Grewia flava, a common, fleshy-fruited shrub species in the southern Kalahari. Understanding the population dynamics of Grewia flava is a crucial task, because it is widely involved in the shrub/bush encroachment process, a major concern for rangeland management due to its adverse effect on livestock carrying capacity and biodiversity.In Southern Africa savannas climate change has been proposed to alter rainfall, the most important environmental driver for woody plants. Woody plants are a major component of savanna vegetation determining rangeland condition and biodiversity. In this study we use a spatially explicit, stochastic computer model to assess the impact of climate change on the population dynamics of z of the time-shrub density relationship to quantify the population trend. For each climate change scenario we then compared the departure of z from typical stable population dynamics under current climatic conditions. Based on the simulation experiments we observed a positive population trend for scenario (1) and a negative trend for scenario (2). In terms of the projected rates of precipitation change for scenario (3) and (4) population dynamics were found to be relatively stable. However, for a larger increase in inter-annual variation or in temporal auto-correlation of rainfall population trends were negative, because favorable rainfall years had a limited positive impact due to the limited shrub carrying capacity.For our study we consider four climate change scenarios that have been proposed for the southern Kalahari for the coming decades: (1) an increase in annual precipitation by 30–40%, (2) a decrease by 5–15%, (3) an increase in variation of extreme rainfall years by 10–20%, (4) and increase in temporal auto-correlation, i.e. increasing length and variation of periodic rainfall oscillations related to El Niño/La Niña phenomena. We evaluate the slope We conclude that a possible increase in precipitation will strongly facilitate shrub encroachment threatening savanna rangeland conditions and regional biodiversity. Furthermore, the negative effects found for positive auto-correlated rainfall support current ecological theory stating that periodically fluctuating environments can reduce population viability because species suffer disproportionately from poor environmental conditions. In order to assess biodiversity response under climate change, Hannah et al.  recentlyGrewia flava DC, a common, fleshy-fruited, woody plant species of South African savannas. Understanding the population dynamics of Grewia flava is a crucial task, because it is widely involved in the shrub/bush encroachment process . Shr. ShrGrewss e.g. -13) and  and Grewss (e.g. ). In the)s Figure . Thus, wnsidered .Grewia flava population trend. Above this threshold frequent droughts kill the offspring and inhibit emergence. Ecological theory generally suggest that population viability decreases with increase in environmental variability or stochasticity [An increase in variability of more than 100% revealed a significant threshold behavior with a negative asticity , becauseasticity . HoweverICV, i.e. intra-cycle variation and PL, i.e. period length to evaluate the impact of climate change related increase in positive auto-correlation of rainfall. In our study region, Kruger [PL value of 22 years between 1955 and 1991. The ICV values used in our simulation experiments refer to an increase in the probability of an extreme rainfall category and are thus difficult to compare with the cyclic variability of actual total rainfall. However, we compared the ICV values from our model with departures of annual rainfall from the inter-annual mean of a typical rainfall oscillation provided by Kruger [ICV value above 60% resembles a realistic rainfall variation between favorable and unfavorable periods of a 22-year oscillation. Although we did not find a negative impact on Grewia flava population dynamics for this scenario, a slight increase in ICV combined with an increasing length of El Niño/La Niña phases might have a significant negative impact on the population viability of Grewia flava  outweigh positive effects in wet years (i.e. increased reproduction). This is noteworthy, as mean annual rainfall and inter-annual variation was kept constant. Until now, this has been primarily shown in solely theoretical studies with under-compensating population dynamics . ,32. 31,3cs (e.g. ) or indics (e.g. ). To ourSGM it is important to discuss the inference of actual precipitation amounts on probability values. Threshold values for extreme rainfall years are based on expert knowledge and may vary depending on the plant species considered. Moreover, percentage changes in extreme rainfall years, as applied in our model, are not necessarily equivalent to percentages changes in mean annual rainfall. For example, an increase in 'high' rainfall years by 10% represents an increase in mean annual rainfall of approximately 10%, depending on actual precipitation amounts in each 'high' rainfall year. Thus, it is important to derive principal population trends from the SGM model results rather than absolute predictions. Another issue regards the temporal dimension: long-term SGM simulation periods deviate from short-term predictions of climate change. However, population trends applied for only a few decades may yield biased results due to the cyclicity of Grewia population dynamics (see [In order to assess the predictive power of ics (see ). Finallics (see ).Grewia flava is relatively long-lived, fire and drought-resistant. Intuitively, this would suggest a low climate change impact on Grewia flava population dynamics. However, we found that despite Grewia flava's capability to survive in a harsh environment, it may be strongly affected when rainfall decreases as predicted, or increases in periodical fluctuations. This may also include possible range shifts in regional distribution which have not been studied here. Based on the model and the climate change scenarios analysed, it would be inappropiate to forecasts changes in the geographical distribution of Grewia flava, because SGM parameters have been estimated and validated for populations in the Kimberley region of the southern Kalahari. Moreover, current geographical distribution suggests that buffer mechanisms may facilitate survival in more arid parts of the southern Kalahari .In this study we have shown that climate change may have severe and sometimes unexpected implications at a regional species level. A woody plant like Grewia flava encroachment in the southern Kalahari. However, increase in rainfall will likely enhance this process. A shift from typical open grassland with solitary trees towards wide spread, homogeneous Grewia flava thickets implies negative cascade effects for other species, resulting in the loss of biodiversity [Decrease in rainfall may reduce the severity of iversity . As a fuSGM and previous results have been presented elsewhere . SGM. SGMSGM Grewia flava typically grows beneath the dominant tree species Acacia erioloba [Grewia flava. Under high cattle grazing a substantial proportion of seeds may be distributed into the open matrix vegetation, since cattle feed on the foliage and fleshy fruits of Grewia flava [Grewia cover, particularly around boreholes . Grewiater fire  and low ter fire . Size clter fire .SGM  (i.e. occupied by Acacia erioloba) or matrix type (M) (i.e. grassland vegetation). However, it switched from T to M status when a tree died after a mean life span of 200 years. The converse occurred where a new tree establishes. Initial tree distribution and spatial recruitment pattern was random with a constant density of 5 trees ha-1 for the entire simulation period. In each time step, SGM simulated important life history stages, environmental conditions and the key ecological processes of Grewia flava, the majority of which are governed by annual rainfall patterns  repr reprSGM ari (see ), an ini-1. Rain falls almost exclusively during summer (November to April) with an inter-annual mean of 417 mm for the period 1940 – 2000 . For this period we defined a threshold value of 150 mm below and above the long-term mean to classify years into 'low', 'average' and 'high' rainfall years  . Similar classifications have been applied in other studies, e.g. in a spatial simulation model of Acacia raddiana in the Negev Desert [Annual rainfall in the southern Kalahari is highly variable, with precipitation ranging between 200 and 700 mm yrly) (see ). Accordv Desert .Grewia size classes and may vary depending on the annual rainfall . Seed dia flava  and reprcal data , the assGrewia flava seeds [M and T. However, depending on annual rainfall conditions, the probability of emergence in the model may vary between 1–2% in average and 2–4% in high rainfall years . As survival of Grewia flava seeds in the soil is very low, seeds that do not emerge are assumed to die.Even though microsite types differ markedly in micro-environmental conditions, they do not differ in emergence rates of va seeds  suggestiSGM, fire was simulated probabilistically for the entire grid and may only occur in high and average rainfall years. Average frequency of 7.9 years in the model is supported by fire intervals reported from similar savannah types [T cells, fire mortality probability is 0% for adults and 75% for seedlings. These model assumptions are realistic, since grass fuel accumulation and fire severity beneath trees is less than in tree inter-spaces . In . In SGM,ption in ). Annualption in  and Schuption in . For seeption in ). All seGrewia flava individuals into categories of small , medium-sized  and large plants  as well as seedlings . We assumed a maximum age for each size class member with MaxAgeS = 5 years for small and MaxAgeM = 25 years for medium-sized shrubs. These estimates are based on annual shoot growth rates and aerial photograph analysis of the study area  = 0.2 for small and Ptransition(M) = 0.1 for medium-sized shrubs. No growth occurs in low rainfall years.In the model, each shrub was assigned a size class with an average canopy cover. For definition of shrub size classes we used the canopy volume to group rea (see ). In eacK = 25 m2 as maximum total cell shrub cover. If shrub cover exceeded K, individuals in the cell died. Density-dependent mortality was simulated annually by removing the smallest individuals first, i.e. in descending order of size class, until shrub cover <K . Inter-rix (see ). For thz-value, i.e. the slope of the year – shrub density relationship. This is a reasonable approach for analyzing time-abundance relationships . When pBased on the standard scenario we performed simulation experiments for each of the four different scenarios of climate change with variation in inter-annual mean of precipitation, coefficient of inter-annual variation, and temporal auto-correlation . Probability of low rainfall years is constant whereas frequency of average years is reduced, accordingly.(1) increase in precipitation, i.e. stepwise increase of Plow resulting in a higher probability of low rainfall years (increase in drought events),(2) decrease in precipitation, i.e. stepwise increase of Plow and Phigh resulting in a higher probability of low and high rainfall years at the cost of average years ,(3) increase in inter-annual variation of precipitation, i.e. stepwise increase of P(4) increase in temporal auto-correlation of rainfall with 'good' and 'poor' phases.For the scenario (4) we varied two parameters:PL, i.e. longer phases of favorable and unfavorable rain conditions and,(4a) introduction of rain cycles with increasing period length ICV, i.e. increasing variation between alternating favorable and unfavorable periods within one rain cycle.(4b) increase in intra-cycle variation PL we applied a period length of 10, 20, 50, 100 and 250 years, respectively, with each period subdivided into a similarly long 'good' and 'poor' phase. For example, a 10-year period length was subdivided into a 5-year period of superior and a 5-year period of poor conditions. For an increase in ICV we alternately increased and decreased high and low rainfall probabilities in each period, respectively (see 4a and 4b in Table ICV value of 100% Plow equaled 0.00 and Phigh 0.26 in a 'good' phase, whereas values were 0.30 and 0.00, respectively for a 'poor' phase. Through this procedure inter-annual mean and variation of rainfall probabilities were identical for each scenario and the default set of rainfall probabilities. Combined increase in PL and ICV resulted in an increase of positive auto-correlation. Near-decadal epochs of above- and below-normal rainfall have been identified for the period 1955–1991 [ICV under predicted climate change.For SGM simulation model, conceived the study and drafted the manuscript. FJ supervised the study and the manuscript writing. Both authors read and approved the final manuscript.JT developed the


