DAT1) has been associated with a range of psychiatric phenotypes, most notably attention-deficit hyperactivity disorder. The mechanism for this association is not yet understood, although several lines of evidence implicate variation in gene expression. In this study we have characterised the genomic structure of the 9- and 10-repeat VNTR alleles, and directly examined the role of the polymorphism in mediating gene expression by measuring comparative in vitro cellular expression using a reporter-gene assay system.The 10-repeat allele of a variable number tandem repeat (VNTR) polymorphism in the 3'-untranslated region of the dopamine transporter gene (Differences in the sequence of the 9- and 10- repeat alleles were confirmed but no polymorphic differences were observed between individuals. There was no difference in expression of reporter gene constructs containing the two alleles.DAT1 expression and that the associations observed with psychiatric phenotypes may be mediated via linkage disequilibrium with other functional polymorphisms.Our data suggests that this VNTR polymorphism may not have a direct effect on  DAT1) is associated with a range of psychiatric phenotypes. In particular, the 10-repeat allele of this polymorphism has been widely associated with attention deficit hyperactivity disorder (ADHD) (e.g. . Furcontrols . Finallythe drug  as wouldt allele ,16. Prev alleles . These dDAT1 VNTR in vitro, although the results from these studies are inconclusive. Michelhaugh et al demonstrated that the human DAT1 9-repeat VNTR enhances transcription in SN4741 cells, obtained from a mouse-embryonic substantia nigra-derived cell line [DAT1 gene. It has been postulated that large repeat motifs in the 3'UTR of genes may alter mRNA stability, but such effects would not be detected in the research presented by Michelhaugh et al. Furthermore, it has been shown that the enhancing effect of sequence elements is largely determined by their relative location [DAT1 [DAT1 VNTR alleles from several primate species transfected into the human neuroblastoma cell-line, SK-N-SH [DAT1 gene expression.A number of groups have investigated the functional role of the ell line . Howeverlocation . Fuke etlocation . They foon [DAT1 . Again,  SK-N-SH . They fo SK-N-SH . They coin vitro cellular expression implementing a reporter-gene assay system in which the VNTR has been inserted in an appropriate 3' configuration, and using human cell-lines known to express DAT1. Analysis was limited specifically to the VNTR sequence itself, so that any functional effect detected could be attributed solely to this candidate polymorphism. Another aim of this study was to fully characterise the genomic structure of the DAT1 VNTR. It is possible that any functional effects of the VNTR are not mediated by repeat length, but instead by variation in the actual sequence of the repeat units. Recent work on the DRD4 exon III VNTR sequence has demonstrated that there can be considerable variation within the sequence of large VNTRs [The aim of this study was to examine directly the role of the VNTR polymorphism in mediating gene expression by measuring comparative ge VNTRs . Such vaDifferences were found between repeat motifs , and nine variants (labelled A-I) were detected in total . Some slight differences were noted between the two cell lines: in both SH-SY5Y cells and HEK-293 cells, the pGL3-basic plasmid, which contains neither promoter nor enhancer sequences, generated the lowest transcriptional activity as expected. In the SH-SY5Y cells, control plasmids containing both an enhancer and promoter showed significantly higher levels of transcriptional activity than the promoter plasmids, although this difference was not seen in the HEK-293 cells.Table DAT1 VNTR; and second to investigate the effect of the 9- and 10-repeat VNTR alleles on levels of transcription. It is possible that any effects, both on behavioural phenotype or levels of DAT1 expression, commonly associated with the VNTR are due to polymorphisms within the repeat rather than the actual length of the VNTR itself. This theory has often been postulated to explain cases of non-replication of association studies with complex disorders. Recent work on the DRD4 exon III VNTR polymorphism has highlighted the existence of both within individual and between individual variation in repeat motif sequence [st and 2nd repeat motifs within a certain length DRD4 VNTR allele may differ in sequence in a single individual, but there is also variation within the 1st motif between different individuals. We sequenced the DAT1 VNTR in individuals homozygous for either the 9- or 10-repeat allele. Differences were found between repeat motifs in both the 9- and 10-repeat alleles, and nine variants were detected in total. Interestingly, although differences were found between the 9- and 10-repeat alleles, the order of the motifs was identical for all examples of each allele size. Given that not one variation in these sequences was observed in the 60 chromosomes sequenced, it can be concluded that any between individual variations that do occur are extremely rare and unlikely to be the cause of common disorders such as ADHD.The aims of this study were twofold: first to characterise the genomic structure of the sequence . In otheDAT1 3'UTR for novel polymorphisms in a Japanese population, but only detected a G>A SNP located upstream of the VNTR at position 2319 [DAT1 VNTR in a small number of Japanese subjects with results identical to those we describe [DraI restriction site, which they claim to be within the 10-repeat allele [Previously, Ueno et al screened the ion 2319 . Fuke etdescribe . Miller t allele . Closer t allele  may be tDAT1 VNTR is perhaps surprising given the number of polymorphisms seen in other large VNTRs . VNTRs Rs (e.g. -29. FurtRs (e.g. ), the avnylation . Alternain vitro possible functional consequences of the DAT1 VNTR polymorphism. We made constructs containing the 9- and 10-repeat alleles of the DAT1 VNTR and the luciferase reporter gene, and transiently transfected them into SH-SY5Y and HEK-293 cells. Constructs containing the VNTR alleles gave slightly lower levels of luciferase activity compared to vectors without the inserts. Although these differences were not statistically significant, they appear to go against the conclusions of a previous study in which the VNTR sequence acted as a strong enhancer of transcription [The second aim of this study was to examine cription . Howevercription . Furthercription . The discription , which dcription  and so icription  is speciDAT1 VNTR alleles. Other transient expression studies that have compared the VNTR alleles have provided mixed results. Fuke et al found that luciferase expression was significantly higher in cells transfected with vectors containing the 10-repeat allele compared to the 7-repeat or 9-repeat alleles [DAT1 [We found no significant differences in luciferase activity between constructs containing the 9- or 10-repeat  alleles . Their ses [DAT1 . Finallyes [DAT1 .not be functional. Unlike the studies of Fuke et al and Miller & Madras [DAT1 gene. Genetic association studies with ADHD, SPECT brain imaging studies, and correlations with levels of DAT protein and DAT1 mRNA all suggest that a variant associated with DAT1 expression is present in this region. Given that in each of these associations, the VNTR has been nominated as the causative polymorphism, it is likely that the real risk variant is in strong LD with it. Ueno et al and Miller et al both report novel SNPs located within the 3'UTR and close to the VNTR [DAT1 and is the real risk variant.Therefore, our data suggest that the VNTR polymorphism itself may & Madras ,21, our the VNTR ,26. It iin vitro studies of gene expression reflect patterns seen in vivo. Future work could employ animal models to characterise more realistically the effect of the VNTR on DAT1 expression. Second, while we ensured that we used cell-lines that naturally express DAT1, and inserted the VNTR into the correct 3'UTR location of the luciferase reporter gene, our constructs could have been improved by using a homologous DAT promoter. Third, we only cloned a very small portion of the DAT1 gene. Even though this was necessary to examine functional effects specific to the VNTR, the fact that the majority of the DAT1 gene was absent means that it is likely that several cis-acting regulatory elements were not present in the constructs and thus the observed expression may not reflect the actual regulation of the gene. Finally these studies have only analysed DAT1 gene expression in the basal state, and complexities such as the induction of expression by factors such as cellular signals would thus be missed. Future work should focus on systematically characterising the remainder of the 3'UTR to discover the functional effects of other polymorphisms in this candidate region.There are several obvious limitations to this study. First, it is not known how well DAT1 VNTR alleles, and directly examined the role of the polymorphism in mediating gene expression by measuring comparative in vitro cellular expression using a reporter-gene assay system. No expression differences were observed between the 9- and 10-repeat alleles suggesting that this polymorphism may not have a direct effect on DAT1 function.In this study we have characterised the genomic structure of the 9- and 10-repeat DAT1 VNTR and performing expression analyses it was imperative to characterise fully its structure. If there was variation within the VNTR between individuals who have repeats of the same length, then these variants could confound expression analyses based simply upon length. Thirty individuals of predominantly Caucasian ethnicity, homozygous for either the 9- or 10-repeat allele of the VNTR, were selected for sequencing. The VNTR region was amplified on an MJ PTC-225 thermal cycler  using the primers 5'- TGT GGT GTA GGG AAC GGC C-3' and 5'- CAT TCG CAA ACA TAA AAA CTG TTG T-3' using a standard PCR protocol with a proofreading polymerase and an annealing temperature of 58°C. PCR products were run on a 2% agarose gel stained with ethidium bromide, and then purified using the QIAquick Gel Extraction Kit . Purified fragments were sequenced using an ABI BigDye Terminator (v3.0) Cycle Sequencing kit  and electrophoresed on an ABI 3100 Genetic Analyzer (PE Applied Biosystems). Sequencing traces were analysed using Sequencher software  and multiple samples aligned to aid mutation detection.Before cloning the Xba I restriction site (TCTAGA) immediately downstream from the luciferase reporter gene, enabling inserts to be cloned into the 3'UTR. Primers were designed to directly flank the DAT1 VNTR and to both of these an Xba I restriction site, along with three extra bases of DAT1 sequence, were added. The primer sequences used were: 5'-TGT TCT AGA TTG TGG TGT AGG GAA CGG C-3' and 5'-AGG TCT AGA AGA GTG TTG GTC TGC AGG CT-3'. The aim of this project was to concentrate solely on the functional significance of the VNTR, so flanking regions around the VNTR were kept as small as possible. Using these primers, the VNTR was amplified in individuals homozygous for the 9- and 10-repeat alleles using standard PCR conditions with an annealing temperature of 55°C. The PCR products were isolated from a 2% agarose gel as described above. The purified fragments were cloned into pCRII TA cloning vectors , which were then transformed into TOP10F E. Coli cells  following the manufacturers protocol. Colonies containing recombinant plasmids were identified using X-gal and IPTG. DNA from colonies containing