Antidepressant prescribing has been increasing in England. Studies in other countries suggest that while this may be associated with reduced suicide rates, it may also be associated with increased fatal poisoning from antidepressant drugs. We therefore conducted an ecological study to assess the association between prescription rates for antidepressants and suicide or fatal antidepressant-related poisoning in England.The Office for National Statistics provided information on the number of suicides, antidepressant-related poisoning deaths and populations for England between 1993 and 2002. The Department of Health supplied data on prescriptions for all antidepressants dispensed in England. Associations between prescriptions and deaths were assessed using Spearman's rank correlation coefficient.s = -0.73, p = 0.02) and fatal poisoning involving tricyclic antidepressants (rs = -0.64, p = 0.05). In contrast, increased prescribing of selective serotonin re-uptake inhibitors and other antidepressants was statistically associated with an increase in fatal poisoning involving these drugs (rs = 0.99, p < 0.001).There were 46,747 suicides, 3,987 deaths involving tricyclic antidepressants and 430 involving selective serotonin re-uptake inhibitors and other antidepressants. Increased antidepressant prescribing was statistically associated with a fall in suicide rates (Spearman's rIncreased prescribing of antidepressants may indicate improved diagnosis and treatment of depression in primary care. Our analysis suggests that this was accompanied by lower suicide rates. A decrease in poisoning deaths involving tricyclic antidepressants may suggest a change in preference for using serotonin reuptake inhibitors and other antidepressant drugs for high-risk patients. This may also partially explain the increase in deaths involving these drugs. Due to the ecological nature of the design, we cannot say conclusively whether reduced suicide rates are a direct consequence of increased antidepressant prescribing rates. To confirm these associations, individual level data on prescribing and suicide is needed. Saving Lives: Our Healthier Nation sets a target to reduce suicide deaths by 20% by 2010 [Around 5000 people take their own lives in England every year . The Gov by 2010 . Treatme by 2010 . Hence, Studies in Sweden , DenmarkIn England, prescriptions for antidepressants have been increasing ,15. We cSuicide deaths were defined as deaths where the coroner has given a verdict of suicide or where an open verdict was reached in a death from injury or poisoning. This is because it is thought that most open verdicts are cases where the harm was self-inflicted but there was insufficient evidence to prove that the deceased deliberately intended to kill themselves . Open veThe Office for National Statistics has stored drug-poisoning mortality data for England and Wales from 1993 onwards in a dedicated database . The datDrug poisoning deaths were defined using the International Classification of Diseases codes shown in Table The Department of Health supplied data on prescriptions for all antidepressants dispensed in England between 1993 and 2002. Prescription information is derived from the Prescription Cost Analysis (PCA) system, which collects data from all prescriptions dispensed in the community . This inDirectly age-standardised rates for suicide and antidepressant-related deaths were calculated using the European Standard Population. Populations for England between 1993 and 2002 were interim revised estimates published by ONS in September 2003. Prescription rates were presented as number of prescriptions per 100 population. Statistical associations between prescription rates and directly age-standardised rates for antidepressant-related poisoning deaths and suicides were assessed using Spearman's rank correlation coefficient.In England between 1993 and 2002, there were 46,747 suicides. Suicide was twice as common in men as in women. There were 3,987 deaths involving tricyclic antidepressants (TCAs, BNF 4.3.1,) and 430 involving selective serotonin re-uptake inhibitors and other antidepressants (SSRIs & others, BNF 4.3.3 & 4.3.4). The number of antidepressant-related deaths was similar for men and women. Changes in age-specific rates were similar across all age groups for suicide and antidepressant-related deaths.Between 1993 and 2002, age-standardised mortality rates for suicide decreased from 98.2 to 84.3 per million population .A previous analysis found that about a third of antidepressant-related deaths also have other drugs or substances mentioned on the death certificate (26% of deaths involving TCAs, 72% involving SSRIs and others) . Hence, Increased prescribing of antidepressants was statistically associated with reduced suicide mortality rates. TCA-related poisoning deaths also decreased during the study period although the evidence for a statistical association with prescribing rates was weaker. Increased prescribing of SSRIs and other antidepressants was statistically associated with increased mortality rates involving SSRI & other-related poisoning, although the rates were much smaller than for TCA-related poisoning or suicide. Because our study uses population level data, we cannot conclude that these associations are necessarily causal.Although the ONS drug poisoning database is the most complete record of drug poisoning statistics available, about 10% of these deaths have no specific information about drug(s) taken . PrescriFurther bias may have been introduced due to prescriber and patient factors. Doctors may perceive SSRIs and other antidepressants to have clinical advantages over TCAs due to their lower toxicity, possibly making them more popular for treating newly diagnosed (an uncontrolled) depression , treatinAssessing the impact of increased antidepressant prescribing is difficult. This is because antidepressants are both a treatment and method for suicidal behaviour. Suicide is also closely linked to availability of methods , generatHowever, our study indicates that at a population level, increased antidepressant prescribing in England was statistically associated with lower rates of suicide and antidepressant-related poisoning. One explanation for our findings is that SSRIs and other antidepressants are being used in preference to TCAs to treat depression. The perceived side effects of TCAs means they are often prescribed or taken at sub-therapeutic doses ,27, leadNevertheless, the clinical benefits of antidepressant drugs for the management of depression and prevention of suicide are still unclear. A recent Cochrane review found little difference between antidepressants and active placebos on improvement of mood .  