Impaired carbohydrate digestion and transport and mucosal dysbiosis in the intestines of children with autism and gastrointestinal disturbances.

Gastrointestinal disturbances are commonly reported in children with autism, complicate clinical management, and may contribute to behavioral impairment. Reports of deficiencies in disaccharidase enzymatic activity and of beneficial responses to probiotic and dietary therapies led us to survey gene expression and the mucoepithelial microbiota in intestinal biopsies from children with autism and gastrointestinal disease and children with gastrointestinal disease alone. Ileal transcripts encoding disaccharidases and hexose transporters were deficient in children with autism, indicating impairment of the primary pathway for carbohydrate digestion and transport in enterocytes. Deficient expression of these enzymes and transporters was associated with expression of the intestinal transcription factor, CDX2. Metagenomic analysis of intestinal bacteria revealed compositional dysbiosis manifest as decreases in Bacteroidetes, increases in the ratio of Firmicutes to Bacteroidetes, and increases in Betaproteobacteria. Expression levels of disaccharidases and transporters were associated with the abundance of affected bacterial phylotypes. These results indicate a relationship between human intestinal gene expression and bacterial community structure and may provide insights into the pathophysiology of gastrointestinal disturbances in children with autism.

Rapid molecular strategy for orbivirus detection and characterization.

Orbiviruses infect a wide range of hosts, including humans. The ability to detect them has been hampered by their diversity. Here we present a simple consensus reverse transcription (RT)-PCR method targeting the polymerase gene for orbivirus recognition and characterization. Phylogenetic assignment is achieved by automated Web-based sequence analysis of amplification products.

Aguacate virus, a new antigenic complex of the genus Phlebovirus (family Bunyaviridae).

Genomic and antigenic characterization of Aguacate virus, a tentative species of the genus Phlebovirus, and three other unclassified viruses, Armero virus, Durania virus and Ixcanal virus, demonstrate a close relationship to one another. They are distinct from the other nine recognized species within the genus Phlebovirus. We propose to designate them as a new (tenth) serogroup or species (Aguacate virus) within the genus. The four viruses were all isolated from phlebotomine sandflies (Lutzomyia sp.) collected in Central and South America. Aguacate virus appears to be a natural reassortant and serves as one more example of the high frequency of reassortment in this genus.

Nucleosome landscape and control of transcription in the human malaria parasite.

In eukaryotic cells, chromatin reorganizes within promoters of active genes to allow the transcription machinery and various transcription factors to access DNA. In this model, promoter-specific transcription factors bind DNA to initiate the production of mRNA in a tightly regulated manner. In the case of the human malaria parasite, Plasmodium falciparum, specific transcription factors are apparently underrepresented with regards to the size of the genome, and mechanisms underlying transcriptional regulation are controversial. Here, we investigate the modulation of DNA accessibility by chromatin remodeling during the parasite infection cycle. We have generated genome-wide maps of nucleosome occupancy across the parasite erythrocytic cycle using two complementary assays--the formaldehyde-assisted isolation of regulatory elements to extract protein-free DNA (FAIRE) and the MNase-mediated purification of mononucleosomes to extract histone-bound DNA (MAINE), both techniques being coupled to high-throughput sequencing. We show that chromatin architecture undergoes drastic upheavals throughout the parasite's cycle, contrasting with targeted chromatin reorganization usually observed in eukaryotes. Chromatin loosens after the invasion of the red blood cell and then repacks prior to the next cycle. Changes in nucleosome occupancy within promoter regions follow this genome-wide pattern, with a few exceptions such as the var genes involved in virulence and genes expressed at early stages of the cycle. We postulate that chromatin structure and nucleosome turnover control massive transcription during the erythrocytic cycle. Our results demonstrate that the processes driving gene expression in Plasmodium challenge the classical eukaryotic model of transcriptional regulation occurring mostly at the transcription initiation level.

Analysis of endocrine disruption in Southern California coastal fish using an aquatic multispecies microarray.

