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PubMed1999

A nucleoside transporter from Trypanosoma brucei involved in drug resistance.

Drug resistance of pathogens is an increasing problem whose underlying mechanisms are not fully understood. Cellular uptake of the major drugs against Trypanosoma brucei spp., the causative agents of sleeping sickness, is thought to occur through an unusual, so far unidentified adenosine transporter. Saccharomyces cerevisiae was used in a functional screen to clone a gene (TbAT1) from Trypanosoma brucei brucei that encodes a nucleoside transporter. When expressed in yeast, TbAT1 enabled adenosine uptake and conferred susceptibility to melaminophenyl arsenicals. Drug-resistant trypanosomes harbor a defective TbAT1 variant. The molecular identification of the entry route of trypanocides opens the way to approaches for diagnosis and treatment of drug-resistant sleeping sickness.

Adenosine↗

PubMed1988

A newly characterized HLA DQ beta allele associated with pemphigus vulgaris.

The inheritance of particular alleles of major histocompatibility complex class II genes increases the risk for various human autoimmune diseases; however, only a small percentage of individuals having an allele associated with susceptibility develop disease. The identification of allelic variants more precisely correlated with disease susceptibility would greatly facilitate clinical screening and diagnosis. Oligonucleotide-primed gene amplification in vitro was used to determine the nucleotide sequence of a class II variant found almost exclusively in patients with the autoimmune skin disease pemphigus vulgaris. In addition to clinical implications, the disease-restricted distribution of this variant should provide insight into the molecular mechanisms underlying associations between diseases and HLA-class II genes.

Alleles↗

PubMed2025

Chromosome-specific centromeric patterns define the centeny map of the human genome.

Centromeres are epigenetically specified by distinct chromatin, whereas their DNA varies between species and individuals. This extensive sequence divergence makes comparative analyses between centromeres challenging. In this study, we identified a chromosome-specific architectural pattern across the human genome, defined by the conserved spacing of a functionally relevant centromeric DNA motif. The distribution of these sites along chromosome arms constitutes the human "centeny map." By using a custom Genomic Centromere Profiling (GCP) pipeline, we leveraged the motif's position, orientation, and organization to construct structural models that enable reclassification of human chromosomal clusters, detection of centromere expansion, and identification of structural variants and misassembled regions. The high-resolution maps derived from this pattern not only provide a framework for comparative analysis of centromeres across evolution and disease but also offer a new dimension for chromosome annotation, assembly, and characterization.

Humans↗

PubMed2000

Serological detection of infection with diverse human and simian immunodeficiency viruses using consensus env peptides.

Cross-species transmission has been shown to play an important role in the emergence of human retroviruses. We developed a generic enzyme immunoassay using synthetic peptides from gp41 and C2V3 consensus sequences (human immunodeficiency virus [HIV] type 1 [HIV-1] groups M, O, and N and the homologous region of simian immunodeficiency virus [SIV] strains from chimpanzees [SIVcpz], SIVcpzGAB1 and SIVcpzANT) to detect divergent HIV and SIV. A cocktail of peptides from gp41 and C2V3 (M-O) detected all HIV-1 group M and O sera and showed cross-reactivity with SIVcpz sera. Further, a mixture of C2V3 peptides (GAB1-ANT) failed to detect HIV-1 infections but reacted with all SIVcpz sera, allowing discrimination of SIVcpz from HIV-1 infections. Since most SIVcpz sera cross-reacted with HIV-1 peptides, we next evaluated SIVcpz serum reactivity with rapid tests for HIV-1/2. SIVcpzANT and SIVcpzUS sera reacted with the Sero-strip and Multispot assays. Both tests are sensitive in detecting group M (97 100%, respectively), although Multispot has lower sensitivity for group O detection (67%) than does Sero-strip (100%). The limited volume and time required to perform these assays make them a generic tool for field screening. The env peptide-based assay and rapid tests should allow for the identification of emerging variants of HIV and SIV.

Animals↗

PubMed2026

Development and evaluation of an ARTIC-based amplicon sequencing assay for whole-genome characterization of respiratory syncytial virus.