declare that they have no competing interests.OM designed the study, conducted the analysis and drafted the manuscript. CG participated in conducting the analysis and drafting the manuscript. AM participated designing the study and drafting the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Serum Response Factor (SRF) is a transcription factor that is required for the expression of many genes including immediate early genes, cytoskeletal genes, and muscle-specific genes. SRF is activated in response to extra-cellular signals by its association with a diverse set of co-activators in different cell types. In the case of the ubiquitously expressed immediate early genes, the two sets of SRF binding proteins that regulate its activity are the TCF family of proteins that include Elk1, SAP1 and SAP2 and the myocardin-related MKL family of proteins that include MKL1 and MKL2 . In response to serum or growth factors these two classes of co-activators are activated by different upstream signal transduction pathways. However, it is not clear how they differentially activate SRF target genes.In order to identify the serum-inducible SRF target genes that are specifically dependent on the MKL pathway, we have performed microarray experiments using a cell line that expresses dominant negative MKL1. This approach was used to identify SRF target genes whose activation is MKL-dependent. Twenty-eight of 150 serum-inducible genes were found to be MKL-dependent. The promoters of the serum-inducible genes were analyzed for SRF binding sites and other common regulatory elements. Putative SRF binding sites were found at a higher rate than in a mouse promoter database but were only identified in 12% of the serum-inducible promoters analyzed. Additional partial matches to the consensus SRF binding site were found at a higher than expected rate in the MKL-dependent gene promoters. The analysis for other common regulatory elements is discussed.These results suggest that a subset of immediate early and SRF target genes are activated by the Rho-MKL pathway. MKL may also contribute to the induction of other SRF target genes however its role is not essential, possibly due to other activation mechanisms such as MAPK phosphorylation of TCFs. The CArG box is specifically bound by Serum Response Factor (SRF) [Quiescent cells exposed to growth factors respond by expressing a variety of immediate early genes (IEG) that do not need new protein synthesis for their expression . Serum oor (SRF) -4. Both or (SRF) . In addior (SRF) .Although the immediate early genes are so named because of their rapid inducibility after growth factor treatment, different kinetics of expression have been observed among the immediate early genes. Expression of the proto-oncogene c-fos peaks at around 30 minutes after stimulation whereas the peak expression of SRF mRNA occurs after 90–120 minutes ,9. Thus Activation of SRF by growth factors occurs through at least two mechanisms – the TCF and RhoA pathways ,11. SeruAn additional pathway that activates SRF is through activation of the small GTPase RhoA . ActivatThe use of pathway specific inhibitors has suggested the presence of two types of SRF target genes – those that are largely dependent on the Rho-actin pathway and those that are largely dependent on the MAP kinase pathway . Use of We and others have recently identified a family of SRF-specific transcriptional co-activators – the MKL family, comprised of MKL1 and MKL2, that function downstream of the RhoA pathway -26. MKL1Chromatin immunoprecipitation experiments have shown that MKL1 binds to the promoters of the cyr61, srf and vinculin genes but not egr-1 or c-fos after swinholide treatment which leads to the activation of the Rho-actin pathway . MoreoveAs a first step towards a better understanding of this differential target gene selection and to demonstrate the importance of the MKL coactivators in immediate early gene induction, we sought to identify the group of SRF target genes that were dependent on the RhoA-MKL pathway. We performed microarray analysis using cell lines expressing dominant negative MKL1 (DN-MKL1) to identify MKL-dependent and -independent serum-induced SRF target genes. The DN-MKL1 protein is effective in blocking both MKL1 and 2 activation of SREs, thus solving the problem of redundancy of these proteins in the cell ,24 as a dominant negative mutant. We previously found that this mutant can prevent activation of SRE reporter genes by MKL1 and MKL2 overexpression  containing 14,824 non-redundant probes were used for hybridization. All data points were done in triplicate and hierarchical clustering was performed using dChip software analysis. We found 229 genes that showed a significant variation across the 8 samples and used the results with these genes for unsupervised clustering [The hierarchical clustering analysis shows classes of differentially regulated genes which we have designated classes 1 to 10 Fig. . We wereTo analyze the serum-inducible genes more carefully, we further dissected the data using more stringent criteria to identify MKL-dependent genes at each time point. We filtered for genes that were serum-induced ≥ 2 fold in WT cells and whose expression in DN-MKL1 cells was at least 35% less than in WT cells Tables . A list To verify the results obtained from the microarray experiments, we checked seven genes by real time quantitative PCR using the SYBR Green method. There was a good, but not perfect correlation, of these methods Fig. . Four MKWe compared the serum inducible genes to known SRF target genes to identify those genes with SRF binding sites. Comparing to a list of SRF targets genes J.M. Mia. We furtWe also searched for promoters with more than one CArG box match since multiple SREs have been reported in several SRF target genes and this could provide a mechanism to distinguish responses. While there were more genes with greater than one CArG box match in the serum-inducible genes than in the DBTSS promoters, the numbers of MKL-independent vs. -dependent genes with greater than one match were too low to show a statistical significance Table .The search for SRF binding sites allowing a one base mismatch was more problematic since there were a high rate of matches in the DBTSS data set, an average of .98 hits per gene Table . There wThe increased frequency of the exact CArG box sequence in the upstream region of many of the MKL-dependent genes gives further confidence that SRF coordinately regulates many immediate early genes. However, the absence of a CArG box does not preclude a gene from being a direct target due to the flexibility of enhancer positioning. The regulatory site may be upstream or downstream of the 1200 bp we have analyzed. For instance, in the case of the jun B gene it has been shown that a CArG box downstream of the gene mediates its response to serum . In addiWe compared the CArG boxes more closely since previous work with altered SREs suggested that changes in the A/T core of the SRF can affect sensitivity to the TCF-independent (i.e. RhoA-MKL) pathway . The knoWe have compared the sequences of the known SRF target genes to determine whether flanking sequences can explain the differential sensitivity to DN-MKL1. We first compared sequence flanking the known CArG boxes. The MEME motif discovery tool was used to identify common elements in 30 bp flanking each side of the CArG boxes. There were no statistically significant matches in the sequences from MKL-dependent of -independent genes. We might have expected to see a TCF site, however this site is very short and flexible. TCF binds to a short site near an SRE and its binding is stabilized by binding to SRF. A consensus of (C/A)(C/A)GGA(A/T) was found, however the orientation and position relative to the SRE was flexible and only the GGA sequence was absolutely required . Visual We used two methods to compare the full promoter sequences of the MKL-dependent and -independent, serum-inducible genes for common elements that might be required for their induction. We first used the MEME tool to compare the promoters (-1000 to +200) derived from the DBTSS database. As in Table -4 to 6.9 × 10-3. These oligonucleotides were not enriched in a test case of 60 random promoter sequences. The related sequence GGGAGG, however, was similarly enriched in the MKL-dependent promoters albeit with less statistical significance (E = 0.8). It is intriguing that these oligonucleotides are similar to the TCF consensus binding site (C/A)(C/A)GGA(A/T). The most significantly enriched oligonucleotide in the 20 MKL-dependent promoters was CCGCGC with an E value of 0.078. This lower significance may partially reflect the smaller size of this data set but also may reflect the lower significance of this enrichment. Given the relatively small number of serum inducible genes and present computational tools, it