PPV species became evident., (Copyright © 2013 Elsevier B.V. All rights reserved.)

To facilitate the specific identification of Echinococcus spp. isolates in endemic countries, a LAMP (loop-mediated isothermal amplification) assay was developed to detect the various agents known to cause cystic echinococcosis (E. granulosus s.s., E. equinus, E. ortleppi, E. canadensis and E. felidis). The infectivity of the different species and the severity of the disease in humans and livestock vary significantly among those species, and correct molecular identification of large numbers of field isolates is crucial to understand their epidemiology. However, funding constraints in many CE endemic countries often prevent PCR-based screening of field isolates. The LAMP method allows the amplification of DNA fragments under isothermal conditions which can be achieved using an ordinary waterbath, and the detection of amplification products only requires a UV light source. In the present study a LAMP assay was developed which allows the detection and differentiation of the 5 CE causing Echinococcus species. The diagnostic power was adjusted to species level, i.e. intraspecific strains (G1-3 within E. granulosus s.s., G6-10 within E. canadensis) are not discriminated. Wherever this would be necessary for epidemiological purposes, the method can be adjusted according to local requirements. The sensitivity of the assay was tested down to one fiftieth of a single protoscolex or egg, respectively. The present study describes a fast and simple method for the differentiation of CE causing Echinococcus species which can facilitate epidemiological studies in endemic countries., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In an effort to clarify the species status of mites of the genus Chorioptes the second internal transcribed spacer of the rRNA gene was characterized in 14 isolates from cattle, horse, sheep and llama of different geographic origins. The genotypes segregated into two clearlyseparated groups of DNA sequences. In addition, two phenotypes couldbe distinguished by the lengths of the outer opisthosomal setae of male adults which had previously been designated as Chorioptes bovis and Chorioptes texanus. The bipartite division of genotypes and phenotypes correlated completely in all isolates. Nine out of ten cattle isolates from three continents were determined to be C. texanus including the first description in Europe and Northern America. Chorioptes texanus appears to have a wider geographic distribution than previously known. Chorioptes bovis was found in four different host species. The apparent lack of host specificity of both species implicates a potential that mites are dispersed freely in a wide range of hosts and this might have contributed to the wide geographic distribution of these species. [ABSTRACT FROM AUTHOR]

Using random amplification of polymorphic DNA (RAPD) a band present with G. muris but not G. intestinalis DNA was detected. This band wascloned and sequenced and this facilitated the selection of a pair ofprimers specific for G. muris. Since G. muris does not infect humans, the use of these primers in conjunction with primers specific for G. intestinalis should enable water to be determined as safe or unsafefor human consumption with respect to the species of Giardia. [ABSTRACT FROM AUTHOR]

The geographical distribution of species differentiation throughout the natural range of two sympatric, closely related species of oaks, Quercus petraea (Matt) Liebl. and Quercus robur L., was investigated.By sampling species in pairs in different European regions, from Spain to Poland and Romania, the differentiation between the two speciescould be subdivided into general and local differentiation. Nine sequence characterized amplified region (SCAR) markers corresponding to genomic regions which discriminate the two species were analysed using single-strand conformation polymorphism (SSCP) or classical electrophoresis (double-stranded DNA on agarose gel) on PCR products providing, respectively, codominant and dominant markers. Similar levels of gene diversity (HE) within the two species were observed, varying generally from 0.3 to 0.5 for dominant markers and from 0..5 to 0.86 forcodominant markers. SSCP loci exhibited numerous alleles that were differently involved in species differentiation. The geographical distribution of species differentiation is heterogeneous between the regions, the north-east populations exhibiting higher differentiation than the others. For most loci, general differentiation was higher than local differentiation and was interpreted as the result of historicalcauses, selection pressures, and intra- and interspecific gene flow. [ABSTRACT FROM AUTHOR]