The use of whole genome amplification in the study of human disease

The availability of large amounts of genomic DNA is of critical importance for many of the molecular biology assays used in the analysis of human disease. However, since the amount of patient tissue available is often limited and as particular foci of interest may consist of only a few hundred cells, the yield of DNA is often insufficient for extensive analysis. To address this problem, several whole genome amplification (WGA) methodologies have been developed. Initial WGA approaches were based on the polymerase chain reaction (PCR). However, recent reports have described the use of non-PCR-based linear amplification protocols for WGA. Using these methods, it is possible to generate microgram quantities of DNA starting with as little as 1mg of genomic DNA. This review will provide an overview of WGA approaches and summarize some of the uses for amplified DNA in various high-throughput genetic applications.     

Taxonomic comparison of three different groups of aflatoxin producers and a new efficient producer of aflatoxin B1, sterigmatocystin and 3-O-methylsterigmatocystin, Aspergillus rambellii sp. nov

Accumulation of the carcinogenic mycotoxin aflatoxin B, has been reported from members of three different groups of Aspergilli (4) Aspergillus flavus, A. flavus var. parvisclerotigenus, A. parasiticus, A. toxicarius, A. nomius, A. pseudotamarii, A. zhaoqingensis, A. bombycis and from the ascomycete genus Petromyces (Aspergillus section Flavi), (2) Emericella astellata and E. venezuelensis from the ascomycete genus Emericella (Aspergillus section Nidulantes) and (3) Aspergillus ochraceoroseus from a new section proposed here: Aspergillus section Ochraceorosei. We here describe a new species, A. rambellii referable to Ochraceorosei, that accumulates very large amounts of sterigmatocystin, 3-O-methylsterigmatocystin and aflatoxin B1, but not any of the other known extrolites produced by members of Aspergillus section Flavi or Nidulantes. G type aflatoxins were only found in some of the species in Aspergillus section Flavi, while the B type aflatoxins are common in all three groups. Based on the cladistic analysis of nucleotide sequences of ITS1 and 2 and 5.8S, it appears that type G aflatoxin producers are paraphyletic and that section Ochraceorosei is a sister group to the sections Flavi, Circumdati and Cervini, with Emericella species being an outgroup to these sister groups. All aflatoxin producing members of section Flavi produce kojic acid and most species, except A. bombycis and A. pseudotamarii, produce aspergillic acid. Species in Flavi, that produce B type aflatoxins, but not G type aflatoxins, often produced cyclopiazonic acid. No strain was found which produce both G type aflatoxins and cyclopiazonic acid. It was confirmed that some strains of A. flavus var. columnaris produce aflatoxin B2, but this extrolite was not detected in the ex type strain of that variety. A. flavus var. parvisclerotigenus is raised to species level based on the specific combination of small sclerotia, profile of extrolites and rDNA sequence differences. A. zhaoqingensis is regarded as a synonym of A. nomius, while A. toxicarius resembles A. parasiticus but differs with at least three base pair differences. At least 10 Aspergillus species can be recognized which are able to biosynthesize aflatoxins, and they are placed in three very different clades.     

An in situ hybridization protocol for planarian embryos: monitoring myosin heavy chain gene expression

The monitoring of gene expression is fundamental for understanding developmental biology. Here we report a successful experimental protocol for in situ hybridization in both whole-mount and sectioned planarian embryos. Conventional in situ hybridization techniques in developmental biology are used on whole-mount preparations. However, given that the inherent lack of external morphological markers in planarian embryos hinders the proper interpretation of gene expression data in whole-mount preparations, here we used sectioned material. We discuss the advantages of sectioned versus whole-mount preparations, namely, better probe penetration, improved tissue preservation, and the possibility to interpret gene expression in relation to internal morphological markers such as the epidermis, the embryonic and definitive pharynges, and the gastrodermis. Optimal fixatives and embedding methods for sectioning are also discussed. A. Cardona and J. Fernández have contributed equally to this work.     

玻璃化与正常苹果试管苗的叶片和茎的显微结构比较

以苹果品种‘鲁加5号’试管苗为试材,其用石蜡切片和电镜扫描观察方法,比较玻璃化与正常试管苗叶片和茎的显微结构的结果表明：苹果玻璃化试管苗的叶片和茎的显微结构与正常试管苗有显著差异,前者的叶片厚度变大,表皮细胞密度极低;表皮细胞体积膨大,液泡化,栅栏组织厚度减小,海绵组织厚度增加;气孔器的长轴变化不明显,短轴变宽,气孔密度极高;茎的维管组织中有空洞和塌陷,导管管壁多皱褶,筛管中无淀粉粒积累。     

Occurrence of Ochratoxin A-Producing Fungi in Commercial Corn Kernels in Argentina

Potentially ochratoxigenic Aspergillus and Penicillium species were identified and the natural occurrence of ochratoxin A (OTA) in corn kernels was evaluated. Likewise, the capacity to produce OTA by Aspergillus section Nigri and Circumdati was investigated. A total of 50 corn samples for human consumption was collected in the south of Córdoba Province. The surface-disinfected method for mycobiota determination was used. The OTA detection was performed by HPLC. OTA production was tested in strains belonging to section Nigri and Circumdati. Statistical analysis demonstrated that the specie A. flavus was isolated in higher frequency (p<0.01) from corn kernels in DRBC and DG18 media. The percentage of corn kernels contaminated by A. niger var. niger was similar in DRBC and DG18 media. The frequency of grains contaminated by A. flavus and A. niger var. awamori was higher than A. niger var. niger and A. japonicus var. japonicus (p<0.01) in DG18 media. The other potentially ochratoxigenic species, A. ochraceus, was isolated between 5% and 10% of the corn kernels in DG18 and DRBC media, respectively. The OTA producing species P. verrucosum was not isolated. All samples of corn were OTA negative (<1 ng g⁻¹). Thirty strains (25%) of the black Aspergillus were OTA producers. From four strains of A. ochraceus isolated, only one produced OTA. Due to the storage variable conditions could not be adequate in this substrate, the presence of ochratoxigenic strains of section Nigri and OTA needs to be evaluated for a longer time to establish the toxicological risk for human beings. The contamination of stored corn kernels with A. flavus and Aspergillus section Nigri was significant.     