appears that more careful experimental mapping of the sequence elements in MKL-dependent and -independent genes will be required to identify the elements that determine their sensitivity to the MKL pathway.A second method we used was to look for common regulatory elements was oligonucleotide analysis which has been used in yeast to identify regulatory sites . This meSerum inducible genes have far ranging effects in many physiological processes such as proliferation, wound healing, migration, and tissue remodeling . SRF hasCluster analysis of the microarray data identified different classes of genes that showed significant variation across the samples. Two classes of genes were either constitutively activated or constitutively repressed. These could be indirect targets of the SRF-MKL pathway or could suggest functions for MKL apart from its role as a transcriptional co-activator of SRF.Among the serum inducible genes, we identified a significant fraction, 28 of 150, that are MKL-dependent. This is not to say that the other 122 genes may not be sensitive to MKL under certain conditions. Rather, they must at least have additional mechanisms for serum induction in NIH3T3 cells. c-fos was identified as an MKL-independent gene although we previously found by RNase protection that its induction was significantly reduced in cells induced with serum for 60 minutes . We did Many of the serum-inducible genes identified here have been previously described as immediate early genes either as single genes or in microarray experiments ,47,48. T6GG, resulted in matches in only about 12% of the serum-inducible genes with similar proportions in the MKL-dependent and -independent classes. While some functional SRF binding sites contain a single base mismatch to the consensus, a broader search for CArG boxes allowing a mismatch resulted in a high number of matches in a broad promoter database. There was a modest, statistically significant increase in the matches in the MKL-dependent promoters suggesting that some of these sites are real SRF targets. Other SRF target sites may lie outside of the promoter regions we have searched (-1000 to +200). This still leaves the possibility that induction of many of the immediate early genes is independent of SRF such as we have found for the c-jun gene [We analyzed the promoter sequences of the MKL-dependent and -independent genes to identify elements that might determine this sensitivity. Exact matches to a simple CArG box consensus, CC(A/T)jun gene . MicroarPossible mechanisms for determining sensitivity to MKL are the sequence of the CArG box, flanking sequence or the context of other promoter elements. Our comparison of the known CArG boxes from MKL-dependent and -independent promoters did not show a strong difference. Similarly we were not able to identify differences in the flanking sequence. Analysis of the more full promoter sequences has also not yielded clear common regulatory elements to date. One set of oligonucleotides, containing the sequence GGAG, was found in both the MKL-dependent and -independent promoters at a significantly higher frequency than in the full promoter database. The significance of these sites remains to be determined though it is notable that they are similar to the TCF consensus site.One possible mechanism for distinguishing MKL target genes is the presence of a TCF site next to a CArG box. This would allow for TCF binding and activation by a MAP kinase pathway instead of the MKL pathway. Since the binding sites for TCF and MKL on SRF are similar , TCF binChromatin immunoprecipitation experiments have shown that MKL1 can bind to the SRF, vinculin and cyr61 promoters in cells treated with the actin inhibitor swinholide A . This isOur results indicate that a subset of serum inducible genes is dependent upon the MKL family for its induction. This genomic classification of MKL-dependent and -independent serum-inducible genes is a significant step for characterizing which pathways are required for induction of each cellular immediate early gene.E. coli DNA polymerase I, and E. coli DNA ligase (Invitrogen). The obtained double-stranded cDNA was then blunted by the addition of 20 units T4 DNA polymerase and incubation for 5 min at 16°C. The material was then purified by phenol:chloroform:isoamyl alcohol extraction followed by precipitation with ammonium acetate and ethanol. The cDNA was then used in an in vitro transcription reaction for 6 hours at 37°C using a T7 in vitro transcription kit (Affymetrix) and biotin-labeled ribonucleotides. The obtained cRNA was purified on an RNeasy column. The eluted cRNA was then fragmented by incubation of the products for 30 min in fragmentation buffer (40 mM Tris-acetate, pH 8.1, 100 mM KOAc, 30 mM MgOAc) at 95°C. The fragmented labeled RNA (15 μg) was hybridized to an Affymetrix mouse MOE430A Genechip at 45°C according to the manufacturer's protocol [NIH3T3 cells stably expressing dominant negative MKL1 (a.a. 1-630)(DN-MKL1) or containing the vector, pBabePuro, were previously described . The celprotocol  and staiThe percent of genes with significant expression  ranged from 50.4 to 56.5% for each microarray. The 3':5' ratio for a control actin gene ranged from 1.0 to 1.31 for each of the 24 microarrays scanned. The scaling factor to account for differences in probe labeling and general changes in hybridization in each microarray was scaled to a target intensity of 250 (using Affymetrix Microarray Suite 5.0 software) and ranged from 1.273–5.542.The dChip software ,53 was u2 distribution model with one degree of freedom [Expression values (MBEIs) at each time point were determined with a standard error of the mean. These values were used to calculate the fold change with 90% confidence intervals. The confidence intervals were derived from the standard errors and fold changes based on a χ freedom . For thet), the cycle at which a statistically significant increase in fluorescence occurs, was derived from the resulting PCR profiles of each sample. Ct is a measure of the amount of template present in the starting reaction. To correct for different amounts of total cDNA in the starting reaction, Ct values for an endogenous control  were subtracted from those of the corresponding sample, resulting in ΔCt. The ΔCt value of the serum starved wt sample was chosen as the reference point and was subtracted from the ΔCt value of all the other samples, resulting in ΔΔCt. The relative quantification value is expressed as 2-ΔΔCt giving the relative difference of the serum induced points compared to the serum-starved cells for expression of a particular gene. All real time PCR data sets shown are the results of two independent mRNA preparations and amplifications. Amplification of only a single species in each PCR reaction was determined by checking for a dissociation curve with a single transition.Total RNA was isolated from the DN-MKL1 and vector containing NIH3T3 cells as described for the microarray analysis and 1 μg was used for first strand cDNA synthesis (Powerscript Reverse Transcriptase, BD Biosciences) using oligo-dT primers according to the manufacturer's protocols. One fiftieth of the reverse transcription reaction was included in a 20 μl PCR reaction. For a quantitative analysis, SYBR green PCR technology was used (Applied Biosystems). Real-time detection of the PCR product was monitored by measuring the increase in fluorescence caused by the binding of SYBR green to double-stranded DNA with an ABI PRISM 7000 Sequence Detector. To calculate relative quantification values a threshold cycle ,54 was uThe CArG box sequences of the known SRE regulated genes were derived from the literature. The motif discovery tool MEME (Multiple EM for Motif Elicitation) ,56 was tPromoter sequences (-1000 to +200) of