Patterns of community integration 2-5 years post-discharge from brain injury rehabilitation

Outcome after traumatic brain injury (TBI) is characterized by a high degree of variability which has often been difficult to capture in traditional outcome studies. The purpose of this study was to describe patterns of community integration 2-5 years after TBI. Participants were 208 patients admitted to a Brain Injury Rehabilitation Unit between 1991-1995 in Brisbane, Australia. The design comprised retrospective data collection and questionnaire follow-up by mail. Mean follow-up was 3.5 years. Demographic, injury severity and functional status variables were retrieved from hospital records. Community integration was assessed using the Community Integration Questionnaire (CIQ), and vocational status measured by a self administered questionnaire. Data was analysed using cluster analysis which divided the data into meaningful subsets. Based on the CIQ subscale scores of home, social and productive integration, a three cluster solution was selected, with groups labelled as working (n = 78), balanced (n = 46) and poorly integrated (n = 84). Although 38% of the sample returned to a high level of productive activity and 22% achieved a balanced lifestyle, overall community integration was poor for the remainder. This poorly integrated group had more severe injury characterized by longer periods of acute care and post-traumatic amnesia (PTA) and greater functional disability on discharge. These findings have implications for service delivery prior to and during the process of reintegration after brain injury.

Recent advances in our knowledge of the Myxozoa

In the last few years two factors have helped to significantly advance our understanding of the Myxozoa. First, the phenomenal increase in fin fish aquaculture in the 1990s has lead to the increased importance of these parasites; in rum this has lead to intensified research efforts, which have increased knowledge of the development, diagnosis, and pathogenesis of myxozoans. The hallmark discovery in the 1980s that the life cycle of Myxobolus cerebralis requires development of an actinosporean stage in the Oligochaete. Tubifex tubifex, led to the elucidation of the life cycles of several other myxozoans. Also, the life cycle and taxonomy of the enigmatic PKX myxozoan has been resolved: it is the alternate stage of the unusual myxozoan. Tetracapsula bryosalmonae, from bryozoans. The 18S rDNA gene of many species has been sequenced, and here we add 22 new sequences to the data set. Phylogenetic analyses using all these sequences indicate that: 1) the Myxozoa are closely related to Cnidaria (also supported by morphological data), 2) marine taxa at the genus level branch separately from genera that usually infect freshwater fishes; 3) taxa cluster more by development and tissue location than by spore morphology; 4) the tetracapsulids branched off early in myxozoan evolution, perhaps reflected by their having bryozoan. rather than annelid hosts; 5) the morphology of actinosporeans offers little information for determining their myxosporean counterparts (assuming that they exist), and 6) the marine actinosporeans from Australia appear to form a clade within the platysporinid myxosporeans. Ribosomal DNA sequences have also enabled development of diagnostic tests for myxozoans. PCR and in situ hybridisation tests based on rDNA sequences have been developed for Myxobolus cerebralis. Ceratomyxa shasta. Kudoa spp,, and Tetracapsula bryosalmonae (PKX). Lectin-based and antibody tests have also been developed for certain myxozoans, such as PKX and C. shasta. We also review important diseases caused by myxozoans. which are emerging or re-emerging. Epizootics of whirling disease in wild rainbow trout (Oncorhynchus mykiss) have recently been reported throughout the Rocky Mountain states of the USA. With a dramatic increase in aquaculture of fishes using marine netpens, several marine myxozoans have been recognized or elevated in status as pathological agents. Kudoa thyrsites infections have caused severe post-harvest myoliquefaction in pen-reared Atlantic salmon (Salmo salar), and Ceratomyxa spp., Sphaerospora spp., and Myxidium leei cause disease in pen-reared sea bass (Dicentrarchus labrax) and sea bream species (family Sparidae) in Mediterranean countries.