recombinant plasmids was prepared using a Qiagen Midi-prep kit , and the presence of insertions was verified using Xba I restriction enzyme digestion and further checked using direct plasmid sequencing. The Xba I digested 9- and 10-repeat VNTR inserts were run on a 2% agarose gel and purified as described above. The fragments were cloned into pGL3-Control and pGL3-Promoter vectors. The pGL3-Control vector contains both SV40 promoter and enhancer sequences whereas the pGL3-Promoter vector contains only an SV40 promoter. Additionally the pGL3-Basic vector which lacks eukaryotic promoter and enhancer elements was tested on its own without any insert, for transcriptional activity and served as a negative control. Five colonies from each of the four cloning reactions  were selected. Following plasmid DNA preparation, the presence and orientation of the inserts was again verified by Xba I digestion and fluorescent sequencing. New DNA stocks for each of the 8 plasmids to be used in subsequent transfection experiments  were prepared, and tested again by Xba I digestion.The pGL3 expression vector family  contains an in vitro studies of gene expression using reporter genes is that the cell-lines used do not naturally express the gene of interest (i.e. DAT1). Before we transfected our constructs into the SH-SY5Y and HEK-293 cell lines they were tested for DAT1 expression using quantitative RT-PCR. Both cell lines were found to naturally express DAT1 making them suitable for our experiments (data not shown). In addition previous in vitro studies have shown regulation of the DAT1 gene in both the HEK-293 and SH-SY5Y cell lines [SH-SY5Y and HEK-293 cells were purchased from ATCC . The SH-SY5Y cell line is a thrice-cloned subline of the neuroblastoma cell line SK-N-SH. The HEK-293 cell line is derived from human embryonic kidney. A problem with many ll lines ,32, sugg2. Transfection of the SH-SY5Y cells was carried out using a calcium phosphate transfection kit . The two different transfection methods used were found to be optimal for each line respectively. The HEK-293 cell line was transiently transfected using Lipofectamine 2000 . Transfection efficiencies were normalised by the co-transfection of the Renilla vector, pRL-SV40 . Following transfection, the cells were allowed to grow for 48 hours. The cells were then washed and 500 μl cell lysis buffer  was added to each well. The firefly luciferase and Renilla luciferase assays were carried out using the Dual-Luciferase Assay System  following the manufacturers protocol. All transfections were done in triplicate, and repeated at least three times. All comparisons between constructs were analysed by one-way analysis of variance (ANOVA), followed by Tukey post-hoc analysis for pairwise comparisons between specific plasmids.Both cell lines were cultured in 6-well tissue culture plates containing Minimun essential medium Eagle (ATCC) supplemented with 10% foetal bovine serum . Cells were grown at 37°C in a humidified atmosphere containing 5% COJM carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. UD participated in the overall design and co-ordination of the study with supervision of cell culture techniques. PA/IC contributed to the interpretation of findings and general points of the experimental design. All authors read and approved the final manuscript


The mechanisms during the initial phase of oxygen toxicity leading to pulmonary tissue damage are incompletely known. Increase of tumour necrosis factor alpha  represents one of the first pulmonary responses to hyperoxia. We hypothesised that, in the initial phase of hyperoxia, TNFalpha activates the caspase cascade in type II pneumocytes (TIIcells).Lung sections or freshly isolated TIIcells of control and hyperoxic treated rats (48 hrs) were used for the determination of TNFalpha (ELISA), TNF-receptor 1 (Western blot) and activity of caspases 8, 3, and 9 . NF-kappaB activation was determined by EMSA, by increase of the p65 subunit in the nuclear fraction, and by immunocytochemistry using a monoclonal anti-NF-kappaB-antibody which selectively stained the activated, nuclear form of NF-kappa B. Apoptotic markers in lung tissue sections (TUNEL) and in TIIcells  were measured using commercially available kits.In vivo, hyperoxia activated NF-kappaB and increased the expression of TNFalpha, TNF-receptor 1 and the activity of caspase 8 and 3 in freshly isolated TIIcells. Intratracheal application of anti-TNFalpha antibodies prevented the increase of TNFRI and of caspase 3 activity. Under hyperoxia, there was neither a significant change of cytosolic cytochrome C or of caspase 9 activity, nor an increase in apoptosis of TIIcells. Hyperoxia-induced activation of caspase 3 gradually decreased over two days of normoxia without increasing apoptosis. Therefore, activation of caspase 3 is a temporary effect in sublethal hyperoxia and did not mark the "point of no return" in TIIcells.In the initiation phase of pulmonary oxygen toxicity, an increase of TNFalpha and its receptor TNFR1 leads to the activation of caspase 8 and 3 in TIIcells. Together with the hyperoxic induced increase of Bax and the decrease of the mitochondrial membrane potential, activation of caspase 3 can be seen as sensitisation for apoptosis. Eliminating the TNFalpha effect in vivo by anti-TNFalpha antibodies prevents the pro-apoptotic sensitisation of TIIcells. Oxidative stress is an important factor of acute lung injury. Prolonged exposure to high concentrations of oxygen (hyperoxia) during mechanical ventilation represents a life-saving intervention for critically ill patients. However, it also induces oxidative stress to the lung. The development of therapeutic strategies, aiming to prevent lung injury depends on a better understanding of the underlying pathways of hyperoxia-induced pulmonary damage.Severe, long lasting hyperoxia causes an inflammatory reaction with an influx of inflammatory cells, cell proliferation and hypertrophy, an increase of cytokines, apoptotic activity and subsequent morphologic evidence of lung injury . The firFollowing the concept that hyperoxic lung injury is a continuous process, we assumed that appropriate metabolic changes of TIIcells start during the initiation phase. This would then, in response to longer lasting severe hyperoxia or an additional stress, merge in apoptosis in the post-initiation phase . FactorsElevation of tumour necrosis factor α (TNFα) represents one of the first pulmonary responses to hyperoxia. Pre-treatment of animals with antibodies directed against TNFα reduces hyperoxia-induced lung injury, strongly suggesting a causal relationship between TNFα and hyperoxic lung . TNFα isIn this paper, we tested the hypothesis that TNFα activates caspases in the initiation phase of pulmonary oxygen toxicity in TIIcells without a significant increase in apoptosis. Since apoptosis is modulated by cell-matrix and cell-cell interactions in cultured TIIcells isolated from animals with acute lung injury ,25. we uad libitum. Preparation of the bronchoalveolar lavage, alveolar macrophages and TIIcells was carried out as previously described -labelled oligonucleotide  which contains DNA binding sites for NF-κB transcription factors was then added to the reaction mixture and incubated for 20 minutes at RT. The complexes were separated on a 4% polyacrylamide gel that was dried and exposed to autoradiography. The specificity of the DNA-binding protein for the putative binding site was established by competition experiments using unlabelled NF-κB consensus oligonucleotide.Wistar rats were lightly anaesthetised by inhalation of ether. The rats obtained intratracheally 50 μg goat IgG  or 50 μg goat polyclonal anti-rat TNFα antibody  per animal, respectively. Thereafter the rats were kept at hyperoxic conditions as described in hyperoxia. After 48 hrs, the TIIcells were isolated and TNFRI expression and caspase-3 activity were determined as described above.For the determination of the TNFα concentration in TIIcells, macrophages, plasma or cell-free bronchoalveolar lavage, we used a commercially available ELISA kit from Biosource . The determination of different apoptotic markers , WesternDifferences between two groups were assessed using the Student's t-test. Probability values < 0.05 (two-tailed) were considered significant .in vivo; at least not in the initiation phase.To test the general usefulness of our hypothesis, we first characterised the oxygen-induced changes in lung-tissue-sections. Immunohistochemically, we observed a clear increase in TNFα, TNFRI, and caspase 3 activities in lung tissue of hyperoxic rats in relation to normoxic animals in the initiation phase Figure . Albeit in vivo. Following our hypothesis, we examined whether TIIcells undergo oxidative stress at our conditions, and whether an increase of TNFα, TNFRI expression, and caspase-3 activity appears in isolated TIIcells.The results indicate that apoptotic parameters as are TNFα content, TNFRI expression and caspase-3 activity increase in lung tissue during the initiation phase but do not induce TIIcell apoptosis The determination of cellular GSH, oxidised GSH (GSSG), and the activity of the GSH-reductase showed the oxidative burdening of TIIcells. In response to hyperoxia, the GSSG content significantly increased and the GSH reductase activity significantly decreased Table . The GSHThe ratio GSH/(GSH+GSSG) is one of the most sensitive parameters to describe oxidative burdening. Our values are comparable to the values found by van Klaveren et al. in freshly isolated type II cells (0.816 versus 0.815) . This raNF-κB activation has been described as an indicator of oxidative stress ,34. In rThe concentration of TNFα significantly increased in response to hyperoxia of rats in plasma, alveolar fluid, lung macrophages and TIIcells Table  as deterin situ that the activation of caspase 3 actually occurs in TIIcells and that caspase activation as a response to an unspecific stress, e.g. isolation, can be excluded , whereas the expression of Fas, a member of the same receptor family, did not change Figure . TNFRI-mAs shown in Table In order to demonstrate the causality between hyperoxia and TNFRI-mediated activation of caspases, we attempted to bind TNFα by anti-TNFα antibodies. Table The content of individual mRNAs in TIIcells was determined by Real-time PCR. In agreement with microarray analysis in total lung of mice  we foundt of -1.15 indicates a 2.2-fold increase of mRNA content.In response to sublethal hyperoxia the mRNA content of TNFα, TNFRI and caspases increased Table . The incAs mentioned above in this section, no increase of apoptosis was detected in TIIcells in response to sublethal hyperoxia by immunohistochemistry Figure . To confAfter hyperoxia for 48 hrs, we detected an activation of caspase 8 and caspase 3. Much to our surprise, we found no increase in apoptosis of TIIcells although the activity of these caspases increased. However, apoptosis might occur later than 48 hrs and will not necessarily arise concomitantly with caspase activation. Thus, following hyperoxia the animals were kept for 24 and 48 hrs under normoxic conditions to test TIIcells for appearance of apoptosis at a later time point.During normoxia, caspase 3 activity gradually decreases compared to control, whereas apoptosis did not change significantly as detected by cell death detection ELISA and by the number of early and late apoptotic cells Table .Short-time hyperoxia, as used in this experiments represents the initiation phase of lung injury . We starFor the first time, we show that sublethal hyperoxia causes not only