the serum inducible genes were also searched for common elements with MEME. Promoters for 20 MKL-dependent, 50 MKL-independent and 50 random promoters were searched. These promoter sequences were additionally searched by oligonucleotide analysis for enriched hexamers ,57 excepAS carried out all experimental sections of the paper. AS and RP conceived of the study and participated in its design and coordination. Both authors read and approved the final manuscript.List of serum inducible MKL-independent genesThis Microsoft Excel spreadsheet file contains a list of genes that were serum inducible (>2-fold) at either the 30, 60 or 120 minute time points and that satisfied the 90% confidence interval criteria for fold-change using the dChip software. The expression of these genes were reduced by less than 35% in the DN-MKL1 cells at each time point. The MKL-dependent genes (reduced by more than 35%) shown in Tables 1 to 3 were removed from this list to give 122 MKL-independent genes. The Affymetrix probe set information, the gene name, Genbank Accession number and Locus link identifier are shown for each of the genes. For each time point the fold induction in serum-treated wt NIH3T3 cells compared to serum-starved wt cells ('fold change') and its upper and lower confidence intervals are shown. The time points where serum induction was deemed significant for each gene are marked by asterisks (*) in the 'filtered' columns.Click here for file


The adrenal gland is one of the common sites of metastasis from primary lung cancer. Adrenal metastases are usually unilateral however bilateral adrenal metastases are seen in 10% of all lung cancer patients; of these 2–3% occurs at the initial presentation of non-small cell lung cancer. Secondary tumors can disrupt the structure and function of the adrenal. This can lead to adrenal hemorrhage, which constitutes a life threatening hazard for the patient.A 59-year-old male presented with persisting abdominal pain. His initial work-up revealed significant anemia, an invasive process in the right upper lobe of the lung and large masses of heterogeneous texture, with hemorrhagic and necrotic elements in both adrenal glands. A biopsy confirmed it to be a large-cell carcinoma of the lungs. The patient developed severe leukocytosis akin to the paraneoplastic syndrome and died suddenly five days after the administration of chemotherapy.Intratumoral hemorrhage is a rare but life threatening complication of adrenal metastases and should be treated as soon as it has been diagnosed. If adrenalectomy is not feasible, combination chemotherapy should be applied as in metastatic disease. For choosing the appropriate chemotherapeutic regimen it is important to accurately achieve the diagnosis. The adrenal gland is a common site for metastases in breast, lung and renal cell carcinomas, melanoma, and lymphoma . AdrenalA 59-year-old male, working in a quarry, was admitted to the hospital complaining of abdominal pain persisting for 45 days, afternoon fever, fatigue and 8 kg weight loss during the last month. Previous medical history was non contributory. He was a heavy smoker for 45 years.At first assessment, the patient presented with disseminated dry rales and decrease of vesicular murmur in the right upper lung field. Following abdominal examination, palpable smooth surface masses were discovered in both renal areas. Laboratory tests demonstrated a significant anemia with normocytic features (hematocrit 24%, Hb 7.2 mg/dl).Chest X-ray revealed a consolidated area in the right upper lobe of the lung. Abdomen ultrasound demonstrated large masses with foci of hemorrhagic necrosis in both adrenal glands Figure .Computed tomographic (CT) -scan revealed an invasive process in the right upper lobe of the lung Figure  and larg1 and T2 weighted sequences. Radiological features were suggestive of adrenal metastasis from lung primary  was performed which showed the masses with disseminated foci of high, heterogeneous signal intensity and pathologic enhancement after intravenous (i.v.) administration of contrast agent in both T Figures , 5. HistAdrenal metastases in the natural history of a malignant neoplasm occur in 20 – 45% of cancer patients, depending on the localization of the primary site . Up to 4The differential diagnosis of an adrenal mass incidentally discovered during imaging examination of the abdomen in patients with non-small cell lung cancer, usually comprises one or more of the characteristics detailed in Table More than 90% of the cases comprising this involve a nonfunctional adenoma or an adrenal metastasis.et al, observed that CT-scan technology has low specificity and sensitivity, which culminates in false positive and negative readings, given that 21% of true adenomas can be mistaken for metastases (density > 10 Hounsfield units) and 11% of true metastases may be mistaken for adenomas (density < 10 Hounsfield units) [Nonfunctional adenomas can be distinguished from adrenal metastasis with precision using modern imaging techniques, especially with MR imaging, where adenoma presents a low intensity signals in T2 sequences compared to hepatic parenchyma. In contrast, the adrenal metastasis presents higher intensity T2 sequence signals than that of the adenoma, relative to hepatic parenchyma. In CT-scan adenoma presents low intensity signals, below 10 Hounsfield units, compared to higher intensity signals in adrenal metastasis. Porte d units) . CT-scand units) .In presence of adrenal metastasis the disease is staged as stage IV, which demonstrates the aggressive biological behavior and systemic dissemination of secondary lesions. These are treated as any other metastatic neoplasm and the appropriate chemotherapy is started. In select patients, where the primary lung site is surgically resectable (T1, T2 and maybe T3), with no involvement or the involvement of only the peribronchial and portal lymph nodes (N0, N1) and where the adrenal metastasis constitutes the unique indication of the disease, simultaneous adrenalectomy along with resection of the primary can increase overall survival . TechnicThe most important complication of adrenal metastases is intratumoral hemorrhage and only nine cases have been reported previously . When heA less common complication of bilateral adrenal metastases is primary adrenal insufficiency, caused by the destruction of adrenal gland architecture and function due to the development of the tumor. The main symptoms resulting from the decreased production of steroid hormones often overlap with the general indicators caused by the primary site, hindering the proper treatment of these patients. Primary adrenal insufficiency is diagnosed when serum cortisol basal values are low (<5μg/dl), and particularly when the values fail to increase within six hours following the i.m. administration of 1 mg of tetracosactride, the synthetic analog of ACTH. The treatment comprises daily administration of glucocorticoid and mineralocorticoid for life.Bilateral adrenal metastases can be found in a small number of patients with non-small cell lung cancer. Early identification of these lesions is necessary, since adrenalectomy may increase survival in select patients. Intratumoral hemorrhage is a rare but life threatening complication and should be treated as soon as it has been diagnosed. If adrenalectomy is not feasible, combination chemotherapy should be applied.The authors declare that they have no competing interests.CK – conceived the study, did the literature search and coordinated the preparation of the manuscript for submissionAT – helped in literature search, reviewed and edited the manuscript for final submissionSM and KV – helped in literature search and drafted the manuscript for submissionAll authors read and approved the manuscript.


