A practical and cost-effective mutation scanning-based approach for investigating genetic variation in Cryptosporidium

In the present study, we used a mutation scanning-targeted sequencing approach to assess variation in part (pgp60) of the 60 kDa glycoprotein (gp60) gene among Cryptosporidium samples from humans in Victoria, Australia. Two nuclear ribosomal loci (the small subunit rRNA gene and the second internal transcribed spacer) were used to identify the samples as Cryptosporidium hominis (n = 74), Cryptosporidium parvum (n = 23) or Cryptosporidium meleagridis (n = 1). In total, nine distinct pgp60 sequences were identified (three C. hominis, five C. parvum and one C. meleagridis). Phylogenetic analyses of the pgp60 sequence data, employing well-defined reference sequences for comparison, allowed the genotypic and subgenotypic classification of samples. The C. hominis samples were classified as Ib A10G2R2, Id A15G1R2, and a new genotype, designated Ib2, was identified subgenotypically as A18G1R4. The C. parvum samples were classified as IIa A18G3R1, IIa A20G3R1, IIa A22G3R1, IIa A23G3R1 and IIc A5G3R2. These findings suggested that the C. hominis metapopulation is largely homogeneous, consisting of a single dominant genotype, Ib A10G2R2, whereas the C. parvum metapopulation is considerably more heterogeneous, with no single dominant genotype. The greater level of genetic heterogeneity found among the C. parvum samples, despite the smaller sample size, may relate to the zoonotic infection pattern of this species, which would be reflective of a greater number of possible infection sources. The present mutation scanning approach, coupled with targeted sequencing of genetically distinct representatives, is a practical, cost-effective tool for large-scale population genetic and epidemiological studies of Cryptosporidium and other eukaryotic organisms.     

Differentiation of Entamoeba histolytica from Entamoeba dispar by PCR-coupled nonisotopic SSCP analysis

Entamoeba histolytica is a pathogenic protozoan parasite, which causes amoebic colitis, dysentery and liver abscesses in humans. Since the cyst and small trophozoite stages of this parasite are indistinguishable by light microscopy from Entamoeba dispar (which is nonpathogenic), specific diagnosis is compromised. To overcome this limitation, a PCR-coupled SSCP approach, utilising a sequence difference of 4.6% in a short region ( approximately 173-174 bp) of the small subunit of nuclear ribosomal DNA, was evaluated for the differentiation of the two species of Entamoeba. Including a range of well-defined control DNA samples (n = 67) to evaluate the specificity of the PCR, 45 DNA samples representing E. histolytica and E. dispar from human faecal samples were tested by SSCP, and unequivocal delineation between the species was achieved. This SSCP approach should provide a practical tool for the specific diagnosis of E. histolytica in humans and for investigating its epidemiology.     

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.     

Elucidating the identity of Anisakis larvae from a broad range of marine fishes from the Yellow Sea,China, using a combined electrophoretic‐sequencing approach

Larval nematodes were collected from marine fishes from the Yellow Sea, China. Specimens (n=1731) of Anisakis type I from 311 fishes (representing 40 species) were each identified based on morphological characters. From the genomic DNA from individual specimens, a region of nuclear ribosomal DNA was amplified by PCR, followed by digestion with restriction endonuclease HinfI, TaqI or HhaI. Subsequently, the ITS‐1 and ITS‐2 regions of selected samples were sequenced. The results revealed three species of Anisakis, namely Anisakis pegreffii (n=1709), A. typica (n=3) and a genotype (n=19) proposed, also based on comparison with previous studies, to be a “hybrid” between A. pegreffii and A. simplex sensu stricto. Thus, A. pegreffii was the dominant species, accounting for 98.7% of the total number of specimens examined herein. This is the first report of A. typica and the “hybrid” genotype from fishes from the Yellow Sea. This study provides important basic information on Anisakis in this region and suggests that the genus Anisakis has substantial host and geographical distributions.     

First genetic analysis of Cryptosporidium from humans from Tasmania,and identification of a new genotype from a traveller to Bali

Little is known about the molecular composition of Cryptosporidium species from humans living in the insular state of Tasmania, Australia. In the present study, we genetically characterized 82 samples of Cryptosporidium from humans following conventional coproscopic testing in a routine, diagnostic laboratory. Using a PCR‐coupled single‐strand conformation polymorphism (SSCP) technique, targeting portions of the small subunit rRNA (SSU), and 60 kDa glycoprotein (gp60) loci, we identified two species of Cryptosporidium, including C. hominis (subgenotypes IbA10G2, IdA16, IeA12G3T3, and IfA19G1) and C. parvum (IIaA16G1R1 and IIaA18G3), and a new operational taxonomic unit (OTU) that genetically closely resembled C. wrairi. This OTU was further characterized using markers in the actin, Cryptosporidium oocyst wall protein (COWP), and 70 kDa heat shock protein (hsp70) genes. This study provides the first characterization of species and genotypes of Cryptosporidium from Tasmania, and presents clear genetic evidence, using five independent genetic loci, for a new genotype or species of Cryptosporidium in a Tasmanian person with a recent history of travelling to Bali, Indonesia. It would be interesting to undertake detailed molecular‐based studies of Cryptosporidium in Indonesia and neighbouring countries, in conjunction with morphological and experimental investigations of new genotypes.     

Application of SNPs with low minor allele frequencies in missing person identification (MPI) through kinship analysis of DNA mixtures

The need to identify a missing person (MP) through kinship analysis of DNA samples found at a crime scene has become increasingly prevalent. DNA samples from MPs can be severely degraded, contain little DNA and mixed with other contributors, which often makes it difficult to apply conventional methods in practice. This study developed a massively parallel sequencing–based panel that contains 1661 single-nucleotide polymorphisms (SNPs) with low minor allele frequencies (MAFs) (averaged at 0.0613) in the Chinese Han population, and the strategy for relationship inference from DNA mixtures comprising different numbers of contributors (NOCs) and of varying allele dropout probabilities. Based on the simulated dataset and genotyping results of 42 artificial DNA mixtures (NOC = 2–4), it was observed that the present SNP panel was sufficient for balanced mixtures when referenced to the closest relatives (parents/offspring and full siblings). When the mixture profiles suffered from dropout, incorrect assignments were markedly associated with relatedness, NOC and the dropout level. We, therefore, indicate that SNPs with low MAFs could be reliably interpreted for MP identification through the kinship analysis of complex DNA mixtures. Further studies should be extended to more possible scenarios to test the feasibility of this present approach.     

GenoMass ‐ a computer software for automated identification of oligonucleotide DNA adducts from LC‐MS analysis of DNA digests

In the investigation of oligonucleotides, DNA and their adducts by LC‐MS, a myriad of data are generated that make manual data processing quite difficult. This paper describes a ‘reversed pseudo‐combinatorial’ approach for fragment identification and the software implementation of this approach. Combinatorial isomer libraries are generated in silico to represent the digestion products of oligonucleotides, DNA or DNA adducts of various sizes. The software automatically calculates ion masses of each isomeric segment of the library, searches for them in complicated LC‐MS data, lists their intensities and plots extracted ion chromatograms (EIC). This customized new data analysis tool has enabled a study of the enzymatic behavior of a nuclease system in the digestion of normal and adducted DNA, and in the recognition of oligomers containing a carcinogen bound to a nucleobase. The software program potentially can be further expanded to postulate unknown DNA sequences and recognize the adduction sites. Copyright © 2008 John Wiley & Sons, Ltd.     

Full-wave analysis of stripping chronopotentiograms at scanned deposition potential (SSCP) as a tool for heavy metal speciation: Theoretical development and application to Cd(II)-phthalate and Cd(II)-iodide systems

A new mathematical treatment has been developed and implemented in an EXCEL spreadsheet in order to determine average equilibrium functions from the full set of data measured by scanning stripping chronopotentiometry (SSCP) in solutions containing different proportions of heavy metal ions and small-sized ligands. It has been applied to the experimental systems Cd(II)-phthalate and Cd(II)-iodide as models of complexation in the absence and in the presence of electrodic adsorption, respectively. The good agreement between the complexation parameters determined in this way, those predicted from literature data and those obtained using a cadmium ion selective electrode (ISE) confirms the validity of the proposed methodology and encourages its further refining for the analysis of macromolecular and heterogeneous systems.     

Inducement and stabilization of G-quadruplex DNA by a thiophene-containing dinuclear ruthenium(II) complex

G-quadruplex structures are attractive targets for the development of anticancer drugs, as their formation in human telomere could impair telomerase activity, thus inducing apoptosis in cancer cells. In this work, a thiophene-containing dinuclear ruthenium(II) complex, [Ru₂(bpy)₄(H₂bipt)]⁴⁺ {bpy = 2,2′-bipyridine, H₂bipt = 2,5-bis[1,10]phenanthrolin[4,5-f]-(imidazol-2-yl)thiophene}, was prepared and the interaction between the complex and human telomeric DNA oligomers 5′-G₃(T₂AG₃)₃-3′ (HTG21) has been investigated by UV-Vis, fluorescence and circular dichroism (CD) spectroscopy, fluorescence resonance energy transfer (FRET) melting assay, polymerase chain reaction (PCR) stop assay, fluorescent intercalator displacement (FID) titrations, Job plot and color reaction studies. The results indicate that the complex can well induce and stabilize the formation of antiparallel G-quadruplex of telomeric DNA in the presence or absence of metal cations, and the ΔT_m value of the G-quadruplex DNA treated with the complex was obtained to be 12.8 °C even at levels of 50-fold molar of duplex DNA (calf-thymus DNA), suggesting that the complex exhibits higher G-quadruplex DNA selectivity over duplex DNA. The complex shows high interaction ability with G-quadruplex DNA at (1.17 ± 0.12) × 10⁷ M^?1 binding affinity using a 2:1 [complex]/[quadruplex] binding mode ratio. A novel visual method has been developed here for making a distinction between G-quadruplex DNA and duplex DNA by our ruthenium complex binding hemin to form the hemin-G-quadruplex DNAzyme.     

采用实时直接分析质谱法原位快速鉴别茶叶

采用近年来发展迅速的常温常压离子化技术——实时直接分析质谱法,建立了对茶叶中主要成分如茶氨酸、咖啡碱等的快速测定方法,通过特征的质谱信号离子,实现了对不同茶叶的快速鉴别。实时直接分析质谱法在大气压下进行,无需对茶叶进行任何的样品处理,大大缩短了分析时间,实现了原位、快速、准确且高通量的检测。     

Protection against DNA damage and inhibition of lipid peroxidation by flavones from Eremosparton songoricum (Litv) Vass

Seven flavonoids were isolated from Eremosparton songoricum (Litv) Vass and their antioxidation actions were examined. To investigate protective ability against DNA damage induced by H₂O₂, human peripheral blood mononuclear cells (PBMCs) were incubated with 5-hydroxy-7,4′-dimethoxyflavone,5,7-dihydroxy-3′,4′-dimethoxyflavone, acacetin, chrysoeriol, pedalitin, flavogadorinin and 5,7,4′-trihydroxy-3′-methoxyflavone-4′-O-D-glucoside separately and then treated by H₂O₂. The percentage of damaged cells were determined by single-cell gel electrophoresis. To examine their inhibition of lipid peroxidation, the compounds were incubated with rat microsomes and the reaction was initiated by Fe²⁺/Vc. The lipid peroxidation product thiobarbituric acid reactive substances (TBARS) was determined. The result showed most of flavonoids had concentration-dependent antioxdative activities. The relationship of antioxidation and the structure is discussed.     

Synthesis,crystal structure and DNA interaction studies of a 2D cadmium(II) coordination polymer constructed from 2-(2-pyridyl)benzimidazole

A new complex associated with Cd²⁺, [Cd(OH-H₂Bdc)(2-Pbim)]_n (1), (OH-H₂Bdc = 5-hydroxyisophthalic acid, 2-Pbim = 2-(2-pyridyl)benzimidazole), has been synthesised under hydrothermal conditions and characterised by elemental analysis, FT-IR spectroscopy, TG/DTG and fluorescence spectrum. Its in vitro cytotoxicity towards four selected tumour cell lines has been evaluated by an microculture tetrozolium assay, the results suggest that complex 1 displays greater inhibition than the free benzimidazole ligand. On the basis of the combination of absorption titration and fluorescence emission titration, the binding mode of complex 1 to calf thymus DNA has been investigated. Complex 1 can interact with the base pairs of double-helical DNA via the combined mode of intercalation and groove binding with larger binding constants.     

