Thinking outside the box: Aboriginal people's suggestions for conducting health studies with Aboriginal communities.

OBJECTIVES: Aboriginal people are under-represented in epidemiological research, largely due to past failures to engage and recruit Aboriginal communities, research fatigue and the use of culturally inappropriate methods. A qualitative study was undertaken in rural and urban Aboriginal communities in north-eastern and south-western Ontario to identify culturally congruent public health research methodologies. STUDY DESIGN: A qualitative participatory research study using focus group discussions. METHODS: This study employed a participatory research framework to elicit methodological suggestions for conducting public health research with Aboriginal communities during focus groups with healthcare providers from six diverse Aboriginal health organizations in Ontario, Canada. RESULTS: Continuing requests for participation in health research studies have led to community exhaustion. Discussions explored appropriate methods to obtain community approval and support for a study, the need for cultural sensitivity training for researchers, the value of conducting studies of interest and benefit to the community, advantages and disadvantages of qualitative and quantitative studies, the benefit of both Aboriginal and non-Aboriginal ethics reviews, the importance of safeguarding trusted information, types of incentives that may enhance study participation, suggestions to improve the collection of questionnaire information and biological specimens, how to resolve contentious issues and dissemination of study results. CONCLUSION: In order to successfully engage Aboriginal people in health studies, researchers need to build rapport with communities, have a community presence, be respectful and collaborative, utilize incentives, and employ flexible and adaptive methodologies of reasonable length. Oral interviews are preferred to self-completed information. The use of more mixed methods methodologies was suggested when quantitative data collection is necessary. Communities expect presentations about research findings.

Parentage testing of racing camels (Camelus dromedarius) using microsatellite DNA typing.

The camel racing industry would have added value in being able to assign parentage with high certainty. This study was aimed at assessing and applying microsatellite multiplexes to construct a parentage testing system for camels. An efficient system of 17 loci from 700 camel samples was used to construct a database of unrelated adults. Based on this, we estimated measures of polymorphism among the markers. In three multiplex reactions, we detected a total of 224 alleles, with 5­23 alleles/locus (mean = 13.18 ± 6.95 SD) and an average heterozygosity (HE) of 0.54 (range 0.032­0.905). The total parentage exclusion probability was 0.99999 for excluding a candidate parent from parentage of an arbitrary offspring, given only the genotype of the offspring, and 0.9999 for excluding a candidate parent from parentage of an arbitrary offspring, given the genotype of the offspring and the other parent. We used 15 juveniles for parentage testing, as well as 17 sires (bull camels) and 21 dams (cows). In the case of parentage assignment, the microsatellite panel assigned all 15 offspring parentage with high confidence. Overall, these findings offer a set of microsatellite markers that are easy, simple and highly informative for parentage testing in camels.

In situ stress testing to identify Australian black tiger prawns (Penaeus monodon) free of gill-associated virus and Mourilyan virus.

OBJECTIVE: To identify whether black tiger prawns (Penaeus monodon) in the Weipa region of the Gulf of Carpentaria, Queensland, are free of gill-associated virus (GAV) and Mourilyan virus (MoV), which are endemic in P. monodon along the east coast of Queensland. PROCEDURE: Preliminary screening suggested that Weipa might be a source of P. monodon that are free of GAV and MoV. To assess this, more than 150 prawns captured near Weipa were maintained locally in tanks for 2 weeks and bled three times as a stressor to promote higher-level infections. The existence of GAV and MoV in lymphoid organ tissue was then determined using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Some prawns were maintained in tanks for an additional 75 days before being tested. RESULTS: Real-time qRT-PCR did not detect GAV in any of 33 pools of RNA isolated from the 166 prawns tested. MoV was detected in five pools of RNA at extremely low viral RNA copy numbers close to the sensitivity threshold of the test. MoV was also detected at a similar low copy number in one of nine pearl-oyster mantle samples used as negative controls. CONCLUSIONS: GAV infection is either absent or, like MoV, potentially present at a very low prevalence in juvenile P. monodon inhabiting the inshore waters at Weipa. This region can thus be recommended as a good source of P. monodon certifiable as specific pathogen-free for GAV and MoV, which is desirable for domestication and selective breeding programs in Australia.

Development of polymorphic expressed sequence tag-derived microsatellites for the extension of the genetic linkage map of the black tiger shrimp (Penaeus monodon).

