A collection of 33 novel human mtDNA homoplasmic variants

Mitochondria are involved in cellular energy production via oxidative phosphorylation and this function may be damaged by any mutation in mitochondrial DNA (mtDNA). To identify novel mtDNA mutations, we have developed a program to systematically screen the entire mitochondrial genome in a large number of individuals with clinical and/or morphological features of mitochondrial dysfunction, but still no genetic diagnosis. The sequence-data were obtained with an automated rapid system, which gave us a series of information: in the eleven mitochondrial genomes analyzed we observed the presence of 33 differences from the revised Cambridge Reference Sequence (Andrews et al., 1999), but they were all homoplasmic in the patients' tissues analyzed (skeletal muscle and blood), suggesting that they are unlikely to be primarily pathogenic though they may be co-responsible in the determination of the disease. This work can therefore help complete the already ample mtDNA polymorphism existent database.     

Antiretroviral prophylaxis of perinatal HIV-1 transmission and the potential impact of antiretroviral resistance

Since 1994, trials of zidovudine, zidovudine and lamivudine, and nevirapine have demonstrated that these antiretroviral drugs can substantially reduce the risk of perinatal HIV-1 transmission. With reductions in drug price, identification of simple, effective antiretroviral regimens to prevent perinatal HIV-1 transmission, and an increasing international commitment to support health care infrastructure, antiretrovirals for both perinatal HIV-1 prevention and HIV-1 treatment will likely become more widely available to HIV-1-infected persons in resource-limited countries.In the United States, widespread antiretroviral usage has been associated with increased antiretroviral drug resistance. This raises concern that drug resistance may reduce the effectiveness of perinatal antiretroviral prophylaxis as well as therapeutic intervention strategies. The purpose of this article is to review what is known about resistance and risk of perinatal HIV transmission, assess the interaction between antiretroviral resistance and the prevention of perinatal HIV-1 transmission, and discuss implications for current global prevention and treatment strategies.     

A compilation of chiropractic and complementary/alternative medicine (CAM) data from public-use national surveys: report on a health-services research resource for the chiropractic and CAM scientific community

BACKGROUND: Federally funded national surveys are routinely conducted to provide reliable, valid, and relevant data on health and health care, and these "public-use" survey data are typically made available for further study by the wider scientific community. The full potential for using such data to examine the delivery, utilization, organization, and costs of chiropractic or complementary/alternative (CAM) health care remains largely untapped. OBJECTIVE: To report on a project that identifies and indexes public-use survey databases that contain explicit reference to chiropractic and CAM health care, and compiles that information into a web-based resource for the scientific community. METHODS: Review of database source collections. RESULTS: The utility and efficiency of secondary analyses as a cost-effective research strategy are well appreciated within the larger health-services research community, creating many possible opportunities for productive cooperative research endeavors across scientific disciplines. CONCLUSION: The Chiropractic and Complementary/Alternative Compilation User's Manual is available for free download at http://w3.palmer.edu/carber/manualhome.asp, or by following the links at the Palmer Center for Chiropractic Research homepage.     

Identification of a novel DNase I hypersensitive site within the far upstream region of the human HLA-DRA gene

The class II products of the major histocompatibility complex have a distribution restricted to certain tissues and cells. For instance, they are constitutively expressed by B lymphocytes, but not by resting T lymphocytes. In this study, we report the identification of a novel DNase I hypersensitive site within a putative regulatory region of the human HLA-DRA gene, the so-called far upstream region. This hypersensitive site was present in the genome of the DRalpha-positive human B-lymphoid Raji cell line, and absent in the DRalpha-negative T-lymphoid Jurkat cell line. In addition, this hypersensitive site was also present in transgenic B lymphocytes isolated from the murine transgenic line TG 53, carrying a single integrated copy of the human HLA-DRA gene per haploid genome. The correlation between DRA expression and the presence of this far upstream hypersensitive site suggests novel long distance chromatin remodeling mechanisms possibly shared by human and murine class II genes.     

