Cardamine amara—to the enormous—124,000 Mb in the lily Fritillaria assyriaca. By comparison, fruitfly and human genomes have 180 Mb and 3,200 Mb, respectively. Genomes of important crops such as sorghum, soybean, maize, and wheat hover between 735 Mb and 16,900 Mb, and determining their complete sequences is daunting and costly.Plant genome sizes span the modest—54 million base pairs (Mb) in the bitter cress Sorghum bicolor), a staple crop in much of the developing world because of its resilience in arid climates.Wide size variations do not necessarily reflect differences in gene content, but rather reflect the presence of repetitive sequence elements that do not generally code for genes. Repetitive elements account for at least 75% of the maize and sorghum genomes. In a new study, Joseph Bedell and his colleagues describe a way to filter away repetitive elements when sequencing the genome of sorghum . BACs offer the most comprehensive representation of the genome because they contain large pieces of unmodified sorghum chromosomal DNA. Of the 148 genes identified on 14 sorghum BACs, 133 appeared in the filtered set. This means that the methylation filtration method captured at least 90% of the genes in the sorghum genome and 96% (131/137) if a repeat cluster of 11 known methylated genes is removed from the analysis.Arabidopsis—a plant model organism—predicted that shotgun approach would yield similar results (67% of the length of 96% of the genes) after sequencing the equivalent of 1.15 times its total length (rather than 1.15 times the length of just the coding regions). Thus, methylation filtration can provide as much information on coding sequences as the shotgun approach, with less investment in sequencing.Methylation filtration also compared favorably to shotgun sequencing, a method that reads the whole genome in small fragments that are progressively assembled into larger pieces by computer analysis. The authors reported that after sequencing 285 Mb of filtered sorghum DNA—approximately 1.15 times the length of the sorghum coding regions—they obtained on average 65% of the length of 96% of the genes. Theoretical calculations and simulation based on the genome of Methylation filtration does not yield a complete genome map, but it offers quicker, more affordable access to genes than most commonly used sequencing approaches. Sorghum is closely related to maize and sugar cane, and more distantly to rice. The availability of its genome sequence offers the chance for more in-depth experiments into the evolution of the grass family, and promises important insights into the genetic control of drought resistance.


Identifying the causative mutation for a disease can be the first step to a potential cure. This task is not always trivial. Often the initial strategy is to look for the variations within a mutated gene that alter its protein coding sequence, as these mutations often alter the gene's function. However, in a growing number of cases, the causative mutation is a “synonymous” mutation—a change in the coding sequence of a gene that doesn't change the sequence of the protein coded by the gene. This type of mutation may be responsible for Seckel syndrome, a human disease characterized by dwarfism. In Seckel syndrome, the mutation doesn't alter the protein sequence itself but instead results in the skipping over of a portion of the protein coding sequence (an exon), a process called altered splicing. The disease-causing potential of this type of splicing mutation has only recently gathered attention.Splicing assembles the exons of a transcribed gene (the RNA copy) into the right order while removing the non-coding sequences of the RNA (introns). This highly regulated process is coordinated by a number of sequences within a gene, including splice sites that precede and follow the exon, as well as by exonic splicing enhancers (ESEs), which help recruit the factors (proteins) necessary to insure proper splicing. Although splice sites have optimal (consensus) sequences, there is some variability amongst individual splice site sequences that allows splicing to take place to a greater or lesser extent. ESEs facilitate splicing, especially when a gene's splice sites vary from the consensus sequence. Candidate ESEs have previously been identified based on their more frequent occurrence in exons that are adjacent to non-consensus splice sites.PLoS Biology, William Fairbrother et al. investigated the functionality of these putative ESEs. If they are functional, the authors reasoned, then mutations that disrupt them would be selected against—that is, these mutations would tend to be discarded—in the human genome.In this issue of To this end Fairbrother et al. developed a computational method, which they call VERIFY , to evaluate the selective pressure on ESEs. They took advantage of a public database of all single nucleotide polymorphisms (DNA changes at a single point) within the human genome and compared them to the chimpanzee genome; this allowed the authors to infer the identity of the ancestral gene . By determining which allele is ancestral and which is the variant, the researchers could then distinguish the mutations that created ESEs from mutations that disrupted ESEs.Mutations that altered or disrupted ESEs were under-represented, leading the authors to conclude that predicted ESE sequences evolve under a more stringent level of selection than exonic sequences with no predicted ESEs. This selective pressure was greater for predicted ESEs located near the splice signals than for ESEs that were located within the exon. This result was consistent with experimental findings that ESE strength diminishes with distance from the splice site.As more vertebrate genomes are sequenced and the public database of single nucleotide polymorphisms continues to grow, this type of computational method will become increasingly valuable. It can help confirm the functionality or role of candidate regulatory elements thought to control various aspects of gene expression, and in so doing, offer insights into the complex machinations required to maintain the healthy operation of the human genome.


Genetic variability in viral populations is usually estimated by means of polymerase chain reaction (PCR) based methods in which the relative abundance of each amplicon is assumed to be proportional to the frequency of the corresponding template in the initial sample. Although bias in template-to-product ratios has been described before, its relevance in describing viral genetic variability at the intrapatient level has not been fully assessed yet.To investigate the role of oligonucleotide design in estimating viral variability within hosts, genetic diversity in hepatitis C virus (HCV) populations from eight infected patients was characterised by two parallel PCR amplifications performed with two slightly different sets of primers, followed by cloning and sequencing (mean = 89 cloned sequences per patient). Population genetics analyses of viral populations recovered by pairs of amplifications revealed that in seven patients statistically significant differences were detected between populations sampled with different set of primers.Genetic variability analyses demonstrates that PCR selection due to the choice of primers, differing in their degeneracy degree at some nucleotide positions, can eclipse totally or partially viral variants, hence yielding significant different estimates of viral variability within a single patient and therefore eventually producing quite different qualitative and quantitative descriptions of viral populations within each host. One of the most difficult tasks faced by virologists is the documentation and evaluation of the genetic variability of viral populations in infected patients. These analyses are greatly facilitated by the use of the polymerase chain reaction (PCR). PCR based techniques do not always produce a highly specific and homogeneous product. When the template is a complex mixture of homologous sequences the aim of the amplification would be to preserve as much as possible the template-to-product ratios of every sequence in order to obtain a good representation of the diversity present in the initial sample. PCR products are derived from templates by a process involving complex chemical kinetics, and the relative abundance of the different homologous genomes among the final products is often a parameter of interest. This is the case, for instance, in experiments aimed at determining natural diversity in microbial communities  or at idThe precise mechanisms involved in the preferential amplification of some templates from non-homogeneous sources are not fully understood and should be differentiated from those related to stochastic or tube-to-tube variations in amplification efficiency. When dealing with heterogeneous templates, two different processes can alter template-to-product ratios: PCR selection and PCR drift . The forGiven the high levels of variability found in RNA virus populations, primers involved in RT-PCR (retrotranscription followed by PCR) are usually designed as degenerate sequences to ensure that the chance of amplifying the different sequences present in an heterogeneous template will be more uniform and, therefore, all will be present in the amplified product in similar proportions to those in the original template. However, the use of even highly degenerate primers does not preclude the possibility of mismatches occurring between a given primer and some of the sequences present in a heterogeneous template, especially for highly variable regions. This would lead to differential amplification of sequences  (i.e. PCHepatitis C virus (HCV) is a positive-sense, single-stranded RNA virus of approximately 9.4 kb, classified in a separate genus (Hepacivirus) of the Flaviviridae family. HCV has been recognised as a major etiological agent of acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma around the world . HCV isoGenetic variability in two different regions of the HCV genome was studied by means of RT-PCR amplification in eight infected patients. A fragment comprising partially E1 and E2 region including HVR1 and HVR2  was amplified in six infected patients. In the other two patients, part of the NS5A region including the ISDR (interferon-sensitivity determining region) and the variable region 3 (V3) was amplified.Each fragment was amplified twice