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 (McNemar, t-test, Bonferroni-Holm corrected for multiple tests). 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 (T0,T2) and after (T1,T3) 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 (p = 0.2 in period 1, P = 0.4 in period 2).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 (first release of NCBI's RefSeq, dated June 30th, 2003), 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 (kinematica. AG, Littau-Luzern, Schwitzerland). 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 (Linux, Microsoft Windows, MacOS X, other UNIX-like operating systems)..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 (Δ8, 17β-E2), 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 (Bhavnani and Zhang, unpublished data), 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) (PKC, crossreacts with PKCα /and PKC δ) purchased from Sigma. Goat polyclonal antibody to Actin (I-19: sc1616) , mouse monoclonal antibody to Fas raised against Fas extracellular domain (CD95:AF435) (R&D System, Inc. Minnepolis, MN), mouse monoclonal antibody cytochrome c (7H8.2C12) (BD PharMingen, Mississauga, ON). Secondary antibodies were obtained from Sigma (Saint Louis, Missouri). SuperSignal® West Pico Chemiluminescent Substrate (Pierce, Rockford, IL). Z-Val-Ala-DL-Asp (OMe)-fluoromethylketone (z-VAD-fmk) and z-Asp (OMe)-Glu (OMe)-Val-DL-Asp (OMe)- fluoromethylketone (z-DEVD-fmk) (Bachem Bioscience Inc., King of Prussia, PA) and the Cytotox 96 Non-radioactive Cytotoxicity Assay Kit (Promega G1780, from VWR, Toronto, Ontario, Canada). Neurobasal, B27, Hanks' buffered salt solution (HBSS) and antibiotics were from Gibco Life technologies (Burlington, Ontario, Canada). Active Caspase-3 Detection FITC kit # 280000 was obtained from Stratagene (La Jolla, CA).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 (MTS assay, Promega G5430) were used to assess cell death and cell viability respectively [During the development of this work, the lactate dehydrogenase (LDH) cytotoxicity assay (Promega G1780, VWR Toronto, Ontario, Canada) 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 (Sigma, Toronto, Canada), 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 (BioRad, Toronto, Canada). Cell lysates containing 10 to 20 μg protein were added in equal volume of 2 X reducing sample buffer (100 mM Tris-CI pH 6.8, 200 mM dithiothretiol, 4% SDS, 20% glycerol, and 0.2% Bromophenol blue) 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 (Schleicher & Schuell Inc., Keene N.H). The blots were blocked with 5% non-fat milk in TBST (20 mM Tris-HCl pH 7.6, 137 mM NaCl, 0.05% Tween-20) 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) (Amersham, Toronto, Canada). 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 (Silk scientific, Inc, Orem, Utah, USA), 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 [Given the fact that signal transduction is achieved by a cascade of protein interactions and activations, one major challenge in dissecting signal transduction pathways is to determine the order in which the signal is transduced. Traditionally, genetic epistasis analysis is used to address this question. In such analysis, the order of gene function can be determined by comparing the phenotype of a double mutant erpreted . ComputaBecause the yeast MAPK pathways involved in pheromone response, filamentous growth, maintenance of cell wall integrity and hypertonic shock response are among the most thoroughly studied pathways, we use them to develop and test our method Fig. . As protTwo expression datasets are used in our analysis, one is composed of 56 conditions relevant to the behavior of MAPK signal transduction and another is the "compendium" set which is composed of 300 diverse mutations and chemical treatments ,11. To i-4 when the threshold is set to 0.7, indicating that protein pairs in adjacent position on a pathway tend to have a higher correlation coefficient value than random protein pairs. This fact is applied in developing our score function that incorporates the gene expression information.where N represents the total number of protein pairs, M represents the number of protein pairs in adjacent positions on a specific MAPK pathway, n is the total number of protein pairs that have an absolute value of correlation coefficient above a given threshold, e.g. 0.7, and k is the number of adjacent protein pairs having an absolute value of correlation coefficient above this threshold. The p-value obtained from the test is 2 × 10For each MAPK pathway, we examine all permuted orders of the pathway components with the starting point (membrane receptor) and the ending point (transcription factor) of each MPAK pathway fixed and calculate the score for each permutation according to the score function defined as in "Method" section. Then, we rank each permutation based on its corresponding score, with the high-ranking orders being the more likely pathway orders.For the pheromone response pathway, the scores based on each individual data set and the scores based on integrating both data sets are shown in Fig. et al. described a time-lagged correlation analysis to infer the interactions among the components on the first few steps of the glycolytic pathway, thus the order of the components on the glycolytic pathway could be deduced [et al. applied this method to identify the cause-effect relationships among genes in the organism Synechocystis in response to different light conditions [et al. used known protein-protein interactions of Saccharomyces cerevisiae as training data and represented the proteins as collections of domains to predict links within the human apoptosis pathway [The rapid accumulation of genomics and proteomics information and the