Flaviviruses isolated from mosquitoes collected during the first recorded outbreak of Japanese encephalitis virus on Cape York Peninsula, Australia

In response to an outbreak of Japanese encephalitis (JE) virus on Cape York Peninsula, Australia, in 1998, mosquitoes were collected using CO2 and octenol-baited Centers for Disease Control and Prevention light traps. A total of 35,235 adult mosquitoes, comprising 31 species, were processed for virus isolation. No isolates of JE virus were recovered from these mosquitoes. However, 18 isolates of Kokobera virus, another flavivirus were obtained from Culex annulirostris. Twelve isolates were from western Cape York (minimum infection rate (MIR) of 0.61: 1,000 mosquitoes) and 6 were from the Northern Peninsula Area (MIR of 1.0:1,000). Potential explanations for the failure to detect JE virus in mosquitoes collected from Cape York Peninsula include the timing of collections, the presence of alternative bloodmeal hosts, differences in pig husbandry, asynchronous porcine seroconversion, and the presence of other flaviviruses.

Development of ligase-assisted spacer addition for the measurement of microsatellites

Conventional methods for detecting differences in microsatellite repeat lengths rely on electrophoretic fractionation on long denaturing polyacrylamide gels, a time-consuming and labor-intensive method. Therefore, there is a need for the development of new and rapid approaches to routinely detect such length polymorphisms. The advent of techniques allowing the coupling of DNA molecules to solid surfaces has provided new prospects in the area of mutation. We describe here the development and optimization of the ligase-assisted spacer addition (LASA) method, a novel and rapid procedure based on an ELISA format to measure microsatellite repeat lengths. The LASA assay was successfully applied to a set of 11 bird samples to assess its capability as a genotyping method.

Phylogenetic relationships of Trypanosoma chelodina and Trypanosoma binneyi from Australian tortoises and platypuses inferred from small subunit rRNA analyses

Trypanosome infections are often difficult to detect by conventional microscopy and their pleomorphy often confounds differential diagnosis. Molecular techniques are now being used to diagnose infections and to determine phylogenetic relationships between species. Complete small subunit rRNA gene sequences were determined for isolates of Trypanosoma chelodina from the Brisbane River tortoise (Emydura signata), the saw-shelled tortoise (Elseya latisternum), and the eastern snake-necked tortoise (Chelodina longicollis) from southeast Queensland, Australia. Partial sequence data were also obtained for T. binneyi from a platypus (Ornithorhynchus anatinus) from Tasmania. Phylogenetic relationships between T. chelodina, T. binneyi and other species were examined by maximum parsimony and likelihood methods. The Australian tortoise and platypus trypanosomes did not exhibit any close phylogenetic relationships with those of mammals, reptiles or amphibians, but were closely related to each other, and to fish trypanosomes. This contra-indicates their co-evolution with their vertebrate hosts but does not exclude co-evolution with different groups of invertebrate vectors, notably insects and leeches.

Genetics of brain function and cognition

There is overwhelming evidence for the existence of substantial genetic influences on individual differences in general and specific cognitive abilities, especially in adults. The actual localization and identification of genes underlying variation in cognitive abilities and intelligence has only just started, however. Successes are currently limited to neurological mutations with rather severe cognitive effects. The current approaches to trace genes responsible for variation in the normal ranges of cognitive ability consist of large scale linkage and association studies. These are hampered by the usual problems of low statistical power to detect quantitative trait loci (QTLs) of small effect. One strategy to boost the power of genomic searches is to employ endophenotypes of cognition derived from the booming field of cognitive neuroscience This special issue of Behavior Genetics reports on one of the first genome-wide association studies for general IQ. A second paper summarizes candidate genes for cognition, based on animal studies. A series of papers then introduces two additional levels of analysis in the ldquoblack boxrdquo between genes and cognitive ability: (1) behavioral measures of information-processing speed (inspection time, reaction time, rapid naming) and working memory capacity (performance on on single or dual tasks of verbal and spatio-visual working memory), and (2) electrophyiosological derived measures of brain function (e.g., event-related potentials). The obvious way to assess the reliability and validity of these endophenotypes and their usefulness in the search for cognitive ability genes is through the examination of their genetic architecture in twin family studies. Papers in this special issue show that much of the association between intelligence and speed-of-information processing/brain function is due to a common gene or set of genes, and thereby demonstrate the usefulness of considering these measures in gene-hunting studies for IQ.