from each HCV infected patient with two slightly different sets of primers Table . DiffereTwo types of population analysis were carried out  with theS) detected in a group of sequences from a particular set of primers ranged from 1 (in patient E04) to 68 (in patient E16). Similarly, for region NS5A, with only two patients analysed, S ranged from 4 to 70 for the same set of primers . Haplotype diversity (HT) also reached both extreme values: in some groups of sequences corresponding to a particular primer set from a single patient , almost every sequence constituted a different haplotype, resulting in HT ~ 1; whereas in other groups  very few haplotypes were detected, with HT ~ 0.1 or even lower. When variability was measured taking both the frequencies of haplotypes and their genetic distances a wider variability range was observed: nucleotide diversity (π) of the viral populations estimated for the eight patients analysed differed by up to three orders of magnitude . Howevek) between both groups of sequences was lower than k obtained for a single primer set (e. g. primer set 2). Moreover, the main haplotype was similarly sampled by the two sets of primers  also showed that the two groups of sequences obtained from each patient were statistically different, with genetic distances (k) between the two groups of sequences from the same patient largely exceeding the genetic distances estimated within a single group of sequences. Moreover, in these four patients, the amount of genetic variability detected also seemed to be deeply affected by the set of primers chosen for PCR .PCR drift (i. e. bias in template-to-product ratios produced by random events occurred in the early cycles of the reaction) and PCR selection  are two processes that can lead to bias in template-to-product ratio of PCR amplifications ,22. In aAttempts to reduce PCR bias caused by primer-template mismatches usually involve designing degenerate primers. Here we have studied the effect of small differences in primer degeneracy on PCR outcome with heterogeneous templates and their implications and extent on viral genetic variability at the intrapatient level. Our results indicate that template-to-product ratios can be significantly biased in standard PCR amplifications of non-homogeneous templates. By means of RT-PCR sampling of HCV sequences from eight infected patients we have found that sequence sampling from a single source varied considerably when two slightly different sets of primers were used in the PCR amplification: both sets of primers were chosen after inspection of HCV-1 aligned sequences from GenBank, and degenerations were introduced in both sets following only slightly different criteria. The use of one set of primers or another not only gave rise to different collections of sequences, but also to their different distribution along phylogenetic trees in most of viral populations studied. These results indicate that bias in template-to-product ratios can severely distort the results of analysis of variability in virus populations, both quantitatively (i. e. amount of genetic variability) and qualitatively (i. e. particular sequence clusters on a phylogenetic tree).Fan et al.  demonstrThe results obtained in this study allow us to strongly suggest that, for PCR-based variability studies, a certain level of primer degeneration, compatible with specific product amplification, would be more than advisable for variability studies in which, as with viral populations, there is no possibility of designing a perfect set of primer pairs that equally amplify all possible templates. For this, it is convenient to align many available related sequences and empirically determine which positions are most polymorphic and therefore susceptible to participate in mismatches.PCR selection  was detected and attributed to differences in degeneracy at some nucleotide positions in the oligonucleotides involved in amplification. Alterations in the template-to-product ratio during PCR amplification significantly affects viral population descriptions to the extent that two PCR outcomes from the same infected patient can result, after genetic population analyses, in two genetically distinct populations. Two important implications can be derived: first, all estimates of genetic variability parameters should be considered always as a minimum, and second, the search for particular existing genomes, such as drug resistant genomes, can be totally or partially eclipsed by others more susceptible to be annealed by the olinucleotides used in the amplification.Serum samples from eight patients infected with HCV were chosen for this study. Six patients infected with HCV-1b  were analysed using two sets of primers that partially amplified the E1E2 region (472 nucleotides) and two patients infected with HCV-1a (N02 and N07) were analysed using two sets of primers that partially amplified the NS5A region (743 nucleotides).The first step in the design of oligonucleotide primers was to collect a representative variety of HCV-1 sequences from GenBank. From 50 homologous sequences, nucleotide positions for primers were chosen with GeneFisher , and twoViral RNA was extracted from 140 μl of serum using High Pure Viral RNA Kit (Roche). In order to prevent any bias during reverse transcription reactions due to oligonucleotide specificity, all reverse transcription reactions were performed using random hexadeoxynucleotides. Reverse transcriptions (RT) were performed in a 20 μl volume containing 5 μl of eluted RNA, 4 μl of 5x RT buffer, 0.5 mM of each deoxynucleotide, 0.5 μg of random hexamers, 100 U of MMLV reverse transcriptase (Promega), and 20 U of RNasin Ribonuclease Inhibitor (Promega). Reactions were incubated at 37°C for 60 min, followed by 2 min at 95°C.Pfu DNA polymerase (Promega). The outer set of primers for the E1E2 region were 1-Eg1  and 1-Ea (or 2-Ea)  and directly cloned into EcoRV-digested pBluescript II SK (+) phagemid (Stratagene). Recombinant plasmid DNA was purified using the High Pure Plasmid Isolation Kit (Roche). Cloned products were sequenced using vector-based primers KS and SK (Stratagene). Sequencing was carried out using ABI PRISM BigDye Terminator v3.0 Ready Reaction Cycle Sequencing KIT (Applied Biosystems) on an ABI 3700 automated sequencer. Sequences were verified and both strands assembled using the Staden package .Sequences were aligned using CLUSTALX v1.81 . The neiS); haplotype diversity (HT) considering as haplotype each different sequence; nucleotide diversity (π) [k). Genetic differentiation between groups of sequences was estimated as the average number of nucleotide substitutions between groups (xyd). The statistical significance of genetic differentiation between groups, as estimated by , was established by the permutation test [Fst, was calculated using the ARLEQUIN program ver. 2.000 [Polymorphism and genetic differentiation were analysed using DNAsp version 4.0 . Estimatsity (π) ; and aveion test . The pror. 2.000 .The authors declare that they have no competing interests.MAB and IG-R co-conceived, designed and coordinated the study, participated in the molecular studies and sequence alignment, interpreted data, oversaw the training of technicians, and co-drafted the manuscript; MAB isolated viral genomes, co-performed population and phylogenetic analyses; NJ and MT-P participated in molecular studies and sequence alignment, interpreted the data and helped draft the manuscript; AM interpreted data and participated in proofreading of the manuscript; FG-C coordinated the study, interpreted data, co-performed population and phylogenetic analyses and participated in proofreading of the manuscript. All authors read and approved the final manuscript.Summary of genetic variability and population differentiation of within patient HCV populations based on viral sequences obtained with two alternative primer set. E1E2 region was analysed in patients E03, E04, E10, E16, E23 and E25, and NS5A region in patients N25 and E02Click here for file


Drosophila melanogaster Malpighian  tubule gives a radically new view of the function of the tubule, emphasising solute transport rather than fluid secretion.Analysis of the transcriptome of the  Drosophila melanogaster Malpighian  tubule, a defined differentiated tissue.Comprehensive, tissue-specific, microarray analysis is a potent tool for the identification of tightly defined expression patterns that might be missed in whole-organism scans. We applied such an analysis to D. melanogaster Malpighian tubule is highly reproducible and significantly different from that obtained from whole-organism arrays. More than 200 genes are more than 10-fold enriched and over 1,000 are significantly enriched. Of the top 200 genes, only 18 have previously been named, and only 45% have even estimates of function. In addition, 30 transcription factors, not previously implicated in tubule development, are shown to be enriched in adult tubule, and their expression patterns respect precisely the domains and cell types previously identified by enhancer trapping. Of Drosophila genes with close human disease homologs, 50 are enriched threefold or more, and eight enriched 10-fold or more, in tubule. Intriguingly, several of these diseases have human renal phenotypes, implying close conservation of renal function across 400 million years of divergent evolution.The transcriptome of the From those genes that are identifiable, a radically new view of the function of the tubule, emphasizing solute transport rather than fluid secretion, can be obtained. The results illustrate the phenotype gap: historically, the effort expended on a model organism has tended to concentrate on a relatively small set of processes, rather than on the spread of genes in the genome. Drosophila [Drosophila, arrays are frequently used for whole-organism studies, but in multicellular organisms the ease of experimentation must be balanced against two potential problems: sensitivity and opposing changes. In the first case, even large changes in gene expression in a small tissue will not significantly influence the overall levels in the whole organism; in the second, changes in opposite directions in roughly balanced populations of cells  will cancel out at an organismal scale. It is thus vital to resolve gene expression not only over time but also over space. In practice, this means looking at gene expression in defined cell types