development of large-scale experiment techniques motivate us to develop computational approaches to dissecting different pathways. Arkin  deduced . Schmittnditions . The lim pathway . HoweverWe note that the utility of integrating yeast protein-protein interaction map and gene expression profiles to predict signal transduction network has previously been described by Steffen and colleagues . In theiWe have shown our method can lead to a good prediction for well-known yeast MAPK signaling pathways. In addition, we have tested our approach on the DNA damage checkpoint pathway that is involved in cell-cycle progression. The "true" pathway ranks 4 among all the 750 possible pathways based on our integrated approach, while it has a rank of 46 and 60 based on protein-protein interaction data alone and gene expression data alone, respectively. Therefore, we conjecture that our approach may be applicable to many other pathways including less well-understood ones.It is worth to note that signaling pathways are not limited to one-dimensional sequence of genes, as our focus in this study. Instead, they should be depicted as multidimensional networks. To make further complicated prediction and modeling of the networks, we need to incorporate more biological information and apply more elaborate statistical approaches.We have demonstrated that our integrated approach can significantly improve the performance of predicting the order of signaling pathway components, without detailed knowledge of all the genes in the pathway or the molecular nature of the gene products. It may be important to incorporate other valuable sources of data, including protein chip data, genomic sequence information and protein domain information if we want to make the transition from a linear one dimension pathway to a multidimensional model of signaling networks, which represents a great challenge in the field of systems biology.For protein-protein interaction data, the score function is defined as follows:i, i+1 = 1 if there is an observed interaction between the ith and the (i+1)th proteins on the pathway and Xi, i+1 = 0 otherwise. Here p represents the false negative rate of the interaction data. In this study, we fixed the false negative rate as 0.4. It was estimated that the total number of interactions between all yeast proteins or the size of yeast interactome is about 20000~30000 [where n is the total number of proteins on the pathway, and X00~30000 ,18. In tFor gene expression data, the score function is defined as:i, i+1 represents the correlation coefficient between the ith and the (i+1)th proteins on the pathway.where ri of all the possible pathways to [0, 1] 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.


The taxonomic name of an organism is a key link between different databases that store information on that organism. However, in the absence of a single, comprehensive database of organism names, individual databases lack an easy means of checking the correctness of a name. Furthermore, the same organism may have more than one name, and the same name may apply to more than one organism.The Taxonomic Search Engine (TSE) is a web application written in PHP that queries multiple taxonomic databases (ITIS, Index Fungorum, IPNI, NCBI, and uBIO) and summarises the results in a consistent format. It supports "drill-down" queries to retrieve a specific record. The TSE can optionally suggest alternative spellings the user can try. It also acts as a Life Science Identifier (LSID) authority for the source taxonomic databases, providing globally unique identifiers (and associated metadata) for each name. and provides a simple demonstration of the potential of the federated approach to providing access to taxonomic names.The Taxonomic Search Engine is available at  Biological taxonomy provides the central link between diverse items of information about an organism. Given the scientific name of an organism, a researcher can query a wide range of databases for information on that organism's genome, development, morphology, geographic distribution, behaviour, phylogeny, and conservation status. However, the utility of taxonomic names as keys to accessing information is hampered by several factors, notably the lack of a single authoritative list of all taxonomic names ,2. In thdocumented API. Taxonomic names themselves have limitations as identifiers in databases [Morus applies to both an animal (the gannet) and a plant (the mulberry tree). Even species names can be identical – a species of wasp and a species of conifer both share the name Agathis montana. Hence, using names alone to link different data sources can be prone to error. As an example, at the time of writing NCBI's LinkOut feature [Loricaria (tax_id = 52085) to the TreeBASE [Loricaria (TaxonID = 1305), which is a plant genus (family Compositae).In the absence of a single database of names, one solution is to use a federated approach  where muatabases  due to t feature  mistakenTreeBASE  taxon LoTo avoid ambiguity some form of identifier other than a taxonomic name needs to be employed, such as Digital Object Identifiers (DOIs)  or Life Given the lack of a central list of names, and the limitations of names as identifiers, there is a clear need for a taxonomy name service that can validate names and provide unique identifiers . The SPIThis paper describes the Taxonomic Search Engine (TSE), which takes federated approach to the problem of searching for taxonomic names. Unlike the SPICE project, the TSE relies solely on the interfaces made available by the data source. A wrapper is created for each source database, but this resides on the same machine as the TSE. In this way, no special demands are made of the source database. The TSE searches multiple