an increase in TNFα concentration in lung tissue , butIt has been widely assumed that macrophages are the source of TNFα in alveolar fluid . Our finCellular effects of TNFα are mediated mainly by its specific receptor, TNFRI. In agreement with our hypothesis, the expression of TNFRI in TIIcells is up-regulated on mRNA and protein level in response to sublethal hyperoxia, while the Fas-expression did not change, although both receptors belong to the same family Figure . It may The intracellular part of the transmembrane TNFRI protein contains the "death domain" which is responsible for the activation of caspase 8. This can trigger the path to the final part of apoptosis via activation of caspase 3 . In lineTo check whether this pro-apoptotic state in TIIcells is indeed triggered by TNFα and whether it may be reversible, anti-TNFα antibodies were administered intratracheally prior to hyperoxic exposure. We show that this treatment completely prevented hyperoxic-induced increase of TNFRI expression and caspase 3 activation.It is a widely accepted concept that activation of caspase 3 marks the "point of no return" in the pathway of apoptotic death of mammalian cells. However, we found that neither in lung tissue nor in freshly isolated TIIcells apoptosis occurs in response to sublethal hyperoxia despite the significant activation of caspases 8 and 3. In other words, the increase of caspase 8 and 3 in response to sublethal hyperoxia did not mark the "point of no return" in TIIcells. This interpretation of our results is strongly corroborated by Perfettini and Kroemer . They suvia strong mitochondrial damage resulting in cytochrome c release and caspase 9 activation in post-initiation phases of pulmonary oxygen toxicity. This concept is supported by recent findings that mitochondrial cytochrome c release is a key event in hyperoxia-induced lung injury [The reason why the activation of caspases did not induce apoptosis of TIIcells is not clear. On the one hand, activation of NF-κB has often been implicated as an anti-apoptotic event -45, and g injury . In factRecently, it has been shown that in response to severe hyperoxia of mice apoptosis and necrosis contribute to an extensive cell death; p53, bax, bcl-x, and Fas increased in mRNA and protein level, but the activity of caspase 3 and caspase 1 did not change . This inIn summary, TIIcells were not killed in the initiation phase of pulmonary oxygen toxicity. More precisely, its pro-apoptotic sensitisation is the background that, together with an additional stress factor, hyperoxia causes lung injury probably by apoptotic elimination of TIIcells. In agreement with this idea, we showed that the combination of the stress factors hyperoxia and vitamin E deficiency increases TIIcell apoptosis . Taking In the initiation phase of pulmonary oxygen toxicity, an increase of TNFalpha and its receptor TNFR1 leads to the activation of caspase 8 and 3 in TIIcells. Together with the hyperoxic induced increase of Bax and the decrease of the mitochondrial membrane potential, activation of caspase 3 can be seen as sensitisation for apoptosis. Eliminating the TNFα effect in vivo by anti-TNFα antibodies prevents the pro-apoptotic sensitisation of TIIcells.DAPI – 4',6-diamidino-2-phenylindole; EMSA – electrophoretic mobility shift assay; GSH – gluthatione; GSSG – oxidised gluthatione; NF-κB – nuclear factor-κB; TIIcell – type II pneumocyte; TNFα – tumour necrosis factor α; TNFR – TNFα receptor; TUNEL – terminal transferase dUTP nick end labellingHW carried out the immunohistochemistry, CS performed the mRNA analysis, AT carried out the protein measurements and participated in data analysis, MR applicated anti TNFα antibodies intratracheally, FS participated in the mRNA analysis and the design of the study, FG and BR conceived of the study, participated in its design and co-ordination, analysed the data and wrote the manuscript. All authors read and approved the final manuscript.


In the absence in the major Australian administrative health record collections of a direct measure of the socioeconomic status of the individual about whom the event is recorded, analysis of the association between the health status, use of health services and socioeconomic status of the population relies an area-based measure of socioeconomic status.This paper explores the reliability of the area of address  as a proxy measure for socioeconomic disadvantage. The Western Australian Data Linkage System was used to show the extent to which hospital inpatient separation rates for residents of Perth vary by socioeconomic status of area of residence, when calculated at various levels of aggregation of area, from smallest (Census Collection District) to largest . Results are also provided of the reliability, over time, of the address as a measure of socioeconomic status.There is a strong association between the socioeconomic status of the usual address of hospital inpatients at the smallest level in Perth, and weaker associations when the data are aggregated to larger areas. The analysis also shows that a higher proportion of people from the most disadvantaged areas are admitted to hospital than from the most well-off areas (13% more), and that these areas have more separations overall (47% more), as a result of larger numbers of multiple admissions.Of people admitted to hospital more than once in a five year period, four out of five had not moved address by the time of their second episode. Of those who moved, the most movement was within, or between, areas of similar socioeconomic status, with people from the most well off areas being the least likely to have moved.Postcode level and SLA level data provide a reliable, although understated, indication of socioeconomic disadvantage of area. The majority of Perth residents admitted to hospital in Western Australia had the same address when admitted again within five years. Of those who moved address, the majority had moved within, or between, areas of similar socioeconomic status.Access to data about individuals from the Western Australian Data Linkage System shows that more people from disadvantaged areas are admitted to a hospital, and that they have more episodes of hospitalisation. Were data to be available across Australia on a similar basis, it would be possible to undertake research of greater policy-relevance than is currently possible with the existing separations-based national database. The majority of work in Australia describing the association between the health status, use of health services and socioeconomic status of the population uses an area-based measure of socioeconomic status. It is necessary to use a proxy measure (the socioeconomic status of the population in the area) because there is no direct measure in the major administrative health record collections of the socioeconomic status of the individual about whom the event is recorded.However, the application of an area-based measure requires a number of assumptions, including that people who move do so between, or within, geographic areas of similar socioeconomic status; and that the (often large) areas used in these analyses provide a reliable indication of the socioeconomic status and health service utilisation of the individuals in the area about whom the event is recorded. Area level socioeconomic status can also be considered as an independent predictor. For example, an individual with low socioeconomic status in an area of higher socioeconomic status is more likely to have better health outcomes than their counterpart in an area of lower socioeconomic status ,2. This In relation to this latter point, Hyndman et al  found thAnother limitation of the majority of Australian health-related datasets is that they record events , rather than individuals.The analysis in this report uses the Western Australian Data Linkage System to explore the reliability of area data as a proxy for socioeconomic disadvantage when analysed for the relatively large geographic units often used in health-related research: it also addresses the limitations of using data about events, rather than individuals. It does this by examining the extent to which hospital inpatient separation rates vary, both overall and by socioeconomic status of area of residence, when calculated at various levels of aggregation, from Census Collection District (CD) – the smallest area level for which a measure of socioeconomic status is available – to the larger units of postcode and Statistical Local Area (SLA). Methods applied include the calculation of correlation coefficients and examination of hospital separation rates by quintile of socioeconomic disadvantage of area, separately for events and individuals.The report also examines the reliability of the socioeconomic status of the address over time, by examining the extent of change in socioeconomic status of area of residence for individuals with repeat hospital episodes over a five year period.The analysis shows that aggregating data to larger area reduces the gap between the index scores for the most disadvantaged and least disadvantaged areas, with the greatest impact on the scores for the most disadvantaged areas. This results in an understatement of the extent of disadvantage in the most disadvantaged areas, as well as an understatement in inequality between the most well off and the poorest areas.Over the five years from 1994 to 1998, a total of 358 948 residents of Perth were admitted to a hospital in Western Australia on one or more occasion, an average of 71 750 individuals admitted per annum. Just over half (53.6%) the individuals admitted were females; 46.4% were males.The rate of individuals admitted was 16.4% higher for females (247.6 separations per 1000 population) than for males, (212.7 separations per 1000 population)  those admitted to hospital had one admission over this period, and more than one third 36.0%) had between two and four admissions, together comprising 90.6% of those admitted  of those admitted once, compared with 59.7% of those admitted more than once. For males, the proportions were 46.4% and 40.3%, respectively.There were 1 665 308 separations of Perth residents from Western Australian hospitals, an average of 2.53 separations per person admitted over the five years from 1994 to 1998. Over half (55.1%) of the separations were of females and 44.9% were of males.Figure The main differences in the profiles of male and female separations are evident at the youngest ages , from ages 20 to 44 years  and from 50 to 79 years . The ages at which the highest rates of admissions of individuals and of multiple admissions (the gap between the separations and admitted profiles) occur are clearly visible in the chart.Unlike the rates for individuals admitted Table , above, As noted, the majority of the analysis by socioeconomic status undertaken in the health sector in Australia is area based, and uses the postcode or SLA as the unit of analysis. This raises the question of the extent to which area based analyses at the postcode or SLA level provide a reliable indication of the socioeconomic status and health service utilisation of the individuals admitted. This report explores the reliability of postcode or SLA level data by examining the extent to which rates of individuals admitted and separations vary when calculated at various levels of aggregation . Ideally, the comparison would be between the socioeconomic status of individuals and of areas; however, the smallest area level for which a measure of socioeconomic status is available is the CD.Variation in the minimum and maximum Index of Relative Socio-economic Disadvantage (IRSD) scores when calculated at the CD, postcode and SLA level is striking and clearly shows the value of the smaller unit in area based analyses Table . The ranThus, the use of larger area aggregates reduces the gap between the index scores for the most disadvantaged and least disadvantaged areas , with the greatest impact on the scores for the most disadvantaged areas . Notably, the difference between the maximum and minimum scores, and the absolute level of the scores, is much less marked between the postcode and SLA.There was a strong association between the IRSD scores for CDs and those for postcode of usual address at the first admission (a Spearman correlation coefficient of 0.74). The correlations were between CDs grouped to quintiles and postcodes grouped to quintiles, ranked by the IRSD, and not between individual CDs and postcodes. A weaker association was found between the quintiles for CDs and those for SLAs (0.64 for people with one separation and 0.63 for people with more than one separation)  than at the CD level   those admitted to hospital had one separation over this period, the proportion varied from 56.3% in Quintile 1 to 51.9% in Quintile 5 Table . This isThere is a substantial difference in the proportion of the population in Quintiles 5 and 1 having two or more separations : the differential for people having one separation is lower, although still notable at 16.1% (a rate of 32 790 separations per 100 000 persons in Quintile 5 and 28 231 separations per 100 000 persons in Quintile 1)  provide a reliable indication as to the socioeconomic status and use of health services of the individuals in the area.Data from the 1996 Census show that 53.5% of Perth's population at the 1996 Census reported that they had a different address to that at the previous Census, five years earlier [Similarly, almost four out of five people admitted to hospital more than once in a five year period had not moved (out of the CD of their address at the first separation) by the time of their second separation. For example, of the 298 809 people admitted to a Perth hospital more than once over the five year period 1994 to 1998, over three quarters  had the same address at the time of the second separation. People were recorded as having 'moved' if the CD of their address changed between the first and last separation over the period from 1994 to 1998. Movement to a different address within a CD was not included.The following table illustrates, for people with multiple admissions, the extent of movement by socioeconomic status. For this part of the analysis, the CD of first and last separation have been allocated to quintiles of socioeconomic disadvantage of area, to provide a comparison of the extent of movement between different levels of socioeconomic status. The construction of the quintiles is described in the section titled 'Methods, Measurement of socioeconomic status' under 'Methods.'Table • people from the most well off areas are less likely to have moved to areas of greatly different socioeconomic status  than are those from the most disadvantaged areas – 40.2% of people in the most advantaged areas (Quintile 1) remained there, despite moving from the CD of their first separation. The proportion in the most disadvantaged (Quintile 5) areas was a lower 30.5%;• while there is movement right across the socioeconomic profile, most movement is between adjacent quintiles. For example, of the 18 875 people who lived in the most disadvantaged areas at their first separation (and moved before a subsequent admission), 71.2% had moved to a CD in the same or next ranked quintile (Quintiles 5 or 4), with just 4.6% moving to the most advantaged areas. Similarly, of the 9 537 people in the most well off areas at their first separation, 63.0% had moved to a CD in the same or next ranked quintile (Quintiles 1 or 2), with a similarly low proportion (4.7%) moving to the most disadvantaged areas;• the most substantial movement between quintiles was of people moving from an address rated as Quintile 5 to one rated as Quintile 4 (40.7%); this was marginally higher than the proportions moving within Quintiles 4 or 1 .There is a strong association between the quintile of socioeconomic disadvantage of area at the first and the last discharge when analysed by CD (a correlation coefficient of 0.88) or SLA (a correlation coefficient of 0.89) of usual address Table . This suThe analysis shows that, for Perth residents admitted to hospital, the use of larger area aggregates reduces the gap between the index scores for the most disadvantaged and least disadvantaged areas, thus understating the extent of inequality between these areas. The greatest impact of aggregation of areas is on the scores for the most disadvantaged areas. This results in an understatement of the extent of disadvantage in the most disadvantaged areas, as well as an understatement in the extent of inequality between the most well off and the poorest areas.Further, the analysis shows that a more people from the most disadvantaged areas are admitted to hospital than from the most well-off areas (13% more), and that these people have more separations overall (47% more), as a result of larger numbers of multiple admissions.As regards the extent of movement, four out of five people admitted to hospital more than once in a five year period had not moved (out of the CD of their address at the first separation) by the time of their second separation. In addition:• people from the most well off areas are less likely to have moved to areas of greatly different socioeconomic status than are those from the most disadvantaged areas;• while there is movement right across the socioeconomic profile, most movement out of a quintile is to areas in adjacent quintiles; and• the most substantial movement between quintiles was of people moving from an address rated as Quintile 5 to an address rated as Quintile 4, although this was only marginally higher than the proportions moving within Quintiles 4 or 1.In summary, postcode level and SLA level data provide a reliable indication of socioeconomic disadvantage of area, when compared with CD-level data. That is, the association between rates of total separations and individuals admitted and socioeconomic disadvantage of area evident at the smallest area level (CD) is also evident in the higher level area aggregates of postcode and SLA.It is reasonable to assume that similar relationships exist in other Australian cities, as well as in other health-related activity .Given the widespread use in Australia of area based analyses at the postcode and SLA level, and the limitations of CDs an area level for the analysis of most health datasets, it is important to know that such analyses provide a reliable indication of the direction and underlying strength of the influence of socioeconomic factors in hospital admissions rates.This is not to imply that the postcode or SLA are the ideal areal unit for analysis, nor that data for Collection District would be. The ideal population size for area-level analysis is likely to vary dependent on the number of cases in the dataset under analysis. For datasets with a large number of cases per capita  the number will be smaller than those with a small number of cases per capita (eg. deaths), even with aggregation of data over a number of years. May SLAs have much larger populations than are necessary to produce reliable results; and the populations of most CDs are too small  for individuals in the Western Australian Data Linkage System to the Australian Bureau of Statistics Population Census has the potential to add considerable value to such analyses. For example, it would be possible to examine an individual's characteristics of education, occupation, labour force status, housing tenure etc., and to more directly examine the relationships between the number of individuals admitted and total separations and these important socioeconomic variables. Linkage to death registration data would also be valuable in understanding more about outcomes related to socioeconomic status. This latter example is a possibility under recently announced plans for the ABS to test the linking of 2006 Census of Housing and Population data to other datasets, such as deaths registrations, held under their Act. This is similar to the approach elsewhere, including New Zealand . It is tThe report addresses differences in the number of individuals admitted and the number of separations they incurred. These are described as 'individuals', or individuals admitted' and separations . 'Separation' is the term describing a completed hospital episode: it is defined in the section titled 'Glossary, Separation' under 'Glossary.'Details of all separations to public and private hospitals in Western Australia for the five years from 1994 to 1998 were extracted from the Western Australian Hospital Morbidity Database (HMDS). Any separation records thought to belong to the same person had previously been linked together within the Data Linkage System, permitting analyses to be performed for both separations and individual persons. The population used in calculating rates is the 1996 Census population.The analysis has been limited to separations of residents of Perth, but includes separations occurring at any public acute or private hospitals in Western Australia.Areas used in the analysis are the Census Collection District (CD), postcode and Statistical Local Area (SLA). See Glossary for definitions of CD, postal area and SLA.The HMDS includes address details for each separation from a hospital in Western Australia since 1993. These addresses have been linked to a Western Australian street address database to assign northing and easting points (geo-codes). These points were then assigned to the appropriate 1991 or 1996 CD using the ABS CData96 mapping tool. The postcode and SLA of the address were then determined by allocation of CDs to postcode or SLA. The boundaries for CDs and SLAs are consistent. However, boundaries for CDs and postcodes are not, so CDs were allocated to postcodes on a 'best fit' basis (see Glossary).Consequently, comparisons can be made between results for CDs and postcode areas, CDs and SLAs and postcode areas and SLAs. This is particularly important, as much of the area analysis undertaken in the health sector in Australia uses the postcode or the SLA, as a majority of data are only available at these area levels, and it is widely accepted that the larger the area, the less homogenous the population is likely to be.There were 2 297 CDs in Perth at the 1996 Census, with 105 postcodes and 37 SLAs. The average population size at each of these area levels is shown in Table In the absence of any direct measure of socioeconomic status in the hospital inpatient data, the socioeconomic status of the area of the address of the individual admitted is used as a proxy measure. The Index of Relative Socio-Economic Disadvantage (IRSD) is the measure used to provide the socioeconomic status of the area of the address. The IRSD is one of five Socio-Economic Indexes for Areas (SEIFA) produced by the Australian Bureau of Statistics (ABS) from data collected at the 1996 Population Census. It is calculated at the CD level and can be produced for other area levels. The postcode and SLA level index scores in this report are the population weighted average of the IRSD scores for the CDs in the postcode or SLA. This calculation is undertaken for all CDs in the postcode or SLA, not just those for which hospital episodes were recorded.Each area level  was allocated