Respiratory syncytial virus (RSV), a ~15.2 kb negative-sense RNA virus, causes acute respiratory infections in infants and older adults. Its two subtypes, RSV-A and RSV-B, evolve rapidly, making ongoing monitoring of circulating strains essential. The Georgia Public Health Laboratory (GPHL) developed and evaluated an amplicon-based whole-genome sequencing (WGS) assay for RSV surveillance. A total of 214 de-identified remnant clinical specimens (102 RSV-A and 112 RSV-B) with RT-PCR Cq values <31 were included. RSV genomes were amplified using ARTIC-style and custom primer sets, with the ARTIC set showing superior performance. Libraries were prepared using a modified Illumina COVIDSeq protocol, sequenced on NextSeq 1000/2000 instruments, and analyzed using the GPHL-RSV-PIPE bioinformatics pipeline. Among genomes meeting validation criteria, sequencing depth was slightly higher for RSV-A (median 53,433×; mean 51,076×) than RSV-B (median 49,699×; mean 46,945×), whereas genomic coverage was slightly lower for RSV-A (median 97.5%; mean 96.6%) than RSV-B (median 98.3%; mean 97.6%). Predominant lineages were A.D.3.1 and A.D.5.2 for RSV-A and B.D.E.1 for RSV-B. For RSV-A, the assay showed 92.8% accuracy, 96.2% sensitivity, 87.2% specificity, 92.6% positive predictive value, and 93.2% negative predictive value. Intra- and inter-run precision assessed using 16 and 53-57 genomes, respectively, showed nearly 100% consensus genome identity with 0-5 nucleotide differences. Specificity testing of 31 non-RSV specimens produced no false-positive detections. Limits of detection were 4.4 TCID50/mL for RSV-A and 18.6 TCID50/mL for RSV-B. These results demonstrate that the ARTIC-based RSV WGS assay enables near real-time surveillance and strengthens data-driven public health responses to future outbreaks.IMPORTANCERSV, with two major subtypes, RSV-A and RSV-B, causes acute respiratory infections that can be severe in infants under 6 months and older adults. Current RSV surveillance at the GPHL relies on the Thermo Fisher TaqMan Gene Expression Capillary assay, which detects and subtypes RSV but lacks resolution for lineage classification and identification of emerging variants. To address this critical gap, GPHL developed and evaluated an amplicon-based WGS assay using 214 de-identified RSV clinical specimens. Genomes were amplified using ARTIC-style and custom-primer sets, with ARTIC primers showing superior performance. The assay demonstrated strong sequencing depth, genomic coverage, specificity, repeatability, reproducibility, and low limits of detection. RSV lineages were accurately determined based on genetic variation. These results establish that the ARTIC-based WGS assay enables near real-time genomic surveillance, supporting monitoring of circulating RSV strains and informing data-driven public health responses.

bioinformatics pipeline↗

PubMed2006

An outbreak of keratitis caused by Mycobacterium immunogenum.

From 8 October to 12 November 2003, 36 patients underwent surgical correction of myopia in a São Paulo, Brazil, clinic. Five patients had clinical signs of infectious keratitis, and a Mycobacterium species with previously unreported patterns determined by PCR restriction enzyme analysis of the hsp65 gene and PCR restriction enzyme analysis of the 16S-23S rRNA internal transcribed spacer (ITS) was isolated from corneal scrapings from four of these patients. Subsequent evaluation by phenotypic tests and partial sequencing of the hsp65, sodA, rpoB, and 16S rRNA genes and the ITS supported the species identification as a variant of Mycobacterium immunogenum. The source of infection was not determined. The outbreak was caused by a single clone, as evidenced by identical pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus-PCR profiles. This is the first report of an outbreak where this species was isolated from infected tissues.

Adult↗

PubMed1985

Comparison of neutralization and DNA restriction enzyme methods for typing clinical isolates of human adenovirus.