databases for a name, and returns the result in a consistent format. For each name, TSE also creates a LSID, so that each name from each source database has a globally unique identifier.The TSE uses five data providers: ITIS, Index Fungorum, IPNI, uBIO, and the NCBI.The Integrated Taxonomic Information System (ITIS)  was estade facto source of taxonomic data in biodiversity informatics projects.ITIS provides a classification of taxonomic names (i.e., a parent-child hierarchy), and where more than one name exists for a taxon, ITIS specifies which name it regards as correct . Every name in the database, regardless of taxonomic status or position in the hierarchy is assigned a unique identifier . The database schema is fully documented, and the entire database is available for downloading by FTP as a SQL schema with the data in delimited text files. As a consequence, ITIS is frequently used as the The International Plant Names Index (IPNI)  combines which returns an XML document. If more than one name exists for a fungus, Index Fungorum designates one name as the "current name." It also reports the basionym (first recorded name) for that taxon. Index Fungorum does support a detailed hierarchical classification in the form of a lineage, but higher level taxa are not assigned records in the database (unlike, for example, ITIS). In fungal taxonomy, names are often assigned to the asexual state (anamorph) of a fungus for which the sexual state (telomorph) is unknown. Names for anamorphs are flagged as such in the database.IndexFungorum  is a dat which returns a nested array data structure.The Universal Biological Indexer and Organizer (uBio)  is a proThe NCBI Taxonomy database  is a curThe basic architecture of the TSE is summarised in Fig. IsAlive, NameSearch, and GetDataForID, which must be overridden in descendant classes. The IsAlive method queries whether the data source is available. The NameSearch method queries a data source for a given string. If one or more names are found, NameSearch returns basic information about that name, including the identifier used by the data source. This identifier is used by the GetDataForID method to query the data source for more details about the name.Each wrapper is derived from the same base class which provides some generic routines for creating XML documents and for caching results (see next section). The wrapper class supports three methods, In order to improve the responsiveness of the search engine, the results of queries to each source database are cached for 24 hours. The results of the query are stored in the format returned by the database (i.e., XML or delimited text), except for uBio where the SOAP response is serialised to disk.The Taxonomic Search Engine seeks exact matches to the user supplied query. In order to accommodate spelling mistakes the web interface to the search engine supports approximate string matching using two techniques. The first employs agrep  to searcThe TSE has a simple web interface Fig. . The use. The service provides two operations: NameSearch which queries the source databases for a user-supplied name, and SpellingSuggestion, which suggests alternative spellings for a name. Hence users can write web service clients that can use the TSE as part of their own applications. The TSE web site provides source code for two simple clients written in perl.The TSE has a SOAP web service that is described by a Web Services Description Language (WSDL) file available at Homo sapiens in the ITIS database would have the LSID:A LSID is a Uniform Resource Name (URN) comprising five parts: the Network Identifier ("lsid"), the root DNS name of the issuing authority, a namespace, an object identifier, and optionally a revision id to indicate the version . TSE genurn:lsid:itis.usda.gov.lsid.zoology.gla.ac.uk:tsn:180092Homo sapiens.where "tsn" is the "taxonomic serial number" used by ITIS as a unique identifier for each taxonomic name, and "180092" is the tsn for The TSE uses the perl library distributed by IBM's Life Science Identifier project  to creatThe 2004 edition of the Species 2000 CD-ROM  was usedTo create a test dataset, 1000 names were selected at random from the Species 2000 dataset. Each name was sent to the TSE web service by a perl script which recorded the time taken for each source database to respond to the query, and whether that source database contained the name. The time recorded is from the time the query was made until the time the response was returned – post processing by the TSE is not included in the measurement. For this experiment, the cache feature was turned off so that for each query the TSE went to the external source database, rather than using a local copy of the query result.The results of the simple performance benchmarks are shown in Table The TSE can be extended to handle additional data sources simply by deriving a new wrapper class from the base class. To date wrappers have only been written for data sources which can return plain text, XML, or SOAP messages. There are many more taxonomic databases that could be queried if wrappers were written to handle HTML output ("screen scraping"). However, this would make the wrapper very vulnerable to changes in web page design . Of courDespite the reasonable performance of TSE, there are obvious limitations in the current design and implementation. The PHP language does not support threads, so each source database is queried sequentially. As additional source databases are added the time to complete the search will get progressively longer. If the performance of additional databases is comparable to those already being queried Table , then eaSome source databases have broad taxonomic coverage such as ITIS, NCBI, and uBio, whereas others are