The major site of hematopoiesis transitions from the fetal liver to the spleen and bone marrow late in fetal development. To date, experiments have not been performed to evaluate functionally the migration and seeding of hematopoietic stem cells (HSCs) during this period in ontogeny. It has been proposed that developmentally timed waves of HSCs enter the bloodstream only during distinct windows to seed the newly forming hematopoietic organs. Using competitive reconstitution assays to measure HSC activity, we determined the localization of HSCs in the mid-to-late gestation fetus. We found that multilineage reconstituting HSCs are present at low numbers in the blood at all timepoints measured. Seeding of fetal bone marrow and spleen occurred over several days, possibly while stem cell niches formed. In addition, using dual-chamber migration assays, we determined that like bone marrow HSCs, fetal liver HSCs migrate in response to stromal cell-derived factor-1α (SDF-1α); however, unlike bone marrow HSCs, the migratory response of fetal liver HSCs to SDF-1α is greatly increased in the presence of Steel factor (SLF), suggesting an important role for SLF in HSC homing to and seeding of the fetal hematopoietic tissues. Together, these data demonstrate that seeding of fetal organs by fetal liver HSCs does not require large fluxes of HSCs entering the fetal bloodstream, and that HSCs constitutively circulate at low levels during the gestational period from 12 to 17 days postconception. Newly forming hematopoietic tissues are seeded gradually by HSCs, suggesting initial seeding is occurring as hematopoietic niches in the spleen and bone marrow form and become capable of supporting HSC self-renewal. We demonstrate that fetal and adult HSCs exhibit specific differences in chemotactic behavior. While both migrate in response to SDF-1α, fetal HSCs also respond significantly to the cytokine SLF. In addition, the combination of SDF-1α and SLF results in substantially enhanced migration of fetal HSCs, leading to migration of nearly all fetal HSCs in this assay. This finding indicates the importance of the combined effects of SLF and SDF-1α in the migration of fetal HSCs, and is, to our knowledge, the first demonstration of a synergistic effect of two chemoattractive agents on HSCs. New results on the migratory behavior of blood cell precursors in the early embryo might be relevant to bone marrow transplants and other clinical therapies During fetal development, the primary anatomical concentration of hematopoietic stem cells (HSCs) changes location several times. The migration of blood-borne progenitors is essential for the establishment of hematopoiesis in subsequent hematopoietic tissues . The speWhile the mechanisms that influence HSC homing and colonization are not completely understood, several experimental models suggest possible regulatory factors. The homing to and colonization of fetal hematopoietic organs by circulating HSCs likely require homing receptor/addressin interactions in the vascular lumen, followed by chemokine/chemokine receptor interactions, integrin/receptor binding, and growth/survival factors. Homing of lymphocytes and leukocytes has been well documented to involve first homing receptor/vascular addressin interactions, resulting in cell tethering and rolling on blood vessel endothelium. The rolling cells respond to a chemoattractant, produced by endothelial or stromal cells within the tissue, by firm adherence to the vessel wall mediated by integrin/receptor interactions. The adhered cells subsequently traverse the vessel wall, migrating toward the increasing gradient of chemoattractant . A simil(Sl), normally expressed in hematopoietic stromal cells, or its receptor gene (W), encoding the c-Kit tyrosine kinase, result in profound but incomplete defects in hematopoiesis (d) activity. Our results indicate that mouse fetal HSCs are found constitutively rather than episodically in fetal circulation and are present at low numbers throughout mid-to-late fetal development. We also measured the seeding of the fetal spleen and fetal bone marrow during this period. The seeding of these organs is a gradual process occurring over several days and does not appear to involve a large influx of HSC. Finally, we found that FL HSCs migrate in response to the chemokine SDF-1α and that this response is substantially enhanced in the presence of SLF. The enhanced chemotactic response of HSCs to the combination of SLF and SDF-1α is a property of FL HSCs, but not adult BM HSCs.+ Thy-1.1lo Sca-1+ Lineage– Mac-1lo , was injected into an adult, lethally irradiated congenic recipient, along with a radioprotective dose of host-type bone marrow. A FE was defined as the amount of blood collected from a single fetus of each age group. The amount of fetal blood transplanted ranged from 4 FE to 0.1 FE. The recipient mice were periodically bled and assayed for donor cells of the B, T, and myeloid lineages.  Mac-1lo  can be d Mac-1lo .+ Thy-1.1lo Sca-1+ Lineage– Mac-1lo  begin to seed the fetal spleen and bone marrow on 14.5–15 dpc and 17.5 dpc, respectively. We propose that fetal HSCs are continuously entering circulation and functionally engraft specialized stem cell niches as they develop  donor and C57BL/Ka-Thy-1.2  recipient mouse strains were bred and maintained at the Stanford University Laboratory Animal Facility, Stanford, California, United States. All mice were routinely maintained on acidified water (pH 2.5). Irradiated recipient mice were more than 8 wk old at the time of irradiation. All protocols were approved by the Administrative Panel on Laboratory Animal Care at Stanford University School of Medicine.A20.1  and AL1-4A2  were used to analyze donor and host cells following reconstitution. Blood analysis included 6B2 (anti-B220), KT31.1 (anti-CD3), GK1.5 (anti-CD4), 53-6.7 (anti-CD8), 8C5 (anti-Gr-1), and M1/70 (anti-Mac-1).The monoclonal antibodies used in immunofluorescence staining for HSC analysis included 2B8 , 19XE5 , E13 , and M1/70 . Lineage marker antibodies included 6B2 (anti-B220), KT31.1 (anti-CD3), GK1.5 (anti-CD4), 53-7.3 (anti-CD5), 53-6.7 (anti-CD8), Ter119 (anti-erythrocyte-specific antigen), 8C5 (anti-Gr-1), and M1/70 (anti-Mac-1). The antibodies were purified and conjugated within our lab. Each antibody was titrated and used at predetermined optimal concentrations .Timed pregnancies of C57BL/Ka-Thy-1.1/Ly-5.2 mice were used to obtain embryos. The day the vaginal plug was observed was designated as 0.5 dpc. The uterus was removed and washed to remove maternal blood. Fetuses were carefully removed to prevent contamination with maternal blood. Fetuses were then decapitated in Hanks' balanced salt solution containing 5 mM EDTA and allowed to bleed out. Fetuses were passaged through several dishes of media until completely pale. Blood was combined and centrifuged. Blood was either prepared for injection by sedimenting RBCs in dextran followed by lysis of erythrocytes in 0.15 M ammonium chloride, 0.01 M potassium bicarbonate solution on ice, or remained unmanipulated and was injected directly into recipients. Results from both preparations were comparable. Spleens were obtained by first removing the spleen and stomach to a dish of Hanks' balanced salt solution containing 2% FBS. The spleens were then peeled from the surface of the stomach and placed in a clean dish of media. Femurs and tibia were removed and cleaned of muscle tissue. Spleens and bone were dissociated using the rubber end of a 1 ml-syringe plunger and filtered through nylon mesh. Blood and tissues were collected from fetuses obtained from at least three pregnant females for each timepoint measured. Fetuses that appeared developmentally advanced or delayed in any age group were discarded.5 CD45 congenic (recipient-type) whole bone marrow cells. Recipient mice were periodically bled to analyze peripheral blood for donor B and T lymphocytes and myeloid cells. Recipients were determined to have LT-MLR if all three of these donor subsets were present for greater than 20 wk. Positive engraftment was determined by comparison to control mice, and in most cases the threshold for positivity was less than 0.1%.Adult recipient mice were lethally irradiated with a split dose of 950 rad as previously described . Recipie6 or 1 × 107 cells/ml) of lineage-depleted cells was placed in the upper chamber. To measure chemokinetic movement, factors were also added to the upper chamber at the same concentration as the lower chamber. Following a 2 h incubation, the upper chamber was removed. A known quantity of fluorescent beads was added to the lower chamber for normalization of migrated HSCs. Migrated cells were removed from the lower chamber and stained with PE-conjugated antirat IgG. After washing, cells were incubated with rat IgG and then labeled with directly conjugated lineage PE, c-Kit APC, Sca-1 Texas Red and Thy-1.1 FITC antibodies. Cells were analyzed by FACS to enumerate migrated HSCs.FL HSCs and adult BM HSCs were prepared as for antibody staining. Cells were stained with a lineage cocktail of the same purified rat IgG monoclonal antibodies used for FACS sorting. The cells were then depleted by magnetic selection using anti-rat IgG beads as per manufacturer's instructions , followed by a 1h incubation in RPMI media  containing 10% FBS in a tissue culture flask at 37°C to remove adherent cells. Dual-chamber chemotaxis assays were performed using 24-well plates with 5-μm pore size inserts , as previously described . SDF-1α-t-test. A p value of <0.05 was considered significant.Results shown in www.ncbi.nlm.nih.gov/LocusLink/) accession numbers of the gene products discussed in this paper are CXCR4 (LocusLink ID 12767), SDF-1α (LocusLink ID 20315), and SLF (LocusLink ID 17311).The LocusLink (