to one of five groups (quintiles). For example, for SLAs, Quintile 1 comprises the SLAs with the highest IRSD scores (most advantaged areas), and Quintile 5 comprises the SLAs with the lowest IRSD score (most disadvantaged areas): each quintile comprises approximately 20% of the Perth population. This process does not provide an exact allocation of population, so the resultant populations are only 'approximately' equal, and the larger the areal units being allocated, the less likely they are to be equal. As shown in Table Three (different) IRSD scores were added to each hospital separation record, based on the CD, postcode or SLA that had been previously assigned to the address on that record. It should be noted that these IRSD scores were actually the average score for the particular CD, postcode or SLA as calculated from 1996 Census data. Quintile ranks for each aggregation level were also applied using population weighting as described above.For analyses involving multiple admissions, the IRSD value used was that for the first separation in the five-year period. These 'first' separations were isolated using the internal links between separation records for the same person and the separation date. Of course, many of these 'first' separations could have been preceded by separations occurring before 1994.Rates are crude rates, per 100 000 population. Ideally the data would have been standardised (by the indirect method). However, access to the source data were limited and to requested tables, and standardisation was not an option.As the data were from a complete enumeration , confidence intervals were only calculated for measures of difference .The Spearman Rank Correlation has been used in the analysis to indicate the degree of correlation between pairs of variables.The Collection District (CD) is the smallest area level in the Australian Bureau of Statistics' statistical geography and is primarily an area used in the five yearly population census.The Index of Relative Socio-Economic Disadvantage (IRSD) is one of five Socio-Economic Indexes for Areas produced by the Australian Bureau of Statistics at recent population censuses. Produced using Principal Components Analysis, it summarises information available from variables related to education, occupation, income, family structure, race (the proportion of Indigenous people), ethnicity (poor proficiency in use of the English language) and housing. The variables are expressed as percentages of the relevant population. The IRSD is available at the Census Collection District level and was then be calculated for postcodes and SLAs by weighting the CD level scores by their population. The IRSD is calculated to show the relativity of areas to the Australian average for the particular set of variables which comprise it; this average score is set at 1000. Scores below 1000 indicate areas with relative disadvantaged populations under this measure, and scores above 1000 indicate areas with relatively advantaged populations. The IRSD scores at the Census Collection District (CD) level have been grouped to postal area, an area developed by ABS for the presentation of population counts and other Census data from the five-yearly population censuses to approximate postcode areas, as the ABS does not collect the postcode at the Census.The term describing a completed hospital episode is a 'separation'. At the time of admission to hospital, the age, sex, address of usual residence and other personal details of the patient are recorded. At the end of the episode, at the time of separation from hospital, details of the episode itself are recorded, including the date, time and method of separation . Consequently, hospital inpatient data collections are based on separations.The postal area is an area developed by ABS for the presentation of population counts and other Census data from the five-yearly population censuses. It approximates postcode areas, as the ABS does not collect the Australia Post postcode at the Census. Postal areas comprise Census Collection Districts (CDs) grouped to approximate postcode areas. Where a CD does not fit entirely within a postcode area, it is allocated to the postcode area into which the population largely falls. Where a CD covers more than one postcode area, the total CD population is allocated to one postcode.The IRSD scores at the Census Collection District (CD) level have been allocated to postal areas as described in the section titled 'Methods, Index of Relative Socio-Economic Disadvantage' under 'Methods.'. Similarly, the postal area of each separation was approximated from the CD of the address.The term postcode, rather than postal area, is used in the text, for ease of reading.See postal area, above.See section titled 'Methods, Measurement of socioeconomic status' under 'Methods.'An SLA in Perth is generally equivalent to a local government area, with additional codes allocated to local government areas split for statistical purposes .


This study describes the results of 18-year of HIV surveillance in Saudi Arabia (SA) and the preventive measures implemented from an Islamic perspective.surveillance for HIV has been underway in SA since 1984. Indications for HIV testing include clinical suspicion, screening of contacts of HIV-infected patients, and routine screening of blood and organ donors, prisoners, intravenous drug users, patients with other sexually transmitted infections, and expatriates pre-employment. This is a case series descriptive study of all confirmed HIV infections diagnosed in SA from 1984 through 2001.a total of 6046 HIV infections were diagnosed, of which 1285 (21.3%) cases were Saudi citizens. Over the 18-year surveillance period the number of HIV infections diagnosed annually among Saudi citizens gradually increased and, over the period 1997–2001, it reached to 84 to 142 cases per year. The number of cases per 100,000 population varied widely between regions with a maximum of 74 cases and a minimum of 2 cases. The infection was most common in the age group 20–40 years (74.6%) and predominantly affected men (71.6%). The modes of transmission among Saudi citizens and expatriates, respectively, were as follows: heterosexual contact, 487 (37.9%) and 1352 (28.4%) cases; blood transfusion, 322 (25.0%) and 186 (3.9%) cases; perinatal transmission, 83 (6.5%) and 19 (0.4%) cases; homosexual contact, 32 (2.5%) and 38 (0.8%) cases; intravenous drug use, 17 (1.3%) and 33 (0.7%) cases; bisexual contact, 10 (0.8%) and 14 (0.3%) cases; unknown, 334 (26.0%) and 3119 (65.5%) cases. The number of HIV infections transmitted by blood or blood products transfusion declined to zero by year 2001 and all such infections occurred due to transfusions administered before 1986. At HIV diagnosis, 4502/6046 (74.5%) patients had no symptoms, 787 (13.0%) patients had non-AIDS defining manifestations, and 757 (12.5%) patients had AIDS. A total of 514/1285 (40%) Saudi patients died by year 2001.the number of HIV cases in SA is limited with heterosexual contact being the main mode of transmission. From an Islamic perspective, preventive strategies include prevention of non-marital sex and intravenous drug use with encouragement of "safe sex" through legal marriage. The human immunodeficiency virus (HIV) continues to be a major health problem worldwide despite enormous efforts to control its spread. As of December 2002, the estimated number of people living with HIV is 42 millions . In yearSaudi Arabia occupies most of the Arabian Peninsula with an area of about 2,240,000 sq km figure . It compInformation on HIV epidemiology in Saudi Arabia and other Islamic countries is limited. Islam prohibits non-marital sex, homosexuality, and intravenous drug use. Therefore, the prevalence and incidence of HIV and other sexually transmitted infections are expected to be low in Islamic communities. This study describes the results of HIV surveillance activities that have been underway in Saudi Arabia from 1984 through 2001 and the preventive measures implemented by the government.Surveillance for HIV infections has been underway since 1984 when the first case of HIV was diagnosed in Saudi Arabia. HIV cases are detected by HIV testing for various indications including clinical suspicion, screening of contacts of HIV-infected patients, routine screening of blood and organ donors, and testing of all prisoners, intravenous drug users, patients with other sexually transmitted infections, and expatriates pre-employment. In addition to being HIV tested in their homeland as a compulsory pre-requisite for employment in Saudi Arabia, expatriates are routinely retested for HIV upon arrival to Saudi Arabia before they are allowed to work and then regularly every 2 years to have their legal residence permits renewed. Only HIV-negative expatriates are allowed to work in Saudi Arabia. Routine screening of blood, blood products, and organ donors for HIV has been a standard procedure in Saudi Arabia since 1986. Enzyme-linked immunosorbent assays (ELISA) are used for routine HIV1/HIV2 testing. Positive ELISA results are confirmed by Western blot test performed in selected referral laboratories. The expanded World Health Organization (WHO) case definition was used to define AIDS [Epidemiological data are collected from patients by the attending physicians on standardized data collection forms. The likely mode of transmission is determined by the attending physician after interviewing the patient and taking complete history regarding high risk behaviors. The mode of transmission is considered to be unknown if the patient fails to admit to any high risk behavior. Collected information is subsequently submitted to the Department of Preventive Medicine in the Central MOH office in Riyadh where all surveillance data are compiled. Annual reports are issued but they are only utilized internally by the concerned officials in the MOH and the Ministry of Interior and they are not made available for the public.From 1984 through 2001, a total of 6046 cases with HIV infection were diagnosed; 1285 (21.3%) cases among Saudi citizens and 4761 (78.7%) cases among expatriates. Table Among 4761 HIV-infected expatriates, 3771 (79.2%) patients were from African countries, 624 (13.1%) patients, from Asian countries, 352 (7.4%) patients, from Middle East countries, and 14 (0.3%) patients, from Western countries. Ninety two percent of patients were from 10 countries, namely, Ethiopia (2214 patients or 46.5%), Nigeria (343 patients or 7.2%), Chad (329 patients or 6.9%), Yemen (309 patients or 6.5%), Sudan (267 patients or 5.6%), Eritrea (248 patients or 5.2%), India (219 patients or 4.6%), Somalia (176 patients or 3.7%), Pakistan (152 patients or 3.2%), and Bangladesh (133 patients or 2.8%), and the remaining 371 (7.8%) patients were from 41 other countries.Table Figure The prevalence and the annually reported HIV infections in Saudi Arabia were limited. More than three quarters of HIV cases were expatriates. Expatriates are routinely tested for HIV upon arrival to Saudi Arabia and then regularly every 2 years to have their legal residence permits renewed. Therefore, one possible explanation for the higher prevalence of HIV infections among expatriates is that HIV testing rates might be higher among expatriates compared to Saudi citizens. The number of HIV infections diagnosed annually among Saudi citizens gradually increased to reach a plateau of 84 to 142 cases per year in the last 5 years of the study period. The infection was more prevalent in Jeddah than it was in other cities in Saudi Arabia. A possible explanation for this increased prevalence in Jeddah is that this city is the main air and sea port for expatriates who come to work in the western province of Saudi Arabia and for those who come to visit the holy places in Makkah and Madinah. Additionally, the population in Jeddah is multi-cultural and inhomogeneous from the religion point of view.HIV infection in Saudi Arabia, as the case worldwide, was more common in the age group 20–40 years and predominantly affected men. All modes of transmission were registered but the main mode was heterosexual contact. Even though blood transfusion caused one quarter of HIV cases among Saudi patients, all such infections occurred due to transfusion of blood or blood products before 1986. This risk factor was virtually eliminated since the introduction of routine HIV screening of donated blood and blood products in 1986. The vast majority of Saudi HIV-infected women with identifiable risk factors acquired the infection through blood transfusion or marital sex with their infected husbands. The large proportion of patients with "unknown" risk factors was likely due to deliberate concealment of the actual risk factors, likely to be illegal sex, by the infected patients as non-marital sex and homosexuality are prohibited in Islam, the religion of all Saudi citizens and the vast majority of expatriates living in Saudi Arabia.The international efforts to control the HIV pandemic have failed to control it on a global scale despite their partial success in many of the developed countries and some of the developing countries. Limitation of resources in underdeveloped countries makes health education and other preventive strategies difficult to implement. Additionally, the limited access to antiretroviral medications in the underdeveloped countries has compounded the problem and perhaps contributed to some extent to the spread of HIV from untreated patients to new victims.Substance abuse is an increasing problem in Saudi Arabia as it is in the rest of the world . SubstanNon-marital sex is expected to remain the main risk factor for acquiring HIV infection in Saudi Arabia as it is worldwide for several reasons. The ever-decreasing religious values, the ever-increasing ease of international transportation and communication, and the increasing poverty and unemployment are the main driving forces for non-marital sex, not only in Saudi Arabia but all over the world. Intravenous drug use is also expected to play a more important role for HIV transmission in Saudi Arabia in the years to come as the population of intravenous drug users increases. Preventive strategies should thus be directed towards these risk factors.Some of the preventive strategies that are advocated and used in other non-Islamic countries such as "Safe Sex" and "Needle Exchange Programs" contradict the Islamic rules and values and as such can not be used as valid and acceptable strategies to prevent the spread of this infection in Islamic countries. The concept of "Safe Sex" basically promotes the use of condoms for non-marital sexual relations, considered in Islamic countries a way of promoting non-marital sex which is absolutely prohibited in Islam. Needle Exchange Programs, likewise, is viewed by Islam as a way of encouraging people engaged in intravenous drug use to continue this prohibited practice.Strategies to prevent HIV infection in Islamic countries have to abide by the Islamic rules and values. Proposed preventive strategies that can be used in Islamic countries include strengthening of both Islamic and health education, encouraging people to follow and implement the Islamic rules and values that prohibit adultery, homosexuality, and intravenous drug use, and to practice safe sex only through legal marriage. Encouragement of legal marriage is a multidisciplinary task that should involve both governmental and non-governmental charitable organizations and the population at large to cut the cost of marriage and support programs that help the youth to get married. There are several such programs in Saudi Arabia. For example the "Charitable project to help the youth to get married" in Jeddah has helped 2500 young men to get married at a cost of 10 million Saudi Riyals (2.7 million US dollars) collected from donations .Other Islamic aspects that are important in preventing non-marital and irresponsible sexual relationships include the fact that men are allowed to be married to up to four women and the fact that there is no age limit for marriage, thus permitting adolescents to get married. Further, Islam obliges women to cover themselves with the so called "Hijab" or veil and to be segregated from men in educational institutes and other mass-gathering. places. Islam also fights poverty, a driving force for commercial sex and prostitution, through a well established system of obligatory charity, known as "Zakat", and voluntary charity, known as "Sadaqa", taken from the rich people and given to the poor and needy. Additionally, Islam obliges the rulers to eliminate all means and factors that are conducive for indulging in non-marital sex and intravenous drug use such as sex trade, prostitution, and drug smuggling and to implement the Islamic penalties on those involved in such illegal acts. It should be noted, however, that HIV preventive strategies in some Islamic countries do not necessarily abide by the Islamic doctrine and that knowledge, attitude, and practice of Muslims in various Islamic societies do not necessarily conform to Islamic norms.Unfortunately, there is little published data on the effectiveness of the Islamic preventive strategies in reducing HIV transmission in Muslim communities. According to the United Nations and the World Health Organization data on HIV prevalence in different countries, however, the prevalence of HIV infection in Islamic countries is strikingly low compared to other countries ,10. A reEven though the number of HIV cases in Saudi Arabia is still limited, there is a potential for a rapid spread of this virus particularly among the youth. Strategies to prevent the spread of this virus in Saudi Arabia as well as in other Islamic countries should conform to the Islamic rules and values to be successful.None declaredTAM designed the study, analyzed the data, and wrote the manuscript. YYM, MHJ, and NSH supervised the data collection and revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Recent interest in the function of the nuclear lamina has been provoked by the discovery of lamin A/C mutations in the laminopathy diseases. However, it is not understood why mutations in lamin A give such a range of tissue-specific phenotypes. Part of the problem in rationalising genotype-phenotype correlations in the laminopathies is our lack of understanding of the function of normal and mutant lamin A. To investigate this we have used photobleaching in human cells to analyse the dynamics of wild-type and mutant lamin A protein at the nuclear periphery.We have found that a large proportion of wild-type lamin A at the nuclear periphery is immobile, but that there is some slow movement of lamin A within the nuclear lamina. The mobility of an R482W mutant lamin A was indistinguishable from wild-type, but increased mobility of L85R and L530P mutant proteins within the nuclear lamina was found. However, the N195K mutant shows the most enhanced protein mobility, both within the nucleoplasm and within the lamina.The slow kinetics of lamin A movement is compatible with its incorporation into a stable polymer that only exchanges subunits very slowly. All of the myopathy-associated lamin A mutants that we have studied show increased protein movement compared with wild-type. In contrast, the dynamic behaviour of the lipodystrophy-associated lamin A mutant was indistinguishable from wild-type. This supports the hypothesis that the underlying defect in lamin A function is quite distinct in the laminopathies that affect striated muscle, compared to the diseases that affect adipose tissue. Our data are consistent with an alteration in the stability of the lamin A molecules within the higher-order polymer at the nuclear lamina in myopathies. The latter is alternatively spliced to produce lamins A and C, as well as other minor species. Lamin B is expressed in all cell types and is essential for cell viability. A-type lamins are expressed in more differentiated cells [The nuclear lamina is a filamentous network of lamin proteins that underlies the inner nuclear membrane (INM). It is thought to make connections between both integral membrane proteins of the INM, and chromatin. It may therefore play a fundamental role in the functional organisation of the nucleus. Lamins are type V intermediate filament (IF) proteins, consisting of a central coiled-coil region, and globular N-terminal and C-terminal domains. The N-terminal domain has a nuclear localisation signal (NLS) and most lamins, except for lamin C, are farnesylated at their carboxy termini via a CaaX motif  Figure . The mamed cells .in vitro into 10 nm filaments that resemble those formed in vivo, lamins assemble in vitro into paracrystalline arrays rather than filaments [in vivo, assembly of correct lamin higher-order structures requires the interaction with other molecules/proteins. Lamin A certainly has the ability to interact with other proteins, and also to influence their localisation. In the absence of lamin A, emerin relocates from the INM to the endoplasmic reticulum [Lamins readily form parallel coiled-coil dimers, which then associate into larger polymers. However, whereas cytoplasmic IF proteins assemble ilaments . This sueticulum ,5,6. Theeticulum ,8. The ceticulum ,10 , whereas residues mutated in other laminopathies are located internally within the hydrophobic core of the domain structure ,26 Figu. The latTo better understand the affects of laminopathy-associated mutations on lamin A function we have used fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) to investigate the protein dynamics of GFP-tagged wild-type and disease-associated mutant lamin As in living cells.To investigate the biological affect of different mutations on lamin A nuclear localisation and dynamics we expressed epitope tagged forms of the protein, carrying disease-associated missense mutations, in human HT1080 cells. The mutations chosen were; L85R (DCM), N195K (DCM), R482W (FPLD), and L530P (AD-EDMD). Although L85R and N195K are both located within the coiled-coil domain of lamin A, and associated with DCM Figure , they haSince these mutations are responsible for autosomal dominant forms of disease they should still exert their molecular phenotype in the cell in the presence of wild-type (wt) protein. Both FLAG-tagged and GFP-tagged prelamin As were transiently transfected into human fibrosarcoma cells. Each protein was processed into mature lamin A  and incoThe lamin A mutations that we have studied are within different domains of the protein Figure , or withIn each case a region at the nuclear lamina was bleached. The fluorescence within a 1.8 × 1.8 μm region of interest (ROI) of this bleach region was then followed every 5 minutes over a period of up to 65 minutes. To calculate the loss of fluorescence attributed to the imaging process alone, the sum of pixel intensities was also calculated for a control (unbleached) cell in each case. This was used to normalise the fluorescence intensity for each ROI . The meaFor wild-type lamin A, fluorescence at the nuclear lamina is visibly bleached . The amino acid residues involved all map to a solvent-exposed surface in the structure of the Ig-like C-terminal domain  Figure . By NMR o assays , and a so assays . We suggThe EDMD-associated L530P mutation is also within the Ig-like domain Figure , but unlMissense mutations in the coiled-coil domain of lamin A are associated with the myopathies, not FPLD Figure . They liGiven the genetically dominant nature of many of the laminopathies, it would be interesting to determine whether the presence of a mutant lamin A has an affect on the mobility of the (GFP-tagged) wild-type protein in the same cells.FLPD is clinically distinct from AD-EDMD and DCM. Patients with FPLD do not have striated muscle pathology, conversely adipose tissue is normal in AD-EDMD and DCM. Whereas we find increased mobility of all the myopathy-associated lamin A mutants we studied, we cannot distinguish between the protein dynamics of wt and an FLPD mutant form of lamin A (R482W). We conclude that in AD-EDMD and DCM laminopathies the structure of the nuclear lamina is perturbed in such a way as to allow for more rapid exchange of lamin A molecules. In contrast, we suggest that, in this respect, the structure of the lamin polymer is normal in FPLD.Green Fluorescent Protein (GFP)-tagged human lamin A cloned in pCDNA-3-EGFP (GFP-HLA) was obtained from L Karnitz . GFP-tagged mutant lamin As were then generated by transfer from N-terminal FLAG-tagged fusion constructs . GFP-HLAHuman HT1080 fibrosarcoma cells were transfected with plasmids using Lipofectamine™2000 according to the manufacturer's recommendations. Cells grown on glass slides were fixed 24 hours later for immunofluorescence or GFP analysis. Cells were fixed for 10 mins in 4% paraformaldehyde and permeabilised for 10 mins in 0.05% Triton-X100. FLAG-tagged proteins were detected with 1:200 dilution of M2 anti-Flag mouse monoclonal antibody (Sigma) and 1:100 anti-mouse Texas Red Fab'2 heavy and light chains (Jackson Labs). Slides were counterstained with DAPI and analysed using a Zeiss Axioplan microscope fitted with a Xillig CCD camera and a focus motor to collect images at 0.5 μm intervals in the z plane .Cells were grown on DeltaT 0.17 mm culture dishes (Bioptechs Inc) and were mounted onto a heated stage (Bioptechs Inc) on a Zeiss LSM510 confocal microscope. An objective warmer (Bioptechs Inc) was also used to help to maintain a stable temperature of the medium in the culture dish.For FRAP, a 1.8 × 1.8 μm region at the nuclear periphery in the mid-focal plane was bleached with 100 iterations at 100% power of the argon laser running at 6.1 mA (50% power). The pinhole size for the confocal was set at 1 Airy unit. The time series software option was used to specify the appropriate time delay between rounds of 3D image stack capture. Each bleached cell was imaged with a ×100 objective, in a window that included other non-bleached cells to allow for relative fluorescence levels to be normalised. Immediately following the bleach, images in the same z-plane were captured at 1s intervals  to correct for nuclear rotation and cell movement. This enabled all images to be superimposed with the pre-bleach image.Because of the length of FRAP analysis, nuclear rotation, cell movement and focus drift presented a problem in registering the bleach ROI between time points. To account for this, the best For FLIP, an ROI at the nuclear periphery was bleached with 10 laser iterations at 100% of 50% total laser output (~6.1 mA). Following the bleach, 5 images were taken at 2 sec intervals using 8% of laser output. The bleach procedure was repeated for 16 rounds.In both FRAP and FLIP, the loss of fluorescence attributed to the imaging process alone was assessed from the sum of pixel intensities in a control (unbleached) cell, in each analysis. The relative fluorescence intensity over time was calculated for each defined ROI using a normalisation equation .SG constructed the GFP-tagged lamin A constructs, did the cell transfections, the fluorescence microscopy and the photobleaching analysis. NG gave assistance and advice in the photobleaching studies. PP advised and assisted in confocal microscopy and wrote the scripts for image registration over the time course of FRAP. CO and HJW constructed the FLAG-tagged lamin A mutants and provided advice. WAB conceived of the study and drafted the manuscript. All authors read and approved the final manuscript


Subventricular microglia (SVMs) are positioned at the interface of the cerebrospinal fluid and brain parenchyma and may play a role in periventricular inflammatory reactions. However, SVMs have not been previously investigated in detail due to the lack of a specific methodology for their study exclusive of deeper parenchymal microglia.in situ complemented the histopathological analysis.We have developed and characterized a novel model for the investigation of subventricular microglial reactions in mice using intracerebroventricular (ICV) injection of high-dose rhodamine dyes. Dynamic studies using timelapse confocal microscopy in situ using timelapse confocal microscopy. Finally, supporting the disease relevance of this event, the triad of ependymal cell death, SVM activation, and infiltrative microgliosis was recapitulated by a single ICV injection of HIV-1 tat protein.We demonstrate that high-dose ICV rhodamine dye injection resulted in selective uptake by the ependyma and ependymal death within hours. Phagocytosis of ependymal debris by activated SVMs was evident by 1d as demonstrated by the appearance of rhodamine-positive SVMs. In the absence of further manipulation, labelled SVMs remained in the subventricular space. However, these cells exhibited the ability to migrate several hundred microns into the parenchyma towards a deafferentation injury of the hippocampus. This "infiltrative microgliosis" was verified Subependymal microglia exhibit robust activation and migration in periventricular inflammatory responses. Further study of this population of microglia may provide insight into neurological diseases with tendencies to involve the ventricular system and periventricular tissues. Microglin vivo . These dImportantly, a population of resident subventricular microglia (SVMs) are found in the subependymal zone ,7 suggesin vivo combined with confocal timelapse techniques for dynamic analyses in adult mouse brain tissue. We hypothesized that SVM activation is a general consequence of periventricular insults that can be caused by diverse circulating substances in the CSF known to damage the ependyma. Our work indicates that activated, phagocytic SVMs are capable of infiltrating deep within the parenchyma.The reaction of SVMs to periventricular damage has not been described in detail. Specifically, the functional repertoire displayed by activated SVMs including phagocytosis and long-distance migration have not been investigated. Here, we directly examine SVM function by exploiting a novel methodology which selectively activates and labels SVMs Adult male C57bl/6 mice (6–8 wks) were obtained commercially from Harlan  and cared for in accordance with Public Health Service and University of Virginia guidelines.9 plaque forming units); 100 mg/ml BSA, 0.1 mM DTT in PBS, or deactivated tat [stratum oriens of the left hippocampus.To damage the ependyma 2–3 μl of 0.2% Sp-DiI  in DMSO; 1:20 rhodamine-conjugated latex microspheres  in sterile PBS; 0.25 U Neuraminidase (Sigma); or 2.0 nM recombinant HIV-1 tat protein in 100 mg/ml BSA, 0.1 mM DTT in PBS were stereotaxically injected into the left lateral ventricle at the following coordinates: L, 1.5 mm; P, -0.5 mm; D, 2.0 mm. GFP-expressing adenovirus .Brains were collected and processed for histopathology as previously described . All ant4 (Molecular Probes) [t-test. Pairwise post-hoc analysis was performed with a t-test and the Bonferroni correction factor. A p < 0.05 was considered statistically significant. Rose plots were created in Kaleidagraph 3.0.200–400 μm live slices were prepared from adult C57BL/6 mice as described previously ,17. Brie Probes) . Values in vivo and in fixed tissues [Rhodamine dyes such as SP-DiI and rhodamine latex microbeads (RhoB) have been classically used for tract tracing studies  tissues ,20. Rece tissues ,21. In p4 lectin from Griffonia simplicifolia . Further, SVMs displayed dynamic behavior consistent with phagocytosis of ependymal debris Fig. ). TherefTo determine if SVM activation is a general response to periventricular damage we injected animals with a sublethal dose of RhoB to label ependymal cells without causing damage. 24 h later we injected 0.25 U neuraminidase to damage the ependyma  via an ain vitro in response to many chemokines and growth factors present in brain lesions and plaques [in vivo we gave mice a deafferenting lesion (FSL) of the hippocampus 24 hours following rhodamine dye injection and allowed survival for up to 28 days. Invasion of the parenchyma by RHO+ cells occurred in the stratum oriens of the denervated hippocampus (cSO) in 21/21 animals but not in sham animals (0/4)  Fig. . Infiltr/4) Fig. . Based o/4) Fig. . This ti/4) Fig. , GSD, ac/4) Fig. , IB4, anin vivo and in situ. We have named this event "infiltrative microgliosis" (IMG).To provide direct evidence for the migration of SVMs into the parenchyma and characterize their general migratory behavior we prepared live brain slices from dye-injected/lesioned mice and rendered confocal time-lapse movies in the cSO Fig. ). RHO+ cin vitro possibly by mimicking, and inducing expression of, chemokines [4 histochemistry (not shown). To determine whether this was due to migration of SVMs into the parenchyma or spreading activation of stationary cells we performed timelapse confocal analysis of live brain slices taken from animals 24 h after ICV tat injection. We found that nearly all activated periventricular microglia were motile and many were locomotory after tat injection . Littlein vivo. For instance, in this study we were able to demonstrate phagocytosis, mitosis, and migration of activated SVMs using histopathological techniques alone. We have shown these cellular activities can be confirmed in situ with timelapse confocal microscopy further validating this versatile protocol. Mechanistic investigations are possible by combining our in vivo and in situ protocols with genetic or pharmacological techniques.While evaluating rhodamine dyes for selective labeling of ependymal cells  for othealone is sufficient to damage the ependyma, activate SVMs, and incite infiltrative microgliosis supports the "cytokine dysregulation hypothesis" [We found SVMs only infiltrated the brain after selective ependymal damage with rhodamine dyes if a distant lesion was also present, likely providing a gradient of chemoattractive cues. CC chemokines are known to be upregulated rapidly after deafferenting injury of the hippocampus . The extothesis" ,22 of daothesis" ,34.in vivo and in situ models for the study of SVM function, further insight into microglial dynamics after brain injury, and novel hypotheses for the role of microglia in periventricular reactions in neurological diseases.In summary, we have shown that SVMs are a highly reactive pool of cells which, when activated, can infiltrate the parenchyma in response to injury cues from damaged brain regions or exposure to HIV-1 tat. These findings provide new stratum oriens of the hippocampus contralateral to stab lesion; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; GFP, green fluorescent protein; GSD, modified Gallyas silver degeneration stain; HIVE, human immune deficiency virus 1 encephalitis; ICV, intracerebroventricular; IFN-γ, interferon gamma; IMG, infiltrative microgliosis; MS, multiple sclerosis; PBS, phosphate-buffered