Wide field of view virtual environments offer some unique features that may be beneficial for use in vestibular rehabilitation. For one, optic flow information extracted from the periphery may be critical for recalibrating the sensory processes used by people with vestibular disorders. However, wide FOV devices also have been found to result in greater simulator sickness. Before a wide FOV device can be used in a clinical setting, its safety must be demonstrated.Symptoms of simulator sickness were recorded by 9 healthy adult subjects after they performed gaze shifting tasks to locate targets superimposed on an optic flow background. Subjects performed 8 trials of gaze shifting on each of the six separate visits.The incidence of symptoms of simulator sickness while subjects performed gaze shifts in an optic flow environment was lower than the average reported incidence for flight simulators. The incidence was greater during the first visit compared with subsequent visits. Furthermore, the incidence showed an increasing trend over the 8 trials.The performance of head unrestrained gaze shifts in a wide FOV optic flow environment is tolerated well by healthy subjects. This finding provides rationale for testing these environments in people with vestibular disorders, and supports the concept of using wide FOV virtual reality for vestibular rehabilitation. One out of three elderly persons and more than one out of five working adults report dizziness,2. ThereThe use of virtual reality (VR) has been explored in many areas of physical and mental rehabilitation -8. ViirrSecondly, people with vestibular disorders complain of what has been called "space and motion discomfort" (SMD) and "visual vertigo" ,18. SituWide field of view (FOV), screen-based projection devices  originated with the CAVE™ at the University of Illinois in Chicago . AlthougThe primary purpose of this paper is to present preliminary evidence for the ability of subjects to tolerate gaze shifting while situated in a wide FOV optic flow environment. We will demonstrate that healthy subjects were able to tolerate the environments without having a large incidence in simulator sickness. The incidence of simulator sickness depended strongly on how much experience the subjects had in the environment, and weakly on the duration of exposure within each visit.Nine adults (22–75 years, mean ± S.D. 39 ± 19 yrs) with no history of vestibular system pathology participated after providing informed consent. Subjects had a visual acuity of 0.3 LogMAR units or better without using corrective lenses, and contrast sensitivity greater than 1.8 (Pelli-Robson Contrast Sensitivity). The protocol was approved by the University of Pittsburgh Institutional Review Board.The Balance NAVE Automatic Virtual Environment (BNAVE), a wide field of view projection-based immersive display system, was developed to investigate the multi-sensory interactions in postural control . Three 2Several environments can be used for vestibular rehabilitation. One environment produces optic flow through the use of moving geometric patterns such as stripes or squares of alternating colors. Scene characteristics such as the spatial frequency, contrast, direction and speed of movement can all be independently prescribed. Furthermore, targets can be inserted into the environment that subjects are requested to look at. Consequently, for vestibular rehabilitation, we can ask a patient to perform unrestrained head gaze shifts to acquire moving targets while a background of moving stripes is moving past the patient, simulating the functional task of looking for a product while moving down the aisle of a grocery store. A virtual grocery store has also been developed Figure . This en2, respectively. During the low contrast conditions, the luminance of the stripes was 15 and 34 cd/m2. The low contrast condition was based on average measurements of luminance obtained from products sampled at a local grocery store, using a luminance meter (LS-100 Luminance Light Meter, Minolta Corp. Ramsey, NJ). The spatial frequencies were set according to common sizes of soup cans (high, 4.2 cycles/meter) and cereal boxes (low, 1.4 cycles/meter) found in the local grocery store. The simulated velocity of the optic flow was 0.5 m/s. The central 25° of the display was masked by a solid region with a luminance of 15 cd/m2 in order to avoid aliasing in the display as the stripes became smaller in the distance.The ability of subjects to perform gaze shifts in response to moving targets superimposed on both static and moving backgrounds was examined. Eight different gaze tasks are performed on each visit Table . Each gaRests of 3 minutes were provided after each task, during which Subjective Units of Discomfort (SUDS, 0–10 range) was rated and the Simulator Sickness Questionnaire (SSQ) was completed . The SSQ2 statistics. Because of the large number of comparisons (5 dependent variables × 2 main effects), the significance level was set at α = 0.05/10 = 0.005.Five dependent variables of interest were examined: SUDS, total SSQ, and 3 SSQ subscales. The three subscales of the SSQ were computed by summing the scores for the component items of each subscale, and multiplying by an appropriate weighting factor (9.54 for Nausea, 7.58 for Oculomotor, and 13.92 for Disorientation) . The totGaze shifts performed by one subject in response to targets moving at least 80° in the yaw plane (Task G) are shown in Figure The tolerance of the subjects to the gaze shifts was assessed using Subjective Units of Discomfort (SUDS) and the Simulator Sickness Questionnaire (SSQ). For each of the measures, the majority of the responses were zero. Therefore, each of the measures was converted into a binary scale consisting of responses equal to zero or greater than zero. The SUDS rating was greater than zero only 25% of the time. For each of the SSQ subscales, a score greater than 0 was given if any of the 7 component items for the subscale was rated greater than 0. The SSQ:Oculomotor subscale had the most non-zero responses, at 29%. The SSQ:Nausea and SSQ:Disorientation subscales had 12% and 5% non-zero responses, respectively. Overall, the SSQ-Total had 31% non-zero responses.2 statistics. The effect of visit number was significant for SUDS, SSQ:Nausea, SSQ:Oculomotor, and SSQ:Total  device. HMD users frequently suffer from eyestrain, and blurred vision and short-term changes in binocular vision possibly due to alterations in the balance between the vergence and accommodation systems ,31. In oThe ability to move one's head and search for targets is a functional task that is often impaired in people with vestibular disorders. In vestibular rehabilitation, patients are encouraged to move their head during daily activities because movement is needed for adaptation and reweighting of the sensory signals. Using optic flow and/or virtual environments, this activity could be restored. Our preliminary analysis demonstrates that healthy people are able to coordinate their head and eye movements in the presence of stationary and moving backgrounds with few side effects. The next step is to perform similar experiments with people who have vestibular disorders.In addition, it is imperative to study if these movements can be tolerated while using narrower FOV HMDs. Head mounted displays will most likely be the desired display system of choice for vestibular rehabilitation because of the relatively modest cost and high portability. However, there are other characteristics of the display systems that need to be considered. For example, it is common for users of HMDs to complain of eyestrain, blurred vision, headache, and nausea ,31. In aThe performance of head unrestrained gaze shifts in a wide FOV optic flow environment is tolerated well by healthy subjects. This finding provides rationale for testing these environments in people with vestibular disorders, and supports the concept of using wide FOV virtual reality for vestibular rehabilitation.