Genetics of cognition: Outline of a collaborative twin study

Amultidisciplinary collaborative study examining cognition in a large sample of twins is outlined. A common experimental protocol and design is used in The Netherlands, Australia and Japan to measure cognitive ability using traditional IQ measures (i.e., psychometric IQ), processing speed (e.g., reaction time [RT] and inspection time [IT]), and working memory (e.g., spatial span, delayed response [DR] performance). The main aim is to investigate the genetic covariation among these cognitive phenotypes in order to use the correlated biological markers in future linkage and association analyses to detect quantitativetrait loci (QTLs). We outline the study and methodology, and report results from our preliminary analyses that examines the heritability of processing speed and working memory indices, and their phenotypic correlation with IQ. Heritability of Full Scale IQ was 87% in the Netherlands, 83% in Australia, and 71% in Japan. Heritability estimates for processing speed and working memory indices ranged from 33–64%. Associations of IQ with RT and IT (−0.28 to −0.36) replicated previous findings with those of higher cognitive ability showing faster speed of processing. Similarly, significant correlations were indicated between IQ and the spatial span working memory task (storage [0.31], executive processing [0.37]) and the DR working memory task (0.25), with those of higher cognitive ability showing better memory performance. These analyses establish the heritability of the processing speed and working memory measures to be used in our collaborative twin study of cognition, and support the findings that individual differences in processing speed and working memory may underlie individual differences in psychometric IQ.

Outcomes of cephalosporin treatment for serious infection due to apparently susceptible organisms producing extended-spectrum b-Lactamases: Implications for the clinical microbiology laboratory

Although extended-spectrum beta-lactamases (ESBLs) hydrolyze cephalosporin antibiotics, some ESBL-producing organisms are not resistant to all cephalosporins when tested in vitro. Some authors have suggested that screening klebsiellae or Escherichia coli for ESBL production is not clinically necessary, and when most recently surveyed the majority of American clinical microbiology laboratories did not make efforts to detect ESBLs, We performed a prospective, multinational study of Klebsiella pneumoniae bacteremia and identified 10 patients who were treated for ESBL-producing K. pneumoniae bacteremia with cephalosporins and whose infecting organisms were not resistant in vitro to the utilized cephalosporin. In addition, we reviewed 26 similar cases of severe infections which had previously been reported. Of these 36 patients, 4 had to be excluded from analysis. Of the remaining 32 patients, 100% (4 of 4) patients experienced clinical failure when MICs of the cephalosporin used for treatment were in the intermediate range and 54% (15 of 28) experienced failure when MICs of the cephalosporin used for treatment were in the susceptible range, Thus, it is clinically important to detect ESBL production by klebsiellae or E, coli even when cephalosporin MICs are in the susceptible range (less than or equal to 8 mug/ml) and to report ESBL-producing organisms as resistant to aztreonam and all cephalosporins (with the exception of cephamycins).

The reliability of immunohistochemistry as a prescreening method for the diagnosis of hereditary nonpolyposis colorectal cancer (HNPCC) - Results of an international collaborative study

Hereditary nonpolyposis colorectal cancer syndrome (HNPCC) is an autosomal dominant condition accounting for 2–5% of all colorectal carcinomas as well as a small subset of endometrial, upper urinary tract and other gastrointestinal cancers. An assay to detect the underlying defect in HNPCC, inactivation of a DNA mismatch repair enzyme, would be useful in identifying HNPCC probands. Monoclonal antibodies against hMLH1 and hMSH2, two DNA mismatch repair proteins which account for most HNPCC cancers, are commercially available. This study sought to investigate the potential utility of these antibodies in determining the expression status of these proteins in paraffin-embedded formalin-fixed tissue and to identify key technical protocol components associated with successful staining. A set of 20 colorectal carcinoma cases of known hMLH1 and hMSH2 mutation and expression status underwent immunoperoxidase staining at multiple institutions, each of which used their own technical protocol. Staining for hMSH2 was successful in most laboratories while staining for hMLH1 proved problematic in multiple labs. However, a significant minority of laboratories demonstrated excellent results including high discriminatory power with both monoclonal antibodies. These laboratories appropriately identified hMLH1 or hMSH2 inactivation with high sensitivity and specificity. The key protocol point associated with successful staining was an antigen retrieval step involving heat treatment and either EDTA or citrate buffer. This study demonstrates the potential utility of immunohistochemistry in detecting HNPCC probands and identifies key technical components for successful staining.