Sorghum bicolor is a close relative of maize and is a staple crop in Africa and much of the developing world because of its superior tolerance of arid growth conditions. We have generated sequence from the hypomethylated portion of the sorghum genome by applying methylation filtration (MF) technology. The evidence suggests that 96% of the genes have been sequence tagged, with an average coverage of 65% across their length. Remarkably, this level of gene discovery was accomplished after generating a raw coverage of less than 300 megabases of the 735-megabase genome. MF preferentially captures exons and introns, promoters, microRNAs, and simple sequence repeats, and minimizes interspersed repeats, thus providing a robust view of the functional parts of the genome. The sorghum MF sequence set is beneficial to research on sorghum and is also a powerful resource for comparative genomics among the grasses and across the entire plant kingdom. Thousands of hypothetical gene predictions in rice and Arabidopsis are supported by the sorghum dataset, and genomic similarities highlight evolutionarily conserved regions that will lead to a better understanding of rice and Arabidopsis. Methylation filtration makes practical the sequencing of large genomes, such as those found in sorghum, by preferentially capturing functionally relevant sequences Sorghum bicolor is a vitally important crop in Africa and much of the developing world. It has a remarkable ability to endure both drought conditions and water-logging and it grows well on marginal lands  and the reassociation time in seconds [t]), allows the separation of low-copy sequences from those of high copy based on annealing rates /[R/N])/(N/UM), or approximately 10%. This is consistent with maize |leucine|WD-repeat|WD repeat|WD40|WD-40| ankyrin|telomere|arm repeat|PPR-repeat|armadillo|tetratricopeptide|TPR-repeat|TPR repeat|Kelch|pentapeptide|C-repeat/”.The This second step was used to replace falsely identified nonrepetitive elements. Removing repeats reduced the database size by 640 sequences to 27,941, which included 4,412 sequences identified as hypothetical by matching the definition line to the case-insensitive regular expression “/hypothetical/.”ftp://ftp.tigr.org/pub/data/Eukaryotic_Projects/o_sativa/annotation_dbs/pseudomolecules/version_2.0/all_chrs/all.seq). The file was dated 30 April 2004 and contained 57,535 sequences . The rice sequence set contains the genomic regions for all predicted rice genes, which includes exons, introns, and untranslated regions where good evidence is provided. No sequences were removed from this database, but they were classified as “repeats,” “hypothetical,” and “known” by the following criteria. (1) Sequences were classified as repeats if the definition line matched a case-insensitive regular expression “/retro|transpos|reverse transcriptase|gag|polyprotein|mutator|maggy|rire|gypsy|copia|bare-1/”; (2) the sequences were classified as hypothetical if the definition line matched a case-insensitive regular expression “/hypothetical/”; and (3) the remaining sequences were classified as known. In total, there were 13,008 repeats, 21,441 hypotheticals, and 25,263 known proteins. The rice chromosomal genomic sequences were used for miRNA identification  and dated 05 September 2003. It contains 12 chromosomes, with sequences comprising 358,546,960 bases.The rice sequence set was downloaded from TIGR . No sequences were removed from this database.Sorghum ESTs were download from the NCBI  of the curated base see . Aside fabulated . For sorThere were 14 finished BAC clones at the time of analysis, with the following accession numbers (and GenInfo identifiers). AC120496.1 (GI:20486389), AF010283.1 (GI:2735839), AF061282.1 (GI:4539654), AF114171.1 (GI:4680196), AF124045.1 (GI:5410347), AF369906.1 (GI:19851516), AF466199.1 (GI:18390096), AF466200.1 (GI:18481699), AF466201.1 (GI:18483227), AF466204.1 (GI:18568251), AF503433.1 (GI:21326110), AF527807.1 (GI:22208458), AF527808.1 (GI:22208471), and AF527809.1 (GI:22208503). The BACs were manually annotated, then reads were mapped to the BACs by BLAST to determine the locations of hits relative to the genes.ftp://ftp.tigr.org/pub/data/TIGR_Plant_Repeats/) and contained 11,043 repeat entries. RepeatMasker was run with the following parameters: “-s; -w; -no_is; -nolow; -lib cereal_repbase.lib”. RepeatMasker parameters also included “-xsmall” to mask in lowercase and “-w” to use the MaskerAid [N=–1; Q=3; R=3; kap; E=1e-10; hspmax=0. To look for potentially novel genes, we used Fgenesh (http://www.softberry.com/berry.phtml) with monocot parameters, Genscan [Arabidopsis parameters, and SNAP [Arabidopsis parameters.We analyzed the BACs with several computational tools in addition to manual editing. Repetitive elements were identified using RepeatMasker  with theaskerAid  enhancemaskerAid ,56 usingaskerAid . The fir Genscan  with Araand SNAP  with Arahttp://www.acedb.org) and edited gene structures by hand. One of the challenges was how to determine when the tools had identified pseudogenes. These are often marked by adjacent repeats, BLASTX alignments containing stop codons, or gene predictions with tiny introns to compensate for frame-shifts. Another challenge was how to use cross-species alignments. Alignments that are nearly identical to genomic sequence are useful for delimiting exon boundaries, but inexact matches pose problems because alignments may terminate because of real exon boundaries or differences between the sequences. Since most of the alignments were from plants other than S. bicolor, we did not employ any programs that align a protein or transcript directly to a genome. Instead, we assigned the position of the splice sites in part by consulting exon predictions, since gene finding algorithms contain probabilistic models of splice sites. We did not report any raw gene predictions. However, some genes do contain exons with no overlapping evidence and are included in the gene structure because they complete an otherwise incomplete gene structure and in some cases are necessary to maintain the reading frame. The BAC annotations are available in GFF format with the supplemental online data.In order to annotate the locations of genes in each BAC, we loaded all the computational results into the ACEDB viewer . A sequence was considered mapped to a BAC if the match was over 90% of the read with 98% identity or higher. A single read was mapped to only one location. A gene was considered tagged if one or more MF sequence(s) overlapped the CDS region by 50 bases or more. The set of Arabidopsis chromosome 1 was compared to the empirical gene tagging results in sorghum. The sequence and annotation of Arabidopsis chromosome 1 was downloaded from TIGR  on 20 February 2004. The chromosome is approximately 30 Mb long with 7,520 genes annotated. The median gene size is 1,960 bp.A computational simulation of shotgun sequencing Computationally generated “reads” of 700 bp in length were created from the chromosome for different levels of raw coverage from 0.5× up to 3.5×. The reads were then mapped back to the chromosome annotation to determine the percentage of the 7,520 genes that were tagged at each level of raw coverage .The lleagues . This daU-BLASTN  search oArabidopsis miRNAs. The shuffling maintains the nucleotide composition of each while scrambling the order [In order to test the specificity of these miRNA matches, we generated shuffled sequences for the 122 rice and 92 he order . The shu−8, which corresponds to a bit score of approximately 61. The rice hits were then counted and categorized.The rice sequences were compared to the sorghum MF using NCBI-BLASTN with the rice sequences as the query and the sorghum MF reads as the database . We counted a rice gene as hit if the E-value was less than or equal to 1 × 10Arabidopsis protein set using NCBI-BLASTX . We counted a rice gene as hit if the E-value was less than or equal to 1 × 10−8, which corresponds to a bit score of approximately 51. The rice genes hit were counted and categorized.To assess how well rice is annotated by a dicot, the rice sequences were also searched against the Arabidopsis protein set was compared the sorghum MF dataset using NCBI-TBLASTN . We counted an Arabidopsis protein as hit if the E-value was less than or equal to 1 × 10−8, which corresponded to a bit score of approximately 57. The Arabidopsis hits were then counted and categorized as shown in The Arabidopsis protein set was also compared to the rice gene sequence dataset using NCBI-TBLASTN . We counted an Arabidopsis protein as hit if the E-value was less than or equal to 1 × 10−8, which corresponded to a bit score of at least 57. The Arabidopsis hits were then counted and categorized as shown in The Arabidopsis proteins that were annotated by sorghum MF but not rice sequence. These 247 proteins were then compared to the entire rice genome using NCBI-TBLASTN . From that set of 247 we removed any Arabidopsis proteins if the E-value was less than or equal to 1 × 10−8, which corresponded to a bit score of at least 59. The remaining 188 proteins were then compared to the entire genome from the O. s. indica cultivar of rice [Arabidopsis proteins if the E-value was less than or equal to 1 × 10−8, which corresponded to a bit score of approximately 60. The resulting set contained 127 Arabidopsis proteins that were supported by sorghum MF but not found in the rice genomes.There were 247  of rice  using NCMethylation was assessed using MethylScreen analysis, which is a real-time PCR technique that reports DNA methylation occupancy information for genomic markers through enzymatic interrogation. MethylScreen analysis compares the cycle thresholds (Cts) of gDNA that has been subjected to various treatments and infers 5′ methylated cytosine (5 mC) occupancy through the changes in Ct mediated by the treatments. The Ct of any locus is a function of the number of copies present within the assay tube. MethylScreen analysis relies upon the simple formula that total gene copies = number of methylated copies + number of unmethylated copies in every sample. Typical sample assays utilize four sample subportions, the first portion of a sample is mock-digested, reporting total copies present. A second  portion is treated with a methylation-sensitive restriction enzyme (MSRE), reporting the number of gene copies that are methylated. The third portion is digested with a methylation-dependent restriction enzyme (MDRE) such as mcrBC, reporting the number of copies that are unmethylated. The fourth reaction is doubly-digested with both the MSRE and MDRE. When working with relatively pure samples, methylated loci have a Ct from MSRE that is the same as the untreated control, and the Ct obtained from the MDRE is greater. Conversely, unmethylated loci have a MDRE Ct that is identical to untreated and a greater Ct in the MSRE reaction.tb2 targeted a 263 bp region from the 5′ end of the gene for the assay. There are four HhaI restriction sites and more than 25 possible mcrBC half-sites (5′-RC-3′) in this region .The gene s region A. We devtb2 gene. Cycling parameters were 95 °C for 3 min, followed by 50 cycles of two-step PCR: 95 °C for 30 s and 56 °C for 30 s. We utilized both a low-temperature (70 °C) and a high-temperature (82 °C) plate read. The input of gDNA was cut to 10 ng per reaction. All amplifications were performed in quadruplicate. Three replicate analyses were performed (digestions and cycling). The threshold was set using a template dilution standard control. For the kafirin