Sulfolobus solfataricus was purified to homogeneity for biophysical characterization.The cluster of orthologous group COG2042 has members in all sequenced Eukaryota as well as in many Archaea. The cellular function of these proteins of ancient origin remains unknown. PSI-BLAST analysis does not indicate a possible link with even remotely-related proteins that have been functionally or structurally characterized. As a prototype among COG2042 orthologs, SSO0551 protein from the hyperthermophilic archaeon 73-Arg78 is hyper-sensitive, and thus accessible and flexible. Lysine labeling with NHS-biotin and cross-linking with DTSSP revealed that the 35 amino acid RLI motif at the N terminus is solvent exposed. Cross-links between Lys10-Lys14 and Lys23-Lys25 indicate that these residues are spatially close and in adequate conformation to be cross-linked. These experimental data have been used to rank multiple three-dimensional models generated by a de novo procedure.The untagged protein is thermostable and behaves as a monomeric protein in gel filtration experiment. Several mass spectrometry-based strategies were combined to obtain a set of low resolution structural information. Kinetic data from limited proteolysis with various endoproteases are concordant in pointing out that region GluOur data indicate that COG2042 proteins may share a novel fold. Combining biophysical, mass-spectrometry data and molecular model is a useful strategy to obtain structural information and to help in prioritizing targets in structural genomics programs. Saccharomyces cerevisiae COG2042 homolog. Deletion of the Yor006c gene was shown to result in a viable phenotype but some apparent moderate growth defects were noticed on a fermentable carbon source [Genomic comparative studies on entirely sequenced genomes from the three domains of life, i.e. Bacteria, Archaea and Eukaryota , evidencn source ,9. Two pn source : Ydl017wSulfolobus solfataricus [A polar region, named RLI, is conserved at the N terminus of COG2042 proteins as well as at the N terminus of another cluster of orthologous proteins, namely COG1245. The latter, exemplified by SSO0287 in ataricus , are larataricus  but coulataricus .In silico modeling (homology modeling, fold recognition, ab initio and de novo modeling) is the alternative to quickly gain the fold of a protein. However, such approach sometimes remains ambiguous in reliably identifying correct structures for protein sequences remotely-related to those found in PDB database. A promising strategy is the use of experimental data (if possible easily obtained) for model discrimination or refinement [Because knowledge of protein structure is of high importance to understand protein function, huge efforts have been recently invested in high-throughput protein structure determination programs . Recent finement -20. For finement . The lowfinement  enables finement -26, in cfinement ,28, can finement  for modeSulfolobus solfataricus was chosen as a prototype because of its thermostability and the probable absence of post-translational modifications when produced as a recombinant form in Escherichia coli. The SSO0551 protein is monomeric with a low molecular mass (19 kDa). This size is easily amenable to characterization by mass spectrometry. Our results reveal that the polar RLI motif at the N terminus is probably structured and solvent exposed, pointing at a common trait between COG2042 and COG1245 proteins, this latter group being also conserved in Eukaryota and Archaea but absent in Bacteria. The accessible and flexible regions defined by limited proteolysis combined with lysine accessibility assessed by NHS-biotin labeling and DTSSP cross-linking allowed us to discriminate among ten top ranking de novo three-dimensional (3D) models.A step further is to attempt such approaches with proteins having no detectable homologs. In order to get insight into the topology of COG2042 members and if possible to use these experimental data to discriminate among structural protein templates, we combined limited proteolysis, lysine labeling and cross-linking strategies. The protein SSO0551 from the hyperthermophilic archaea S. solfataricus was used as query in a PSI-BLAST database search to identify homologous proteins. A constant cutoff expectation value of 10-15 resulted after three iterations in selection of 40 sequences (15 from Archaea and 25 from Eukaryota) that were all aligned over their full length. No close homologs  with full-length sequence matching to SSO0551 were found among Bacteria. Remarkably, all completely sequenced Eukaryal organisms were found to have one SSO0551 homolog. Fig. S. solfataricus (NC_002754) should be a more appropriate start codon than atg starting at nucleotide 484916 and mentioned erroneously in current database. From the unrooted phylogram  was engineered. As we could not exclude that the 42 amino acids extension at the N terminus was not an annotation artifact, we intended to check experimentally whether this putative extension could have some influence on SSO0551. A second construct (pSBTN-AB30) was simultaneously engineered supposedly allowing production of a 26 kDa N-terminal 6His variant. Unexpectedly, no major difference in expression was detected between the two cellular extracts when they were resolved on SDS-PAGE. Two overexpressed products with both an apparent molecular weight of approximately 20 kDa were obtained upon addition of IPTG (data not shown). Fingerprint identification of these two products was carried out by trypsin proteolysis and mass spectrometry. Table From multiple sequence alignments, E. coli Rosetta(DE3)(pLysS)(pSBTN-AB30) cells was heated at different temperatures. Proteins that remained soluble were analyzed on SDS-PAGE. Most of E. coli contaminants were removed by such treatment. SSO0551 polypeptide remained soluble even when cell extract was heated to 80°C and therefore this protein was considered as thermostable. This protein was purified to homogeneity by a three-step purification protocol. A 20 min heat treatment at 70°C . This measurement unequivocally confirmed that a truncated protein is produced using E. coli Rosetta(DE3)pLysS transformed with pSBTN-AB30. Both SDS-PAGE  and 222 nm (-12.7 103 deg cm2 dmol-1). Deconvolution of the CD spectrum leads to an estimation of secondary structural element content of about 28–29 % of α-helices and 14–16 % of β-sheets using K2D neural-software. Predictions of SSO0551 secondary structures by PSIPRED and Jpred web servers gave values of 10–11 % of β-sheets in relative agreement with the circular dichroïsm data, but overestimated the α-helix average content (54 %). PSIPRED and Jpred predictions are based on neural networks trained on known folds. The overestimation of the α-helix content may be due to the novel fold of these COG2042 proteins as discussed here below.Content of secondary structure elements in SSO0551 was estimated by far-UV circular dichroïsm. Fig. Native molecular mass of SSO0551 was determined by size-exclusion chromatography on a Superdex 200 HR10/30 calibrated column. Pure protein eluted as a peak centered at 39.1 mL in the assay conditions corresponding to an apparent molecular mass lower than 20 kDa. This elution profile indicates that this structured protein behaves as a compact monomer.m/z 19198.4, but mixed with signals corresponding to 8 different large fragments. Among these, 7 peptides arose from an N-terminal proteolysis: [32–166], [35–166], [56–166], [57–166], [76–166], [79–166], [101–166] . MALDI-TOF mass spectrometry was used to determine cleavage sites by following the time course generation of peptides. Several protease/substrate ratios were assessed to confirm which preferential sites on entire protein were first attacked (earliest cleavage), thus corresponding to a native state of the protein. The two fragments generated by such cleavage may be more vulnerable to subsequent attacks than native protein and therefore late proteolytic sites are considered less informative. Both small and large peptides generated during proteolysis were evaluated. Partial proteolyzed products obtained with trypsin were first resolved by reverse-phase chromatography and analyzed by MALDI-TOF mass spectrometry. Results recorded from direct analysis of the digestions without prior separation were almost similar to those obtained with separation. Therefore, the latter cost-effective strategy was used for analyzing the numerous conditions tested. Figure m/z: 8614.6 amu, 10603.4 amu, 6489.8 amu, and 12724.1 amu, were attributed to fragments [1–75] (Δmass: -178 ppm), [76–166] (+89 ppm), [1–56] (+145 ppm), [57–166] (+200 ppm), respectively (data not shown). These data clearly indicate that Lys75 and Arg56 are two sites of early cleavage by trypsin. Identification of peptides Val32-Lys166 (15478.7 amu, -53 ppm) and Gly35-Lys166 (15191.2 amu, +153 ppm) also indicates that Arg31 and Lys34 could be two other initial nick-sites.During the earliest events of the trypsin proteolysis analyzed in various conditions for detection of large products but also smaller peptides, monocharged cations with following m/z of 1920.9 amu ([1-15] +70 ppm) and 17296.8 amu ([16–166], -95 ppm) on one hand, 8998.1 amu ([1–78], -176 ppm) and 10217.5 amu ([79–166], +332 ppm) on the other hand. These data indicated that Arg78 and Arg15 are the main proteolyzed sites when ArgC enzyme was used. Chymotrypsin attacks SSO0551 native protein mainly at Phe74 because two complementary peptides with m/z of 8487.0 amu ([1–74], -249 ppm) and 10734.2 amu ([75–166], -157 ppm) were clearly evidenced. Glu73 is the main proteolyzed site when GluC protease was used, as peptides with m/z of 8338.7 amu ([1–73], -118 ppm) and 10880.0 amu ([74–166], +28 ppm) were detected. For all these analysis, smaller peptidic fragments that accumulated over time could be attributed from further proteolysis of the products arising from initial attacks (data not shown). All these results are concordant in pointing out that Glu73-Arg78 and Glu28-Arg31 are two accessible solvent-exposed regions of the protein as they can be proteolyzed by several endopeptidases, the first cited being definitively hyper-sensitive. Local unfolding not just surface exposure is necessary for efficient in vitro proteolysis because the polypeptide segment being cleaved must form a specific structure with the associated protease [73-Arg78 region should also correspond to a flexible region, i.e. a protruding loop.Similar experiments with endoproteinase Arg-C resulted in observation of two pairs of complementary peptides with protease . For thiThe SSO0551 protein contains 21 lysine residues (12 %) distributed along the whole polypeptide sequence. Under mild conditions that should keep the native conformation of the protein, specific labeling of these residues with NHS-biotin may give further details about their respective surface accessibility and/or their interactions with other residues . After r10, Lys14, Lys20, Lys23, Lys25, Lys51, Lys75, Lys128, and Lys154, assuming that proteases do not cleave after a modified residue. Other residues, such as Lys34 and Lys49 might be also labeled  and compared to untreated samples. SSO0551 sequence coverage was estimated to be 92 % with all 21 lysine residues included in this coverage. Peptides (Δmass below 120 ppm) detected with NHS-biotin treated samples but not detected with untreated samples are listed in Table m/z 1169.55, 2077.04, 2715.11 and 2871.14, detected for SSO0551 treated by DTSSP  correspond in mass to addition of a DTSSP moiety on peptides [24-31] (Δmass tolerance: +16 ppm), [123–138] (-7 ppm), [57–78] (+61 ppm) and [56–78] (+82 ppm). Since trypsin does not cleave after a modified lysine, we conclude that Lys25, Lys75 and Lys128 were modified. After DTT treatment, peaks corresponding to the expected products  were detected at m/z 1065.46 (-109 ppm), 1973.03 (0 ppm), 2611.21 (-28 ppm) and 2767.26 (-45 ppm), respectively. Fig. + ions at m/z 1491.76 and 1835.84 corresponding to intrapeptide cross-linked peptides: [21-31] (+31 ppm) and [3-15] (+3 ppm), respectively. These peptides contain two proximal lysine residues (Lys23-Lys25 and Lys10-Lys14). As shown in Fig. m/z 1493.69 and 1837.81 appears at the expected increment . An additional peak at m/z 2502.22 could be attributed to peptide [35–55] (+37 ppm) with an intrapeptide cross-link between Lys49 and Lys51. However, the corresponding reduced peak was not detected. Strikingly, every lysines that were reactive with DTSSP were also detected by NHS-biotin labeling. Most modifications are located at the N terminus of the protein where five modified residues belong to the RLI motif.Using a lysine cross-linking reagent, DTSSP, it is possible to assess intra- or inter-molecular protein contacts ,32. DTSS73-Arg78 region being revealed hyper-sensitive to various proteases . The RLI domain is also present at the N terminus of another group of orthologous proteins, namely COG1245. Remarkably, COG1245 proteins only occur in two domains of life (Archaea and Eukarya) similarly to COG2042 proteins. Although co-occurrence of protein members is not strictly identical (for example, pyrococci encompass the information for COG1245 but not for COG2042 polypeptides), such occurrence pattern may reflect a functional link between the two protein families.Although COG2042 proteins are distributed among a large number of organisms, no experimental evidences have yet been reported concerning their biochemical characterization and function. As they are not related, even remotely, to any other family of proteins, COG2042 members can be phylogenetically considered as orphans. Figure ses Fig. . It probses Fig. . The lenab initio models of SSO0551 using the fully-automated ROBETTA server based on ROSETTA procedures [73-Arg78 is solvent exposed (data not shown). Among these three models, M4 and M8 respect the ranking of preferential nick-sites for trypsin, chymotrypsin, ArgC and GluC proteases. Solvent accessibility for lysine side chain was evaluated for models M4, M7 and M8 and compared with experimental data (data not shown). All the lysine residues labeled with NHS-biotin are found solvent-exposed in model M8. Manual inspection of cross-linked lysines (Lys10-Lys14 and Lys23-Lys25) revealed that model M4 is not valid because of the opposite orientation of Lys10 and Lys14. Figure 10-Lys14 and Lys23-Lys25 pairs are 12.7 Å and 13.3 Å, respectively. Search with DALI for structural homologs using model M8 did not result in significant scores with any known PDB structures. This is consistent with the PSI-BLAST results and may indicate that COG2042 proteins share a novel fold. COG2042 proteins are thus a target of choice for genomic structural studies.Our initial objective was to obtain about SSO0551 as much low-resolution structural information as possible in order to discriminate among putative three-dimensional models representing COG2042 protein structure. However, currently available threading tools applied on SSO0551 failed to detect any structurally related-proteins. Alternatively, we obtained ten different ocedures . On thesocedures . Models In conclusion, we have presented a strategy consisting in obtaining low-resolution structural information (determination of nick-sites, solvent exposed residues, and residue-residue distances) that can be used to distinguish among a large set of theoretical molecular models. Lack of remotely-related structural templates or lack of adequacy between experimental