Sixty-five adenovirus isolates collected over a 3.5-year period were typed by both standard microneutralization techniques and restriction endonuclease digestion of viral DNA. Of the 65 isolates, 47 (72.3%) representing six adenovirus types could be typed by microneutralization. Eighteen isolates demonstrated partial neutralization with standard antisera to two or more adenovirus serotypes and thus could not be definitively typed. DNA analysis permitted typing of 64 of the 65 isolates (98.5%) (including four isolates which contained mixtures of two adenovirus types), and 12 different types were identified. Neutralization and DNA typing disagreed for five isolates, and in each case, digestion with multiple restriction endonucleases and DNA hybridization studies were consistent with the type assigned by DNA analysis. In addition, the DNA analysis method allowed the identification of genomic variants (genome types) of five adenovirus types. We conclude that typing clinical isolates of adenovirus by restriction endonuclease digestion of viral DNA can be done rapidly, provides additional epidemiological and typing information, and provides fewer ambiguous results than does typing by neutralization.

Adenoviruses, Human↗

PubMed1993

Recognition of a highly conserved region of human immunodeficiency virus type 1 gp120 by an HLA-Cw4-restricted cytotoxic T-lymphocyte clone.

Human immunodeficiency virus type 1 (HIV-1) isolates exhibit extensive sequence variation, particularly in the gp120 subunit of the envelope glycoprotein, and the degree of this variation has raised questions as to whether conserved regions of the HIV-1 envelope can be recognized by the host immune response. A CD8+ cytotoxic T-lymphocyte (CTL) clone specific for the HIV-1 envelope was derived by culturing peripheral blood mononuclear cells from an HIV-1 seropositive subject in the presence of a CD3-specific monoclonal antibody, interleukin-2, and irradiated allogeneic peripheral blood mononuclear cells. Lysis of target cells was restricted by an HLA-C molecule, Cw4, which has not been previously shown to present viral antigen to CTL. Mapping of the specificity of this CTL clone by using synthetic HIV-1 peptides localized the epitope to an 8-amino-acid region of gp120 (amino acids 376 to 383) which is conserved among approximately 90% of sequenced viral isolates. Examination of the recognition of variant peptides by this CTL clone demonstrated that a single, nonconservative amino acid substitution within the 8-amino-acid minimal epitope could abrogate lysis of targets incubated with the variant peptide. The identification of a CTL epitope in a highly conserved region of gp120 documents the ability of cellular immune responses of infected persons to respond to relatively invariant portions of this highly variable envelope glycoprotein. However, the ability of even a single-amino-acid change in gp120 to abolish lysis by CTL supports the hypothesis that sequence variation in HIV-1 may serve as a mechanism of immune escape. In addition, the identification of an HLA-C molecule presenting viral antigen to CTL supports a functional role for these molecules.

Amino Acid Sequence↗

PubMed1993

Within-family selection at an otherwise unselected locus in dairy cattle.

Identification of allelic variants with economic importance is feasible via molecular genetic techniques. This information can be used to increase the frequency of favourable alleles in dairy cattle. The effect of selection on the genotype within families in the early stages of life is examined. Three different strategies are considered: (1) random mating of bull sires with bull dams and with cows, with embryo selection of young bulls and all cows; (2) random mating of bull sires with bull dams, with embryo selection of young bulls only; (3) minimizing or avoiding matings between homozygotes for the unfavourable allele, with embryo selection of young bulls. Selection strategies assume the use of reproductive technologies such as embryo transfer to produce large family sizes for within-family selection to be practiced. All the three strategies increase the frequency of the favourable allele rapidly. Strategy 1 gives the fastest increase in the frequency of the favourable allele. The increase in the frequency of the favourable allele is slower under random mating (strategy 2) than under a negative assortative mating (strategy 3). This is a novel example of increased selection response with negative assortative mating.

Alleles↗

PubMed1990

Motion sickness: a synthesis and evaluation of the sensory conflict theory.

"Motion sickness" is the general term describing a group of common nausea syndromes originally attributed to motion-induced cerebral ischemia, stimulation of abdominal organ afferents, or overstimulation of the vestibular organs of the inner ear. Seasickness, car sickness, and airsickness are commonly experienced examples. However, the identification of other variants such as spectacle sickness and flight simulator sickness in which the physical motion of the head and body is normal or even absent has led to a succession of "sensory conflict" theories that offer a more comprehensive etiologic perspective. Implicit in the conflict theory is the hypothesis that neural and (or) humoral signals originate in regions of the brain subserving spatial orientation, and that these signals somehow traverse to other centers mediating sickness symptoms. Unfortunately, our present understanding of the neurophysiological basis of motion sickness is incomplete. No sensory conflict neuron or process has yet been physiologically identified. This paper reviews the types of stimuli that cause sickness and synthesizes a mathematical statement of the sensory conflict hypothesis based on observer theory from control engineering. A revised mathematical model is presented that describes the dynamic coupling between the putative conflict signals and nausea magnitude estimates. Based on the model, what properties would a conflict neuron be expected to have?

