Multilocus mutation scanning for the analysis of genetic variation within Malassezia (Basidiomycota: Malasseziales)

Members of the genus Malassezia are budding yeasts, characterized by a thick cell wall. Recently, these yeasts have received attention as emerging pathogens. They are common commensals on the skin of animals and can become pathogenic under the influence of various predisposing factors. Central to studying their taxonomy, systematics, and ecology and to diagnosis is the accurate identification of species or operational taxonomic units. To overcome the limitations of current phenotypic and biochemical methods of identification, a PCR-coupled SSCP approach, utilizing sequence variation (0.4-33.5%) in short regions (approximately 250-270 bp) of the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA and the chitin synthase-2 gene (chs-2), was assessed for the identification and differentiation of different species/genotypes of Malassezia, characterized previously by DNA sequencing. Genomic DNA samples (n = 30) from Malassezia isolates cultured from canine skin scrapings were assessed by SSCP analysis of the two different genetic loci, and unequivocal delineation between genotypes and species was achieved. This SSCP approach is considered to provide a practical tool for the rapid and reliable genetic characterization of Malassezia genotypes/species from dogs and for investigating their population genetics and ecology. It will also provide a powerful tool for studies of Malassezia isolates from other animal species.     

Analysis of the genetic diversity within Cryptosporidium hominis and Cryptosporidium parvum from imported and autochtonous cases of human cryptosporidiosis by mutation scanning

The present study investigated sequence variation in part of the 60 kilodalton glycoprotein (pgp60) gene among Cryptosporidium hominis and Cryptosporidium parvum isolates (n=115) from citizens of the UK inferred to have been infected whilst travelling abroad (to 25 countries) or in the UK. The genomic DNA samples from these isolates were subjected to PCR-coupled single-strand conformation polymorphism analysis, followed by targeted sequencing of pgp60. Individual samples were classified to the genotypic and subgenotypic levels based on phylogenetic analysis (Bayesian inference) of pgp60 data, including published sequences for comparison. Based on this analysis, five C. hominis (Ia-If) and four C. parvum (IIa, IIc-IIe) genotypes were identified, equating to 16 and 10 subgenotypes, respectively. Of these genotypes, C. hominis Ib was predominant (n=82). Interestingly, one subgenotype (C. hominis Ib A10G2R2) accounted for the majority of the samples examined and was identified in travellers to 14 countries; the examination of published records suggested that C. hominis Ib A10G2R2 has a global distribution. Numerous new and seemingly rare subgenotypes (eight for C. hominis and six for C. parvum) were also discovered. In conclusion, the present study revealed substantial genetic variation in pgp60 within both C. hominis and C. parvum and emphasizes the need to undertake investigations of human and animal populations in countries for which there is no information on the genetic make-up of Cryptosporidium infecting humans.     

Specific and genotypic identification of Cryptosporidium from a broad range of host species by nonisotopic SSCP analysis of nuclear ribosomal DNA

The accurate identification of Cryptosporidium (Protozoa: Apicomplexa) species and genotypes is central to the understanding of the transmission and to the diagnosis and control of cryptosporidiosis. In this study, we demonstrate the effectiveness of nonisotopic SSCP analysis of a approximately 300 bp region of the small subunit (pSSU) of ribosomal DNA for the specific identification of and delineation among 18 different Cryptosporidium species and genotypes from a wide range of hosts. This mutation scanning approach allowed the rapid and reliable differentiation between species/genotypes differing by as little as 1.3% in the pSSU sequence, with the capacity to detect point mutations. The present findings confirm the usefulness of this tool for the rapid genetic screening of Cryptosporidium samples from any host species, providing a foundation for detailed systematic, epidemiological and ecological studies. Although applied herein to pSSU, this low cost approach should be applicable to a wide range of genetic loci for population genetic investigations of Cryptosporidium.     