A strategy for detection of known and unknown SNP using a minimum number of oligonucleotides applicable in the clinical settings

Detection of unknown single nucleotide polymorphism (SNP) relies on large scale sequencing expeditions of genomic fragments or complex high-throughput chip technology. We describe a simplified strategy for fluorimetric detection of known and unknown SNP by proportional hybridization to oligonucleotide arrays based on optimization of the established principle of signal loss or gain that requires a drastically reduced number of matched or mismatched probes. The array consists of two sets of 18-mer oligonucleotide probes. One set includes overlapping oligos with 4-nucleotide tiling representing an arbitrarily selected "consensus" sequence (consensus-oligos), the other includes oligos specific for known SNP within the same genomic region (variant-oligos). Fluorescence-labeled DNA amplified from a homozygous source identical to the consensus represents the reference target and is co-hybridized with a differentially-labeled test sample. Lack of hybridization of the test sample to consensus- with simultaneous hybridization to variant-oligos designates a known allele. Lack of hybridization to consensus- and variant-oligos indicates a new allele. Detection of unknown variants in heterozygous samples depends upon fluorimetric analysis of signal intensity based on the principle that homozygous samples generate twice the amount of signal. This method can identify unknown SNP in heterozygous conditions with a sensitivity of 82% and specificity of 90%. This strategy should dramatically increase the efficiency of SNP detection throughout the human genome and will decrease the cost and complexity of applying genomic wide analysis in the context of clinical trials.     

Evaluation of Toxoplasma gondii recombinant proteins for the diagnosis of recently acquired toxoplasmosis by an immunoglobulin G analysis

The value of T. gondii recombinant antigens rRop2, rGra4, rGra7 and rSAG1m (mature version) or rSAG1ct (C-terminal version) in differentiating recently acquired from chronic infections was determined by IgG-ELISA. The general highest sensitivity was observed with rRop2 whereas rSAG1m was not recognized by any of the serum samples, suggesting an incorrect folding. rGra4 and rGra7 showed significant higher sensitivity and absorbance values with serum samples from recently infected individuals compared to those with chronic infection. In contrast, rRop2 and rSAG1ct did not show differences in the reactivity pattern between both groups of serum samples.     

Effect of triethylamine on the recovery of selected South American alphaviruses,flaviviruses, and bunyaviruses from mosquito (Diptera: Culicidae) pools

We evaluated the effect of triethylamine (TEA) on the recovery of infectious virus from pools of mosquitoes for two South American alphaviruses (eastern equine encephalomyelitis and Venezuelan equine encephalomyelitis subtypes IIIC and ID), one flavivirus (Ilheus) and two bunyaviruses (Mirim [Guama group] and Itaqui [group C]). Mosquitoes were inoculated intrathoracically with virus, held for 7-10 d at 26 degrees C, and handled under one of four regimens before testing for the presence of virus by plaque assay. Mosquitoes were killed by freezing at - 70 degrees C for 3 min and tested immediately for the presence of virus; killed by freezing at -70 degrees C for 3 min and then held at room temperature for 1 h before testing for the presence of virus; anesthetized with TEA and assayed immediately for the presence of virus; or anesthetized with TEA and then held at room temperature for 1 h before being assayed for the presence of virus. For each of the viruses tested, viral titers in mosquitoes anesthetized with TEA were similar to those in mosquitoes killed by freezing at-70 degrees C. Likewise, there was no significant difference in viral titers in mosquitoes anesthetized with TEA and held at room temperature for 1 h or in mosquitoes frozen at -70 degrees C and held at room temperature for 1 h before being processed for virus by isolation. Triethylamine is advantageous for the handling of mosquitoes in a field environment. The elimination of the need for a cold chain, without compromising virus recovery, increases the feasibility of conducting research projects requiring the isolation of live virus from mosquitoes in remote tropical environments.     

Molecular characterization of 22q11 deletion in a three-generation family with maternal transmission

Haploinsufficiency of chromosome 22q11.2 is a well-established cause of both the DiGeorge anomaly and the velocardiofacial syndrome. This condition shows a continuous spectrum of phenotypic manifestations with a considerable inter- and intrafamilial variability. We report on a three-generation family with four members sharing the same 3 Mb long deletion but showing different phenotypic expression. In the first generation, the deleted patient has hypernasal speech and suffers from recurrent psychotic episodes. Two of her offspring inherited the deletion. One of these, a male, has hypernasal speech, low-set ears, hypocalcemia, severe development delay, and tetralogy of Fallot. The other, a female, has hypernasal speech, minor facial anomalies, and very mild mental retardation. Her daughter has tetralogy of Fallot, velopharyngeal insufficiency, and mild facial anomalies. This family is an example of the widely variable phenotypic expressivity of the 22q11.2 deletion. There is no correlation between the size of the deletion and the phenotypic manifestations. Genetic background and/or environmental factors could explain the different phenotypes observed in the affected members of the family.