Evidence for three tumor suppressor loci on chromosome 9p involved in melanoma development

Cytogenetic and loss of heterozygosity (LOH) studies have long indicated the presence of a tumor suppressor gene (TSG) on 90 involved in the development of melanoma, Although LOH at 90 has been reported in approximately 60% of melanoma tumors, only 5-10% of these tumors have been shown to carry CDKN2A mutations, raising the possibility that another TSG involved in melanoma maps to chromosome 90. To investigate this possibility, a panel of 37 melanomas derived from 35 individuals was analyzed for CDKN2A mutations hy single-strand conformation polymorphism analysis and sequencing. The melanoma samples were then typed for 15 markers that map to 9p13-24 to investigate LOH trends in this region. In those tumors demonstrating retention of heterozygosity at markers flanking CDKN2A and LOH on one or both sides of the gene, multiplex microsatellite PCR was performed to rule out homozygous deletion of the region encompassing CDKN2A. CDKN2A mutations were found in tumors from 5 patients [5 (14%) of 35], 4 of which demonstrated LOH across the entire region examined. The remaining tumor with no observed LOH carried two point mutations, one on each allele, Although LOH was identified at one or more markers in 22 (59%) of 37 melanoma tumors corresponding to 20 (57%) of 35 individuals, only 11 tumors from 9 individuals [9 (26%) of 35] demonstrated LOH at D9S942 and D9S1748, the markers closest to CDKN2A. Of the remaining 11 tumors with LOH, 9 demonstrated LOH at two or more contiguous markers either centromeric and/or telomeric to CDKN2A while retaining heterozygosity at several markers adjacent to CDKN2A. Multiplex PCR revealed one tumor carried a homozygous deletion extending from D9S1748 to the IFN-alpha locus. In the remaining eight tumors, multiplex PCR demonstrated that the observed heterozygosity was not attributable to homozygous deletion and stromal contamination at D9S1748, D9S942, or D9S974, as measured by comparative amplification strengths, which indicates that retention of heterozygosity with flanking LOH does not always indicate a homozygous deletion, This report supports the conclusions of previous studies that at least two TSGs involved in melanoma development in addition to CDKN2A may reside on chromosome 9p.

A single nucleotide polymorphism in the 5' untranslated region of RAD51 and risk of cancer among BRCA1/2 mutation carriers

RAD51 colocalizes with both BRCA1 and BRCA2, and genetic variants in RAD51 would be candidate BRCA1/2 modifiers. We searched for RAD51 polymorphisms by sequencing 20 individuals. We compared the polymorphism allele frequencies between female BRCA1/2 mutation carriers with and without breast or ovarian cancer and between population-based ovarian cancer cases with BRCA1/2 mutations to cases and controls without mutations. We discovered two single nucleotide polymorphisms (SNPs) at positions 135 g-->c and 172 g-->t of the 5' untranslated region. In an initial group of BRCA1/2 mutation carriers, 14 (21%) of 67 breast cancer cases carried a c allele at RAD51:135 g-->c, whereas 8 (7%) of 119 women without breast cancer carried this allele. In a second set of 466 mutation carriers from three centers, the association of RAD51:135 g-->c with breast cancer risk was not confirmed. Analyses restricted to the 216 BRCA2 mutation carriers, however, showed a statistically significant association of the 135 c allele with the risk of breast cancer (adjusted odds ratio, 3.2; 95% confidence limit, 1.4-40). BRCA1/2 mutation carriers with ovarian cancer were only about one half as likely to carry the RAD51:135 g-->c SNP. Analysis of the RAD51:135 g-->c SNP in 738 subjects from an Israeli ovarian cancer case-control study was consistent with a lower risk of ovarian cancer among BRCA1/2 mutation carriers with the c allele. We have identified a RAD51 5' untranslated region SNP that may be associated with an increased risk of breast cancer and a lower risk of ovarian cancer among BRCA2 mutation carriers. The biochemical basis of this risk modifier is currently unknown.