Impact on growth and aflatoxin B1 accumulation by Kluyveromyces isolates at different water activity conditions

This study showed the impact on germination, mycelial growth and aflatoxin B₁ accumulation when interacting Aspergillus aflatoxigenic strains with Kluyveromyces isolates and the effect of water activity on this relationship. Isolates Y₁₄ and Y₁₆ reduced the percentage of germination of all Aspergillus strains and decrease germ tube elongation rate at majority of water activity assayed. Similarly they produced an increase of germination lag phase and lag phase of growth beside decreased growth rate of all Aspergillus strains. At water activities 0.994, 0.982, 0.955 and 0.937, no aflatoxins were produced in paired cultures with isolates Y_25, Y₂₂, Y₁₆, and Y₁₄, and Kluyveromyces isolates Y₁₄ and Y₁₆ impact both growth and aflatoxin accumulation at wide range of water activity.     

The influence of pre- and post-zygotic barriers on interspecific Corymbia hybridization

Background and Aims

Corymbia species from different sections hybridize readily, with some of increasing economic importance to plantation forestry. This study explores the locations of reproductive barriers between interspecific Corymbia hybrids and investigates the reproductive success of a wide taxonomic range of C. torelliana hybrid crosses.

Methods

Pollen, pistil and embryo development were investigated for four C. torelliana crosses (×C. torelliana, ×C. citriodora subsp. citriodora, ×C. tessellaris and ×C. intermedia) using fluorescent and standard microscopy to identify the locations of interspecific reproductive isolating barriers. Corymbia torelliana was also crossed with 16 taxa, representing six of the seven Corymbia sections, both Corymbia subgenera and one species each from the related genera, Angophora and Eucalyptus. All crosses were assessed for capsule and seed yields.

Key Results

Interspecific C. torelliana hybridization was controlled by pre-zygotic reproductive isolating barriers inhibiting pollen adhesion to the stigma, pollen germination, pollen tube growth in the style and pollen tube penetration of the micropyle. Corymbia torelliana (subgenus Blakella, sect. Torellianae) was successfully hybridized with Corymbia species from subgenus Blakella, particularly C. citriodora subsp. citriodora, C. citriodora subsp. variegata, C. henryi (sect. Maculatae) and C. tessellaris (sect. Abbreviatae), and subgenus Corymbia, particularly C. clarksoniana and C. erythrophloia (sect. Septentrionales). Attempted intergeneric hybrids between C. torelliana and either Angophora floribunda or Eucalyptus pellita were unsuccessful.

Conclusions

Corymbia hybrids were formed between species from different sections and subgenera, but not with species from the related genera Angophora or Eucalyptus. Reproductive isolation between the interspecific Corymbia hybrid crosses was controlled by early- and late-acting pre-zygotic isolating barriers, with reproductive success generally decreasing with increasing taxonomic distance between parent species. These findings support the monophyly of Corymbia and the close relationships of infrageneric clades. The hybridizing propensity of Corymbia species provides opportunities for breeding but suggests risks of environmental gene flow.     

The health implications of birth by Caesarean section

Since the first mention of fetal programming of adult health and disease, a plethora of programming events in early life has been suggested. These have included intrauterine and postnatal events, but limited attention has been given to the potential contribution of the birth process to normal physiology and long‐term health. Over the last 30 years a growing number of studies have demonstrated that babies born at term by vaginal delivery (VD) have significantly different physiology at birth to those born by Caesarean section (CS), particularly when there has been no exposure to labour, i.e. pre‐labour CS (PLCS). This literature is reviewed here and the processes involved in VD that might programme post‐natal development are discussed. Some of the effects of CS are short term, but longer term problems are also apparent. We suggest that VD initiates important physiological trajectories and the absence of this stimulus in CS has implications for adult health. There are a number of factors that might plausibly contribute to this programming, one of which is the hormonal surge or “stress response” of VD. Given the increasing incidence of elective PLCS, an understanding of the effects of VD on normal development is crucial.     

‘寒富’苹果二倍体及其同源四倍体叶片超微结构和叶绿素荧光参数特征

采用扫描电镜和石蜡切片法,以‘寒富’苹果二倍体及经秋水仙素加倍获得的同源四倍体植株为材料,比较两种倍性植株叶片超微结构、叶绿素含量及叶绿素荧光参数的日变化规律。结果显示:(1)与二倍体植株相比较,其同源四倍体叶片厚度、栅海比、气孔长、气孔宽、分别增加了15.1%、16.1%、70.5%、27.2%,而气孔密度显著减少了58.7%;其同源四倍体叶保卫细胞中叶绿体数和叶绿素含量分别比二倍体植株高出125.3%、37.7%。(2)‘寒富’苹果同源四倍体与其二倍体的叶绿素荧光参数PSⅡ的原初光能转化效率(Fv/Fm)、PSⅡ的潜在光化学效率(Fv/F0)和以吸收光能为基础的光合性能指数(PI)值的日变化趋势相似,但PI平均值比二倍体显著高出38.6%。研究表明,同源四倍体较二倍体叶片在形态上更大、更厚,气孔更大、密度更小,栅海比更大,表现出抗病的叶片结构;同时同源四倍体较二倍体含有更高的叶绿素含量,表现更优良的光合特性。