genes, the average difference in Ct between the mcrBC single and the HhaI + McrBC double-digests is 2.46 cycles (22.08 ± 0.34 HhaI + McrBC - 19.62 ± 0.19 McrBC).Each of the 11 genes was broken into approximately 1.5-kb pieces, which were aligned to create a consensus kafirin assembly B. The coPCR products from the kafirin cycling reactions were cloned using the topoisomerase-assisted method . Libraries of insert-bearing clones were generated using standard techniques. From each library, 200 lacZ-negative clones were selected for characterization. The clones were sequenced with a single read using the M13 priming site on the pCR2.1 plasmid (Invitrogen). All seven subfamilies were discovered from both the treated and untreated genomic samples (unpublished data), indicating that all 11 genes were amplified and recoverable, even in the mcrBC-digested fractions.Arabidopsis DREB genes DREB1A, DREB1B, DREB1C, DREB2A, and DREB2B were used in a TBLASTN search of an assembly the sorghum dataset using WU-BLAST . Matches to the sorghum assembly with an E-value of 1 × 10−8 or less were analyzed with FGENESH (monocot) to select assemblies with a full-length protein. Out of 67 full length proteins identified in this manner, five sorghum proteins were identified as DREB1 genes based on conservation of the AP2 domain and a conserved C-terminal motif, LWSY [The five if, LWSY .Table S1Arabidopsis proteins that have matches to the sorghum MF set at a TBLASTN E-value less than or equal to 1 × 10−8, but are not found in the O. s. japonica or O. s. indica genomes at the same cutoff.Shown is a list of 127 (120 KB DOC).Click here for additional data file.http://www.ncbi.nlm.nih.gov/). On 6 January 2004, Orion deposited 50,161 of the sequences under accession numbers CL147592–CL197752. The 36,825 Cold Spring Harbor Laboratories MF sequences were previously deposited in GenBank under the accession numbers CC058553–CC059980, BZ329127–BZ342789, BZ342901–BZ352342, BZ365856–BZ368372, BZ369686–BZ370012, BZ421595–BZ424357, BZ625682–BZ629992, and BZ779555–BZ78192. The remaining 481,989 MF sequences from Orion Genomics are deposited in GenBank under accession numbers CL147592–CL197752 and CW020594–CW502582. The Orion UF sequences are deposited in GenBank's Genome Survey Sequence under accession numbers CW512190–CW514008. The University of Oklahoma UF sequences are deposited in the NCBI trace archive under accession numbers TI566112507–TI566128395. GenBank accession numbers for other genes discussed in this paper are sorghum Cs1 (AF206660); Arabidopsis DREB genes DREB1A (Q9M0L0), DREB1B (P93835), DREB1C (Q9SYS6), DREB2A (O82132), and DREB2B (O82133). Genbank accession numbers for BAC clones (with GenInfo identifiers) are AC120496.1 (GI:20486389), AF010283.1 (GI:2735839), AF061282.1 (GI:4539654), AF114171.1 (GI:4680196), AF124045.1 (GI:5410347), AF369906.1 (GI:19851516), AF466199.1 (GI:18390096), AF466200.1 (GI:18481699), AF466201.1 (GI:18483227), AF466204.1 (GI:18568251), AF503433.1 (GI:21326110), AF527807.1 (GI:22208458), AF527808.1 (GI:22208471), and AF527809.1 (GI:22208503). The GenBank accession number for the protein CAPRICE is NP_182164. Accession numbers for genes used in phylogenetic analysis of sorghum DREB are as follows. Rice genes are OsDREB1A (AF300970), OsDREB1B (AF300972), OsDREB1C , and OsDREB1D ; AP2 domains from other Arabidopsis proteins are also included: APETALA2 , AtERF-1 (BAA32418), LEAFY PETIOLE (AAF32292), and TINY (Q39127).The sorghum MF sequence set is deposited in the Genome Survey Sequence division of GenBank (


Complex Regional Pain Syndrome type one (CRPS I) or formerly Reflex Sympathetic Dystrophy (RSD) is a disabling syndrome, in which a painful limb is accompanied by varying symptoms. Neuropathic pain is a prominent feature of CRPS I, and is often refractory to treatment. Since gabapentin is an anticonvulsant with a proven analgesic effect in various neuropathic pain syndromes, we sought to study the efficacy of the anticonvulsant gabapentin as treatment for pain in patients with CRPS I.We did a randomized double blind placebo controlled crossover study with two three-weeks treatment periods with gabapentin and placebo separated by a two-weeks washout period. Patients started at random with gabapentin or placebo, which was administered in identical capsules three times daily. We included 58 patients with CRPS type 1.Patients reported significant pain relief in favor of gabapentin in the first period. Therapy effect in the second period was less; finally resulting in no significant effect combining results of both periods. The CRPS patients had sensory deficits at baseline. We found that this sensory deficit was significantly reversed in gabapentin users in comparison to placebo users.Gabapentin had a mild effect on pain in CRPS I. It significantly reduced the sensory deficit in the affected limb. A subpopulation of CRPS patients may benefit from gabapentin. Spontaneous recovery does occur and several therapies have been described, but for some patients CRPS I is a chronic disabling disease. Neuropayndromes -15. Anecyndromes ,8,16-27.This study complied with the Declaration of Helsinki regarding investigations in humans after approval of the protocol by the Institutional Review Board of the University Hospital Maastricht, the Netherlands. Patients were recruited from a database with patients who, in recent years, had been diagnosed with complex regional pain syndrome type I in our hospital. All patients had been treated in our pain management and research center  and had received sympathetic blocks, mannitoSince our patient population consisted of chronic CRPS I patients with a multiple years' duration of pain complaints refractory to various treatments, we assumed that their pain complaints would be more or less stable. We therefore undertook a double blind, randomized crossover study. Randomization of patients took place after baseline measurements and written informed consent. The assignment scheme was generated by our hospital pharmacy from a table of random numbers. The closed envelopes containing the assignments were prenumbered and kept at the pharmacy. The first treatment group received gabapentin, followed by a washout period and placebo treatment. The second treatment group received placebo treatment, which was followed by a washout period and gabapentin treatment. Each medication period lasted three weeks separated by the two-weeks washout period. Medication was stocked and delivered to the patient at the hospital pharmacy. Both the gabapentin capsules and the identical placebo capsules were delivered immediately before the start of the two medication periods. Left over medication was recollected and counted.The gabapentin (GBP) dose was slowly increased to reduce adverse side effects:600 mg's GBP AN once a day on day 1–2600 mg's GBP b.i.d. on day 3–4600 mg's GBP t.i.d on day 5–21Placebo dose was identically titrated. Patients were allowed to take their usual analgesics and were told preferably not to change the usual dose.The patients were reevaluated at the hospital three weeks (T1), five weeks (T2) and eight weeks (T3) after randomization. During the trial, the patient noted her/his pain rate of the past 24 hr (VAS) and the use of additional analgesics in a diary. During each hospital visit the following assessments were done:1. Global perceived effect (GPE) on pain indicating: worst ever; much worse; somewhat worse; not improved/not worse; somewhat improved; much improved and best ever. GPE on function was scored on an analogous scale.2. Neuropathic pain scale (NPS), a 10 item qualitative evaluation of neuropathic pain.3. Sensibility through Von Frey monofilament skin application each on 9 areas corresponding to cutaneous nerve branches and dermatomes of either both hands or both feet. Stimulu4. Mechanic allodynia test with brush strokes and static pressure with the finger tip, on 9 ar5. Edema, discoloration, and changed skin temperature were scored after physical examination on a three point scale indicating no, some or overt presence of each sign, the latter two signs in comparison to the healthy or healthiest limb. Physical examination in CRPS is well comparable to instrumental evaluation of signs with volumeter, infrared thermometer and goniometer.90-R)[6. Impairment and disability tests: Symptom Checklist-90-Revised (SCL-90-R), Brief P90-R), adaptedA blinded independent investigator (STvdB) did sensibility, allodynia and range of motion tests (see above). A physician (AvdV), who examined each patient, did all the other measurements throughout the trial. Patient, investigator and physician were unaware of the treatment received. We tested blinding by questioning physician and participants after each medication period.The statistical analysis of VAS-scores was determined per patient using estimating medication and period effect through linear regression analyses. Possible relationship of patient characteristics and outcome was tested by Pearson R's test. Mann-Whitney analyses were used for monofilament sensitivity on log-transformed data. Three point scales and seven point scales were dichotomized and like the SCL-90-R, NPS and CRPS-Brief Inventory questionnaires intra-individual paired tested . Student t-tests and regression analysis were used to test treatment effect, which is calculated in crossover studies as ((AT1-AT0)-(AT3-AT2))/2+((BT3-BT2))-BT1-BT0)/2, where A represents data of placebo starters and B data of gabapentin starters both before  and after  treatments. BlindinAfter randomization 58 patients were enrolled, with a mean age of 44.0 range 24–75) resulting in 29 patients in the gabapentin-placebo arm and 29 patients in the placebo-gabapentin arm; 49 patients completed the gabapentin period, 50 patients the placebo period, 46 patients completed both periods and were used for further within-patient paired analysis Fig. . Twelve 4–75 resuAfter each medication period both patient and physician were asked about their ideas concerning study medication in the past period. The treating physician guessed the used medication correctly more often after both phases than can be explained by coincidence (p = 0.000). Blinding for patients was sufficient in the first phase, but not anymore after the second phase (p = 0.2 versus p = 0.000).Comparing gabapentin and placebo users in terms of pain relief, there was a significant pain relief in favor of gabapentin in the first period. Therapy effect in the second period was less, finally resulting in no significant effect combining results of both periods. There was an unexpected increase of pain level above baseline in the washout period for both the gabapentin starters and placebo starters Figure .Global perceived pain relief as measured by the seven-point scale showed a significant effect for gabapentin, and also more pronounced in the first period. This measurement also found a significant effect in the second period, with an effect being defined as a patient scoring 'much improvement'. Statistical analysis of global perceived effect showed significant more treatment effect (p = 0.002) with 43 % versus 17 % reported pain relief respectively during gabapentin compared to placebo treatment. 