In this study, microsatellite markers were developed for the genetic linkage mapping and breeding program of the black tiger shrimp Penaeus monodon. A total of 997 unique microsatellite-containing expressed sequence tags (ESTs) were identified from 10 100 EST sequences in the P. monodon EST database. AT-rich microsatellite types were predominant in the EST sequences. Homology searching by the blastn and blastx programs revealed that these 997 ESTs represented 8.6% known gene products, 27.8% hypothetical proteins and 63.6% unknown gene products. Characterization of 50 markers on a panel of 35-48 unrelated shrimp indicated an average number of alleles of 12.6 and an average polymorphic information content of 0.723. These EST microsatellite markers along with 208 other markers (185 amplified fragment length polymorphisms, one exon-primed intron-crossing, six single strand conformation polymorphisms, one single nucleotide polymorphism, 13 non-EST-associated microsatellites and two EST-associated microsatellites) were analysed across the international P. monodon mapping family. A total of 144 new markers were added to the P. monodon maps, including 36 of the microsatellite-containing ESTs. The current P. monodon male and female linkage maps have 47 and 36 linkage groups respectively with coverage across half the P. monodon genome.

Predictors for non-adherence to antiretroviral therapy.

OBJECTIVES: To determine the risk factors for non-adherence to antiretroviral therapy. METHODS: Two hundred clients attending the Melbourne Sexual Health Centre completed a questionnaire about lifestyle, self-efficacy, depression, drug or alcohol use, social supports, and attitudes to health care. Self-reported adherence (SRA) was measured by missed doses in the last 4, 7 and 28 days. Routinely collected viral load levels were reviewed. RESULTS: Two hundred (85%) out of 231 eligible clients participated in the study. Viral load was most strongly associated with SRA for the last 28 days (P < 0.001). Non-adherence was defined as <98.2% SRA. Non-adherence was most strongly associated with having regular daily routines [odds ratio and 95% confidence interval = 0.4 (0.2, 0.7)], having set times for getting up and going to bed [0.5 (0.3, 1.0)], using marijuana more than 4 times per week [0.4 (0.2, 1.0)] and lower self-efficacy which included; being sure that you will be able to take medications as directed [0.2 (0.1, 0.6)] and being sure that missing doses of HIV medication will result in drug resistance [0.4 (0.2, 0.7)]. When significant questions were combined into a composite score to screen for non-adherence, the sensitivity to predict non-adherence was as high as 71% with a specificity of 59%. CONCLUSIONS: This study showed that a 10-min questionnaire was associated with clients past non-adherence to antiretroviral therapy and may be useful for predicting future adherence.

Structure-based design, synthesis and SAR of a novel series of thiopheneamidine urokinase plasminogen activator inhibitors.

The serine protease urokinase plasminogen activator (uPA) is thought to play a central role in tumor metastasis and angiogenesis. Molecular modeling studies suggest that 5-thiomethylthiopheneamidine inhibits uPA by binding at the S1 pocket of the active site. Further structure based elaboration of this residue resulted in a novel class of potent and selective inhibitors of uPA.

Synthesis of thiophene-2-carboxamidines containing 2-aminothiazoles and their biological evaluation as urokinase inhibitors.

The serine protease urokinase (uPa) has been implicated in the progression of both breast and prostate cancer. Utilizing structure based design, the synthesis of a series of substituted 4-[2-amino-1,3-thiazolyl]-thiophene-2-carboxamidines is described. Further optimization of this series by substitution of the terminal amine yielded urokinase inhibitors with excellent activities.

Research and development to improve naval shipboard waste management using a compact closed-loop-controlled waste incinerator.

Research has been conducted into the application of forced acoustics for enhancing the performance of a pyrolyzed waste afterburner configured as a dump combustor. Subscale studies showed that acoustic forcing of an air jet entering a dump chamber could trigger the formation of coherent vortices generated by entrainment of ambient gases. Subsequent studies showed that combustible gases could be introduced into the coherent vortices, and with additional modulation this configuration would lead to an enhanced combustion rate with low emissions of pollutants. The acoustically forced burner concept was scaled up to practical levels and tested as an afterburner on a commercial waste incinerator operating in pyrolysis mode. Results show that the afterburner can promote both compactness, due to the rapid combustion rate, and low pollutant emissions resulting from enhanced mixing prior to combustion.