Mutation scanning-coupled analysis of haplotypic variability in mitochondrial DNA regions reveals low gene flow between human and porcine Ascaris in endemic regions of China

Haplotypic variation within and among the Ascaris populations representing six provinces in China was investigated. Mitochondrial DNA regions in the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes were amplified by PCR from total genomic DNA samples (n > 720) from Ascaris individuals from humans and pigs, and subjected to mutation scanning and subsequent selective sequencing. For the cox1, ten different electrophoretic profiles were recorded for human Ascaris, and the same number for pig Ascaris, one of them being common to both host species. For the nad1, 11 different profiles were detected for human Ascaris, and 15 for pig Ascaris. Having defined all haplotypes (20 for pcox1 and 26 for pnad1) by sequencing, their frequencies were estimated in each of the two host species and each of the six provinces. For each mitochondrial region, the frequency of the different haplotypes varied considerably, depending on host species and geographical origin. Analysis of the sequence data (representing all haplotypes for each mitochondrial locus) by F-statistics indicated restricted gene flow between human Ascaris and pig Ascaris, and supported the conclusions from previous molecular epidemiological investigations that pigs are not a significant source of Ascaris infection in humans in endemic regions.     

Bioactive conformation analysis of anthranilic diamide insecticides: DFT-based potential energy surface scanning and 3D-QSAR investigations

Anthranilic diamides are fasting growing class insecticides in modern crop protection for their high activity, low ecotoxicity, and broad insecticidal spectra. However, the bioactive conformations of anthranilic diamides are still unclear until now. In the present study, DFT-based potential energy surface scanning was used to detect the low energy conformations of chlorantraniliprole, then were used respectively in the structure alignment for a series of anthranilic diamide compounds followed by detailed CoMFA and CoMSIA analyses. Finally, the bioactive conformations of anthranilic diamide insecticides were revealed from a series of low energy conformations, which might provide some clues for future insecticide design.     

Characterization and properties of chemically modified Corchorus capsularis jute fiber via pulping and grafting: Infrared,thermogravimetric analysis,differential scanning calorimetry,scanning electron microscopy,X‐ray diffraction,biodegradation, and superabsorbency

Jute fiber (Corchorus capsularis, JRC‐321 variety), an environmentally and ecologically friendly product, was chemically modified by the cooking alkaline sulfite process to unbleached and bleached pulps, which were further modified via graft copolymerization with acrylamide monomer with a complex initiating system: CuSO₄/glycine/KHSO₅. The above samples were characterized and morphologically analyzed by IR, thermogravimetric analysis, differential scanning calorimetry, scanning electron microscopy, and X‐ray diffraction. The biodegradability and superabsorbency of the samples were also evaluated for their novel commercial importance as jute‐based superabsorbents. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 2696–2703, 2003     

A comparative study on specific and nonspecific interactions in bovine serum albumin: thermal and volume effects of halothane and palmitic acid

Interaction modes of halothane and palmitic acid with bovine serum albumin (BSA) were studied from the thermal and volumetric viewpoints. The thermal stability of BSA was increased by increasing both ligand concentrations. However, the stronger effect of palmitic acid than halothane on BSA was observed at lower concentrations irrespective of the pH-dependent BSA structure. On the other hand, the volume of BSA in the solution shrunk by adding halothane independent of its structure while it expanded by adding palmitic acid. The molar ratio of halothane to BSA at the effective concentration was not consistent with the binding numbers on human serum albumin determined from the X-ray analysis, whereas that of palmitic acid was in good agreement with the numbers. We judged from these facts that halothane is a nonspecific binder to BSA; by contrast, palmitic acid is a specific binder. The stabilization mechanisms of the BSA structure were also revealed.     

Compound-specific stable isotope analysis of organic contaminants in natural environments: a critical review of the state of the art,prospects, and future challenges

Compound-specific stable isotope analysis (CSIA) using gas chromatography-isotope ratio mass spectrometry (GC/IRMS) has developed into a mature analytical method in many application areas over the last decade. This is in particular true for carbon isotope analysis, whereas measurements of the other elements amenable to CSIA (hydrogen, nitrogen, oxygen) are much less routine. In environmental sciences, successful applications to date include (i) the allocation of contaminant sources on a local, regional, and global scale, (ii) the identification and quantification of (bio)transformation reactions on scales ranging from batch experiments to contaminated field sites, and (iii) the characterization of elementary reaction mechanisms that govern product formation. These three application areas are discussed in detail. The investigated spectrum of compounds comprises mainly n-alkanes, monoaromatics such as benzene and toluene, methyl tert-butyl ether (MTBE), polycyclic aromatic hydrocarbons (PAHs), and chlorinated hydrocarbons such as tetrachloromethane, trichloroethylene, and polychlorinated biphenyls (PCBs). Future research directions are primarily set by the state of the art in analytical instrumentation and method development. Approaches to utilize HPLC separation in CSIA, the enhancement of sensitivity of CSIA to allow field investigations in the µg L^–1 range, and the development of methods for CSIA of other elements are reviewed. Furthermore, an alternative scheme to evaluate isotope data is outlined that would enable estimates of position-specific kinetic isotope effects and, thus, allow one to extract mechanistic chemical and biochemical information.Abbreviations BTEX benzene, toluene, ethylbenzene, xylenes - MTBE methyl tert-butyl ether - PAHs polycyclic aromatic hydrocarbons - VOCs volatile compounds - PCBs polychlorinated biphenyls - CSIA compound-specific (stable) isotope (ratio) analysis - GC-IRMS, GC/IRMS or GCIRMS gas chromatography-isotope ratio mass spectrometry - GC-C-IRMS, GC/C/IRMS or GCC-IRMS gas chromatography-combustion-isotope ratio mass spectrometry - irmGC/MS isotope ratio monitoring gas chromatograph-mass spectrometry - GC/P/IRMS gas chromatography-pyrolysis-isotope ratio mass spectrometry (used for D/H) - KIE kinetic isotope effect - PSIA position-specific isotope analysis (for intramolecular isotope distribution) - SNIF-NMR site-specific natural isotopic fractionation by nuclear magnetic resonance spectroscopy     

Size fractionation of aquatic colloids and particles by cross-flow filtration: analysis by scanning electron and atomic force microscopy

The suitability of the combined application of environmental scanning electron microscopy (ESEM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) for the evaluation of the ability of cross-flow filtration (CFF) to perform adequate size fractionation of freshwater colloids and particles was examined. ESEM and SEM imaging provided reference images of the CFF-generated fractions and, in estimating the experimental cut-off diameter of the membrane, provided evidence that separation was not consistent with nominal pore sizes of the membranes. However, analysis of the images showed that size distribution of CFF-generated fractions and the estimated cut-off diameter of the membranes were dependent on the advantages and limitations of the two imaging techniques. With both ESEM and SEM, best estimates of size cut-offs were lower than the nominal pore size of the membrane in the case of 0.45 μm membranes, but roughly accurate in the case of 0.1 μm pore size membranes. The results also suggested that the effectiveness of CFF may benefit from a pre-separation step using a minimally perturbing technique such as split thin-flow fractionation. AFM demonstrated the presence of colloids smaller than 50 nm in all fractions including the retentates, showing that CFF fractionation is not fully quantitative and not based on size alone. The results indicate that previous studies investigating trace element partitioning using CFF may need re-evaluation as the importance of particles and large colloids may be over-estimated.     

A comparative study on the single-scan and multiple-scan techniques in differential scanning calorimetry: Application to the crystallization of the semiconducting Ge0.13Sb0.23Se0.64 alloy

A method has been developed for analysing the evolution with time of the volume fraction transformed and for calculating the kinetic parameters at non-isothermal reactions in materials involving formation and growth of nuclei. By considering the assumptions of extended volume and random nucleation, a general expression of the fraction transformed as a function of time has been obtained in isothermal crystallization processes. Considering the mutual interference of regions growing from separate nuclei the Johnson–Mehl–Avrami equation has been deduced as a particular case. The application of the transformation rate equation to the non-isothermal processes has been carried out under the restriction of a nucleation which takes place early in the transformation and the nucleation frequency is zero thereafter. Under these conditions, the kinetic parameters have been deduced by using the techniques of data analysis of single-scan and multiple-scan. The theoretical method developed has been applied to the glass-crystal transformation kinetics of the semiconducting Ge_0.13Sb_0.23Se_0.64 alloy. The kinetic parameters obtained according to both techniques differ by only about 2.5%, which confirms the reliability and accuracy of the single-scan technique when calculating the above-mentioned parameters in non-isothermal transformation processes. The phases at which the above-mentioned semiconducting glass crystallizes after the thermal process have been identified by X-ray diffraction. The diffractogram of the transformed material shows that microcrystallites of Sb₂Se₃ and GeSe are associated with the crystallization process, remaining a residual amorphous matrix.