Post-transcriptional regulation of the GLI1 oncogene by the expression of alternative 5 ' untranslated regions

The oncogene GLI1 is involved in the formation of basal cell carcinoma and other tumor types as a result of the aberrant signaling of the Sonic hedgehog-Patched pathway. In this study, we have identified alternative GLI1 transcripts that differ in their 5' untranslated regions (UTRs) and are generated by exon skipping. These are denoted (alpha -UTR, beta -UTR, and gamma -UTR according to the number of noncoding exons possessed (three, two, and one, respectively). The alpha- and beta -UTR forms represent the major Gli1 transcripts expressed in mouse tissues, whereas the gamma -UTR is present at relatively low levels but is markedly induced in mouse skin treated with 12-O-tetradecanoylphorbol 13-acetate, Transcripts corresponding to the murine beta and gamma forms were identified in human tissues, but significantly, only the gamma -UTR form was present in basal cell carcinomas and in proliferating cultures of a keratinocyte cell line. Flow cytometry analysis determined that the gamma -UTR variant expresses a heterologous reporter gene 14-23-fold higher than the alpha -UTR and 5-13-fold higher than the beta -UTR in a variety of cell types. Because expression of the gamma -UTR variant correlates with proliferation, consistent with a role for GLI1 in growth promotion, up-regulation of GLI1 expression through skipping of 5' noncoding exons may be an important tumorigenic mechanism.

Development of a real-time RT-PCR assay for plasma membrane calcium ATPase isoform 1 (PMCA1) mRNA levels in a human breast epithelial cell line.

The plasma membrane Ca2+ pump is a key regulator of cytosolic free Ca2+. Recent studies have demonstrated the dynamic expression of the plasma membrane Ca2+ pump in a variety of cell types. Furthermore, alterations in plasma membrane calcium pump activity have now been implicated in human disease. In this study, the development of a technique to quantitatively assess mRNA expression of the human plasma membrane Ca2+ ATPase (PMCA1) isoform of the plasma membrane Ca2+ pump, using a real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) assay in a human breast epithelial cell line (MCF-7) is described. The sequences of the PMCA1 primers and probe for real-time RT-PCR are presented. The results also indicate that PMCA1 mRNA can be normalized to both 18S ribosomal RNA (18S rRNA) and human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) in MCF-7 cells. Real-time RT-PCR will be most useful in assessing PMCA1 mRNA expression in cases where only low amounts of RNA are available and/or when numerous samples must be assessed simultaneously. (C) 2001 Elsevier Science Inc. All rights reserved.

A DNA fingerprinting procedure for ultra high-throughput genetic analysis of insects

Existing procedures for the generation of polymorphic DNA markers are not optimal for insect studies in which the organisms are often tiny and background molecular Information is often non-existent. We have used a new high throughput DNA marker generation protocol called randomly amplified DNA fingerprints (RAF) to analyse the genetic variability In three separate strains of the stored grain pest, Rhyzopertha dominica. This protocol is quick, robust and reliable even though it requires minimal sample preparation, minute amounts of DNA and no prior molecular analysis of the organism. Arbitrarily selected oligonucleotide primers routinely produced similar to 50 scoreable polymorphic DNA markers, between individuals of three Independent field isolates of R. dominica. Multivariate cluster analysis using forty-nine arbitrarily selected polymorphisms generated from a single primer reliably separated individuals into three clades corresponding to their geographical origin. The resulting clades were quite distinct, with an average genetic difference of 37.5 +/- 6.0% between clades and of 21.0 +/- 7.1% between individuals within clades. As a prelude to future gene mapping efforts, we have also assessed the performance of RAF under conditions commonly used in gene mapping. In this analysis, fingerprints from pooled DNA samples accurately and reproducibly reflected RAF profiles obtained from Individual DNA samples that had been combined to create the bulked samples.

Detection and differentiation of human polyomaviruses JC and BK by LightCycler PCR