restricted to particular groups, such as fungi (Index Fungorum) and vascular plants (IPNI). Hence, it makes little sense to query Index Fungorum or IPNI for an animal name . An option to select the databases to query could be easily added to the TSE web interface. However, it would be more efficient if the TSE could determine which databases were relevant to the user's query. If the TSE knew that the query string was the name of a fungus, it could send the query to the appropriate database. In practice, however, this is problematic. In order to know what organism a name refers to the TSE would have to have access to a databases of names and their classification – the very lack of such a database is the motivation behind the TSE in the first place. Furthermore, as discussed above, the same name can apply to different organisms. A user searching using the term "Morus" might be looking for a plant name, or an animal name (or perhaps both). There is some scope for more intelligent querying, such as looking for aspects of the name that are specific to one of the codes of nomenclature (e.g., most plant family names end in "-aceae"), but any such effort needs to be done with care – for example, "Compositae" is a family of plants.The Taxonomic Search Engine is a simple tool for querying multiple taxonomic databases. Typically, results of querying five major databases are returned in a few seconds. In addition to providing basic information about a name, the TSE acts as a LSID authority, providing globally unique identifiers for each name. The TSE provides a simple demonstration of the potential of the federated approach to providing access to taxonomic names..The source code for the TSE, the web site, and the LSID authorities is available from the TSE site ), and a developer key from Google [TSE requires a web server and the PHP scripting language. It has been developed and tested under Red Hat Linux 8.0 with the Apache web server version 2.0.40 and PHP version 4.2.2, and Mac OS X 10.2.8 with Apache version 1.3.29 and PHP version 4.3.4. If PHP does not have the XSLT extension enabled then the user will either have to recompile PHP, or install the Sablotron toolkit . The codm Google .


Molecular motors harness the energy of ATP (or GTP, a related energy currency) and transform it into mechanical force. Well-known examples of motors include myosin and dynein, proteins that use ATP to ferry intracellular cargo along fibers made of actin or tubulin proteins. The ATP-dependent assembly of actin or tubulin fibers itself can work as a motor: for instance, the march of white blood cells toward pathogens is powered by the growth of actin filaments pushing against the cells' membranes. In all cases, coherent motion implies a coordinated and polarized use of energy.Now, Julia Cox, Oleg Tsodikov, and Michael Cox present evidence indicating that filaments of the bacterial RecA protein, long known for their role in homologous recombination and DNA repair, have properties reminiscent of a molecular motor as well. RecA filaments consist of DNA helices lined with RecA protein. RecA filaments invade a region of double-stranded DNA with similar nucleotide sequence, displacing one strand to pair with the other. Strand invasion can lead to a re-assortment—known as recombination—of DNA regions on either side of the shared sequence. It can also initiate the repair of DNA lesions during replication—the process by which a DNA molecule is copied to make two.RecA is also an ATPase, an enzyme capable of hydrolyzing (breaking down) ATP, when bound to DNA. RecA uses ATP to carry out strand exchange over long sequences and impose direction to the exchange, to bypass short sequence heterogeneities, and to stall replication so DNA lesions can be mended. But how RecA molecules within a filament coordinate and organize their activities to carry out these functions has remained obscure.Cox et al. addressed this problem in the test tube, by examining RecA filaments grown from mixing RecA protein with DNA. Previous experiments have shown that filament assembly spreads rapidly in the 5′-to-3′ direction once the first RecA molecule is loaded onto DNA. At the same time, ATP hydrolysis causes the release of RecA from DNA, but the exact rate of RecA dissociation is not known. Experiments suggest, however, that RecA molecules only dissociate from DNA when they are at the fiber's 5′ end, while ATP hydrolysis occurs all along its length.Under their experimental conditions, the authors found that a RecA molecule hydrolyzed 20 ATPs per minute—or one ATP every three seconds. If ATP hydrolysis occurred randomly in the fiber, one would expect a RecA molecule to dissociate about once every 1.5 seconds, or 40 RecA molecules to dissociate per minute. But this is not what the authors found—instead they estimated the dissociation to be at a rate of 120 RecA molecules per minute. Hence, six RecA molecules dissociate from a fiber in the time—three seconds—it takes for an individual RecA molecule to burn one ATP. This implies a filament organization in which every RecA molecule hydrolyzes ATP in synchrony with the sixth RecA molecule to its left and the sixth RecA molecule to its right.The authors note that there are approximately six RecA molecules per helical turn in a RecA filament. They propose that the RecA molecules hydrolyzing ATP at any given moment are aligned in a “stripe” that runs along the side of the filament. This stripe of ATP hydrolysis moves around the fiber in a repeating pattern of six steps. At the 5′ end of the fiber, ATP hydrolysis leads to RecA release. But in the middle of the fiber, it could work as a rotary motor, with the power to wind or unwind DNA and drive strand invasion through difficult passages of damaged DNA.