saline; pERK, phosphorylated/activated extracellular signal-regulated kinase; PI, post-injury; RHO+, rhodamine-positive; RhoB, rhodamine latex microbeads; SEM, standard error of the mean; Sp-DiI, 1,1'-dioctadecyl-6,6'-di(4-sulfophenyl)-3,3,3',3'-tetramethylindocarbocyanine; SVM, subventricular microglia; TNF-α, tumor necrosis factor alpha.BrdU, 5-bromo 2-deoxyuridine; BSA, bovine serum albumin; CSF, cerebrospinal fluid; cSO, The authors declare that they have no competing interests.WSC conceived of and designed the study, carried out all experiments, performed data analysis, and drafted the manuscript. S-IM participated in study design especially with regards to the timelapse experiments. AFH participated in study design and coordination and provided the confocal facilities, equipment, and expertise for timelapse experiments. JWM participated in study design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.SVM phagocytosis of ependymal debris. Activity of SVMs suggestive of phagocytosis of dye-labeled ependymal cell debris 24 h following injection. SVMs can be seen extending processes towards debris. Examples of ependymal debris are pseudocolored yellow. Each frame is a 2D projection representing a stack of 6 images 8 μm apart. Original magnification, 40×.Click here for filestratum oriens from the subependymal region of the posterior lateral ventricle. Note highly directed migration into the hippocampus. Each frame is a 2D projection representing a stack of 4 images 10 μm apart taken every 3 minutes. Original magnification, 20×.Dynamics of infiltrative microgliosis. Infiltrative microgliosis of SVMs into the hippocampal Click here for fileSVM dynamics in response to HIV-1 tat protein. Extensive migratory activation of periventricular microglia in response to 2.0 nM ICV HIV-1 tat protein. This migratory reaction extends several hundred microns into the parenchyma. v3v, ventral third ventricle. Each frame is a 2D projection representing a stack of 6 images 8 μm apart taken every 90 seconds. Original magnification, 20×. Field measures 700 × 700 μm.Click here for fileControl video for HIV-1 tat protein. Lack of activation of SVMs and migration with ICV injection of deactivated HIV-1 tat protein . The paucity of IB4 labeling indicates the limited microglial activation. Note blood vessel endothelial cell labeling and gradual photobleaching. Each frame is a 2D projection representing a stack of 6 images 8 μm apart taken every 90 seconds. Original magnification, 20×. Field measures 700 × 700 μm.Click here for fileHIV-1 tat incites infiltrative microgliosis. HIV-1 tat activated subventricular microglia infiltrate the parenchyma. Three highlighted cells correspond to those in Figure Click here for file


C. elegans development, behavior and physiology. A thorough understanding of the ways in which genes control these aspects of biology depends, in part, on the accuracy of phenotypic analysis. While worms that move poorly are relatively easy to describe, description of hyperactive movement and movement modulation presents more of a challenge. An enhanced capability to analyze all the complexities of nematode movement will thus help our understanding of how genes control behavior.Nematode sinusoidal movement has been used as a phenotype in many studies of We have developed a user-friendly system to analyze nematode movement in an automated and quantitative manner. In this system nematodes are automatically recognized and a computer-controlled microscope stage ensures that the nematode is kept within the camera field of view while video images from the camera are stored on videotape. In a second step, the images from the videotapes are processed to recognize the worm and to extract its changing position and posture over time. From this information, a variety of movement parameters are calculated. These parameters include the velocity of the worm's centroid, the velocity of the worm along its track, the extent and frequency of body bending, the amplitude and wavelength of the sinusoidal movement, and the propagation of the contraction wave along the body. The length of the worm is also determined and used to normalize the amplitude and wavelength measurements.goa-1 gene, which encodes Go-alpha). We also demonstrate the use of this system for analyzing the effects of toxic agents. Concentration-response curves for the toxicants arsenite and aldicarb, both of which affect motility, were determined for wild-type and several mutant strains, identifying P-glycoprotein mutants as not significantly more sensitive to either compound, while cat-4 mutants are more sensitive to arsenite but not aldicarb.To demonstrate the utility of this system, we report here a comparison of movement parameters for a small set of mutants affecting the Go/Gq mediated signaling network that controls acetylcholine release at the neuromuscular junction. The system allows comparison of distinct genotypes that affect movement similarly , as well as of different mutant alleles at a single locus (null and dominant negative alleles of the C. elegans movement, as well as a comparison of gene function. Concentration-response curves will allow rigorous analysis of toxic agents as well as of pharmacological agents. This type of system thus represents a powerful analytical tool that can be readily coupled with the molecular genetics of nematodes.Automated analysis of nematode movement facilitates a broad spectrum of experiments. Detailed genetic analysis of multiple alleles and of distinct genes in a regulatory network is now possible. These studies will facilitate quantitative modeling of  C. elegans as an experimental molecular genetic system was to understand how genes control behavior, especially locomotion, since the uncoordinated (Unc) mutants were discovered early in the history of C. elegans genetics whereV1 = first segment vector andV2 = second segment vectordifference during each possible 32 frame (~6 second) time window; that is, the most positive angle minus the most negative angle.We define the FLEX at each articulation point as the maximum angle specgram calculates the spectrogram for a given signal as follows: 1. It splits the signal into overlapping sections and applies the window specified by the window parameter to each section. 2. It computes the discrete-time Fourier transform of each section with a length nfft FFT to produce an estimate of the short-term frequency content of the signal; these transforms make up the columns of B. The quantity (length(window) – numoverlap) specifies by how many samples specgram shifts the window. 3. For real input, specgram truncates the spectrogram to the first nfft/2 + 1 points for nfft even and (nfft + 1)/2 for nfft odd." The magnitude of the function indicates the relative energies of the signal's component frequencies. We take the highest magnitude (non-constant) component frequency as the characteristic frequency of that time-window of angles. We apply the spectrogram function ("specgram") from Matlab's Signal Processing Toolbox to the matrix of bend angles calculated for FLEX. Specgram calculates a windowed discrete-time Fourier transform (short-time Fourier transform) for the changing angles for each articulation point using a sliding window 32 frames (~6 seconds) wide. We quote from Matlab's documentation for specgram: "For the time delay calculation, the program correlates anterior bend angles with posterior bend angles occurring at later time using a Dynamic Time Warping function. The program then uses these correlations to calculate the time required for the posterior bend to reach the same angle as its anterior neighbor.The program aligns the major axis of a best-fit bounding box with the worm's instantaneous velocity vector. The width of the bounding box (its minor axis) is taken as the instantaneous wormtrack waveform amplitude.We apply a rotation and translation transform to the spine of the worm from every frame to mathematically align each worm with y = 0 using the instantaneous velocity vector as the worm's centerline:We create a matrix w containing the XY coordinates of the 13 points for a worm's spine and create a translation transform that we will use to center the worm's midpoint at the XY origin:We also create a rotation transform to align the instantaneous velocity vector (with angle theta), to y = 0:We multiply the two matrices to create a convenient combined transformC = B*A;and finally multiply our matrix by the combined transform matrixww = C*w;which yields a matrix ww with the rotated and aligned coordinates for the 13 points. We perform a spatial Fast Fourier Transform  on the rotated "spine" of each worm, using the varying y-values as the "signal" with their corresponding x-position values defining the signal to be in a spatial  domain. By working in the spatial domain, the result from our FFT is in cycles per mm. The inverse of this result is the track wavelength, in mm per cycle.2 + Δ y2)0.5Sum of the distances between the points distributed along the worm's spine with each point-to-point distance calculated by  each worm's velocity data into discrete ranges (or 'bins'). For velocity we use 'bins' that are 0.03333 mm/sec wide, i.e. bins would represent 0 to 0.03333 mm/sec, another 0.03333 to 0.06666 mm/sec, and so on. Bin sizes for each parameter are as follows: Point and Centroid Velocity, 0.03333 mm/sec; Flex, 0.1 radian; Frequency, 0.16665 Hz; Time Delay, 0.075 sec; Track Amplitude, 0.01 mm; Track Wavelength, 0.05 mm; Length-Normalized Track Amplitude, 1% mean body length; Length-Normalized Track Wavelength, 5% mean body length.Again using our velocity example, the frequency of occurrence for the number of velocity values in each bin is normalized to a percent of velocities observed for that worm, and the normalized velocity distribution is added to a list of the other normalized velocity distributions for that population.  The final histogram curve for the population is generated by plotting the mean value of each data bin's normalized frequency of occurrence (on the y-axis) versus the bin value (on the x-axis) which shows us the proportion of worms that exhibited each velocity. The same method is used for creating the histogram curves for each metric, naturally selecting bin sizes appropriate for the data in question:Standard statistical tests were performed using Matlab functions. Each p-value reported was from a one-way analysis of variance (ANOVA) comparing the distribution of mean values  from each population of worms against that of each other population; each ANOVA tested the null hypothesis that the mean of the mean values from each populations were the same. Unless otherwise noted, statistical tests were performed on data from worms only when moving forward. For the toxicant concentration-response data, curves were fit by non-linear regression using Prism (GraphPad Software) sigmoidal dose-response equation with variable slope.Source code is available through a GPL at Documentation of this software is available as a pdf from C. elegans movement for toxicology studies, YK performed most of the toxicology experiments. JM, CC, RS and PS analyzed the data. CC, JM, and PS wrote the paper. All authors read and approved the final manuscript.SM, JB, JM and PS conceived and designed the original automated system for tracking and movement measurement. SM developed a working prototype system and brought in the key algorithms used. CC wrote most of the code in the current release. JM developed the protocol for the plate assay, and devised the dose-response and genetic experiments. RS conceived of applying automated analysis of