Improving the robustness of a partial least squares (PLS) model based on pure component selectivity analysis and range optimization: case study for the analysis of an etching solution containing hydrogen peroxide

Pure component selectivity analysis (PCSA) was successfully utilized to enhance the robustness of a partial least squares (PLS) model by examining the selectivity of a given component to other components. The samples used in this study were composed of NH₄OH, H₂O₂ and H₂O, a popular etchant solution in the electronic industry. Corresponding near-infrared (NIR) spectra (9000-7500 cm⁻¹) were used to build PLS models. The selective determination of H₂O₂ without influences from NH₄OH and H₂O was a key issue since its molecular structure is similar to that of H₂O and NH₄OH also has a hydroxyl functional group. The best spectral ranges for the determination of NH₄OH and H₂O₂ were found with the use of moving window PLS (MW-PLS) and corresponding selectivity was examined by pure component selectivity analysis. The PLS calibration for NH₄OH was free from interferences from the other components due to the presence of its unique NH absorption bands. Since the spectral variation from H₂O₂ was broadly overlapping and much less distinct than that from NH₄OH, the selectivity and prediction performance for the H₂O₂ calibration were sensitively varied depending on the spectral ranges and number of factors used. PCSA, based on the comparison between regression vectors from PLS and the net analyte signal (NAS), was an effective method to prevent over-fitting of the H₂O₂ calibration. A robust H₂O₂ calibration model with minimal interferences from other components was developed. PCSA should be included as a standard method in PLS calibrations where prediction error only is the usual measure of performance.     

Synthesis,DFT analysis and DNA studies,cytotoxicity and luminescence properties of a dinuclear copper(II) complex with 1,10-phenanthroline and 4-aminobenzoate

The dinuclear copper(II) complex, [Cu₂(phen)₂(4-aminobenzoate)₂(H₂O)₂](NO₃)₂·2(4-aminobenzoic acid)·3H₂O (phen = 1,10-phenanthroline), has been synthesized and structurally characterized by elemental analyses, IR, EPR, UV-visible and single-crystal X-ray crystallography. The complex crystallized in a monoclinic system with space group C2/c, a?=?26.0022(10) Å, b?=?10.2524(4) Å, c?=?20.9983(7) Å, α?=?90°, β?=?106.9550(10)° and γ?=?90°. The Cu(II) ion adopts a distorted square-pyramidal geometry formed by two N atoms from the phen ligand and two O atoms of the two 4-aminobenzoic acid ligands and one water O atom. The Cu…Cu separation is 3.0570(5) Å. A twofold axis passes through the midpoint of the Cu-Cu vector. The complex has intraligand (π–π*) fluorescence properties. The binding of this dinuclear copper(II) complex with calf thymus DNA (CT-DNA) was investigated by UV-vis absorption, fluorescence spectroscopic, cyclic voltammetric and viscosity techniques. Also, the cleavage of pBR322 DNA with dinuclear copper(II) complex was studied using gel electrophoresis method. The exhibited potent cytotoxic effects against human cell line (HepG2) and it was found to have good antimicrobial activities. The primary coordination sphere of dinuclear copper(II) complex is optimized, structural parameters are calculated and energy gaps of frontier orbital (HOMO-LUMO) have been calculated with B3LYP/6-31G/LANL2DZ level of theory in the gaseous phase. The calculated geometric and spectral results reproduced the experimental data with well agreement. Theoretical calculated molecular orbitals (HOMO-LUMO) and their energies have been calculated that suggest charge transfer occurs within the complex.     

Study and validity of 13C stable carbon isotopic ratio analysis by mass spectrometry and 2H site-specific natural isotopic fractionation by nuclear magnetic resonance isotopic measurements to characterize and control the authenticity of honey

Honey samples were analyzed by stable carbon isotopic ratio analysis by mass spectrometry (SCIRA-MS) and site-specific natural isotopic fractionation measured by nuclear magnetic resonance (SNIF-NMR) to first determine their potentials for characterizing the substance and then to combat adulteration. Honey samples from several geographic and botanical origins were analyzed. The δ¹³C parameter was not significant for characterizing an origin, while the (D/H)_I ratio could be used to differentiate certain single-flower varieties. Application of the official control method of adding a C₄ syrup (AOAC official method 998.12) to our authentic samples revealed anomalies resulting from SCIRA indices that were more negative than −1‰ (permil). A filtration step was added to the experimental procedure and provided results that were compliant with the natural origin of our honey samples. In addition, spiking with a C₄ syrup could be detected starting at 9-10%. The use of SNIF-NMR is limited by the detection of a syrup spike starting only at 20%, which is far from satisfying.     

Quantitative analysis of DNA methylation in the promoter region of the methylguanine‐O6‐DNA‐methyltransferase gene by COBRA and subsequent native capillary gel electrophoresis

Along with histone modifications, RNA interference and delayed replication timing, DNA methylation belongs to the key processes in epigenetic regulation of gene expression. Therefore, reliable information about the methylation level of particular DNA fragments is of major interest. Herein the methylation level at two positions of the promoter region of the gene methylguanine‐O⁶‐DNA‐Methyltransferase (MGMT) was investigated. Previously, it was demonstrated that the epigenetic status of this DNA region correlates with response to alkylating anticancer agents. An automated CGE method with LIF detection was established to separate the six DNA fragments resulting from combined bisulfite restriction analysis of the methylated and non‐methylated MGMT promoter. In COBRA, the DNA was treated with bisulfite converting cytosine into uracil. During PCR uracil pairs with adenine, which changes the original recognition site of the restriction enzyme Taql. Artificial probes generated by mixing appropriate amounts of DNA after bisulfite treatment and PCR amplification were used for validation of the method. The methylation levels of these samples could be determined with high accuracy and precision. DNA samples prepared by mixing the corresponding clones first and then performing PCR amplification led to non‐linear correlation between the corrected peak areas and the methylation levels. This effect is explained by slightly different PCR amplification of DNA with different sequences present in the mixture. The superiority of CGE over PAGE was clearly demonstrated. Finally, the established method was used to analyze the methylation levels of human brain tumor tissue samples.     

Rapid collection and identification of a novel component from Clausena lansium Skeels leaves by means of three-dimensional preparative gas chromatography and nuclear magnetic resonance/infrared/mass spectrometric analysis

The present research reports the use of a three-dimensional preparative gas chromatography (prep GC) system, equipped with three Deans-switch devices and 5%diphenyl/wax/mid-polarity ionic liquid stationary phases, for the isolation of volatile components from a complex natural source, namely wampee essential oil (derived from Clausena lansium Skeels leaves). Collection was performed by using a simple and effective lab-constructed trapping device.     

Absolute quantification of superoxide dismutase in cytosol and mitochondria of mice hepatic cells exposed to mercury by a novel metallomic approach

In the last years, the development of new methods for analyzing accurate and precise individual metalloproteins is of increasing importance, since numerous metalloproteins are excellent biomarkers of oxidative stress and diseases. In that way, methods based on the use of post column isotopic dilution analysis (IDA) or enriched protein standards are required to obtain a sufficient degree of accuracy, precision and high limits of detection. This paper reports the identification and absolute quantification of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in cytosol and mitochondria from mice hepatic cells using a innovative column switching analytical approach. The method consisted of orthogonal chromatographic systems coupled to inductively coupling plasma-mass spectrometry equipped with a octopole reaction systems (ICP-ORS-MS) and UV detectors: size exclusion fractionation (SEC) of the cytosolic and mitochondrial extracts followed by online anion exchange chromatographic (AEC) separation of Cu/Zn containing species. After purification, Cu,Zn-SOD was identified after tryptic digestion by molecular mass spectrometry (MS). The MS/MS spectrum of a doubly charged peptide was used to obtain the sequence of the protein using the MASCOT searching engine. This optimized methodology reduces the time of analysis and avoids the use of sample preconcentration and clean-up procedures, such as cut-off centrifuged filters, solid phase extraction (SPE), precipitation procedures, off-line fractions insolates, etc. In this sense, the method is robust, reliable and fast with typical chromatographic run time less than 20 min. Precision in terms of relative standard deviation (n = 5) is of 3–5% and detection limits is 0.21 ng Cu g⁻¹.