Animals↗

PubMed2005

Genetic classification of benign and malignant thyroid follicular neoplasia based on a three-gene combination.

Thyroid carcinoma is a common endocrine cancer with a favorable prognosis if subjected to timely treatment. However, the clinical identification of follicular thyroid carcinoma (FTC) among patients with benign thyroid nodules is still a challenge. Preoperative fine needle aspiration-based cytology cannot always differentiate follicular carcinomas from benign follicular neoplasias. Because current methods fail to improve preoperative diagnosis of thyroid nodules, new molecular-based diagnoses should be explored. We conducted a microarray-based study to reveal the genetic profiles unique to FTC and follicular adenomas (FAs), to identify the most parsimonious number of genes that could accurately differentiate between benign and malignant follicular thyroid neoplasia. We confirmed our data by quantitative RT-PCR and immunohistochemistry in two independent validation sets with a total of 114 samples. We were able to identify three genes, cyclin D2 (CCND2), protein convertase 2 (PCSK2), and prostate differentiation factor (PLAB), that allow the accurate molecular classification of FTC and FA. Two independent validation sets revealed that the combination of these three genes could differentiate FTC from FA with a sensitivity of 100%, specificity of 94.7%, and accuracy of 96.7%. In addition, our model allowed the identification of follicular variants of papillary thyroid carcinoma with an accuracy of 85.7%. Three-gene profiling of thyroid nodules can accurately predict the diagnosis of FTC and FA with high sensitivity and specificity, thus identifying promising targets for further investigation to ultimately improve preoperative diagnosis.

Adenocarcinoma, Follicular↗

PubMed1981

Kluver-Bucy syndrome in Pick disease: clinical and pathologic correlations.

The clinical and neuropathologic findings of five cases of Pick disease were studied. All had severe anterior temporal atrophy and abnormal neurons with highly argyrophilic cytoplasm of Pick bodies. The amygdala was abnormal in every case and had severe involvement of all nuclear subdivisions. Behaviorally, the patients exhibited elements of Kluver-Bucy syndrome early in the disease. Language abnormalities were also prominent. Memory and spatial orientation were frequently spared until late. Computed tomography (CT) demonstrated marked lobar atrophy in one of two patients examined. The early appearance of Kluver-Bucy syndrome and the late occurrence of amnesia and spatial disorientation allow clinical identification of this variant of Pick disease.

Aged↗

PubMed2006

Spurious hemoglobin Barts caused by bilirubin: a common interference mimicking an uncommon hemoglobinopathy.

High-performance liquid chromatography (HPLC) is replacing electrophoresis for identification of hemoglobin variants. Our objective was to identify unknown tall peaks with elution times and shapes of hemoglobin Barts found on hemoglobin chromatograms that could not be confirmed by alkaline and acid gel electrophoresis. Of 90 specimens identified with this peak, 86 were from patients with hemoglobin SS. Regression of the height of the unknown peaks to serum bilirubin concentrations, diminution of the unknown peaks by washing the specimens, and chromatographic similarity of a total bilirubin serum calibrator, a bilirubin proficiency testing specimen, and 3 patients' serum samples with markedly elevated bilirubin to hemoglobin Barts provide evidence the peak was bilirubin. We suggest exclusion of bilirubin before HPLC results are reported as consistent with hemoglobin Barts.

Adolescent↗

PubMed2005

Parkinson's disease: assays for the ubiquitin ligase activity of neural Parkin.

The identification of monogenic variants of Parkinson's disease (PD) has provided novel insights into its unknown pathogenesis. As the first protein linked to autosomal-recessive forms of PD, Parkin became a welcome tool to explain biochemical and neuropathological observations that had suggested involvement of the ubiquitin-proteasome system (UPS) in PD. Based on cellular expression studies and biochemical in vitro experiments, several researchers ascribed an E3-type, E2-dependent ubiquitin protein ligase activity to wild-type (but not mutant) Parkin proteins. Although the individual components of the proposed Parkin ubiquitin ligase complex in the normal human brain remain to be identified and the E3 ligase effect of Parkin function has not yet been confirmed in an animal model, the scientific exploration of a protein with several links to the UPS has provided many leads in PD research. This chapter describes assays that the authors have used to examine the cellular and in vitro effects of neural Parkin.

Animals↗

PubMed2003

Leprosy susceptibility revealed.

In order for these findings to have practical significance in terms of leprosy control and prevention, it will be necessary to extend the linkage of chromosome 6q25 to another region endemic for leprosy. Replicative findings would likely mean that the chromosome 6q25 susceptibility gene is a variant of a common gene that promotes susceptibility to infection per se. Identification of the gene variant will hopefully reveal insight about transmission and disease incidence--the longstanding enigmas of leprosy. Whether a more effective, universal MDT treatment or another type of prevention (either vaccine or environmental) could be based on this knowledge, is an exciting prospect to contemplate.

Chromosomes, Human, Pair 6↗

PubMed2000

Mutations in the insulin promoter factor-1 gene in late-onset type 2 diabetes mellitus.

OBJECTIVE: Insulin promoter factor-1 (IPF-1) is a transcription factor expressed in pancreatic beta cells. Following the identification of missense variants in the coding regions of the IPF-1 gene, in subjects selected for a strong family history of type 2 diabetes, the aim of our study was to evaluate the prevalence of these variants in the common form of type 2 diabetes. METHODS: Three variants (C18R, Q59L and D76N) were screened by PCR-RFLP in a group of 296 unrelated French late-onset type 2 diabetic subjects consecutively recruited in a diabetes department of a university hospital, regardless of family history of diabetes. RESULTS: The C18R and Q59L variants were each found in 0.37% of the diabetic patients, and in none of 147 controls. We did not detect the D76N variant, which was the most frequent variant in subjects with a strong family history of diabetes, in patients or controls. CONCLUSIONS: We have observed a combined prevalence of missense variants in the coding region of the IPF-1 gene of around 1%, in unselected patients with the common form of late-onset type 2 diabetes. The prevalence of these variants in subjects with a strong family history of type 2 diabetes had been found to be as high as approximately 6%. These differences in prevalence might be related to differences in the clinical profile of patients, such as age of onset of diabetes and associated obesity, as well as a family history of diabetes.

Age of Onset↗

PubMed2001

Molecular diagnosis of animal diseases: some experiences over the past decade.

The experiences of a veterinary research and diagnostic laboratory are summarized on the development and application of the PCR to diagnose a wide range of viral diseases in animals. The group started the routine diagnostic application of the PCR as early as 1988 and today a total of 35 nested PCR assays are in routine use for the detection of 15 DNA and 20 RNA viruses. Special tools and laboratory practice were applied to avoid false-positive results, while false-negatives are avoided by internal controls (mimics). At present, the classical nested PCR methods are being replaced by real-time TaqMan and molecular beacon assays and the multiplex real-time PCR detection of viruses is also under development. By direct sequencing of the PCR products, phylogeny studies are performed and molecular epizootiology results are provided for rapid and exact identification of virus variants. Molecular epizootiology also contributes to trace the routes of virus spreading on large geographic areas. Recently, large efforts have been made to follow the recommendations of Office International des Epizooties for the standardization and international harmonization of the molecular diagnostic assays.

Animal Diseases↗

PubMedDate not supplied

Comparative studies of Yersinia pestis outer membrane isolation techniques and their potential use in plaque epidemiology.

In the present study three techniques for obtaining outer membrane enriched fractions from Yersinia pestis were evaluated. The techniques analysed were: differential solubilization of the cytoplasmic membrane with Sarkosyl or Triton X-100, and centrifugation in sucrose density gradients. The sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of outer membrane isolated by the different methods resulted in similar protein patterns. The measurement of NADH-dehydrogenase and succinate dehydrogenase (inner membrane enzymes) indicated that the outer membrane preparations obtained by the three methods were pure enough for analytical studies. In addition, preliminary evidences on the potential use of outer membrane proteins for the identification of geographic variants of Y. pestis wild isolates are presented.

Bacterial Outer Membrane Proteins↗