Human polyomaviruses JC and BK may cause several clinical manifestations in immunocompromised hosts, including progressive multifocal leukoencephalopathy and hemorrhagic cystitis. Molecular detection by PCR is recognized as a sensitive and specific method for detecting human polyomaviruses in clinical samples. In this study, a real-time PCR assay using the LightCycler platform was evaluated and compared to an in-house PCR assay using a conventional detection method. A total of 122 urine specimens were tested, and human polyomavirus was detected in 49 specimens (40%) by both conventional PCR and LightCycler PCR. The remaining 73 specimens (60%) were found negative by both assays. For 46 of the 49 positive specimens, LightCycler PCR and conventional PCR identified the same polyomavirus type. These samples included 30 samples with JC virus (JCV), 14 samples with BK virus (BKV), and 2 samples in which both viruses were detected. In the remaining three samples, both JCV and BKV were detected by the conventional assay, but only JCV was detected by the LightCycler assay. The results of this study show that the LightCycler PCR assay displays sensitivity and specificity similar to those of a conventional PCR assay. These data, combined with its rapid turnaround time for results and decreased hands-on time, make the LightCycler PCR assay highly suitable for the rapid detection and differentiation of JCV and BKV in the clinical laboratory.

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.

Adult mouse intrinsic laryngeal muscles express high levels of the myogenic regulatory factor, MYF-5

The intrinsic laryngeal muscles display unique structural and functional characteristics that distinguish them from the skeletal muscle of the trunk and limbs. These features include relatively small muscle fibers, super-fast contraction speed, and fatigue resistance. The molecular basis of tissue-specific functions and other characteristics is differential gene expression. Accordingly, we have investigated the molecular basis of the functional specialization of the intrinsic laryngeal muscles by examining the expression of two key genes in the larynx, known to be important for skeletal muscle development and function: (a) the muscle regulatory factor, Myf-5, and (b) the superfast-contracting myosin heavy chain (EO-MyHC). We have found that the adult thyroarytenoid muscles express much higher levels of both Myf-5 and EO-MyHC messenger ribonucleic acid (mRNA), compared to lower hindlimb skeletal muscle where Myf-5 mRNA levels are very low and EO-MyHC is not detectable. These findings suggest that the unique functional characteristics of the intrinsic laryngeal muscles may be based in laryngeal muscle-specific gene expression directed by a unique combination of muscle regulatory factors. Such laryngeal muscle-specific genes may allow the future development of new treatments for laryngeal muscle dysfunction.

Rapid transplacental infection with bovine pestivirus following intranasal inoculation of ewes in early pregnancy

Despite the importance of congenital viral infections in both veterinary and human medicine, only limited experimental work has been carried out to elucidate the mechanisms involved in transplacental virus infections. To further an understanding of fetal infection with pestiviruses, the distribution of bovine pestivirus in the uterine and fetal tissues of ewes in early pregnancy, following a natural route of infection, was investigated. On the 18th day of pregnancy, nine ewes were inoculated by the intranasal route with 1 X 10(5) 50% tissue culture infective doses of an Australian isolate of noncytopathic bovine pestivirus (bovine viral diarrhea virus genotype 1). All ewes were ovariohysterectomized at approximately 100 hours postinfection. Samples from the reproductive tract and conceptus were examined histologically and tested for bovine pestivirus by nested reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry and for interferon-tau mRNA expression by nonnested RT-PCR. Although no histopathologic changes were observed in the maternal or fetal tissues, virus was detected in the reproductive tract of all nine ewes and in all of the conceptuses examined. Al; the time of surgery, only two of the nine ewes were demonstrably viremic. This study demonstrates that bovine pestivirus can spread from a natural site of infection to the ovine fetus within 4 days in the absence of maternal immunity and despite the presence of interferon expression in the reproductive tract.

Eutectic solidification mode in sodium modified Al-7 mass %Si-3.5 mass %Cu-0.2 mass %Mg casting alloys

The influence of sodium (Na) on nucleation and growth of the Al-Si eutectic in a commercial hypoeutectic Al-Si-Cu-Mg foundry alloy has been investigated. The microstructural evolution during eutectic solidification was studied by a quenching technique. By comparing the orientation of the aluminium in the eutectic to that of the surrounding primary aluminium dendrites by EBSD, the eutectic solidification mode could be determined. The results show that the eutectic solidification starts near the mould wall and evolves with front growth opposite the thermal gradient on a macro-scale, and on a micro-scale with independent heterogeneous nucleation of eutectic grains in interdendritic spaces. Na-modified alloys therefore behave significantly differently from those modified by other elemental additions.