data and most theoretical models indicates that such family of proteins should become a priority in structural genomic projects.Most chemicals used in this study were obtained from Sigma and were of analytical grade. Oligonucleotide primers were purchased from Genset. N-hydroxysuccinimide-biotin (NHS-biotin) and 3,3'-dithio-bis [sulfosuccinimidyl-propionate] (DTSSP) were obtained from Pierce. Matrices for Matrix-assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) mass spectrometry and calibration standards were purchased from Bruker Daltonics. Sequencing grade proteolytic enzymes were from Roche Applied Science.S. solfataricus P2 genome (NC_002754)) and an N-terminal extended version of SSO0551 (starting with an ATG codon at nucleotide 484916). For both proteins, an N-terminal 6His tag was added to render the purification of the recombinant products easier. For this purpose, synthetic oligonucleotide primers were oAB22 (5'-gctagcATGAAGCCCAAACCC-3') and oAB49 (5'-gctagcATGAAGGTATATATTATAGAC-3') that both contain an engineered NheI site, oAC34 (5'-cggatcctacTCATTTTTCAAGTATTTTC-3') and oAE62 (5'-ggatcctcaTCATTTTTCA AGTATTTTCTC-3') that both contain an engineered BamHI site . Oligonucleotide pairs oAB22/oAC34 and oAB49/oAC34 were used for two distinct PCR amplifications of SSO0551 with S. sulfolobus total DNA as template. A 643-bp fragment (N-ter 6His-tag extended version of SSO0551) and a 517-bp fragment (N-ter 6His-tag SSO0551) were obtained, respectively. They were cloned into pCRScript-cam (Stratagene), resulting in plasmids pSBTN-AB36 and pSBTN-AB37, respectively. The two inserts were removed by digestion with NheI and BamHI and ligated with T4 DNA ligase into plasmid pSBTN-AB23 (Armengaud J. & Chaumont V., unpublished data), a derivative of pCR T7/NT-topo (Invitrogen) containing a T7 promoter and 6 His-tag, previously digested with the same endonucleases. The resulting plasmids pSBTN-AB30 and pSBTN-AB31, respectively, were verified by DNA sequencing in order to ascertain the integrity of the nucleotide sequence. Hyperexpression of the recombinant SSO0551 constructs was achieved with E. coli Rosetta(DE3)pLysS strain (Novagen), freshly transformed with the plasmids described above. Cultures were carried out at 30°C as described earlier [Two constructs were designed in order to get overexpression of the SSO0551 ORF (starting with an ATG codon at nucleotide 484790 on the Crick strand of 2HPO4/KH2PO4 buffer (pH 7.2) containing 400 mM K-glutamate. The pellet was thawed on ice and resuspended in 120 mL of buffer A. The cells were disrupted by sonication with a total energy delivered of 71 kJ. The cell-extract was then centrifuged at 30,000 g for 20 min at 4°C to remove cellular debris and aggregated proteins. The supernatant was subjected to a 20 min heat treatment using a water bath maintained at 70°C, and immediately centrifuged a second time at 30,000 g for 20 min at 4°C. Chromatographic steps were performed at room temperature using an Äkta Purifier FPLC system (Amersham Biosciences). The 135 mL supernatant was applied at a flow rate of 2.8 mL/min onto a XK 26 × 20 column (Amersham Biosciences) containing 50 mL of Chelating Sepharose Fast Flow (Amersham Biosciences) and previously loaded with 200 mM NiSO4, washed with milliQ water and equilibrated with Buffer A containing 50 mM imidazole. The fraction collected during the IMAC loading was shown to contain the SSO0551 protein. This 222 mL fraction was concentrated to a volume of 56 mL by means of Centricon Plus-20 filtration units (Millipore) and then dialyzed overnight at 4°C against 20 mM K2HPO4/KH2PO4 buffer (pH 7.2) containing 20 mM NaCl (buffer B). The 78 mL supernatant obtained after centrifugation at 30,000 g for 10 min at 4°C was divided and applied in two separate runs onto a 6 mLResource-S ion-exchange column (30 mm × 16 mm, 15 μm) from Amersham Biosciences, previously equilibrated with buffer B and operated at a flow rate of 3 mL/min. After a 10 column volume wash with buffer B, proteins were resolved with a 25 column volume linear gradient from 20 to 500 mM NaCl in buffer B. Recombinant SSO0551 was eluted at approximately 250 mM NaCl and desalted by overnight dialysis against Buffer B. The resulting 20 mL protein solution was concentrated to a volume of 8 mL by means of Centricon Plus-20 filtration units (Millipore). The sample was again divided and applied in two separate runs onto a superdex75 gel filtration packed into a HR 16/50 column at a flow rate of 1.5 mL/min in 20 mM K2HPO4/KH2PO4 buffer (pH 7.2) containing 100 mM NaCl. The fractions obtained with the two runs were pooled and dialyzed overnight at 4°C against 10 mM HEPES buffer (pH 7.2). After dialysis, the fraction was centrifuged at 26,000 g for 20 min at 4°C and the protein concentration was measured by spectrophotometry using a molar absorption coefficient of 19060 M-1 cm-1 at 280 nm. The purified protein was flash frozen in liquid nitrogen and stored at -80°C at a concentration of 0.48 mg/mL.The purification of recombinant SSO0551 was performed from 44 g  packed cells. Buffer A consisted of 50 mM KFar- and near-UV circular dichroism spectra were recorded at 20°C between 200 and 300 nm on a J-810 Jasco spectropolarimeter equipped with a PTC-424S Jasco Peltier, using a quartz cuvette of 1 mm path length, with a 20 nm/min scanning speed and a band-width of 1 nm. Three spectra of purified SSO0551 at 1.92 μM in 10 mM HEPES buffer (pH 7.2) were averaged and corrected from the baseline for buffer solvent contribution. Experimental data were analyzed using the program K2D  describeThe native molecular mass of SSO0551 was estimated by gel filtration chromatography on a Superdex 200 gel packed into a HR10/30 column (Amersham Biosciences) with a final bed volume of 24 mL. The column was equilibrated at room temperature at a flow rate of 0.5 mL/min with 50 mM Tris/HCl buffer, pH 8.3, containing 50 mM NaCl and eluted with the same buffer. Protein standards used to calibrate the column were ribonuclease A (15.8 kDa), chymotrypsinogen A (21.2 kDa), ovalbumin (49.4 kDa), albumin (69.8 kDa), aldolase (191 kDa) and catalase (215 kDa), all from Amersham Biosciences. Exclusion limit was evaluated with dextran blue 2000 (Amersham Biosciences). A sample consisting of 90 μL of SSO0551 at 25.2 μM was injected and specific absorptions at 280 and 266 nm were followed.Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) mass measurements were performed using a Biflex IV instrument  in positive ionization mode. Protein samples and large peptidic fragments (>3500 Da) were applied to the target using sinapinic acid prepared as saturated solution in 30 % acetonitrile, 70 % milli-Q water and 0.1 % TFA as matrix. Samples were prepared using the dried droplet method and measured in linear mode. Small peptide samples were measured in reflectron mode using α-cyano-4-hydroxycinnamic acid in 30% acetonitrile containing 0.1% trifluoroacetic acid as matrix. Mass spectra were obtained by summation of 100–210 laser shots. The instrument was calibrated for determination of entire protein masses using either a mixture of chymotrypsin and bovine serum albumine, or apomyoglobin and aldolase. For peptides, the instrument was calibrated using a pepmix calibration kit . When necessary, the mass spectrometer was also internally calibrated using some of the theoretical peptide masses.in-solution partial digestion, 0.2 nmol of pure SSO0551 were diluted into buffer D1 (20 mM TRIS/HCl, pH 7.8), buffer D2 (20 mM NH4HCO3, pH 7.8) or buffer D3 (20 mM TRIS/HCl, pH 7.8, containing 10 mM CaCl2 and 5 mM DTT). Trypsin or chymotrypsin was added to SSO0551 diluted into buffer D1, whereas Glu-C or Arg-C was added to the protein diluted into buffer D2 or D3, respectively. