Does 'imprinting' with low prenatal vitamin D contribute to the risk of various adult disorders?

Hypovitaminosis D is a candidate risk-modifying factor for a diverse range of disorders apart from rickets and osteoporosis. Based on epidemiology, and on in vitro and animal experiment, vitamin D has been linked to multiple sclerosis, certain cancers (prostate, breast and colorectal), insulin-dependent diabetes mellitus and schizophrenia. I hypothesise that low pre- and perinatal vitamin D levels imprint on the functional characteristics of various tissues throughout the body, leaving the affected individual at increased risk of developing a range of adult-onset disorders. The hypothesis draws from recent advances in our understanding of the early origin of adult disease and proposes a 'critical window' during which vitamin D levels may have a persisting impact on adult health outcomes. Methods to test the hypothesis are outlined. If correct, the hypothesis has important implications for public health. Careful attention to maternal vitamin D status could translate into diverse improvements in health outcomes for the following generation. (C) 2001 Harcourt Publishers Ltd.

An algorithm to predict 3 ' intron splice sites in Plasmodium falciparum genomic sequences

A new algorithm, PfAGSS, for predicting 3' splice sites in Plasmodium falciparum genomic sequences is described. Application of this program to the published P. falciparum chromosome 2 and 3 data suggests that existing programs result in a high error rate in assigning 3' intron boundaries. (C) 2001 Elsevier Science B.V. All rights reserved.

Identification of vaccine candidate antigens from a genomic analysis of Porphyomonas gingivalis

Porphyromonas gingivalis is a key periodontal pathogen which has been implicated in the etiology of chronic adult periodontitis. Our aim was to develop a protein based vaccine for the prevention and or treatment of this disease. We used a whole genome sequencing approach to identify potential vaccine candidates. From a genomic sequence, we selected 120 genes using a series of bioinformatics methods. The selected genes were cloned for expression in Escherichia coli and screened with P. gingivalis antisera before purification and testing in an animal model. Two of these recombinant proteins (PG32 and PG33) demonstrated significant protection in the animal model, while a number were reactive with various antisera. This process allows the rapid identification of vaccine candidates from genomic data. (C) 2001 Elsevier Science Ltd. All rights reserved.

Rapid isolation of total RNA and genomic DNA from Hakea actities

Tissues of the Australian native plant species Hakea actities (Proteaceae) contain numerous metabolites and structural compounds that hinder the isolation of nucleic acids. Separate RNA and genomic DNA extraction procedures were developed to isolate high quality nucleic acids from H. actities. Total RNA was extracted from leaves, roots and cluster roots of H. actities grown in low nutrient levels. Cluster root formation in H. actities only occurs when the plants are grown in low nutrient concentrations. However, under these conditions, nucleic acid extraction becomes increasingly difficult. The new procedures are faster than many of the published nucleic acid extraction protocols, and avoid the use of hazardous chemicals. The RNA extraction method was used successfully on another Australian species and a crop species, suggesting that the procedure is useful for molecular studies of a broad range of plants.

Phytophthora sojae avirulence genes Avr4 and Avr6 are located in a 24kb, recombination-rich region of genomic DNA

A cross between two different races (race 7 x race 25) of the soybean root and stem rot pathogen Phytophthora sojae was analyzed to characterize the genomic region flanking two cosegregating avirulence genes, Anur4 and Anur6. Both genes cosegregated in the ratio of 82:17 (avirulent:virulent) in an F-2 population, suggestive of a single locus controlling both phenotypes. A chromosome walk was commenced from RAPD marker OPE7.1C, 2.0 cM distant from the Anur4/6 locus. Three overlapping cosmids were isolated which included genetic markers that flank the Anur4/6 locus. The chromosome walk spanned a physical distance of 67 kb which represented a genetic map distance of 22.3cM, an average recombination frequency of 3.0kb/cM and 11.7-fold greater than the predicted average recombination frequency of 35.3 kb/cM for the entire P. sojae genome. Six genes (cDNA clones) expressed from the Anur4/6 genomic region encompassed by the cosmid contig were identified. Single nucleotide polymorphisms and restriction fragment length polymorphisms showed these six genes were closely linked to the Anur4/6 locus. Physical mapping of the cDNA clones within the cosmid contig made it possible to deduce the precise linkage order of the cDNAs. None of the six cDNA clones appear to be candidates for Anur4/6. We conclude that two of these cDNA clones flank a physical region of approximately 24 kb and 4.3 cM that appears to include the Anur4/6 locus. (C) 2003 Elsevier Inc. All rights reserved.

Genomic screen for genes involved in mammalian craniofacial development

Using a subtractive hybridisation approach, we enriched for genes likely to play a role in embryonic development of the mammalian face and other structures. This was achieved by subtracting cDNA derived from adult mouse liver from that derived from 10.5 dpc mouse embryonic branchial arches 1 and 2. Random sequencing of clones from the resultant library revealed that a high percentage correspond to genes with a previously established role in embryonic development and disease, while 15% represent novel or uncharacterised genes. Whole mount in situ hybridisation analysis of novel genes revealed that approximately 50% have restricted expression during embryonic development. In addition to expression in branchial arches, these genes showed a range of expression domains commonly including neural tube and somites. Notably, all genes analysed were found to be expressed not only in the branchial arches but also in the developing limb buds, providing support for the hypothesis that development of the limbs and face is likely to involve analogous molecular processes. (C) 2003 Wiley-Liss, Inc.

Chromosomal gains and losses in ocular melanoma detected by comparative genomic hybridization in an Australian population-based study

To define the location of potential oncogenes and tumor suppressor genes in ocular melanoma we carried out comparative genomic hybridization (CGH) analysis on a population-based series of 25 formalin-fixed, paraffin-embedded primary tumors comprising 17 choroidal, 2 ciliary body, 4 iris, and 2 conjunctival melanomas. Twelve (48%) of the 25 melanomas showed no chromosomal changes and 13 (52%) had at least one chromosomal gain or loss. The mean number of CGH changes in all tumors was 3.3, with similar mean numbers of chromosomal gains (1.5) and losses (1.8). The highest number of chromosomal changes (i.e., nine) occurred in a conjunctival melanoma and included four changes not observed in tumors at any other ocular site (gains in 22q and 11p and losses in 6p and 17p). The most frequent gains in all primary ocular melanomas were on chromosome arm 8q (69%), 6p (31%) and 8p (23%) and the most frequent losses were on 6q (38%), 10q (23%), and 16q (23%). The most common pairing was gain in 8p and gain in 8q, implying a whole chromosome copy number increase; gains in 8p occurred only in conjunction with gains in 8q. The smallest regions of copy number alteration were mapped to gain of 8q21 and loss of 6q21, 10q21, and 16q22. Sublocalization of these chromosomal changes to single-band resolution should accelerate the identification of genes involved in the genesis of ocular melanoma.

Complete genomic sequence of the Australian south-west genotype of Sindbis virus: Comparisons with other Sindbis strains and identification of a unique deletion in the 3 '-untranslated region

Our previous studies have shown that two distinct genotypes of Sindbis (SIN) virus occur in Australia. One of these, the Oriental/Australian type, circulates throughout most of the Australian continent, whereas the recently identified south-west (SW) genetic type appears to be restricted to a distinct geographic region located in the temperate south-west of Australia. We have now determined the complete nucleotide and translated amino acid sequences of a SW isolate of SIN virus (SW6562) and performed comparative analyses with other SIN viruses at the genomic level. The genome of SW6562 is 11,569 nucleotides in length, excluding the cap nucleotide and poly (A) tail. Overall this virus differs from the prototype SIN virus (strain AR339) by 23% in nucleotide sequence and 12.5% in amino acid sequence. Partial sequences of four regions of the genome of four SW isolates were determined and compared with the corresponding sequences from a number of SIN isolates from different regions of the World. These regions are the non-structural protein (nsP3), the E2 gene, the capsid gene, and the repeated sequence elements (RSE) of the 3'UTR. These comparisons revealed that the SW SIN viruses were more closely related to South African and European strains than to other Australian isolates of SIN virus. Thus the SW genotype of SIN virus may have been introduced into this region of Australia by viremic humans or migratory birds and subsequently evolved independently in the region. The sequence data also revealed that the SW genotype contains a unique deletion in the RSE of the 3'UTR region of the genome. Previous studies have shown that deletions in this region of the SIN genome can have significant effects on virus replication in mosquito and avian cells, which may explain the restricted distribution of this genotype of SIN virus.

Genomic and transcriptomic features of Chlamydia trachomatis. Tracking the basis for the ecological success

Dissertation presented to obtain a Ph.D degree in Biology, speciality in Molecular Genetics, by Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Genomic characterization of adenovirus serotype 7 isolated in Brazil from acute respiratory disease patients during the period from 1980 to 1991

Forty isolates of adenovirus type 7 were analized by restriction enzyme digestion with BamHI, SmaI, EcoRI and HindIII. These isolates were obtained from acute respiratory disease patients during the years 1980 to 1991. Only two genomic types were found: Ad7b and Ad7e, with Ad7b (87.5%) being more frequent than Ad7e (12.5%). The genomic type Ad7e appeared in the years 1980, 1981 and 1983. Ad7b appeared in 1982 and it was the only genomic type found from 1984 to 1991. Both genomic types were responsible for lower (LRTI) and upper (URTI) respiratory tract infection, but the proportion LRTI/URTI is higher for Ad7b (25/6) than for Ad7e (1/4).

Penicillium glabrum cork colonising isolates – preliminary analysis of their genomic similari

Cork stopper manufacturing process includes an operation, known as stabilisation, by which humid cork slabs are extensively colonised by fungi. The effects of fungal growth on cork are yet to be completely understood and are considered to be involved in the so called “cork taint” of bottled wine. It is essential to identify environmental constraints which define the appearance of the colonising fungal species and to trace their origin to the forest and/or as residents in the manufacturing space. The present article correlates two sets of data, from consecutive years and the same season, of systematic biologic sampling of two manufacturing units, located in the North and South of Portugal. Chrysonilia sitophila dominance was identified, followed by a high diversity of Penicillium species. Penicillium glabrum, found in all samples, was the most frequent isolated species. P. glabrum intra-species variability was investigated using DNA fingerprinting techniques revealing highly discriminative polymorphic markers in the genome. Cluster analysis of P. glabrum data was discussed in relation to the geographical location of strains, and results suggest that P. glabrum arise from predominantly the manufacturing space, although cork resident fungi can also contrib

Backside-surface imprinting as a new strategy to generate specific plastic antibody materials

A backside protein-surface imprinting process is presented herein as a novel way to generate specific synthetic antibody materials. The template is covalently bonded to a carboxylated-PVC supporting film previously cast on gold, let to interact with charged monomers and surrounded next by another thick polymer. This polymer is then covalently attached to a transducing element and the backside of this structure (supporting film plus template) is removed as a regular “tape”. The new sensing layer is exposed after the full template removal, showing a high density of re-binding positions, as evidenced by SEM. To ensure that the templates have been efficiently removed, this re-binding layer was cleaned further with a proteolytic enzyme and solution washout. The final material was named MAPS, as in the back-side reading of SPAM, because it acts as a back-side imprinting of this recent approach. It was able to generate, for the first time, a specific response to a complex biomolecule from a synthetic material. Non-imprinted materials (NIMs) were also produced as blank and were used as a control of the imprinting process. All chemical modifications were followed by electrochemical techniques. This was done on a supporting film and transducing element of both MAPS and NIM. Only the MAPS-based device responded to oxLDL and the sensing layer was insensitive to other serum proteins, such as myoglobin and haemoglobin. Linear behaviour between log(C, μg mL−1) versus charged tranfer resistance (RCT, Ω) was observed by electrochemical impedance spectroscopy (EIS). Calibrations made in Fetal Calf Serum (FCS) were linear from 2.5 to 12.5 μg mL−1 (RCT = 946.12 × log C + 1590.7) with an R-squared of 0.9966. Overall, these were promising results towards the design of materials acting close to the natural antibodies and applied to practical use of clinical interest.

Surface Imprinting Approach on Screen Printed Electrodes Coated with Carboxylated PVC for Myoglobin detection with Electrochemical Transduction

A novel surface molecularly-imprinted (MI) material to detect myoglobin (Myo) using gold screen printed electrodes (SPE) was developed. The sensitive detection was carry out by introducing a carboxylic polyvinyl chloride (PVC-COOH) layer on gold SPE surface. Myo was attached to the surface of gold SPE/PVC-COOH and the vacant spaces around it were filled by polymerizing acrylamide and N,N-methylenebisacrylamide (cross-linker). This polymerization was initiated by ammonium persulphate. After removing the template, the obtained material was able to rebind Myo and discriminate it among other interfering species. Various characterization techniques including electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) confirmed the surface modification. This sensor seemed a promising tool for screening Myo in point-of-care.

Artificial antibodies for troponin T by its imprinting on the surface of multiwalled carbon nanotubes: Its use as sensory surfaces

A novel artificial antibody for troponin T (TnT) was synthesized by molecular imprint (MI) on the surface of multiwalled carbon nanotubes (MWCNT). This was done by attaching TnT to the MWCNT surface, and filling the vacant spaces by polymerizing under mild conditions acrylamide (monomer) in N,N′-methylenebisacrylamide (cross-linker) and ammonium persulphate (initiator). After removing the template, the obtained biomaterial was able to rebind TnT and discriminate it among other interfering species. Stereochemical recognition of TnT was confirmed by the non-rebinding ability displayed by non-imprinted (NI) materials, obtained by imprinting without a template. SEM and FTIR analysis confirmed the surface modification of the MWCNT. The ability of this biomaterial to rebind TnT was confirmed by including it as electroactive compound in a PVC/plasticizer mixture coating a wire of silver, gold or titanium. Anionic slopes of 50 mV decade−1 were obtained for the gold wire coated with MI-based membranes dipped in HEPES buffer of pH 7. The limit of detection was 0.16 μg mL−1. Neither the NI-MWCNT nor the MWCNT showed the ability to recognize the template. Good selectivity was observed against creatinine, sucrose, fructose, myoglobin, sodium glutamate, thiamine and urea. The sensor was tested successfully on serum samples. It is expected that this work opens new horizons on the design of new artificial antibodies for complex protein structures.