BACKGROUND: Endocrine disruptors include plasticizers, pesticides, detergents, and pharmaceuticals. Turbot and other flatfish are used to characterize the presence of chemicals in the marine environment. Unfortunately, there are relatively few genes of turbot and other flatfish in GenBank, which limits the use of molecular tools such as microarrays and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) to study disruption of endocrine responses in sentinel fish captured by regulatory agencies. OBJECTIVES: We fabricated a multigene cross-species microarray as a diagnostic tool to screen the effects of environmental chemicals in fish, for which there is minimal genomic information. The array included genes that are involved in the actions of adrenal and sex steroids, thyroid hormone, and xenobiotic responses. This microarray will provide a sensitive tool for screening for the presence of chemicals with adverse effects on endocrine responses in coastal fish species. METHODS: We used a custom multispecies microarray to study gene expression in wild hornyhead turbot (Pleuronichthys verticalis) collected from polluted and clean coastal waters and in laboratory male zebrafish (Danio rerio) after exposure to estradiol and 4-nonylphenol. We measured gene-specific expression in turbot liver by qRT-PCR and correlated it to microarray data. RESULTS: Microarray and qRT-PCR analyses of livers from turbot collected from polluted areas revealed altered gene expression profiles compared with those from nonaffected areas. CONCLUSIONS: The agreement between the array data and qRT-PCR analyses validates this multispecies microarray. The microarray measurement of gene expression in zebrafish, which are phylogenetically distant from turbot, indicates that this multispecies microarray will be useful for measuring endocrine responses in other fish.

Frequency of mitochondrial 12S ribosomal RNA variants in an adult cystic fibrosis population.

In adult cystic fibrosis patient populations, gram-negative bacteria, particularly Pseudomonas aeruginosa, frequently require aggressive therapy including systemic antibiotics, bronchodilators and airway clearance techniques. Aminoglycosides including tobramycin are used frequently to control these chronic airway infections. They, however, cause important nephrotoxic and ototoxic effects that can significantly alter the quality of life. We investigated the genetic predisposition to aminoglycoside ototoxicity in a typical unscreened North American cystic fibrosis population by screening for variants in mitochondrial 12S ribosomal RNA and noted several polymorphisms occurred at higher frequencies than expected and were associated with clinically significant cases of hearing loss. In the population studied, both patients possessing the 1555A>G transition exhibited profound ototoxicity after nontoxic dosing of tobramycin. We also identified new homoplasmic genetic variations in the mitochondrial 12S ribosomal RNA, several of which occurred in highly conserved regions of the gene and were present in patients with moderate-to-severe ototoxicity after exposure to aminoglycosides.

Development and application of a microarray meter tool to optimize microarray experiments.

BACKGROUND: Successful microarray experimentation requires a complex interplay between the slide chemistry, the printing pins, the nucleic acid probes and targets, and the hybridization milieu. Optimization of these parameters and a careful evaluation of emerging slide chemistries are a prerequisite to any large scale array fabrication effort. We have developed a 'microarray meter' tool which assesses the inherent variations associated with microarray measurement prior to embarking on large scale projects. FINDINGS: The microarray meter consists of nucleic acid targets (reference and dynamic range control) and probe components. Different plate designs containing identical probe material were formulated to accommodate different robotic and pin designs. We examined the variability in probe quality and quantity (as judged by the amount of DNA printed and remaining post-hybridization) using three robots equipped with capillary printing pins. DISCUSSION: The generation of microarray data with minimal variation requires consistent quality control of the (DNA microarray) manufacturing and experimental processes. Spot reproducibility is a measure primarily of the variations associated with printing. The microarray meter assesses array quality by measuring the DNA content for every feature. It provides a post-hybridization analysis of array quality by scoring probe performance using three metrics, a) a measure of variability in the signal intensities, b) a measure of the signal dynamic range and c) a measure of variability of the spot morphologies.

Biochip platforms as functional genomics tools for drug discovery.

Improvements in DNA microarray technology have resulted in the generation of data on a scale that, for the first time, permits detailed scrutiny of the human genome. These data provide the foundation for understanding not only the connections between genes and the purpose of genes in the human genome, but also the molecular basis of genetic defects. These advances have the potential to significantly improve healthcare management by improving disease diagnosis and specifically targeting molecular therapy. Herein, the current state of the technology is reviewed, the commercial platforms used by the biopharmaceutical industry are compared and contrasted, and recent efforts in cross-platform data integration are explored.