and tissues as well as in the whole organism. Our assumption is that the expression of many putative genes will go undetected until such tissue-specific studies are performed [Microarrays allow the interrogation of the transcriptome, the set of genes transcribed in a particular cell type under a particular condition . Arrays osophila . Commonlosophila , of rhytosophila  or of reosophila ,6. In Drerformed  - with oDrosophila melanogaster. The tubule is a valuable model for studies of both epithelial development and function. Developmentally, the tissue is derived from two distinct origins: an ectodermal outpushing of the hindgut and subsequent invasion (late in embryogenesis) by mesodermal cells [We applied Affymetrix arrays in the context of a defined tissue with extensive physiological characterization, the Malpighian  tubule of al cells . Tubule al cells . The traal cells -12. The al cells ,13,14. Cal cells -17, and al cells ,18-20.This wealth of physiological knowledge provides a framework for the analysis of the results, and thus - unusually in genetic model organisms - a reality check on the usefulness of the experiment.Drosophila melanogaster Malpighian  tubules, for which defined state there is a wealth of physiological data, with matched whole flies. As described in Materials and methods, data were analyzed by Affymetrix MAS 5.0 software, or by dChip, or dChip and Significance Analysis of Microarrays (SAM) software. Both methods of identifying differentially expressed genes from dChip-normalized data gave virtually the same results. Indeed, SAM analysis followed by further filtering produced 1,465 differentially expressed genes compared to 1,455 genes identified within filtering by dChip alone. Furthermore, the latter list is indeed a subset of the former one. For that reason we report only the list generated by dChip in comparison with MAS data.The principle of the experiment was to compare the transcriptome of 7-day adult r = 0.6, p < 0.0001). Taking genes called as significant by both systems, MAS5 'up' call or dChip t-test p-value of 0.01, and narrowing the list by setting an arbitrary cutoff of twofold enrichment and minimum mean difference of 100, MAS5 reported 683 genes and dChip reported 671. Furthermore, the dChip-reported genes overlap with 77% of MAS5-reported genes and this number increases to 91% if only the top 500 MAS5-reported genes are considered. Our confidence in the quality of the dataset is thus high. For simplicity, and because the two analyses produce concordant results, further analysis is restricted to the MAS5 results.Both MAS and dChip/SAM gave comparable views of the data, despite the radically different approaches to analysis. It has been shown that the average absolute log ratios between replicate arrays calculated with dChip are significantly lower than one calculated with Affymetrix software (Li and Wong ). This bThe full microarray data have been deposited in ArrayExpress . The flyFour genes were selected from each of three fly tubule expression classes: very highly enriched; uniformly expressed; and very highly depleted. The expression of each gene was verified by quantitative reverse transcription PCR (RT-PCR) and the data are presented in Table rp49 , and compared with the globally normalized Affymetrix signal, the agreement is seen to be excellent . With appropriate caution, the normalized Affymetrix signal can thus be taken as a reasonable estimate of expression levels between genes.These data can also be used to validate the use of the normalized Affymetrix signal as a semi-quantitative measure of RNA abundance Table . If the t Figure , with a +, K+ ATPase is also highly abundant: this is more surprising, and is discussed below. Two organic cation transporters are also very abundant. Alcohol dehydrogenase, long known to be expressed in tubules [Table  tubules ,24, is a tubules -30. Howe tubules ,32. The Table Drosophila, and shows that the tubule phenotype may go some way to closing it. Around 20% of Drosophila genes have been studied in sufficient detail to attract names (beyond the standard 'CG' notation for computer-annotated genes). Figure Drosophila and other species, will rely increasingly on the study of specific tissues, as it is only in this context that expression of genes will be either measurable or explicable.Another prominent feature of the signal data in Table Many microarray experiments merely classify enriched genes to their Gene Ontology families. However, the uniquely detailed physiological data available on the Malpighian tubule allows a much more informative approach. The dataset can be validated by inspection, based on known molecular functions in the tissue and new functions can be inferred from abundant or enriched transcripts in the dataset. As the array is relatively comprehensive , the results are also relatively authoritative.Drosophila eye-color mutants also have an effect on tubule color, and have since been shown to encode genes for transport of eye-pigment precursors [scarlet and white, and among the monocarboxylic acid transporters is CG12286, which we have recently argued to correspond to karmoisin, a probable kynurenine tranporter [The housekeeping ribosomal transcripts vanish from the enrichment list Table , which iecursors ,36. Thesanporter . Glucoseanporter ,39, and As well as specific transporters, the tubule is enriched for several families of broad-specificity transporters . When combined these would be capable of excreting a huge majority of organic solutes. These results invite a substantial revision of our interpretation of the role of the tubule. Classically, it is considered to be the tissue that excretes waste material, both metabolites and xenobiotics, and provides the first stage of osmoregulation. However, nearly all work on insect tubules in the last half-century has focused on the ionic basis of fluid secretion and its control, as these are easily measured experimentally. Although there have been sporadic reports on the active transport of organic solutes such as dyes -42, the Drosophila genes [Physiological analysis of the tubule has concentrated on the secretion of primary urine, and the energizing transporter is a plasma membrane proton pump, the V-ATPase ,49-51. Tla genes ,53. V-ATla genes . In suchla genes . It is cThe array data thus allow a rapid and authoritative prediction to be made on the subunit composition of the plasma membrane V-ATPase. It will be interesting to extend these data to other epithelia in which V-ATPase is known to be functionally significant.+, K+-ATPase in tubule is enigmatic. In nearly all animal epithelia, transport is energized by a basolateral Na+, K+-ATPase, which establishes a sodium gradient that drives secondary transport processes. By contrast, insect epithelia are energized by a proton gradient from the apical V-ATPase [+, K+-ATPase inhibitor [+, K+-ATPase. It is thus interesting to note that both Atpalpha and Nervana 1  are among the most abundant transcripts in tubule (Table CG3701), and both Nrv2  and other novel β-like subunits are at near-zero levels. As Na+, K+-ATPase has previously been documented as being particularly abundant in Drosophila tubule [+, K+-ATPase as an important part of models of tubule function.The role of the classical NaV-ATPase ,57 and, nhibitor . Accordile Table . Both ara tubule , it may Drosophila, there are at least 28, and in human 255, K+-channel genes [Irk3, Ir, Irk2 and NCKQ are all both very abundant and highly enriched in tubule. Irk3 in particular is 80-fold enriched over the rest of the fly, implying a unique role in tubule. Three of these genes are members of the inward rectifier family of potassium channels: supporting the hypothesis that they are critical for potassium entry, these channels are known to be highly barium-sensitive [SUR mRNA is present at extremely low abundance . However, CG9270, a gene with very close similarity to SUR (1 × 10-28 by BLASTP) is very abundant in tubule , is very much less abundant . We therefore predict that novel inward rectifiers, formed between Irk3, Ir or Ir2 and CG9270, may provide the major basolateral K+ entry path in tubule. In contrast, the other classes of K+ channel, and the Na/K/Cl co-transporter that has been documented in tubule, are all relatively low in both abundance and enrichment.Potassium is actively pumped across the tubule, and the main basolateral entry step is via barium-sensitive potassium channels, both in tubule ,60,61 anel genes . Inspectensitive . An inwaensitive ,67. In tDrosophila tubules, a hormonally regulated chloride conductance pathway has been shown to occur in the stellate cells, although the molecular correlate of the currents has not been determined. There are three ClC-type chloride channels in the Drosophila genome, and RT-PCR has shown that all three are expressed in tubule [CG6942) is both very abundant and enriched in tubule . It is thus an obvious candidate partner to provide a shunt pathway for the epithelial V-ATPase.In a fluid-secreting epithelium, a necessary correlate of the active transport of cations must be the provision of a shunt pathway for anions and a relatively high permeability to water. In n tubule . The arrDrosophila tubule [CG7777 , but tubule-specific roles to CG4019, CG17664 and DRIP. In particular, CG17664, is both highly abundant and very highly enriched .Water flux through the tubule is also phenomenally fast: each cell can clear its own volume of fluid every 10 seconds . Althouga tubule , and AQPa tubule . Table 9The hormonal control of fluid secretion is well understood. The major urine-producinig region of the tubule is the main segment , and is There are many other receptors that are reasonably abundant and enriched in tubule. As well as candidate receptors for calcitonin-like and other neuropeptides, there are two glycine/GABA-like receptors that might be expected to form ligand-gated chloride channels, together with good matches to vascular endothelial growth factor-like, insulin-like and bombesin-like receptors. The localization of, ligands for, and functional roles of these receptors will be of great interest. It should be noted in this context that all hormones characterized so far act on one of the two main cell types in the principal section of the tubule. There are, however, six genetically defined cell types and six regions in the adult tubule , and it Overall, the main surprise from