13 % of patients reported aggravation of pain during gabapentin vs. 9 % during placebo treatment . This difference was found in patients with upper and lower extremity CRPS, but was still significant in the subgroup of lower extremity CRPS (p = 0.011) as seen in table Mechanical allodynia to static and dynamic stimuli (soft touch and brush) was measured by a mean of 11-point scales (range 0–10) of 9 areas of the hand/feet corresponding to cutaneous nerve branches. We found no effect of gabapentin on allodynia in comparison to placebo.No difference was found on the parameters edema, discoloration, range of motion of wrist/ankle and fingers/toes between placebo and GBP. 10 patients out of 45 improved in relative skin temperature during placebo use compared to 18 patients out of 45 in gabapentin, which is two sided tested not significantly different .The reported function improvement was, with 10 positive responders during GBP versus 7 positive responders during placebo, not significantly different (N = 46) between the two treatments. The SCL-90 showed no significantly better scores during gabapentin treatment. CRPS-BI showed improvement of sleep between placebo treatment and gabapentin treatment., but this effect disappeared after Bonferroni-Holm correction.Dizziness, somnolence and lethargy were significantly more often reported during gabapentin use than during placebo. Before washout 95 % of patients (n = 21) reported side effects during gabapentin use versus 58 % in placebo treatment (n = 14). After washout this was respectively 63% (n = 15) in GBP and 32% (n = 7) in placebo use. For more details on side effects see table 6. Since a high incidence of side effects can produce a stronger placebo effect, we analyzed the possible correlation between side effects and pain relief. There was a small relation between perceived side effects and pain relief in placebo users in period 2 , but, whether in period 1 or period 2, no relationship was found during the use of gabapentin .To evaluate gabapentin treatment as a treatment for pain in CRPS, we conducted a placebo-controlled crossover study. We conclude from our trial that overall, gabapentin did not relieve pain as compared to placebo on pain visual analogue scores, our primary outcome measure. Gabapentin relieved pain in a subgroup of patients and gave a significant global perceived pain relief. The effect was mild and there was no patient in which gabapentin completely eliminated pain. Moreover, the frequency of side effects as dizziness, somnolence and lethargy was higher during gabapentin treatment than with placebo. These side effects probably also account for the relative lack of blinding we observed in our study. This does not mean that the study was biased: our population was chronic CRPS patients who all had undergone numerous unsuccessful treatments, and clearly wanted the drug to work. Any possible bias would therefore have been positive towards gabapentin.Although we did not find a significant pain reducing effect when analyzing the complete trial, we did find a significant effect in the first half of the trial. In fact, the difference in outcome between the two trial halves is striking. There was a reverse carry-over effect resulting in increasing pain above baseline after the washout period for both gabapentin and placebo starters. The increase of pain intensity above baseline level in the second period (before the start of placebo treatment) cannot be explained pharmacologically. Gabapentin has no known biological dependency or tolerance mechanism. It can be a period effect, although this would more likely result in a regression to the middle instead of increasing pain. Perhaps this is a reversed placebo effect in which the expectation and/or the actual perception of not receiving gabapentin anymore might increase pain intensity. Kemler and de Vet found that treatment allocation in a trial could influence pain intensity in CRPS. The decWe found a decreased sensory deficit in gabapentin users compared to placebo users. We did not expect this, but found in the literature several cases in which gabapentin decreased the area of hypesthesia in neuropathic pain syndromes. This haGabapentin had a mild effect on pain in patients CRPS I. It significantly reduced the sensory deficit in the affected limb. A subpopulation of CRPS patients may benefit from gabapentin, but then for each individual patient the benefit has to be weighed against the frequently occurring side effects.Parke-Davis (now a Warner-Lambert/Pfizer division) supplied gabapentin and matching placebo capsules for this trial. Drs. Van de Vusse and Weber have received financial support from Parke-Davis to attend one congress. Parke-Davis has had no role in the writing of this manuscriptAvdV initiated the trial and wrote, with WEJW and AHFK, the protocol. The study and its data management was done by AvdV and SS-vdB. AHFK did the statistical analyses. AvdV wrote the first draft of the manuscript, which was finished in its final form by WEJW.The pre-publication history for this paper can be accessed here:


Short oligonucleotide arrays have several probes measuring the expression level of each target transcript. Therefore the selection of probes is a key component for the quality of measurements. However, once probes have been selected and synthesized on an array, it is still possible to re-evaluate the results using an updated mapping of probes to genes, taking into account the latest biological knowledge available.We investigated how probes found on recent commercial microarrays for human genes (Affymetrix HG-U133A) were matching a recent curated collection of human transcripts: the NCBI RefSeq database. We also built mappings and used them in place of the original probe to genes associations provided by the manufacturer of the arrays.In a large number of cases, 36%, the probes matching a reference sequence were consistent with the grouping of probes by the manufacturer of the chips. For the remaining cases there were discrepancies and we show how that can affect the analysis of data.While the probes on Affymetrix arrays remain the same for several years, the biological knowledge concerning the genomic sequences evolves rapidly. Using up-to-date knowledge can apparently change the outcome of an analysis. In a relatively short time microarrays have become a well established technique, widely used by researchers. Microarrays offer nothing less than to be able to monitor simultaneously the expression levels for thousands of genes.targets, and the polymers of nucleic acids fixed on the surface are called probes. The very large number of genes represented on each microarray requires the use of computer based approaches. Although such approaches currently constitute a very rich and active area of research, for many data analyses this step can be summarized simply: under the prior assumption that for the large majority of the genes represented on a microarray the expression will not vary significantly across experiments, the main focus is be to isolate the few genes of interest from the rest. Short oligonucleotide arrays are a particular type of microarrays. Short oligonucleotide arrays are constituted of short probes (oligonucleotides) with several probes designed to match different part of the target sequences. The use of techniques originating from the micro-electronics industry proved very successful in the making of short oligonucleotides arrays [Affymetrix company establish itself as one of the primary manufacturers for microarrays. The pre-processing of oligonucleotide array data differs from other microarray data, specifically because probe intensities associated with each gene are generally summarized by an expression value, or expression index. As it contributes to the computation of the expression values, this step alone is of importance. Different algorithms have been suggested to replace and improve Affymetrix's original algorithms [et al.'s playerout [The RNA molecules from the biological sample are called s arrays , helpinggorithms , includilayerout , Li and layerout , medianplayerout , and Afflayerout . Howeverlayerout ,8.The annotation for sequenced genomes have progressed considerably since the design of the Affymetrix chips, even the most recent ones, and matching the latest transcriptomic (or genomic) data available with the chip designs is an obvious thing to do.We have performed such a remapping for a few Affymetrix chips, and we show that the resulting probe-to-gene mapping can differ substantially from the original Affymetrix mapping. This can affect the interpretation of experimental data.As annotations of genomes continue to evolve, it is also desirable to have a framework to perform and handle up-to-date probe-to-gene mapping. We provide an open source and documented implementation to do so.The results obtained are subdivided in two main categories: the matches between probes and reference sequence obtained, and the difference in the outcome of an analysis when using an alternative mapping.A fair number of probes were found to match several reference sequences, as shown in Figure NM_001544.2 is found to have 21 matching probes. Eleven of these matching probes also match another reference sequence: NM_022377.1. A quick look at the annotation reveals that both reference sequences are two different transcripts variants of the same gene 'Homo sapiens intercellular adhesion molecule 4, Landsteiner-Wiener blood group (ICAM4)' and that the same probes are found matching these two sequences. However the ten remaining probes matching NM_001544.2 are also found matching a fairly large number of other reference sequences . We found that these probes are designed to match sub-sequences frequently found in mRNA: ALU repeats. All the probes matching ALU repeats are in the official mapping grouped in a common probe set, called 'human ALU'.For example, the RefSeq NM_017445.1, NM_003519.3, NM_003520.3, NM_003521.2, NM_003525.2, NM_003528.2, NM_080593.1 and XM_301109.1, annotated 'H2B histone family, member S (H2BFS), mRNA', 'histone 1, H2bl (HIST1H2BL), mRNA', 'histone 1, H2bn (HIST1H2BN), mRNA', 'histone 1, H2bm (HIST1H2BM), mRNA', 'histone 1, H2bi (HIST1H2BI), mRNA', 'histone 2, H2be (HIST2H2BE), mRNA', 'histone 1, H2bk (HIST1H2BK), mRNA' and 'similar to Histone H2B 291B (LOC350694), mRNA' respectively.Besides 'human ALU' probes, other probes matching multiple reference sequences were found. In that case, the reference sequences matching a given probe have closely related annotations, or even identical annotations. Complex mixtures of partial overlaps for the probe sets can then be observed. As an example, the probe 88322 is found matching the reference sequences alternative mappings, so we chose to simply ignore the probes matching several reference sequences in the rest of this study.Such matches in each case require expert annotators to curate the official mapping, were found to be legitimate perfect match probes in the alternative mapping.Other multiple matches are more easy to handle, and potentially more harmful when included in an analysis. Some probes are found to hybridize to several unrelated reference sequences, as shown in Figure official mapping. When a 'one to one' association can be established between a probe set of the official mapping and a probe set in an alternative mapping, which means that a given reference sequence matches all the probes associated with one Affymetrix ID, we conclude a complete agreement between the alternative mapping and the official one , while the alternative mappings made from matching NCBI's reference sequences was used to obtain two more sets of expression values (sets Alt1 and Set Alt2), using all the matching probes or all the probes matching uniquely respectively. In other words, the set Alt2 differs from the set Alt1 in the sense that probes in Alt1 matching several reference sequences were removed from Alt2. The set Affy describes 22283 probe sets, the set Alt1 describes 18076 probe sets  and the set Alt2 11640 probe sets . The number of mismatch probes in the official mapping that are found in the alternative mappings is low: 290 in Alt1 and 87 in Alt2.Naturally the expression values, or expression indexes, computed from the probes intensities are sensitive to differences in the mapping: different probes will give different summary expression values, which can have an effect on the outcome of an analysis. To verify it, we performed a standard exploratory analysis of Affymetrix data . The probe level intensities were pre-processed and expression values computed. The For each one of the three sets, 'significantly differentially expressed genes' (SDEGs) are searched for: a Welch's two-sample t-test is performed on all the expression values in each set, and the selection for significant p-values done as described by Ventura and collaborators ,10 .The environment for alternative mappings we present could also be of use when using We built new 'probe to gene' mappings by matching the sequences for the probes found on human Affymetrix GeneChip arrays against NCBI's RefSeq database of sequences. Solely observing the distribution of the probes matching reference sequences, and comparing the results with the mapping provided by the manufacturer of the chips, we found potential problems such as probes matching several reference sequences and probes from different probe sets in the official mapping matching all the same reference sequence. Depending on the mapping used, the outcome of the data analysis will change as different genes may be selected as differentially expressed. We suggest that a good mapping of probe to genes changes in time, follows the most recent updates in sequence databases . We like to picture the current situation, where the mappings are frozen, as a Dorian Gray-like syndrome: the apparent eternal youth of the mapping does not reflect that somewhere the 'picture of it' decays. We developed a set of open-source tools, perfectly integrated to an already existing working environment for Affymetrix arrays. The tools are documented and made available to the research community. They let one reproduce our results, and build other mappings.Homo sapiens mRNA are considered.The database of reference sequences used for remapping the Affymetrix chips is NCBI's RefSeq , freely available for download on the RefSeq website . Only seThe Affymetrix chip types HG-U95Av2 and HG-U133A, both designed for the study of the human transcriptome, were used. While similar features were observed for both, we focus on the HG-U133A to describe our results, as this is the chip with the most recent design.The probe sequences for the chips are freely available on the Affymetrix website , as wellAffymetrix GeneChips have several probes per probe set, and a probe set usually represents a gene. Each probe is 25 bases long, and on most arrays probes are grouped in pairs. A probe pair is constituted of a perfect match (pm) probe, designed to match perfect a target gene sequence, and a mismatch (mm) probe, designed to measure non-specific hybridization. The mismatch probe differs from its associated perfect match probe only in the 13th base. In some cases, a probe set represents a fragment of a gene, e.g, 3-prime and 5-prime extremities of the same gene are used as internal control for the efficiency of the reverse transcription. We prefer the term 'probe set ID' to 'gene' since probe sets are not always genes. We call the association 'probes – probe set ID' a mapping. We refer to the grouping of probes in probe sets given by Affymetrix as the official mapping, while a grouping of the probes matching a reference sequence into a probe set is referred to as an alternative mapping. The short length of the probes, as well as several authors reporting a successful use of perfect matches only [mm) as potential pm probes to perform the matching. The matching of a probe to a sequence is done using the Bioconductor package matchprobes. It performs an exact string matching, as done by the standard C library string: only complete sequence identity between a probe and a fragment of a reference sequence is counted as a match.hes only , suggestWe collected pancreatic tumor tissue from 8 patients and normal pancreatic tissue from 5. Tumor samples were collected from patients undergoing surgery for pancreatic tumors, quick frozen in liquid nitrogen and kept at -80°C until RNA extraction . The tissue was homogenized using a Polytron . 5 μg RNA was extracted from each sample and labeled. The RNA was extracted according to the Trizol protocol (Invitrogen). The samples were applied to Affymetrix HG-U133A GeneChips according to manufacturer's instruction. Clinical results from this study will be published elsewhere (manuscript in preparation).vsn [medianpolish, using only perfect match probes. The exclusive use of perfect match probes is needed to have an identical pre-processing step for the official mapping and alternative mappings, to allow comparison of the results obtained.The probe level data are pre-processed using the affy software package . No backvsn  normalizAll the datasets and software used are freely available, and on a wide range of platforms ..The original material used to obtain our results, software and data, is available from the web page: Alternative mappings The package altcdfenvs, new with the Bioconductor release 1.4 of May 2004 and available on the Bioconductor website, is designed to help researchers to build their own alternative mappings, using their own set of reference sequences and the probe sequences for the Affymetrix chip of their choice.LG conceived, developed and implemented the concept of alternative mapping for Affymetrix chips, and drafted the manuscript. MM and LFH provided original experimental data. SK provided guidance with the choice of a source for reference sequences, and with comments and input on the manuscript.


Apoptosis plays a key role in cell death observed in neurodegenerative diseases marked by a progressive loss of neurons as seen in Alzheimer's disease. Although the exact cause of apoptosis is not known, a number of factors such as free radicals, insufficient levels of nerve growth factors and excessive levels of glutamate have been implicated. We and others, have previously reported that in a stable HT22 neuronal cell line, glutamate induces apoptosis as indicated by DNA fragmentation and up- and down-regulation of Bax (pro-apoptotic), and Bcl-2 (anti-apoptotic) genes respectively. Furthermore, these changes were reversed/inhibited by estrogens. Several lines of evidence also indicate that a family of cysteine proteases (caspases) appear to play a critical role in neuronal apoptosis. The purpose of the present study is to determine in primary cultures of cortical cells, if glutamate-induced neuronal apoptosis and its inhibition by estrogens involve changes in caspase-3 protease and whether this process is mediated by Fas receptor and/or mitochondrial signal transduction pathways involving release of cytochrome c.8, 17β-estradiol (10 nM-10 μM) resulted in the prevention of cell death and was associated with a significant dose-dependent decrease in caspase-3 protein levels, with Δ8, 17β-E2 being more potent than 17β-E2. Protein levels of Fas receptor remained unchanged in the presence of glutamate. In contrast, treatment with glutamate induced, in a time-dependent manner, the release of cytochrome c into the cytosol. Cytosolic cytochrome c increased as early as 1.5 h after glutamate treatment and these levels were 5 fold higher after 6 h, compared to levels in the untreated cells. Concomitant with these changes, the levels of cytochrome c in mitochondria decreased significantly. Both 17β-E2 and Δ8, 17β-E2 reduced the release of cytochrome c from mitochondria into the cytosol and this decrease in cytosolic cytochrome c was associated with inhibition of glutamate-induced cell death.In primary cultures of rat cortical cells, glutamate induced apoptosis that was associated with enhanced DNA fragmentation, morphological changes, and up-regulation of pro-caspase-3. Exposure of cortical cells to glutamate resulted in a time-dependent cell death and an increase in caspase-3 protein levels. Although the increase in caspase-3 levels was evident after 3 h, cell death was only significantly increased after 6 h. Treatment of cells for 6 h with 1 to 20 mM glutamate resulted in a 35 to 45% cell death that was associated with a 45 to 65% increase in the expression of caspase-3 protein. Pretreatment with caspase-3-protease inhibitor z-DEVD or pan-caspase inhibitor z-VAD significantly decreased glutamate-induced cell death of cortical cells. Exposure of cells to glutamate for 6 h in the presence or absence of 17β-estradiol or Δ8, 17β-E2 being more potent than 17β-E2. To our knowledge, this is the first study to demonstrate that equine estrogens can prevent glutamate-induced translocation of cytochrome c from mitochondria to cytosol in rat primary cortical cells.In the primary cortical cells, glutamate-induced apoptosis is accompanied by up-regulation of caspase-3 and its activity is blocked by caspase protease inhibitors. These effects of glutamate on caspase-3 appear to be independent of changes in Fas receptor, but are associated with the rapid release of mitochondrial cytochrome c, which precedes changes in caspase-3 protein levels leading to apoptotic cell death. This process was differentially inhibited by estrogens with the novel equine estrogen Δ A num neurons ,6.The mechanisms by which glutamate-induced neurotoxicity or excitotoxicity is mediated, has not been established, however, a substantial body of evidence suggests that glutamate toxicity involves oxidative stress and apoptosis (programmed cell death) ,7-9. Thiin vitro cell death induced by various neurotoxins [8-estrogens were the most effective neuroprotectors [8-estrogens may to some extent, be due to their greater antioxidant properties [2) which has high affinity for both estrogen receptors ERα and ERβ and Δ8, 17β-estradiol , an estrogen that is a more potent antioxidant than 17β-E2 and whose activity appears to be mediated to a greater extent via ERβ [A number of studies have demonstrated that estrogens are potent antioxidants capable of inhibiting some of the neurotoxic effects of oxidative stress ,26,27. Wrotoxins -31. We hoperties ,29,31. H via ERβ .4) cultured for 7 days in 96-well plates were treated with increasing concentrations (0.2 – 100 mM) of glutamate for 18 h. As depicted in Figure Cortical cells 2.5 × 1004 cultur2  for 7 days. The medium was then changed and the effects of various concentrations (1–20 mM) of glutamate on lactate dehydrogenase (LDH) release were measured for 6 h. The results indicate that glutamate induced a significant (30–40%) increase in LDH release compared to the control untreated cells. No significant difference in LDH release was observed in 6 h at glutamate concentration between 1 to 20 mM, (data not shown). Based on these observations and the cell viability data .Cortical cells were cultured in 6-well plates  in the levels of caspase-3 occurred at 10 times lower concentration (1 μM)  of PKC ,36. PrimWestern blot analysis had indicated the presence of pro-caspase-3 in primary cortical cells treated with glutamate Figure . To deteTo determine whether the regulation of caspase-3 in glutamate-induced cell death in cortical cells is modulated by Fas receptor mediated apoptotic pathway, expression of Fas receptor protein was evaluated by Western blot analysis. The results indicate Figure  that ant2 or Δ8, 17β-E2 for 6 hours resulted in a significant decrease in the release of cytochrome c into the cytosol  polymerase (PARP), sterol-regulatory element-binding protein (SREBPs), PKC, DNA-dependent protein kinase, DNA-fragmentation factor (DFF) and a number of others ,25,35,36i. a polyclonal goat antibody caspase-3 p11 (K-19) against the p11 fragment of caspase-3, ii. a polyclonal rabbit antibody against caspase-3 (H-227) and iii. a monoclonal mouse antibody (E-8) (data not shown with this latter antibody). These observations suggest that either our antibodies are incapable of detecting these fragments or that cells may clear them rapidly as been observed in other studies [When cells undergo apoptosis, caspase-3 is initially present as pro-enzyme (32 kDa precursor protein) that is subsequently transformed into the active heterodimeric complexes through a cascade of proteolytic events ,18. The  studies . Since w studies ,36. The  studies ,36. The  studies . Taken t studies . Since i studies . WhetherSeveral signaling pathways are implicated in the initiation of the caspase-3 cascade; one of the well defined pathways for activation of apoptosis is Fas receptor-mediated pathway -43. Fas Another recently characterized mechanism for pro-caspase-3 activation involves translocation of the respiratory chain protein, cytochrome c (Apaf-2), from mitochondria to the cytosol during the induction of apoptosis by a variety of different agents in non-neuronal and neuronal cells such as cerebellar granule cells ,50. Cyto2 induces release of cytochrome c from heart mitochondria [2+) and Bcl-2 expression [2+ in cortical cells [We have previously reported that a number of equine estrogens, which are components of the drug CEE (conjugated equine estrogen) used extensively for management of vasomotor symptoms and osteoporosis in postmenopausal women, are potent antioxidants and protect neuronal cells against cell death induced by oxidized LDL and glutamate ,29,56,57chondria  and alsochondria . These opression . Exposural cells ,53 and ual cells . Whether2 has higher affinity for ERα and ERβ, it is possible that some of these effects are due to the antioxidant properties of estrogens. However, we have also demonstrated [8, 17β-E2 expresses its biological effects to a two fold greater extent via ERβ than 17β-E2 [The cortical neuronal cells used in the present study contain both estrogen receptors ERα and ERβ , and therefore whether the mechanism by means of which estrogens exert their neuroprotective effects remain to be elucidated. In general, the current evidence indicates that there are at least two mechanisms for estrogen action: i) the genomic mechanism mediated via two nuclear receptors ERα and ERβ, and ii) the non-genomic mechanism mediated via putative membrane receptors and these have been recently reviewed . Althougnstrated  that Δ8,n 17β-E2 . Since tn 17β-E2  and refe2+ and mitochondrial trans-membrane potential [Recent studies further report that estrogen receptor (ERβ) is localized in the mitochondrial membrane and estrogen can directly modulate the mitochondrial content of Caotential ,62. The otential  or β-amyotential  via modu8, 17β-E2 being more potent than 17β-E2, To our knowledge, this is the first study to demonstrate that equine estrogens can prevent glutamate-induced translocation of cytochrome c from mitochondria to cytosol in rat primary cortical cells.In the primary cortical cells, glutamate-induced apoptosis is accompanied by up-regulation of caspase-3 that can be blocked by caspase protease inhibitors. These effects of glutamate on caspase-3 appear to be independent of changes in Fas receptor, but are associated with the rapid release of mitochondrial cytochrome c, which precedes changes of caspase-3 protein levels leading to apoptotic cell death. This process was differentially inhibited by estrogens with the novel equine estrogen Δsc 7148) and goat polyclonal antibody to caspase-3 p11 (K-19: sc-1224). Both of these antibodies react specifically with precursor protein: Mr 32 kDa and the active caspase-3: Mr 20 (p 20) and 11 kDa (p 11) fragments. Rabbit polyclonal antibody to protein kinase c (P4334)  purchased from Sigma. Goat polyclonal antibody to Actin (I-19: sc1616) , mouse monoclonal antibody to Fas raised against Fas extracellular domain (CD95:AF435) , mouse monoclonal antibody cytochrome c (7H8.2C12) . Secondary antibodies were obtained from Sigma . SuperSignal® West Pico Chemiluminescent Substrate . Z-Val-Ala-DL-Asp (OMe)-fluoromethylketone (z-VAD-fmk) and z-Asp (OMe)-Glu (OMe)-Val-DL-Asp (OMe)- fluoromethylketone (z-DEVD-fmk)  and the Cytotox 96 Non-radioactive Cytotoxicity Assay Kit . Neurobasal, B27, Hanks' buffered salt solution (HBSS) and antibiotics were from Gibco Life technologies . Active Caspase-3 Detection FITC kit # 280000 was obtained from Stratagene .All primary and secondary antibodies and other reagents were purchased from commercial sources: rabbit polyclonal antibody to caspase-3 . Following dissociation, cells were suspended in 2 X volume of HBSS with Ca2+ and Mg2+ and allowed to settle for 3 to 5 minutes. The supernatant was transferred to new tubes and centrifuged at 1100 rpm (200 g) for 1 min. The pellets were suspended in HBSS buffer. Fresh cell suspensions were plated onto poly-L-lysine-coated 96-well plates or 6-well plates containing neurobasal medium supplemented with 2% B27, 0.5 mM L-glutamine, 25 μM glutamic acid, 120 mg/L penicillin and 260 mg/L streptomycin and incubated at 37 C in a humidified incubator with 5% CO2/95% air. Culture medium was replaced every other day. Experiments were performed on days 7 to 8 of cultures in neurobasal medium containing 2% antioxidant free B27 supplement. Under these serum-free culture conditions, only neuronal cells survive.Eighteen to twenty day old pregnant Sprague-Dawley rats were sacrificed by cervical dislocation. The fetuses were delivered and fetal skulls were carefully opened and the meninges were removed and the brain processed essentially as described by Brewer et al. . Briefly® Aqueous Non-radioactive cell proliferation assay  were used to assess cell death and cell viability respectively [During the development of this work, the lactate dehydrogenase (LDH) cytotoxicity assay  and the Cell Titer 96ectively . In MTS ectively  a strongectively . Since wectively . In all Apoptotic cells produce characteristic DNA ladders made up of nucleotide fragments which are visualized by staining with ethidium bromide following DNA-agarose gel electrophoresis. We used DNA fragmentation as one of the criteria for apoptotic cell death and this was determined as described previously . Briefly2PO4, 1.7 mM NaHPO4 150 mM NaCl, pH 7.4), containing 1% Nonidet P-40 , 0.5% sodium deoxycholate, 0.1% SDS and 1 mM phenyl methyl sulfonyl fluoride) for 20 min on ice. Cell lysates were centrifuged at 10,000 g at 4 C for 10 min. The protein concentration was determined by Bradford method . Cell lysates containing 10 to 20 μg protein were added in equal volume of 2 X reducing sample buffer  and heated at 100 C for 3 min. The samples were electrophoresed on discontinuous 10% polyacrylamide gel electrophoresis under constant current (14–15 mA). Separated proteins were electrotransfered onto a Protan nitrocellulose membrane . The blots were blocked with 5% non-fat milk in TBST  at 4 C overnight and then incubated with primary antibody for 1 to 2 h at room temperature, washed three times with TBST and incubated (1–2 h) with appropriate horse radish peroxidase (HRP)-conjugated second antibody. The membranes were washed three times with TBST and the immunoblots were visualized on X-ray films after exposure to enhanced chemiluminescence reagent (ECL) . Actin bands were monitored on the same blot to verify consistency of protein loading. Briefly, the Immunoblots were stripped with TBST containing 0.04% sodium azide for 30 min at room temperature. The blots were probed with anti-actin primary antibody and second antibody (anti-goat) as described above. The molecular size of protein was determined by running pre-stained protein markers in an adjacent lane, omitting primary antibody as a procedural control. The band intensity was measured from scanned images using UN-SCAN IT Gel Automated Digitizing system, Version 5.1 , software.Protein levels of Caspase-3, Fas or PKC were determined by Western blotting using a polyclonal and monoclonal antibody raised against caspase-3 or Fas or PKC as described previously . In briet- test. Analysis of variance (ANOVA) when appropriate, was used for multiple comparisons. Significance was set at P < 0.05.Each experiment was repeated at least three times and combined data were compared using the Student's paired 2, 1 mM Na-EDTA, 1 mM Na-EGTA, 1 mM dithiothretiol and protease inhibitors-complete cocktail). After 15 min incubation on ice, cells were homogenized using a glass homogenizer (25 stokes) at 4 C. Cell homogenates were spun at 750 g for 10 minutes and the pellets containing the nuclei and unbroken cells were discarded. The supernatant was then centrifuged at 10,000 g for 15 minutes. The pellets containing mitochondria was stored at -80 C until processed. The 10,000 supernatant was further centrifuged at 100,000 g for 1 hour at 4 C to obtain the cytosolic fraction (S-100). The intactness of the mitochondrial membranes was checked by assaying mitochondrial specific enzyme glutamate dehydrogenase in mitochondria, mitochondria treated with Triton X-100 and in cytosol (S-100) as described previously [The cell homogenates, mitochondria and cytosolic fractions were prepared essentially as described by Atlante et al. . The celeviously . GDH wasCaspase inhibitors were used as described previously  caspase-The primary cortical cells were cultured for 7 days in 96-well microplates and then treated with or without glutamate for 3 h and 6 h. Cells were then processed essentially as described by the manufacturer of the assay kit (Stratagene). Briefly, the microplates were centrifuged (200 × g) for 2 minutes and the supernatant discarded. The cells were fixed for 15 minutes following which the fixative was aspirated and the cells washed twice with phosphate buffered saline (PBS) containing 1% saponin. The cells were then incubated with the primary anti-cleaved caspase-3 antibody in PBS + 1% saponin for 30 minutes at room temperature. The cells were then rewashed and incubated for 30 minutes with secondary FITC-goat anti-rabbit antibody and then washed twice and the relative fluorescence measured using a microplate reader .YMZ is a postdoctoral fellow who participated in the development of the hypothesis, study design and carried out most of the experimental work and preparation of the manuscript.BB conceived the study and participated in the development of the hypothesis, the study design, and overall direction of the study and preparation of the manuscript.


Signal transduction is one of the most important biological processes by which cells convert an external signal into a response. Novel computational approaches to mapping proteins onto signaling pathways are needed to fully take advantage of the rapid accumulation of genomic and proteomics information. However, despite their importance, research on signaling pathways reconstruction utilizing large-scale genomics and proteomics information has been limited.We have developed an approach for predicting the order of signaling pathway components, assuming all the components on the pathways are known. Our method is built on a score function that integrates protein-protein interaction data and microarray gene expression data. Compared to the individual datasets, either protein interactions or gene transcript abundance measurements, the integrated approach leads to better identification of the order of the pathway components.As demonstrated in our study on the yeast MAPK signaling pathways, the integration analysis of high-throughput genomics and proteomics data can be a powerful means to infer the order of pathway components, enabling the transformation from molecular data into knowledge of cellular mechanisms. Signal transduction is the primary means by which eukaryotic cells respond to external signals from their environment and coordinate complex cellular changes. It plays an important role in the control of most fundamental cellular processes including cell proliferation, metabolism, differentiation, and survival . ExtraceRecently, with the accumulation of genome sequence information, large-scale genomic and proteomic techniques have offered insights into the components of signal transduction pathways and the molecular and cellular responses to cell signaling. For example, large-scale yeast two-hybrid screening methods and Co-IP technique have been used to identify physical interactions between proteins -5. Synthab to that of a single mutant a, or a single mutant b. However, this analysis is time-consuming, expensive and sometimes the results can be misinterpreted  byThe two data sources are considered with equal importance, so we rescale the score Smin and Smax are the minimum and the maximum scores of all the possible pathways respectively for either protein-protein interaction data or gene expression data. The rescaling procedure is performed on both data sets. The integrated score is the sum of the rescaled scores for each individual data set.where SYL designed the study, performed the pathway analysis, and drafted the manuscript. HZ conceived and guided the study. Both authors read and approved the final manuscript.


Thermotoga maritima, and describe how it catalyzes the formation of the amino acid methionine.If a cell is a complex symphony of chemical reactions, its enzymes are the instruments through which this elemental music is played. Each reaction is catalyzed by a specific enzyme, whose uniquely shaped active site not only binds reactants, but, by forming weak and temporary bonds, coaxes them into new orientations with new partners, thus creating the products. Determining exactly how any individual enzyme accomplishes this task—which amino acids make up the active site, which bonds form where when enzyme meets substrate, which electrons switch partners as new bonds form—is the work of the structural biochemist. In this issue, Martha Ludwig and Robert Pejchal elucidate the structure of cobalamin-independent methionine synthase (MetE) from the bacterium Methionine synthases actually come in two forms, which use somewhat different mechanisms to accomplish the same task: transfer of a methyl group (CH3) from methyltetrahydrofolate to the terminal sulfur of homocysteine. The cobalamin-dependent form, MetH, relies on the cofactor cobalamin (vitamin B12), which pulls the methyl away at one active site, and then donates it at a second active site. Here, a central zinc atom binds and activates homocysteine, enabling it to attack the incoming methyl group that is attached to cobalamin. MetE, on the other hand, has no cofactor and only one active site, which sits at the junction of two eight-stranded barrels. The structure and sequence of these barrels indicate they arose through duplication of a primordial zinc-bearing, homocysteine-binding protein. This unique duplex now bears only one zinc atom, deep within the cleft separating the two barrels.10.1371/journal.pbio.0030031.sv001). There must be subsequent conformational changes within the active site that serve to bring the two substrates together and promote transfer of the methyl group. Exactly how methyltetrahydrofolate reorients within the cleft to complete the reaction is not yet clear. The reaction catalyzed by MetE proceeds slowly, at only 1%–2% of the speed of that catalyzed by MetH. One reason for this rather sluggish activity is that homocysteine, even when activated by binding to zinc, is much poorer than cobalamin at displacing the methyl group of methyltetrahydrofolate.As in MetH, the role of the zinc is to bind homocysteine, but in MetE, this event also induces a conformation change around the zinc. The zinc and its coordinating partners form an umbrella; entering from the handle end, the homocysteine sulfur pulls the zinc toward it and turns the umbrella inside out. Methyltetrahydrofolate initially binds along the edge of the cleft, with the methyl group on the folate oriented far from the sulfur on the homocysteine, as can be seen in the research article's Video S1 (DOI: While MetE's unique active-site structure was made possible by gene duplication, the two barrels are no longer identical. Through evolution, the second, N-terminal, barrel has lost the ability to bind zinc or homocysteine, and indeed appears to contribute little to the active function of the enzyme. Nonetheless, this barrel may be necessary to temporarily isolate the substrates from solvent and to form the hydrophobic environment in which the reaction is more favorable. Further research may indicate more about the function of this unequal partner, and provide more detail on the exact atomic movements within the cleft at the moment of reaction.


A recent television series in the UK celebrates the genetic diversity of human life C. elegans. Those mutants won him a Nobel Prize, some thirty years later. Armand Leroi is another aficionado of C. elegans mutants, but he decided to write a book—and then to make a television series—on mutants of humanity, not of worms. He says at the beginning and end of the series: “We are all mutants, but some of us are more mutant than others.” This is a good slogan, and very proper for embracing humanity as a whole. He backs it up with an aphorism from Etienne Geoffroy Saint-Hilaire, pioneer of teratology, who proclaimed: “There are no monsters and Nature is one.” What Geoffroy meant was that abnormalities provide clues to normal processes, and hence are invaluable to science, if they can be properly understood. But monsters and mutants are, undeniably, fascinating in their own right.My graduate adviser, Sydney Brenner, used to exclaim ‘Revenons à nos mutants!’ as he sat down at the bench to search for yet more genetic variants of Mutants, the book, is excellent: impressively researched and illustrated and extremely well written. The resulting television series on Channel 4 in the United Kingdom has a distinctly different impact. The series covers only a few of the subjects dealt with in the book, and handles the material in a different way. Inevitably, television can't include much science or scholarly detail, but it compensates with human images that are wholly gripping—both the preserved specimens and the living subjects who talk about the strange conditions that they live with. It's a freak show, but a freak show with thoughtful scientific commentary.Each of the three programmes in the series has a particular theme. The first, ‘The Mystery of Growth’, is devoted mainly to skeletal disorders. We meet Carole Ozel, who copes with extraordinary courage with a terrible disease called fibrodysplasia ossificans progressiva (FOP), in which bony tissue forms throughout the body, gradually immobilizing the body in a second skeleton. Later we encounter a crew of charming and articulate dwarfs taking time out from a disco at the Reno convention of the Little People of America. They are happy to be called dwarfs or little people, but midget is no longer an acceptable term. Being a dwarf, in fact, is sometimes a ticket to fame and fortune, as in the case of Joseph Boruwlawski, last of the court dwarfs, who enchanted European royalty, married a beautiful woman, and lived happily to the age of 98.The second part, ‘The Dangerous Womb’, is about birth defects, conjoined twins, and basic embryology. The makers of the piece went to the trouble of getting the developmental biologist Eddie de Robertis to reconstruct the classic experiment of Mangold and Spemann that revealed the underlying basis of some of these defects. The programme goes into some detail about what is now known about the molecular basis of normal and abnormal development, and how we can begin to explain such extraordinary forms as that of the Parodi twins, who had distinct heads and shoulders, but merged into a single torso and a single pair of legs.The third programme, ‘The Meaning of Beauty’, deals with lesser but still striking abnormalities such as albinism and hypertrichosis (excess hair). It also moves into contentious areas, in a frank discussion of race genetics. Leroi makes the essential point loudly and clearly: there is much more genetic variation within any village on earth than there is between different human populations. We are extraordinarily unified, from a genetic standpoint. But, as he notes, the idea of race persists, and about 7% of global genetic variation in human DNA includes AIMs, ancestry informative markers. Some of these distinguish, for example, Africans and Europeans and provide objective information about the ancestry of different human populations.Newsweek's idea of human facial perfection, as an exemplar. Yes, she looks nice, but nice can be boring. Beauty, above all other human attributes, is profoundly influenced by culture, and it is hard to take this interpretation of beauty as an adequate explanation, rather than just a pretty way to finish the series.This being television, the series closes with a discussion of beauty, which Leroi proposes to be simply the absence of visible mutant defects, using Saira Mohan, For the most part, Leroi makes an agreeable and humane commentator, though he is not immune to the slight self-satisfaction that seems to overcome all scientists on television. The camera also spends an excessive amount of time dwelling on him, to a point where it becomes irritating to see him walking—frequently in slow motion— into yet another museum or laboratory. Sometimes the focus on the presenter pays off, as when we see the six-foot scientist looking like a small child beside Chris Greener, the tallest man in Britain, or witness Leroi's faint chagrin at discovering that his DNA is mostly European, despite his cosmopolitan family history. More questionable are the bits where he paddles casually through a tub of preserved viscera from some long-gone sufferer from situs inversus (mirror-reversed organs) and succumbs to laughter at the sight of Ditto, the amazing two-faced pig. This may be an honest attempt at portraying the conflicted reactions we all have to abnormality, but it seems bound to cause trouble.Alien movie, in which the audience was never allowed to see the monster directly.Arty camerawork is a running feature of the programmes, and there is a great deal of smoke and mirrors about the whole production. Perhaps this is deliberate, reminding us that it is hard to look directly at extreme deformity, but there is an air of ‘I wants to make your flesh creep’ about the many sidelong shots of mutant babies in bottles in the Vrolik Museum, to which we return again and again throughout the series. Viewing the already distorted fetuses through further distorting camera angles and under green lighting doesn't really achieve anything. The treatment begins to resemble the first In the end, what is most memorable about these programmes is the living people themselves, and how they have coped with their various genetic disorders. It is very touching to see the home movies of Tiffany York, born with mermaid syndrome, or sirenomelia (in which the legs are fused together), taking her first tottery steps after corrective surgery, and to listen to her talking philosophically about her life as she floats in a Florida swimming pool. It is similarly cheering to hear from the dwarfs and albinos, or from Chuy Aceves, who has hypertrichosis and looks like the original Hollywood wolfman but suffers no ill effects and is proud of his rare condition. For these sections alone, let alone the serious and well-explained scientific background, the series is well worth seeing. It makes one feel surprisingly good about the human race and the human spirit.