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.     

Genetic characterization of Cryptosporidium parvum from calves by mutation scanning and targeted sequencing – zoonotic implications

This study explored the genetic make‐up of Cryptosporidium in fecal samples from 268 individual calves on pasture‐based dairy farms in three regions of Victoria, Australia. An integrated approach, using PCR‐coupled single‐strand conformation polymorphism, targeted sequencing and phylogenetic analysis, was employed to classify the genetic variants (i.e. genotypes and subgenotypes) of Cryptosporidium parvum present in 124 (46.3%) samples and to infer their zoonotic potential. Genotypic and subgenotypic classification was achieved using a portion of the 60 kDa glycoprotein gene (designated pgp60); specific identity was verified using a region within the small subunit of the nuclear ribosomal RNA (pSSU). Twelve sequence types representing ten distinct subgenotypes were defined within genotype IIa, namely IIaA16G3R1 (n=7), IIaA17G2R1 (1), IIaA18G2R1a (2), IIaA18G2R1b (1), IIaA18G4R1 (1), IIaA19G3R1a (80), IIaA19G3R1b (1), IIaA20G2R1 (9), IIaA20G3R1 (1), IIaA20G4R1 (9), IIaA21G3R1 (1) and IIaA23G3R1 (9), of which IIaA18G2R1b, IIaA18G4R1 and IIaA19G3R1b are new records. All of the subgenotypes, except IIaA16G3R1, IIaA18G4R1 and IIaA20G4R1, have been detected previously in humans and are thus considered to be of zoonotic relevance. (Nucleotide sequences reported in this paper are available in the GenBank database under accession numbers FJ825018‐FJ825029).     

Evaluation of the quality of clinical laboratories in the Slovak Republic: conceptual and contextual remarks

The paper demonstrates conceptual parallels and relationships between intellectual capital measurement methods and the evaluation of quality in clinical laboratories in the Slovak Republic. It explores further the contextual links of those parallels with the tangibility (or intangibility) of quality indicators of laboratory diagnostics. It also highlights the problems which laboratory staff in Slovakia are confronted with. Presented at the conference Quality in the Spotlight, March 2007, Antwerp, Belgium.     

Highly sensitive non‐isotopic restriction endonuclease fingerprinting of nucleotide variability in the gp60 gene within Cryptosporidium species,genotypes and subgenotypes infective to humans,and its implications

The high‐resolution analysis of genetic variation has major implications for the identification of parasites and micro‐organisms to species and subspecies as well as for population genetic and epidemiological studies. In this study, we critically assessed the effectiveness of a PCR‐based restriction endonuclease fingerprinting (REF) method for the detection of mutations in the 60 kDa glycoprotein gene (gp60) of Cryptosporidium, a genus of parasitic protists of major human and animal health importance globally. This gene displays substantial intraspecific variability in sequence, particularly in a TCA (perfect and imperfect) microsatellite region, is present as a single copy in the nuclear genome and is used widely as a marker in molecular epidemiological studies of Cryptosporidium hominis and C. parvum, the two predominant species that infect humans. The results of this study demonstrated an exquisite capacity of REF to detect nucleotide variability in the gp60 gene within each of the two species. The differentiation of genotypes/subgenotypes based on REF analysis was supported by targeted sequencing, allowing the detection of levels of variation as low as a single‐nucleotide transversion for amplicons of ∼1 kb in size. The high‐throughput potential and relatively low‐cost of REF make it a particularly useful tool for large‐scale genetic analyses of C. hominis and C. parvum. REF could also be utilized for comparative surveys of genetic variability across large nuclear genomic regions. Such analyses of Cryptosporidium in clinical and environmental samples by REF have important implications for identifying sources of infection, modes of transmission and/or possible infectivity to humans, thus assisting in the surveillance and control of cryptosporidiosis. Given its excellent mutation detection capacity, REF should find broad applicability to various single‐copy genes as well as a wide range of other protozoan and metazoan parasites. (The nucleotide sequences reported in this article are available in the GenBank database under accession numbers GU214343–GU214371).     

Intramolecular controls for the generation of clinical-quality SNP genotypes and the assessment of normal heterozygote imbalance

We present an inventive method that generates intramolecular controls for SNP analysis, termed Mirror SNPs. Using the ovine diseases of callipyge and scrapie as examples, we describe the PCR-driven production of balanced heterozygote copies of the various SNPs implicated in these diseases. In the absence of a callipyge-positive control DNA, we generated a balanced heterozygote Mirror SNP that represents both the wild-type and mutant forms of the causal polymorphism. Simultaneous analysis of this artificial Mirror SNP and the Real (target) SNP was used to prove the absence of the mutant form of the nucleotide at the Real SNP position in tested samples. Scrapie susceptibility was assessed using a PCR-driven system which generated four separate Mirror SNPs, and these enabled the confirmation of an apparent departure from 'balanced' heterozygote appearance at Real SNPs tested. Mirror SNP technology is generic and will enable the accurate assessment of rare and medically important SNP variants, more accurate frequency determinations, and the potential assessment of SNPs in 'mixed template' samples common in forensic analyses.     