these data is the sheer range of candidate ligands that could be inferred; this more than doubles the size of the endocrine repertoire so far postulated for insect tubules.Drosophila tubule (Table dunce). For cGMP, there are both soluble and membrane guanylate cyclases, implying that the tubules may produce cGMP directly in response to novel ligands, as has recently been suggested [Drosophila genes encoding protein kinase G are expressed in tubule, and one is highly enriched. This is consistent with the renal phenotype observed both in foraging mutants [Rack1).On a more general level, it is possible to trace out the key genes in all three intracellular signaling pathways that have been studied in detail in le Table . The resuggested . Both Dr mutants , and in  mutants . There iThe developmental origin of the tubule has been reviewed in detail -82. Briecut, which is known to be required for development of, and expressed in adult Malpighian tubules [forkhead and homothorax, both implicated by expression or mutational analysis to be involved in tubule development [Teashirt, which has recently been shown to be stellate-cell specific in the late embryo [Some of these transcription factors are already known to be present in tubule, and their presence is confirmed:  tubules ; and forelopment ,85. TeasETS21C, CG4548, bowl, sequoia, tap, CG1162, pnt, shaven, forkhead domain 59A, sloppy paired 2, lim3) as important in adult. Significantly, these mainly encode transcription factors implicated in development of the nervous system , so their reuse in the adult tubule is not too surprising. Once the binding sites for these factors are known, it will be interesting to model gene expression in different tubule regions.The array results also implicate a further set of transcription factor genes  are both insertions in alkaline phosphatase 4, a gene which is only expressed in lower tubule and the ellipsoid bodies of brain (next to the mushroom bodies) [corto is found only in stellate cells. Several other transcription factors show ubiquitous, rather than patterned, expression in the tubule, but this is nonetheless consistent with their identification in the microarray dataset.As transcription factors have been studied experimentally in some detail, they are relatively well represented by enhancer trap and other st Table . Accordi bodies) . There iAnother interesting aspect of the data in Table trp and trpl are normally considered to be eye-specific, and have an essential role in phototransduction [The whole premise of microarray work is that an abundant or enriched signal indicates the importance of a gene product in a particular context. This hypothesis is normally both untested and unchallenged. The unusual depth of functional understanding of the tubule allows a more rigorous appraisal. In fact, the majority of the genes implicated in tubule function are found well up the list. There are, however, several conspicuous exceptions Table . The calsduction -90. It isduction ,72. NoneDrosophila genes with known human disease homologs. The results  encodes xanthine oxidase, and mutation in either human or fly produces severe nephrolithiasis with concomitant distortion of tubules (reviewed in [ir); Dent disease, caused by mutation in ClC5 (homolog CG5284); proximal renal tubular acidosis, caused by mutation in the NDAE co-transport (homolog ndae1); nephrophatic cystinosis, caused by mutation in a lysosomal cystine transporter (homolog CG17119); mucopolysaccharidosis type IV, caused by mutation in galactosamine-6-sulphatase, an enzyme enriched in both human and fly kidney (homolog CG7402). Overall, there is a clear message that human and fly renal function may be relatively similar over quite a wide range of properties.Consequent to the demonstration of the phenotype gap, there are some intriguing, abundant and enriched genes which by virtue of their non-uniform expression, are likely to be important in (and best studied in) tubule. A systematic approach was taken by combining the tubule-enriched gene list with the homophila database of ts Table  show theiewed in ). The dibc10) is highly abundant (893 ± 50) and moderately enriched (1.9 ± 0.09) in tubule, and a P-element insertion within the gene P{GT1}BG02443, is available from stock centers.The tubule phenotype may also prove highly informative for other genes implicated in disease. Recently, a small 10 kDa protein, bc10, was shown to be downregulated in the transition from early-stage to invasive bladder carcinoma . The norCG4928 represents an abundant and enriched transcript , that is highly similar (1.9 × 10-75) to the C. elegans gene unc-93 [This comparative approach can be extended to non-human species. For example, These data have value at two distinct levels: specific and general. Specifically, we have found out more about the operation of the Malpighian tubule than in any single published piece of work since the very first pioneering days: a summary is given in Figure trp and trpl channels and NOS. It is, however, probably significant that these are cell-signaling molecules, where a relatively small number of molecules can have a disproportionate influence on cell behavior. By contrast, the transport genes for which the tubule is so enriched are much more likely to exert effects proportional to their abundance.There are two areas where microarray data deserve comment. Firstly, more than 300 genes are expressed in tubule but called as absent in whole-fly samples. Although there is an obvious convenience and consistency in employing whole-organism samples for array studies, it is important to recognize that the approach is very likely to suppress the detection of those interesting genes that are not widely expressed. Secondly, the premise that abundance on an array  necessarily correlates with functional significance can be spectacularly refuted by three examples, the Reverse genetics is a vital tool in functional genomics, but the 'phenotype gap' has hampered widespread implementation of this approach . As the Drosophila melanogaster were kept on standard diet at 25°C and 55% relative humidity on a 12:12 h photoperiod. Malpighian tubules were dissected from 7-day-old adults, for compatibility with the extensive physiological literature on the tubule [e tubule ,75,94-96in vitro incubation. Care was taken to sever the tubules from the gut at the lower ureter so that no other tissue was included in the sample. For each experimental point, whole flies from the same culture were homogenized in Trizol in batches of 100, to permit a matched pair comparison. Six repeats were performed. RNA was extracted according to standard protocols, and quality was assessed with an Agilent RNA Bioanalyzer. Samples of 20 μg total RNA were reverse-transcribed, then in vitro transcribed, according to Affymetrix standard protocols. The quality of the ccomplementary RNA (cRNA) was also checked on an Agilent RNA Bioanalyzer, with a sample in which the broad cRNA peak exceeded the height of the low molecular weight degradation peak taken to be satisfactory. Samples were then run on the Affymetrix Drosophila genome array under standard conditions. Quality control was at several levels: the Affymetrix MAS 5.0 software provided evidence of successful sample preparation, with test genes providing a 3':5' signal ratio of less than 3. dChip [Tubules were dissected in batches of 1,000 by a group of eight experimenters. Tubules were aggregated into Trizol every 15 min to minimize the distortion of the transcriptome by the trauma of dissection and 3. dChip  providedp < 0.05. In this analysis method, tubule and fly samples were taken as matched pairs, reflecting their biological origin. The dChip-based low-level analysis consisted of background correction followed by the multi-chip, 'invariant-set' nonlinear normalization at probe level followed by the calculation of model-based expression indices using PM expression values only. Differentially expressed genes between two groups of five replicates were identified within dChip by filtering data using the following criteria: lower 90% confidence bound of fold-change [p-value for t-test of equal group means < 0.01. Alternatively, the differentially expressed genes were identified using SAM software with 1,000 sample permutations and false-discovery rate cutoff of 1%. These were then post-filtered using two first criteria from the dChip analysis mentioned above. Fold change was calculated as a ratio of group means. Outputs were saved as Excel files, and parsed by hand-coded Perl scripts.Microarray samples were analyzed by two independent routes. The first was low-level analysis with the Affymetrix MAS 5.0 suite and identification of differentially expressed genes using the Affymetrix Data Mining Tool. The second was low-level analysis using dChip software  followedd-change  > 2; difA list of genes (Additional data file A list of genes called as upregulated in tubule by Affymetrix SAM 5 software, and with more than two-fold enrichment Click here for additional data file


The combination of spinal manipulation and various physiotherapeutic procedures used to correct the curvatures associated with scoliosis have been largely unsuccessful. Typically, the goals of these procedures are often to relax, strengthen, or stretch musculotendinous and/or ligamentous structures. In this study, we investigate the possible benefits of combining spinal manipulation, positional traction, and neuromuscular reeducation in the treatment of idiopathic scoliosis.A total of 22 patient files were selected to participate in the protocol. Of these, 19 met the study criterion required for analysis of treatment benefits. Anteroposterior radiographs were taken of each subject prior to treatment intervention and 4–6 weeks following the intervention. A Cobb angle was drawn and analyzed on each radiograph, so pre and post comparisons could be made.After 4–6 weeks of treatment, the treatment group averaged a 17° reduction in their Cobb angle measurements. None of the patients' Cobb angles increased. A total of 3 subjects were dismissed from the study for noncompliance relating to home care instructions, leaving 19 subjects to be evaluated post-intervention.The combined use of spinal manipulation and postural therapy appeared to significantly reduce the severity of the Cobb angle in all 19 subjects. These results warrant further testing of this protocol. In the MEDLINE- indexed literature, chiropractic treatment has shown to be largely ineffective at significantly reducing scoliotic curvatures. Chiropractic treatment for scoliosis typically consists of spinal manipulation, electric stimulation, some form of isotonic, active exercises, and shoe lifts . HoweverThe treatment in this study focuses on the reduction of scoliosis by manipulative and rehabilitative methods not commonly used by most chiropractors. The major difference in this treatment compared to others is that stimulation of the involuntary postural reflexes is utilized in the clinic setting as well as in home care. Many of the proposed etiologies of idiopathic scoliosis are neurological in origin, including brain asymmetry , neural A nonrandomized set of 22 patients participated in the study. The age range of the subject group was 15–65 years of age. The patients were selected from 3 different chiropractic facilities in the United States. Patients were evaluated according to their chief complaint at initial presentation. Patients were excluded from the study if neoplasm, malignancy, fracture, scoliosis secondary to genetic disorders, or previous arthrodesis were identified.Each patient was examined radiographically for location and severity of scoliosis with standing anteroposterior full spine imaging. All patients removed their shoes for the imaging. Cobb angles were drawn on each radiograph to identify the degree of curvature present. A specific treatment plan was created based upon the results of each patient's radiographic measurements before and after a sample trial of the proposed clinical procedures. Initially, standing lateral cervical, nasium, lateral lumbar, and anteroposterior lumbopelvic views were taken. These views were taken to quantify forward head posture, cervical lordosis, lumbar lordosis, the sacral base angle, and the Cobb angle of the major lateral curvature. We decided to use the radiographic positioning and analysis outlined by Harrison et al -16, due The specific manipulative and rehabilitative procedures used in this study are designed to both reduce the scoliotic curvature and theoretically retrain the involuntary neuromuscular, reflexive control of posture and balance. However, the specific neurological effects, if any, remain to be investigated. Some of the procedures have been separately introduced or tested ,18,24-26The manipulative procedures included an upper cervical adjustment designed to mobilize the atlantal-occipital joint with the use of a percussive instrument. This technique is shown in Figure ® and a Posturomed® [Certain traction procedures are also employed. These procedures are delivered using high-density foam blocks to pre-stress the spine into specific positions so ligament deformation and stress-relaxation can take place. Supine pelvic blocking was performed on each patient for 15 minutes. The position of the blocks was determined by each patient's pelvic rotation on radiograph and posture analysis. One block is placed under the iliac crest of the posterior ilium, and the other block is placed under the femoral head of the opposite, anteriorly-rotated ilium. Figure turomed® . Tjernstturomed®  showed tThe position of the body weighting was also determined radiographically for each patient. Initially, hipweights and shoulderweights were applied according to each patient's posture analysis. Anteroposterior cervicothoracic and lumbopelvic views were taken while wearing the head and body weighting. Since changes in spinal position are not reliably seen by visualization ,29, thesThe attending physician treated each patient 3 times per week for the first 4–6 weeks. A total of 3 physicians performed the treatment intervention for all patients. However, each patient did not receive identical treatment at all visits. The physicians performed only those manipulative procedures that were deemed necessary based upon a visual posture analysis at the beginning of each treatment session. However, the rehabilitative procedures remained constant throughout the study for all patients.Specific home care exercise programs were taught to each patient. These exercises were performed on a daily basis. Each patient was instructed to wear the head and body weighting twice daily for 15 minutes each time. Secondly, each patient was given a set of triangular foam blocks to lie on once daily for 20 minutes, immediately prior to going to bed at night. The foam blocks were positioned under the cervicothoracic and thoracolumbar regions simultaneously. The position of these blocks is shown in Figure At the conclusion of the trial period, a post-intervention radiographic study was conducted. The same anteroposterior full spine view was taken, and Cobb angles were again measured at the same vertebral levels. The average starting Cobb angle was found to be 28°, while the post-intervention Cobb angles measured an average of 11°, for an overall average reduction of 17°. Every patient made at least a 25% improvement. The largest improvement measured 33°, and the smallest improvement measured 8°. Table It is important to mention that these patients were initially treated prior to this study. Because of this, the pre and post treatment radiographs had previously been analyzed for sagittal curve and Cobb angle measurements. For purposes of this study, however, all of the radiographs were sent to a single chiropractic physician to analyze each of the patient files. This physician did not participate in the treatment process, nor did this physician have contact with any of the patients. This was performed to separate examiner bias from the treatment results.While only radiographic procedures were reported for this study, other physiologic parameters were utilized to document patient progress. Unfortunately, since the patient files were extracted from 3 different spine clinics, a consistent functional or symptomatic measure was not used in all 22 cases. A functional rating index, a visual analog scale, and SF-36 were used on the patients here. As a result, these values are not reported to avoid variability in outcome interpretation.Scoliosis has recently been associated with a lower quality of life -32, loweSpinal manipulation alone does not appear to significantly alter spinal structure when administered as a sole treatment modality ,40. TherThe outcome measures for this study are divided into a series of both short-term and long-term goals. The outcome of the initial stage of care is to reduce forward head posture and improve the sagittal cervical and lumbar curves. As the position of the head migrates forward, or away from the body's vertical axis, increased strain is placed upon the muscles of the head, neck and shoulders. Cailliet and Zohn indicated that an additional 10 inch/lbs of leverage is added to the spinal system in a forward head posture ,42. AddiThe effects of the loss of cervical and lumbar lordosis have been previously reported ,35-37. ROnce the cervical and lumbar lordoses are corrected, coronal reduction of the scoliotic curvatures begins. Here this was accomplished by adding a shoulderweight to the right shoulder and a hipweight to the anterior right ilium and posterior left ilium. Wu and Essien  have preLearning a new motor coordination skill can be divided into 3 phases: cognitive, associative, and autonomous . In the The study design used here does present specific limitations. Due to the lack of a control group, comparative data and conclusions cannot be made. Additionally, a retrospective design does not blind the practitioners to treatment. Although we attempted to select patient files at random from 3 separate spine clinics, nonrandomized sample populations such as ours do not necessarily reflect the potential outcomes in a general population. Therefore, future studies in this area should incorporate a control group and a randomized patient population. Follow-up studies should also focus on the potential long-term benefits of conservative scoliosis treatment, given the relative scarcity of biomedical literature available on long-term benefits from any scoliosis treatment.Within the design limitations of the present study, the combined use of manipulative and neuromuscular rehabilitation seemed to reduce scoliotic curvatures in 19 subjects by an average of 17°. This reduction took place within a 4 to 6-week period. Although this treatment was not tested over the long term, the magnitude of the present results warrants further studies into its effectiveness. This treatment should also be tested on specific types of scoliosis in follow-up trials. A long-term investigation of this protocol is desirable.This manuscript was submitted by Spinal Technologies, a BioMed Central institutional member. The rehabilitation equipment used in this study is patented by Burl R Pettibon, DC and Spinal Technologies. MWM is the Director of Research for the Pettibon Biomechanics Institute, and an active postgraduate instructor for Spinal Technologies. MWM does not receive monetary compensation for this position. Rather, he is granted funding from Spinal Technologies to obtain biomedical literature and statistician services. DW is a past postgraduate instructor for Spinal Technologies, founder and director of the CLEAR Institute, and CEO of the Flex Neck Company. GL is an active postgraduate instructor for Spinal Technologies. The authors receive lecture fees for each continuing education seminar conducted. All 3 authors maintain private chiropractic practices from where all of the patient files in this study were taken. None of the above companies donated, funded, or reimbursed any monies or equipment for this study. None of the authors have any ownership in Spinal Technologies or its subsidiary companies, and none will gain any financial interest as a result of this paper.Each author worked on one-third of the patient population. The first author was responsible for collecting the data and putting our findings into written format.The pre-publication history for this paper can be accessed here:


It is supplied with input files designed for this purpose. More broadly, the program is applicable for finding specific target regions (referred to as "variable regions") in DNA sequence. A variable region is any specific sequence fragment of interest, such as an indel region, a codon or codons, or sequence coding for a particular RNA secondary structure.The program InDeVal was originally developed to help researchers find known regions of insertion/deletion activity (with the exception of isolated single-base indels) in newly determined Poaceae InDeVal input is DNA sequence and a template file (sequence flanking each variable region). Additional files contain the variable regions and user-defined messages about the sequence found within them .Variable regions are found by determining the position of flanking sequence (referred to as "conserved regions") using the LPAM (Length-Preserving Alignment Method) algorithm. This algorithm was designed for InDeVal and is described here for the first time.InDeVal output is an interactive display of the analyzed sequence, broken into user-defined units. Once the user is satisfied with the organization of the display, the information can be exported to an annotated text file.trnL-F files) and display user-selected messages specific to the sequence variants found. InDeVal output is designed to facilitate comparison between the analyzed sequence and previously evaluated sequence. The program's sensitivity to different levels of nucleotide and/or length variation in conserved regions can be adjusted. InDeVal is currently available for Windows in .InDeVal can find multiple variable regions simultaneously (28 indel regions in the Poaceae  Gaps caused by insertion and deletion events (indels) are often important features of DNA sequence data, which is widely used in phylogenetic studies -13. AlthPhylogenetic estimation depends on accurate character homology assessment  ,8,18, whSequence comparisons are complicated by the ambiguities gaps introduce into alignments. Finding a target region recognized from one alignment within another can be time consuming and difficult to perform accurately. Because of the limits of computer screen size and human analytical ability, alignments of hundreds of sequences are difficult to evaluate, even when they can be prepared.trnL (UAA) 5' exon and trnF (GAA), hereafter called trnL-F [trnL-F sequences. Comparing indel regions across these sequences can reveal patterns in indel behavior and aid researchers in creating accurate alignments. A discussion of the indel regions in these sequences is being prepared separately for publication.Poaceae is one of the largest families of flowering plants and is economically important . Lower-ld trnL-F ,23-28. Ad trnL-F  containetrnL-F sequences and simultaneously compare them with 533 previously determined sequences . It is supplied with input files designed for this purpose. More broadly, the program is applicable for finding specific target regions (referred to as "variable regions") in DNA sequence. A variable region is any specific sequence fragment of interest, such as an indel region, a codon or codons, or sequence coding for a particular RNA secondary structure. The LPAM algorithm, which was specifically designed for InDeVal, is used to find sequence (referred to as "conserved regions") flanking the variable region in the analyzed sequence. InDeVal can find multiple variable regions simultaneously (28 indel regions in the Poaceae trnL-F files). The program's sensitivity to different levels of nucleotide and/or length variation in conserved regions can be adjusted.The program InDeVal was originally developed to help researchers find known indel regions (with the exception of isolated single-base indels) in newly determined Poaceae InDeVal uses three types of input files: one conserved region file, separate variable region files for each variable region, and a DNA sequence file Table . The contrnL-F sequences to accommodate single, large deletions that spanned otherwise conserved regions. Treating them the same as the other indels would have resulted in a few large, difficult-to-interpret variable regions. InDeVal performance is improved by designing templates with conserved regions at least 20 bases long on either side of each variable region. However, the program still functions using templates with only one conserved region, conserved regions only 4 bases long, and variable regions flanked by other variable regions . Although InDeVal parameters can be adjusted to reflect different degrees of nucleotide and length variation in conserved regions, it is a good idea to use a representative sequence for template design, especially if some conserved regions are short. Additional templates can be designed to accommodate distantly related taxa. .A conserved region file contains at least one template, created by taking a representative sequence, removing the variable regions, and replacing them with variable region file names . MultipltrnL-F files, the output information is the list of taxa with each variation. For coding sequence, the output information could be the amino acids for which the variations code. If the user is interested only in knowing if a particular variation is present, the output could be simply "Yes". Variants not found in the variable region file are also reported by InDeVal. The program works if the variable region file is completely blank , but it obviously cannot provide output messages in this case. Symbols can be used in variable region sequence to draw attention to specific features of interest. They are ignored during alignment, but are displayed in the Variable Region Sequence List Box  of various alignments of these sequences using the web-based programs BLAST 2 SEQUENCES [A conserved region file (TemplatePtrnLF.txt) and 28 variable region data files, designed from 533 Poaceae EQUENCES  and/or CEQUENCES .InDeVal begins by sorting a template's conserved regions by length. It then searches for each conserved region in the analyzed sequence (using LPAM – see below), proceeding from longest to shortest. Found conserved regions are used to limit the search space for future searches Figure .Sometimes conserved regions are not found or are found at multiple locations within their subsequence. The program records this information and proceeds to the next longest sequence. A conserved region that cannot be aligned is recorded as "not found." A conserved region with multiple possible alignments is recorded as "found", but none of its possible alignments is used to limit the searches for the remaining regions.This template alignment algorithm preserves the ordering found in the template, giving priority to the alignment suggested by the longer conserved regions, which are assumed to be more reliable. Using the alignment of longer regions to reduce the search space for smaller regions minimizes the probability of finding ambiguous or incorrect alignments.InDeVal uses the LPAM algorithm, designed specifically for InDeVal and described here for the first time, to align the conserved regions within their subsequences. LPAM divides a conserved region into overlapping "words", strings of sequence of a user-defined length. That is, given a 4-base word length, the sequence "caatgt" would be represented as "caat", "aatg", "atgt". (Conserved regions shorter than the word length are found only if they match exactly.) LPAM searches the analyzed sequence for each of these words and notes multiple finds, single finds, and missing words. Each word is allowed one "vote" for a possible alignment, i.e., for the base in the analyzed sequence that begins the conserved region. If the word occurs once, it casts its vote for that alignment. If it occurs multiple times, its vote is divided equally among the possibilities. Words that are not found cast no vote. The alignment that receives the most votes is assumed to be the most probable.LPAM permits a (user-definable) degree of length variation. A word's possible alignments are evaluated according to whether or not they agree with the most probable alignment for the region as a whole. Suppose, for example, that the most probable ungapped conserved region alignment would begin at base 53. If the tolerance is set to 3, LPAM will allow the first word to begin at any base from 50 to 56, preferring the possibility closest to 53. If the first word in the conserved region sequence has no acceptable alignment, LPAM searches for the first word that does have an acceptable alignment. All bases preceding this first found word are aligned with no gaps. In the above example, if the first and second words are not found and the third word aligns beginning at base 57, the first two bases of the conserved region will be aligned with bases 55 and 56.Each subsequent base is aligned similarly. If the word it begins has no acceptable alignment, the base is aligned according to the previous acceptable word. If the word it begins has multiple acceptable alignments, the one closest to the previous alignment is chosen. Note that the range of acceptable alignments remains constant; it is a function of the initial voting and does not depend on any of the choices made in aligning individual bases.The aligned bases from the analyzed sequence are compared with the template conserved region sequence, and a percentage similarity is established. If this is greater than the user-defined cut-off value, the conserved region is listed as found.InDeVal then checks the most probable alignment that clearly differs from the first.  If this alignment also yields a percentage similarity greater than the cut-off value, neither is selected, but the region is still considered to be "found" for purposes of measuring similarity to the template (see below).trnL-F sequences, where conserved regions are by definition length-conserved. In 533 sequences, there were only three instances where an indistinguishable mismatch was caused by an indel instead of a point mutation and resulted in misalignment. It is important to design templates so that the ends of conserved regions are not in sequence prone to indel activity.The LPAM algorithm is able to align conserved regions despite both point mutations and indels. However, deletions in the first or last word of a conserved region are indistinguishable from point mutations (and are interpreted as such). LPAM proved effective in the Poaceae The alignment suggested by LPAM is not necessarily locally maximal, i.e., there are cases where a slight adjustment of the alignment would produce a higher percentage of matched bases. Furthermore, a number of factors can prevent LPAM from finding a correct alignment. Repetitive sequence, resulting in multiple matches, can be very problematic. InDeVal may not be appropriate for analyzing repetitive sequence, and if it is used for this purpose, templates must be designed with great care. If every word contains a point mutation, the conserved region cannot be found. Indels can cause problems if they are dispersed so that several words are disrupted. In general, LPAM will correctly align regions if the mutations are clustered and the first and last base can be assigned correctly (which will be the case if they have no indels between them and the undisrupted word that will position them). Though hypothetical situations where LPAM would create incorrect alignments are easy to envision, in practice, the algorithm proved able to reliably determine where the conserved regions of a sequence begin and end. This is sufficient for the purposes of InDeVal. It is important to note that even when LPAM does not find a conserved region alignment, a possible alignment is displayed, and the user can rearrange it (to a limited extent) and interpret it.Once conserved regions have been found, InDeVal calculates a "found conserved region score" for each template. Each matched conserved region is given a value equal to the number of bases in that region in the template , and these values are summed to give a score for the template. This prevents a template with a major deletion from being selected over a template with more matching conserved regions (as could happen if the deletion template were a better match at the base-per-base level). Templates with higher scores are ranked above those with lower scores Figure .If two templates have roughly equal found conserved region scores (to within a user-selected tolerance), the one with the higher "score percentage"  is ranked higher. This ensures that sequences that actually do have deletions will be matched with deletion templates, even though the templates  have an equal number of bases in found conserved regions Figure .All the templates are ranked in this way and listed in the Template List Box in order. The highest ranked template is automatically selected, and its variable regions are analyzed. The user has the option of comparing the sequence to a template other than the one selected by InDeVal.Each variable region is defined as the sequence between two specific conserved regions. If both flanking conserved regions are found, the bases between them are recorded, and the appropriate variable region file is searched for a matching string of bases.trnL-F file, one- and two-base conserved regions were incorporated into adjacent variable region files because they were impossible to find.Sequence between two found conserved regions is classified as a confused region if the template indicates that it should contain one or more conserved regions that were not found. Because the variable regions in a confused region are not clearly delimited, InDeVal searches the entire region for each variable region sequence in each applicable variable region file, and all potential matches are recorded. The user is able to select from among these possibilities, rearrange the display to reflect them, and study different sequence interpretations. In this manner, InDeVal is able to deal satisfactorily with most short conserved regions that cannot be found because of point mutations. In the Poaceae A short conserved region will be misaligned if a point mutation prevents it from being found in the proper place and a perfect match is found nearby. If this occurs, it is possible to set the program to disregard conserved regions of this size. (This situation has only been observed for 4- and 5-base conserved regions). The region can then be satisfactorily parsed using the confused region algorithm.InDeVal has two output windows Table . The SeqThe Variable Region Analysis Window displays the variable region file name, variants proposed by InDeVal, and the sequence and length of the line currently selected in the Sequence Analysis Window (which can be any line from any sequence). The user can request displays of some or all of the sequences in the variable region file.A text file can be created once the user is satisfied with the display in the Sequence Analysis Window. This file gives information about the sequence and template, lists the positions of the various conserved and variable regions, and shows the entire analyzed sequence. The user can choose whether or not variable region file information is displayed.InDeVal for the Windows platform is available in InDeVal is a program designed to search DNA sequence for target regions and to display information about them. It can find multiple target regions simultaneously and is relatively robust when challenged by conserved region variation and differences among analyzed sequences in length, spanned region, and format. InDeVal differs from other alignment software in that it breaks the analyzed sequence into user-defined units and emphasizes regions that are of most interest to the user. This makes it possible to quickly compare specific features among many hundreds of sequences. An advantage of InDeVal is that, while it can be used to quickly skim and classify hundreds of sequences, it displays all surrounding sequence. Therefore, if at any time questions arise about the initial classification, the researcher can recreate the InDeVal alignment, instantly find the area in question, and study it for alternative explanations.Project name: InDeValProject home page: Operating system(s): Windows PlatformProgramming language: Microsoft Visual Basic 6.0Other requirements: NoneLicense: NoneAny restrictions to use by non-academics: No restrictionsSDSH planned and oversaw the project, collected the data, prepared the data files, tested the program, and wrote the documentation. JAH suggested the project and designed, wrote, and debugged the program. Both authors read and approved the final manuscript.InDeVal 1.0 installation version InDeVal is currently available for the Windows platform. Instructions for use can be found in the help files, which are included with the program and are accessible from the Sequence Analysis Window. An installation version of InDeVal 1.0 with accessory files can be obtained by clicking on the link below or by visiting . Running the installation program installs (in addition to system files) an InDeVal program directory that contains the Indeval.exe file, the 7 InDeValHelp.txt files, the InDeValOptions.txt file, the InDeValParams.txt (parameters) file, and a Templates subdirectory containing TemplatePtrnLF.txt and a Vr subdirectory with 28 Poaceae trnL-F variable region files. The program is archived using WinZip® 9.0. WinZip is available from . The zipped package has 3.2 MB.Click here for fileAnnotated illustrations of InDeVal files and displays This file (386 kB) is a 9-page pdf that contains annotated illustrations and explanations of InDeVal files and displays. The structure and format of the conserved region, variable region, sequence, and output files is illustrated. Annotated screenshots of the Sequence Analysis Window and Variable Region Analysis Window are also included. These illustrations, combined with the InDeVal help files, serve as the InDeVal Manual.Click here for fileInDeVal source code The source code for InDeVal in Microsoft Visual Basic 6.0 can be obtained by clicking on the link below or by visiting . The files are archived using WinZip® 9.0. The package (61 kB) includes the 32 code files, the InDeVal icon and the bitmap from which it was constructed, and InDeValSourceCodeHelp.txt, a file with advice on orienting within the source code files.Click here for file


Viruses are obligate intracellular parasites and rely upon the host cell for different steps in their life cycles. The characterization of cellular genes required for virus infection and/or cell killing will be essential for understanding viral life cycles, and may provide cellular targets for new antiviral therapies.Igf2r), a protease associated with insulin growth factor binding protein 5 (Prss11), and the CTCF transcriptional regulator (Ctcf). The disruption of Ctcf, which encodes a repressor of Igf2, was associated with enhanced Igf2 gene expression. Plasmids expressing either the IGF-II pro-hormone or IGF-II without the carboxy terminal extension (E)-peptide sequence independently conferred high levels of cellular resistance to reovirus infection. Forced IGF-II expression results in a block in virus disassembly. In addition, Ctcf disruption and forced Igf2 expression both enabled cells to proliferate in soft agar, a phenotype associated with malignant growth in vivo.A gene entrapment approach was used to identify candidate cellular genes that affect reovirus infection or virus induced cell lysis. Four of the 111 genes disrupted in clones selected for resistance to infection by reovirus type 1 involved the insulin growth factor-2 (IGF-II) pathway, including: the mannose-6-phosphate/IGF2 receptor (These results indicate that IGF-II, and by inference other components of the IGF-II signalling pathway, can confer resistance to lytic reovirus infection. This report represents the first use of gene entrapment to identify host factors affecting virus infection. Concomitant transformation observed in some virus resistant cells illustrates a potential mechanism of carcinogenesis associated with chronic virus infection. Viruses as obligate intracellular parasites rely upon the host cell for different steps in their life cycle, including attachment, disassembly, transcription, translation, reassembly, and egress. Consequently, characterization of these cellular processes will be essential for any understanding of viral life cycles, and may provide cellular targets for new antiviral therapies.Igf2r, locus ID 25151), the IGF binding protein 5 protease , and an electron coupling reagent  were added at 20 μl per well . Plates were incubated for 2 hours, and then the absorbance was determined at 490 nm. Each set of conditions was repeated in triplicate.RIE-1, 6B72, or JS, ELO, and CH conducted most of the laboratory work. KSW assisted in the analysis of fluorescein-labelled virus preparations. HER provided the vectors and advice on their use. DHR discovered that persistently infected cells require serum to survive, allowing the selection of genetically resistant cell clones. HER and DHR provided funding and supervision for the research, and prepared the manuscript. All authors have read and approved the final manuscript.


In the 1980's and 1990's HIV/AIDS was the emerging infectious disease. In 2003–2004 we saw the emergence of SARS, Avian influenza and Anthrax in a man made form used for bioterrorism. Emergency powers legislation in Australia is a patchwork of Commonwealth quarantine laws and State and Territory based emergency powers in public health legislation. It is time for a review of such legislation and time for consideration of the efficacy of such legislation from a country wide perspective in an age when we have to consider the possibility of mass outbreaks of communicable diseases which ignore jurisdictional boundaries. The management of infectious diseases in an increasingly complex world of mass international travel, globalization and terrorism heightens challenges for Federal, State and Territory Governments in ensuring that Australia's laws are sufficiently flexible to address the types of problems that may emerge.Health Act 1958. These sections emphasize the need to protect the privacy of the infected individual and to undertake a staged response which is proportional to the risk presented by the infected individual. The legislation has been very effective with HIV and has been praised for its progressive approach  in the symptoms (though these may vary in severity) or treatment of the virus Australia is a federal system. There are two parallel sets of laws in operation. The Commonwealth Constitution sets out the legislative powers of the Commonwealth. Specific powers are listed in the Commonwealth constitution but State constitutions have broad powers covering matters such as peace, order and good governance. As the Commonwealth has no specific power to legislate with respect to health, other than the quarantine power, national legislative schemes in public health which rely upon a cooperative approach from all States and Territories are cumbersome and difficult.Without a specific head of power, the Commonwealth has limited ability to legislate with respect to health. "That is, the legislative powers of the Commonwealth are specified in the Constitution and do not include expressly most of the activities that together comprise the field of public health" For thisThe quarantine power would be the most likely candidate for a head of power on which to base development of commonwealth laws for the management of public health emergencies. Another possibility may be the external affairs power, if there was a relevant treaty or international agreement which could be given effect to in domestic law. However the legislation would have to be limited to laws giving effect to the treaty.Health Act 1911 (WA), Public Health Act 1952 (NT) based on an 1898 Ordinance (Both these Acts are currently under review). Health emergency powers vary from one jurisdiction to another, but include powers to support disease surveillance, contact tracing and orders to restrict behavior or movement of individuals with an infectious disease in certain circumstances. There are also powers to recall food, search premises and seize property, close buildings and a range of other substantial and intrusive powers.States and territories have a range of emergency powers available to them in their existing public health legislation. Some are relatively old. For example, the It is suggested that it is time to consider whether state and territory public health legislation contains sufficient measures to manage the outbreak of an infectious disease in a modern environment which includes mass travel, swift spread of infection and additional complexity raised by fears of bioterrorism.Currently, in a public health emergency caused by the spread of an emerging infectious disease, Australia could need to rely on a patchwork of legislative measures to assist it to cope. Commonwealth quarantine laws and State and Territory powers in public health legislation may all be needed to address the problem. If an outbreak occurred on a border, or in some area where jurisdiction may be in doubt such as airspace or offshore and a state or territory response was required in addition to any quarantine measures, there could be confusion over jurisdiction for the application of State and Territory powers. State and Territory public health acts do not adequately provide for interjurisdictional communication and cooperation. There could also be difficulties if an infectious disease caused overseas deaths of people from more than one State or Territory in circumstances where an Australian coronial investigation was considered desirable. In such a situation, the jurisdiction of more than one Australian coroner would be triggered. Several State and Territory coronial laws could apply and there could be different inquests under different laws undertaken by different coroners into the same incident.It is suggested that it is time to look at the efficiency of the emergency powers laws of Australia as a whole: to map the laws in each jurisdiction and the Commonwealth quarantine laws and to consider their effectiveness in the face of the outbreak of a fast moving, easily spread infectious disease. The efficacy of Australia's laws should also be considered in relation to bioterrorism. While there were no infections from anthrax in 2003 despite a great deal of media coverage and infections and deaths in the US, a responsible legislature ought to acknowledge the possibility and ensure that the law is ready to support a swift and effective response.It is not enough to consider whether the individual pieces of legislation are up to the task of managing outbreaks of newly emerging infectious diseases. Indeed many of the jurisdictions are currently reviewing their public health legislation and will no doubt give proper consideration to this issue as part of the review. But who is thinking about how the legislation of all jurisdictions and the Commonwealth quarantine fits together? What powers enable communication and cooperation between jurisdictions about the outbreak of infectious disease? What kind of opportunity is there for a coordinated response? Can public health orders made in one jurisdiction travel to another jurisdiction when the infected individual travels? What arrangements can be made if an outbreak occurs on or close to a interstate border? What if there is an outbreak on a bus carrying passengers from Victoria, through South Australia to the Northern Territory?It is encouraging to note that, even without specific legislation, there has been a mechanism to achieve communication and cooperation between jurisdictions through the Communicable Disease Network of Australia (CDNA). This Network has in fact been quite successful in fostering regular communication between the Communicable Disease Units across the country and has been involved in coordinated actions during a number of multistate outbreaks.Despite the existence of this network and other good working relationships between government officials and various agencies in different jurisdictions, a serious outbreak of communicable disease would require the existence of legislative powers. Public health emergencies generate confusion, even panic. Clarity of powers and the way those powers interact with each other would be crucial in an emergency. It became apparent after the Bali tragedy in 2002 that coroner's jurisdiction was triggered differently in different jurisdictions and some acts did not support communication and cooperation when inquests might be needed for deaths of people ordinarily resident in several jurisdictions. The time to find the shortcomings in the legislation is well before the crisis.A review of the efficacy of how these laws work together to protect the public health of all Australians should be undertaken. It has been possible to overcome the hangovers of federation for the betterment of all Australians in relation to corporations law. When doubts were recently raised about the constitutional basis of the corporations law scheme, the States and Territories were able to cooperate and refer the necessary powers to the Commonwealth to provide certainty about the laws which govern our corporations. Is our public health any less important than governance of our corporations? Could we cooperate to give ourselves certainty, flexibility and a consistent approach which protects the rights of those subject to some very broad powers?The States and Territories are generally reluctant to refer powers to the Commonwealth. It may be time to seriously discuss referral of powers in the context of health emergency powers. At the very least, it is time that the Commonwealth, States and Territories recognised the need for the laws to work as a set of laws to protect the whole country, not simply individual laws to protect individual jurisdictions.State Emergency Health Powers Act has been developed in the US in 2001 [There has been work done internationally in this area. A model  in 2001  In the pThe development and adoption of the model food legislation provides a useful model of a cooperative uniform approach. A model act was developed in consultation with all jurisdictions. It covered areas agreed to be core areas of the Act which ought to be the subject of a national approach and other provisions which were considered to be administrative and were to be adopted at the discretion of each jurisdiction. An intergovernmental agreement was signed as a mechanism to protect the uniformity of the legislation. The agreement sets up a Ministerial Council, supported by a Food Regulation Standing Committee. The Council has responsibility for deciding on proposals to amend the model  If a decThe law is an important tool in supporting the management of the outbreak of infectious diseases. The existence of our Federal system has meant that we have a different approach in each State and Territory together with Commonwealth control of quarantine. Newly emerging infectious diseases creating real threats to public health in an era of easy mass travel, and the present threat of bioterrorism mean that it is time Australia examined all laws to contain and manage infectious disease outbreak. The laws should be examined both for their effectiveness in the areas they cover, and as part of a whole which ought enable a response which protects the health of all Australians, and crosses borders as easily as SARS or avian influenza.


We are pleased that Dr. Maitland and colleagues consider our data on volume status (intra- and extracellular) of Gabonese children to be important. We did not consider our children with severe malaria to have intravascular volume depletion for the following reasons. When we measured central venous pressures in a proportion of children on admission, there was no evidence of intravascular volume depletion (median [interquartile range] = 6.5 [3–7.5] cm water), and these values did not change significantly over 24 h, suggesting that our severely ill children had adequate filling pressures. Consistent with this observation, our severely ill children improved rapidly when markers of tissue hypoxia  were serially monitored and children were managed with a relatively conservative fluid replacement regimen. Interestingly, extracellular volume was not increased at admission or afterwards either. Capillary leakage, which commonly accompanies hypovolaemia associated with septic shock, was therefore unlikely to be a significant pathophysiological process in these children with malaria. There may be differences in the severe syndromes of malaria seen in different geographical locations, perhaps accounting for the clinical features attributable to compensated hypovolemic shock reported by Maitland and colleagues. Such differences can be assessed using simple and recently calibrated bioelectrical impedance analysis methodology as well as other techniques that monitor intravascular volumes. The design of optimal fluid management regimens for children with severe malaria can thus be informed not only by theoretical considerations, but also by appropriate physiological assessments.