Phylogenetic diversity of bacteria associated with the marine sponge Rhopaloeides odorabile.

Molecular techniques were employed to document the microbial diversity associated with the marine sponge Rhopaloeides odorabile. The phylogenetic affiliation of sponge-associated bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rDNA analysis. The community structure was extremely diverse with representatives of the Actinobacteria, low-G+C gram-positive bacteria, the beta- and gamma-subdivisions of the Proteobacteria, Cytophaga/Flavobacterium, green sulfur bacteria, green nonsulfur bacteria, planctomycetes, and other sequence types with no known close relatives. FISH probes revealed the spatial location of these bacteria within the sponge tissue, in some cases suggesting possible symbiotic functions. The high proportion of 16S rRNA sequences derived from novel actinomycetes is good evidence for the presence of an indigenous marine actinomycete assemblage in R. odorabile. High microbial diversity was inferred from low duplication of clones in a library with 70 representatives. Determining the phylogenetic affiliation of sponge-associated microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation conditions to target specific groups of well-represented bacteria for laboratory culture. Novel media incorporating sponge extracts were used to isolate bacteria not previously recovered from this sponge.

Down-regulation of poison ivy/oak-induced contact sensitivity by treatment with a class II MHC binding peptide:hapten conjugate.

Immune regulation of contact sensitivity to the poison ivy/oak catechol was studied at the level of class II MHC-restricted T cell recognition of hapten:peptide conjugates. In this study we have shown that 1) T cells from C3H/HeN (H-2k) mice, immunized with a synthetic I-Ak binding peptide coupled to 3-pentadecyl-catechol (PDC; a representative catechol in urushiol), recognized peptides derived from syngeneic cells linked to the same catechol; 2) T cells from draining lymph nodes of C3H/HeN mice skin-painted with PDC proliferated in response to a peptide carrier:PDC conjugate only when it was linked at the 7th, but not the 4th or the 10th, position on the peptide carrier; and 3) tolerization studies confirmed down-regulation of PDC-induced delayed-type hypersensitivity following treatment with a single I-Ak binding peptide carrying PDC covalently bound to a lysine residue at the middle (7th) TCR contact position. Tolerization with peptide:PDC conjugate resulted in abrogation of hapten-specific T cell proliferative responses that correlated with diminished IL-2 secretion. On the basis of these data we propose that it may be sufficient to couple the hapten at a single, well-chosen position on a carrier peptide to target a relevant population of T cells involved in contact sensitivity.

Effect of ovariectomy and steroid hormone replacement on the recovery of arterial blood pressure following haemorrhage in anaesthetized Brattleboro rats.

There is increasing evidence that ovarian steroids inhibit vascular responsiveness to the neurohypophysial hormone vasopressin. The present study examined the recovery of the arterial blood pressure following a single (2 ml/100 g body weight) haemorrhage in ovariectomized (OVX) Brattleboro rats with hereditary hypothalamic diabetes insipidus (BDI) and rats of the parent Long Evans (LE) strain. Some groups of OVX rats received subcutaneous implants of either 17 beta-oestradiol (E2) or progesterone 7 days prior to haemorrhage. The arterial blood pressure recovery immediately following haemorrhage was significantly impaired in both groups of steroid-treated OVX LE rats compared with the OVX controls (both comparisons P < 0.05). The impairment in blood pressure recovery seen in the steroid-replaced OVX LE rats was similar to that seen in pro-oestrous rats (when ovarian steroid levels are raised) compared with male rats of this strain (P < 0.05). In contrast, ovariectomy with or without steroid replacement in BDI rats had no further effect on the already attenuated recovery of arterial blood pressure after haemorrhage in this strain. Heart rate responses to haemorrhage also showed strain differences, which were dependent on steroid treatment. Pro-oestrous female LE rats showed a small decrease in heart rate after haemorrhage, followed by a recovery process, and this initial bradycardia was markedly enhanced in the OVX steroid-treated animals. In contrast, untreated OVX LE rats showed an initial and sustained increase in heart rate which was significantly higher than in the steroid-treated OVX animals (P < 0.05). All BDI rats, irrespective of treatment, consistently showed an increased heart rate after haemorrhage. In conclusion, ovarian steroid replacement in OVX LE, but not vasopressin-deficient BDI, rats was associated with an attenuated pressor recovery after haemorrhage. This provides further evidence for the existence of an important inhibitory interaction between ovarian steroids and vasopressin. The initial decrease in heart rate observed in pro-oestrous and steroid-treated OVX LE rats after haemorrhage also appears to be related to an ovarian steroid-vasopressin interaction.

Simultaneous detection of different Rhizobium strains marked with either the Escherichia coli gusA gene or the Pyrococcus furiosus celB gene.

A new marker system for gram-negative bacteria was developed on the basis of the celB gene from the hyperthermophilic archaeon Pyrococcus furiosus, which encodes a thermostable beta-glucosidase with a high level of beta-galactosidase activity. The celB gene is highly suitable as a marker for studying plant-bacterium interaction because endogenous background beta-glucosidase and beta-galactosidase enzyme activity can readily be inactivated by heat and because inexpensive substrates for detection are commercially available. Two celB-expressing transposons were constructed for use in ecological studies of a variety of gram-negative bacteria. The combined use of the gusA marker gene and celB allowed the simultaneous detection of several Rhizobium strains on a plant, and multiple-strain occupancy of individual modules also could be easily detected.

Diversity of Rhizobia Nodulating Phaseolus vulgaris L. in Two Kenyan Soils with Contrasting pHs.

Rhizobia were isolated from two Kenyan soils with pHs of 4.5 and 6.8 and characterized on the basis of their host ranges for nodulation and nitrogen fixation, colony morphologies, restriction fragment fingerprints, and hybridization with a nifH probe. The populations of rhizobia nodulating Phaseolus vulgaris in the two soils were similar in numbers and in effectiveness of N(inf2) fixation but were markedly different in composition. The population in the Naivasha soil (pH 6.8) was dominated by isolates specific in host range for nodulation to P. vulgaris; these all had multiple copies, in most cases four, of the structural nitrogenase gene nifH. Only one of the isolates from this soil formed effective nodules on Leucaena leucocephala, and this isolate had only a single copy of nifH. By contrast, the population in the acid Daka-ini soil (pH 4.5) was composed largely of broad-host-range isolates which had single copies of nifH. The isolates from the Daka-ini soil which were specific to P. vulgaris generally had three copies of nifH, although one isolate had only two copies. These rhizobial isolates are indigenous to Kenyan soils and yet have marked similarities to previously described Rhizobium species from other continents.

Differential stability of HLA-DR alleles independent of endogenous peptides.

Purified HLA DRB1*0101 was shown to be inherently more stable to dissociation than DRB1*0401. The residues responsible for the differential stability were defined by constructing hybrid molecules, which contained a small number of residues from DRB1*0101 substituted into the framework of DRB1*0401. One of the hybrid molecules, containing six substituted amino acids, was as stable as DRB1*0101, but exhibited the binding specificity of DRB1*0401. This result indicated that the differential stability between the alleles arose from structural differences, and was not due solely to varying populations of endogenous peptides.

beta-Glucuronidase (GUS) transposons for ecological and genetic studies of rhizobia and other gram-negative bacteria.

A series of transposons are described which contain the gusA gene, encoding beta-glucuronidase (GUS), expressed from a variety of promoters, both regulated and constitutive. The regulated promoters include the tac promoter which can be induced by IPTG, and nifH promoters which are symbiotically activated in legume nodules. One transposon contains gusA with a strong Shine-Dalgarno translation initiation context, but no promoter, and thus acts as a promoter-probe transposon. In addition, a gus operon deletion strain of Escherichia coli, and a transposon designed for use in chromosomal mapping using PFGE, are described. The GUS transposons are constructed in a mini-Tn5 system which can be transferred to Gram-negative bacteria by conjugation, and will form stable genomic insertions. Due to the absence of GUS activity in plants and many bacteria of economic importance, these transposons constitute powerful new tools for studying the ecology and population biology of bacteria in the environment and in association with plants, as well as for studies of the fundamental molecular basis of such interactions. The variety of assays available for GUS enable both quantitative assays and spatial localization of marked bacteria to be carried out.

Prediction of peptide affinity to HLA DRB1*0401.