Molecular characterization and antioxidant assay of pigment producing bacteria,Sphingomonas paucimobilis and Microbacterium arborescens isolated from fresh water sediments

Abstract

This study focuses on isolation of pigment producing bacteria from fresh water sediment. The isolated bacteria were grown in nutrient broth and the maximum absorbance of 2.512 was obtained for the extracted pigment at 500 nm. The effective strains were optimized, pH 11 and temperature 30 °C was found to be more favorable for its maximum growth. The isolates were identified based on their molecular characterestics as Microbacterium arborescens and Sphingomonas paucimobilis, molecular size of the amplified 16S rRNA gene sequence was found to be approximately 1270 and 765 bp respectively. The antioxidant property of the pigment was analyzed using DPPH and ABTS assay. The IC₅₀ value of Microbacterium arborescens was higher in all the three assays in comparison with Sphingomonas paucimobilis. The extracted pigment was characterized for the presence of compounds using GC-MS and FTIR analysis to determine the functional groups. As the pigment obtained from M. arborescens had shown better antioxidant activity it may be used as colorant in food industrial applications.     

Molecular deformation and vibrational structure of the first U.V. band in linear polyenes

We try to reproduce the vibrational structure of the first U.V. band of linear polyenes, using the simple model proposed by McCoy and Ross. The Hückel orbitals give satisfactory results for the first terms of the series but a bad asymptotic behaviour when the dimension of the system increases. Bond alternation improves slightly the agreement with experimental spectra, but a discrepancy remains: we discuss its possible origin.

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Zusammenfassung Es wird versucht, die Schwingungsstruktur der ersten UV-Bande der linearen Polyene mit Hilfe des Modells von McCoy und Ross wiederzugeben. Die Hückel-Orbitale geben zufriedenstellende Resultate nur für die ersten Glieder der Serie, aber schlechtes asymptotisches Verhalten für große n. Bindungsalternierung verbessert die Übereinstimmung geringfügig. Der Ursprung der Diskrepanz wird zu erklären versucht.

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Résumé Nous essayons de rendre compte de l'intensité des bandes vibrationnelles de la première transition U.V. des polyenes linéaires, à l'aide du modèle simple de McCoy et Ross. La méthode de Hückel donne des résultats satisfaisants pour les premiers composés de la série, mais un mauvais comportement asymptotique quand la dimension du systeme augmente. L'alternance des liaisons améliore légèrement les résultats mais laisse subsister une divergence pour les chaînes longues: nous discutons l'origine possible de cette divergence.

     

人类端粒DNA的分子识别及其作用机制探讨

核酸中富含短的G-碱基重复的序列可以形成一种复杂的高级结构,称为G-四链体（G-quadruplex）.在基因组中,借助生物信息学发现这类富G序列广泛分布在基因的启动子区,特别是那些参与到复制中去的基因,例如癌基因.同时发现这类序列在mRNA的5′非翻译区（5′UTR）也广泛存在.这类序列在染色体末段端粒部位的存在及功能已得到充分阐明.已知端粒富含G-碱基序列,其3′末端以单链状态存在,这使得在一些小分子的选择性作用下端粒序列很容易形成G-四链体结构,进而破坏端粒结构,影响端粒酶活性.已知端粒酶在超过85%的肿瘤中过量表达,因此,端粒酶已经成为抗癌药物设计的特殊靶点,是目前本领域的研究热点之一.已发现系列配体通过有效抑制端粒酶而表现高的抗肿瘤活性.本文主要综述了近年来端粒G-四链体分子识别及其药物靶向的最新进展,并对其作用机理做了进一步的分析和探讨.     

Assessment of Genetic Stability in Traditional Ginger Cultivated in Manipur,India Based on Molecular and Chemical Markers

Randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to analyze the genetic stability of ten local cultivars collected from nine districts of Manipur, India with the released ginger variety Nadia. A total of 15 RAPD and 8 ISSR primers resulted in 107 and 53 distinct and reproducible bands, respectively. A lack of polymorphism revealed the genetic stability among the local cultivars. Unlike molecular markers, analysis of essential oil composition by gas chromatography–mass spectrometry (GC-MS) revealed variation among 11 clones. Among eight major constituents obtained by GC-MS technique, cinnamyl acetate was found only in IBSD/Z-41d cultivars, whereas, in IBSD/Z-41o no trace of trans-geraniol was observed. Moreover, concentration of 6-gingerol determined by high-performance liquid chromatographic (HPLC) method shows that IBSD/Z-41r contains the highest and IBSD/Z-41i contains the lowest gingerol percentage.     

白石湖煤THF萃取物中硫、氮化合物赋存形态

采用四氢呋喃(THF)对白石湖煤进行索氏萃取,萃取液在四氟硼酸银存在下与碘甲烷反应,转化成甲基磺酸盐。采用电喷雾电离(ESI)傅里叶变换离子回旋共振质谱(FT-ICR MS)对甲基硫盐和含氮化合物进行了表征,结合硫、氮化学发光检测器(GC-SCD和GC-NCD)分析,研究THF萃取物中硫、氮化合物形态。结果表明,THF萃取物中主要是S_1、S_2、N_1、N_1O_1类化合物,含量较高的硫、氮化合物分别是二苯并噻吩类化合物及咔唑类化合物。     

Identification of deletion and duplication genotypes of the PMP22 gene using PCR-RFLP, competitive multiplex PCR, and multiplex ligation-dependent probe amplification: a comparison

We evaluated the efficacy of PCR-RFLP, competitive multiplex PCR, and a commercially available system of multiplex ligation-dependent probe amplification (MLPA) for the determination of deletion and duplication genotypes of the PMP22 gene. We compared the methods for efficiency, sensitivity, and specificity. We determined the gene dosage of the PMP22 gene via PCR-RFLP, competitive multiplex PCR, and MLPA. To demonstrate the sensitivity and accuracy of these three methods, a total of 185 samples from 42 patients with hereditary neuropathy with liability to pressure palsies (HNPP), 57 patients with Charcot-Marie-Tooth disease type 1A (CMT1A), and 86 unaffected individuals, were analyzed. Molecular diagnosis by PCR-RFLP was performed on all 185 samples; 24 HNPP deletions and 33 CMT1A duplications were identified. In contrast, 25 HNPP deletions and 38 CMT1A duplications were identified correctly using competitive multiplex PCR and MLPA. Six samples were incorrectly identified by PCR-RFLP (one HNPP deletion and five CMT1A duplications). Competitive multiplex PCR and MLPA demonstrated reliability and relative speed compared to PCR-RFLP; they were superior to PCR-RFLP for gene dosage quantification. Multiplex PCR and MLPA should be the methods of choice for detection of deletion and duplication genotypes in molecular genetic diagnoses.     

Development of the 16 X‐STR loci typing system and genetic analysis in a Shanghai Han population from China

This study developed a new multiplex PCR system that simultaneously amplifies 16 X‐STR loci in the same PCR reaction, and the polymorphism and mutation rates of these 16 X‐STR loci were explored in a Shanghai Han population from China. These loci included DXS10134, DXS10159, DXS6789, DXS6795, DXS6800, DXS6803, DXS6807, DXS6810, DXS7132, DXS7424, DXS8378, DXS9902, GATA165B12, GATA172D05, GATA31E08, and HPRTB. Samples from 591 unrelated individuals (293 males and 298 females) and 400 two‐generation families were successfully analyzed using this multiplex system. Allele frequencies and mutation rates of the 16 loci were investigated, with the comparison of allele frequency distributions among different populations performed. Polymorphism information contents of these loci were all >0.6440 except the locus DXS6800 (0.4706). Nine cases of mutations were detected in the 16 loci from the investigation of 9232 meioses. Pairwise comparisons of allele frequency distributions showed significant differences for most loci among populations from different countries and ethnic groups but not among the Han population living in other areas of China. These results suggest that the 16 X‐STR loci system provides highly informative polymorphic data for paternity testing and forensic identification in the Han population in Shanghai, China, as a complementary tool.     

Development and validation of a rapid ultra high performance liquid chromatography with tandem mass spectrometry method for the simultaneous determination of darunavir,ritonavir, and tenofovir in human plasma: Application to human pharmacokinetics

A sensitive ultra high performance liquid chromatography with tandem mass spectrometry method was developed for the simultaneous determination of darunavir, ritonavir and tenofovir in human plasma. Sample preparation involved a simple liquid–liquid extraction using 200 μL of human plasma extracted with methyl tert‐butyl ether for three analytes and internal standard. The separation was accomplished on an Acquity UPLC BEH C₁₈ (50 mm x 2.1 mm, 1.7 μm) analytical column using gradient elution of acetonitrile/methanol (80:20, v/v) and 5.0 mM ammonium acetate containing 0.01% formic acid at a flow rate of 0.4 mL/min. The linearity of the method ranged between 20.0 and 12 000 ng/mL for darunavir, 2.0 and 2280 ng/mL for ritonavir, and 14.0 and 1600 ng/mL for tenofovir using 200 μL of plasma. The method was completely validated for its selectivity, sensitivity, linearity, precision and accuracy, recovery, matrix effect, stability, and dilution integrity. The extraction recoveries were consistent and ranged between 79.91 and 90.04% for all three analytes and internal standard. The method exhibited good intra‐day and inter‐day precision between 1.78 and 6.27%. Finally the method was successfully applied for human pharmacokinetic study in eight healthy male volunteers after the oral administration of 600 mg darunavir along with 100 mg ritonavir and 100 mg tenofovir as boosters.     

Identification and genetic characterization of Anisakis larvae from marine fishes in the South China Sea using an electrophoretic‐guided approach

From 35 species of marine fishes (n = 327) from the South China Sea, 237 nematode larvae were collected and identified morphologically as Anisakis. Genomic DNA was isolated from each larva and subjected to PCR‐based RFLP and targeted sequencing of a nuclear ribosomal DNA region between the 3′‐end of the small subunit and 5′‐end of the large subunit of the rRNA genes (= internal transcribed spacers, ITS+). Four different RFLP profile combinations (sets) were detected for all restriction endonucleases (HinfI, HhaI, and TaqI), of which three were characteristic of Anisakis typica, A. pegreffii, and A. physeteris, respectively. One profile set (for sample CA‐2012) was linked to an ITS+ sequence that was identical to a previously published sequence of Anisakis sp. (sample HC‐2005; originating from the African shelf) and another sequence (PH‐2010; Madeira, Portugal). Phylogenetic analysis was carried out using the ITS+ sequence data from this study and reference sequences from the GenBank database. Neighbor joining and maximum parsimony trees displayed three clades. Clades I and II included nine described species of Anisakis, including all type I and type II larvae; clade III represented some undescribed species of Anisakis. Morphological comparison showed that Anisakis sp. CA‐2012 was distinct from type I and type II larvae based on its tail shape and ratio of tail length to body length. The phylogenetic analysis and morphological characters suggest that Anisakis sp. CA‐2012 represents a new record, now called Anisakis type III larvae.     

分子生物学手段检测转Bt基因棉花中Cry杀虫蛋白的定性和定量方法

建立了转Bt基因棉花中Cry杀虫蛋白的提取、样品前处理以及酶联免疫(ELISA)定量分析方法,并使用凝胶电泳、普通聚合酶链式反应(PCR)和实时荧光定量PCR等分子生物学手段对转基因棉花中的Bt基因进行定性和定量检测.所建立的苏云金芽孢杆菌杀虫晶体蛋白(Cry1Ab蛋白和Cry1Ac蛋白)标准曲线线性关系良好,相关系数r2均大于0.999,相对标准偏差RSD均小于2.0%.方法简单、快速、重现性和精密度好,可为农业食品行业和环境领域科研人员提供一种简便快速地从转基因棉花中检测Bt毒蛋白的分析方法.     

A framework for the development of STR genotyping in domestic animal species: Characterization and population study of 12 canine X‐chromosome loci

This study reports the methodology used to search, select and characterize STR loci on the canine X chromosome using publicly available genome resources and following the current guidelines for human and non‐human forensic testing. After several rounds of selection, 12 X‐STR markers were optimized for simultaneous co‐amplification in a single PCR, and genetic profiles were determined in a sample of 103 unrelated dogs. Mendelian inheritance was verified and mutation rates were assessed using family groups. Alleles that varied in size were sequenced to create a standardized nomenclature proposal based on the number of repeats. All loci conformed to Hardy–Weinberg expectations. The resulting panel showed high forensic efficiency, presenting high values of power of discrimination (in males and females) and mean exclusion chance, both in trios involving female offspring and in duos composed of dam and male offspring. Its use may complement the information obtained by autosomal STR analysis and contribute to the resolution of complex cases of kinship in dogs. The presented methodology for the de novo construction of an STR multiplex may also provide a helpful framework for analogous work in other animal species. As an increasing number of reference genomes become available, convenient tools for individual identification and parentage testing based on STR loci selected from autosomes or sex chromosomes' sequences may be created following this strategy.