On surrogate methods for detecting lateral gene transfer

Surrogate methods for detecting lateral gene transfer are those that do not require inference of phylogenetic trees. Herein I apply four such methods to identify open reading frames (ORFs) in the genome of Escherichia coli K12 that may have arisen by lateral gene transfer. Only two of these methods detect the same ORFs more frequently than expected by chance, whereas several intersections contain many fewer ORFs than expected. Each of the four methods detects a different non-random set of ORFs. The methods may detect lateral ORFs of different relative ages; testing this hypothesis will require rigorous inference of trees. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science BN. All rights reserved.

Quantitative PCR-ELAHA for the determination of retroviral vector transduction efficiency

Current methods to detect transduction efficiency during the routine use of integrating retroviral vectors in gene therapy applications may require the use of radioactivity and usually rely upon subjective determination of the results. We have developed two competitive quantitative assays that use an enzyme-linked, amplicon hybridization assay (ELAHA) to detect the products of PCR-amplified regions of transgene from cells transduced with Moloney murine leukemia virus vectors. The quantitative assays (PCR-ELAHA) proved to be simple, rapid, and sensitive, avoiding the need for Southern hybridization, complex histochemical stains, or often subjective and time-consuming tissue culture and immunofluorescence assays. The PCR-ELAHA systems can rapidly detect proviral DNA from any retroviral vector carrying the common selective and marker genes neomycin phosphotransferase and green fluorescent protein, and the methods described are equally applicable to other sequences of interest, providing a cheaper alternative to the evolving real-time PCR methods. The results revealed the number of copies of retrovector provirus present per stably transduced cell using vectors containing either one or both qPCR targets.

Primary aldosteronism: Are we diagnosing and operating on too few patients?

Many cases of potentially curable primary aldosteronism are currently likely to be diagnosed as essential hypertension unless screening tests based on suppression of renin are tarried out in all hypertensive patients. More than half of the patients with primary aldosteronism detected in this way have normal circulating potassium levels, so measurement of potassium is not enough to exclude primary aldosteronism. When primary aldosteronism is diagnosed, fewer than one-third of patients are suitable for surgery as initial treatment, but this still represents a significant percentage of hypertensive patients. After excluding glucocorticoid-suppressible primary aldosteronism, adrenal venous sampling is essential to detect unilateral production of aldosterone and diagnose angiotensin-responsive aldosterone-producing adenoma. One cannot rely on the computed tomography scan. If all hypertensive patients are screened for primary aldosteronism and the workup is continued methodically in those with a positive screening test, patients with unilateral overproduction of aldosterone who potentially can be cured surgically are not denied the possibility of cure.

Olfaction in the Queensland fruit fly, Bactrocera tryoni. II: Response spectra and temporal encoding characteristics of the carbon dioxide receptors

Single-unit electrophysiology was used to record the nerve impulses from the carbon dioxide receptors of female Queensland fruit flies, Bactrocera tryoni. The receptors responded to stimulation in a phasic-tonic manner and also had a period of inhibition of the nerve impulses after the end of stimulation, at high stimulus intensities. The cell responding to carbon dioxide was presented with a range of environmental odorants and found to respond to methyl butyrate and 2-butanone. The coding characteristics of the carbon dioxide cell and the ability to detect other odorants are discussed, with particular reference to the known behavior of the fly.

Power of the joint segregation analysis method for testing mixed major-gene and polygene inheritance models of quantitative traits

Understanding the genetic architecture of quantitative traits can greatly assist the design of strategies for their manipulation in plant-breeding programs. For a number of traits, genetic variation can be the result of segregation of a few major genes and many polygenes (minor genes). The joint segregation analysis (JSA) is a maximum-likelihood approach for fitting segregation models through the simultaneous use of phenotypic information from multiple generations. Our objective in this paper was to use computer simulation to quantify the power of the JSA method for testing the mixed-inheritance model for quantitative traits when it was applied to the six basic generations: both parents (P-1 and P-2), F-1, F-2, and both backcross generations (B-1 and B-2) derived from crossing the F-1 to each parent. A total of 1968 genetic model-experiment scenarios were considered in the simulation study to quantify the power of the method. Factors that interacted to influence the power of the JSA method to correctly detect genetic models were: (1) whether there were one or two major genes in combination with polygenes, (2) the heritability of the major genes and polygenes, (3) the level of dispersion of the major genes and polygenes between the two parents, and (4) the number of individuals examined in each generation (population size). The greatest levels of power were observed for the genetic models defined with simple inheritance; e.g., the power was greater than 90% for the one major gene model, regardless of the population size and major-gene heritability. Lower levels of power were observed for the genetic models with complex inheritance (major genes and polygenes), low heritability, small population sizes and a large dispersion of favourable genes among the two parents; e.g., the power was less than 5% for the two major-gene model with a heritability value of 0.3 and population sizes of 100 individuals. The JSA methodology was then applied to a previously studied sorghum data-set to investigate the genetic control of the putative drought resistance-trait osmotic adjustment in three crosses. The previous study concluded that there were two major genes segregating for osmotic adjustment in the three crosses. Application of the JSA method resulted in a change in the proposed genetic model. The presence of the two major genes was confirmed with the addition of an unspecified number of polygenes.

Granulocyte-colony-stimulating factor (G-CSF)-primed allogeneic bone marrow: significantly less graft-versus-host disease and comparable engraftment to G-CSF-mobilized peripheral blood stem cells

Prospective studies have shown rapid engraftment using granulocyte-colony-stimulating factor-mobilized peripheral blood stem cells (G-PBSCs) for allogeneic transplantation, though the risks for graft-versus-host disease (GVHD) may be increased. It was hypothesized that the use of G-CSF to prime bone marrow (GBM) would allow rapid engraftment without increased risk for GVHD compared with G-PBSC. Patients were randomized to receive G-BM or G-PBSCs for allogeneic stem cell transplantation. The study was designed (beta < .8) to detect a difference in the incidence of chronic GVHD of 33% ( < .05). The plan was to recruit 100 patients and to conduct an interim analysis when the 6-month follow-up point was reached for the first 50 patients. Fifty-seven consecutive patients were recruited (G-BM, n = 28; G-PBSC, n = 29). Patients in the G-PBSC group received 3-fold more CD34(+) and 9-fold more CD3(+) cells. Median times to neutrophil (G-BM, 16 days; G-PBSC, 14 days; P < .1) and platelet engraftment (G-BM, 14 days; G-PBSC, 12 days; P < .1) were similar. The use of G-PBSC was associated with steroid refractory acute GVHD (G-BM, 0%; G-PBSC, 32%; P < .001), chronic GVHD (G-BM, 22%; G-PBSC, 80%; P < .02), and prolonged requirement for immunosuppressive therapy (G-BM, 173 days; G-PBSC, 680 days; P < .009). Survival was similar for the 2 groups. Compared with G-PBSC the use of G-BM resulted in comparable engraftment, reduced severity of acute GVHD, and less subsequent chronic GVHD. (Blood. 2001;98:3186-3191) (C) 2001 by The American Society of Hematology.

The use of perineal ultrasound to quantify levator activity and teach pelvic floor muscle exercises

Perineal ultrasound was used to detect and quantify levator activity by measuring the displacement of the internal urethral meatus against the inferoposterior margin of the symphysis pubis, Women who had previously been instructed in pelvic floor muscle exercises were more likely to contract the levator muscle when asked to do so than were those without previous instruction (P

Detection of dengue viral RNA in Aedes aegypti (Diptera : Culicidae) exposed to sticky lures using reverse-transcriptase polymerase chain reaction

Active surveillance for dengue (DEN) virus infected mosquitoes can be an effective way to predict the risk of dengue infection in a given area. However, doing so may pose logistical problems if mosquitoes must be kept alive or frozen fresh to detect DEN virus. In an attempt to simplify mosquito processing, we evaluated the usefulness of a sticky lure and a seminested reverse-transcriptase polymerase chain reaction assay (RT-PCR) for detecting DEN virus RNA under laboratory conditions using experimentally infected Aedes aegypti (L.) mosquitoes. In the first experiment, 40 male mosquitoes were inoculated with 0.13 mul of a 10(4) pfu/ml DEN-2 stock solution. After a 7-d incubation period, the mosquitoes were applied to the sticky lure and kept at room temperatures of 23-30 degreesC. Following 7,10,14, and 28 d application, 10 mosquitoes each were removed from the lure pooled and assayed for virus. DEN virus nucleic acid was clearly detectable in all pools up to 28 d after death. A second study evaluated sensitivity and specificity using one, two, and five DEN-infected mosquitoes removed after 7, 10, 14, 21 and 30 d application and tested by RT-PCR. All four DEN serotypes were individually inoculated in mosquitoes and evaluated using the same procedures as experiment 1. The four serotypes were detectable in as few as one mosquito 30 d after application to the lure with no evidence of cross-reactivity. The combination of sticky lures and RT-PCR show promise for mosquito and dengue virus surveillance and warrant further evaluation.