A method to predict quantitatively peptide binding to HLA DRB1*0401 has been developed using a data set of the relative contributions of each of the naturally occurring amino acids in the context of a simplified peptide back-bone. The prediction assumed that the relative role of each of the peptide side chains could be treated independently and could be measured by assaying each of the 20 naturally occurring amino acids at the central 11 positions of a 13-residue peptide previously shown to contain the minimal requirements for high-affinity binding to HLA-DR proteins. The resultant database was shown to have predictive value when tested on a set of 13 unrelated peptides known to bind DRB1*0401 with a wide range of apparent affinity. The database was tested further by analyzing myelin basic protein. All 13 amino acid peptides containing a hydrophobic amino acid at the third position were synthesized and assayed for binding purified DRB1*0401. In every case, the measured affinity correlated with the predictive values within the experimental error of the assays. Finally, the ability to predict peptide binding to MHC class II molecules was shown to help in identifying T cell determinants. The specificity of DRB1*0401-restricted T cell hybridomas against human serum albumin corresponded to two peptides, predicted and shown to bind the class II protein with high affinity.

Specific T cell recognition of minimally homologous peptides: evidence for multiple endogenous ligands.

The T cell receptor (TCR) can interact with a spectrum of peptides as part of its ligand, including the immunogenic peptide, variants of this peptide,and apparently unrelated peptides. The basis of this broad specificity for ligand was investigated by substitution analysis of a peptide antigen and functional testing using a B cell apoptosis assay. A peptide containing as few as 1 aa in common with this peptide could stimulate a specific T cell response. Two endogenous ligands, an agonist and a partial agonist, were readily identified from a search of the SwissProt database, indicating that multiple endogenous ligands likely exist for a given T cell. These findings strongly support the concept that one TCR has the ability to interact productively with multiple different ligands, and provide evidence that such ligands exist in the endogenous peptide repertoire.

Characterization of epitopes recognized by hapten-specific CD4+ T cells.

Although protein-derived nominal Ags have, in many instances, been precisely determined, the epitopes recognized by hapten-specific CD4+ T cells responsible for contact sensitization have not been defined. To better understand the nature of the precise epitopes generated after hapten interaction with Langerhans cells (LC), we assessed the ability of TNP-modified I-Ak- and I-Au-binding peptides to activate hapten-specific CD4+ T cells obtained respectively from TNCB-primed C3H (H-2k) and PL/j (H-2u) mice. Using LC as APC, I-Ak-restricted TNP-specific CD4+ T cells proliferated in the presence of the synthetic peptide hen egg lysozyme 52-61 derivatized with TNP at position 56, and less so when TNP was coupled at positions 53 or 59. Similarly, I-Au-restricted TNP-specific CD4+ T cells from PL/j mice were triggered by the synthetic I-Au-binding 13 mer poly(A)-Y5-R6 TNP-modified at position 4, and to a limited extent with TNP coupled in positions 7 or 10. Our results indicate that hapten-modified MHC class II binding nonautologous peptides are recognized by hapten-specific CD4+ T cells and that precise positioning of hapten molecules on peptides binding MHC class II molecules is required for optimal CD4+ T cell recognition. These findings provide insight into the manner in which haptens are recognized by T cells involved in contact sensitivity and should facilitate the study and design of specific therapies for the manipulation of hapten-specific CD4+ T cell responses.

Prediction of peptide affinity to HLA DR molecules.

A method to quantitatively predict peptide binding to HLA DRB1*0401, B1*0101, and B1*1501 has been developed using a dataset of the relative contributions of each of the naturally occurring amino acids in the context of a simplified peptide backbone. The prediction assumed that the relative role of each of the peptide sidechains could be treated independently and could be measured by assaying each of the twenty naturally occurring amino acids at the central eleven positions of a 13 residue peptide previously shown to contain the minimal requirements for high affinity binding to HLA DR proteins. Three separate databases were generated. They were shown to have predictive value when tested on a set of 13 unrelated peptides known to bind the DR proteins with a wide range of apparent affinity. The DRB1*0401 database was tested further by analyzing myelin basic protein. All 13 amino acid peptides containing a hydrophobic amino acid at the third position were synthesized and assayed for binding purified DRB1*0401. In every case, the measured affinity correlated with the predictive values within the experimental error of the assays. Finally, the ability to predict peptide binding to MHC class II molecules was shown to help in identifying T cell determinants. The specificity of DRB1*0401 restricted T cell hybridomas against human serum albumin corresponded to two peptides, predicted, and shown to bind the class II protein with high affinity.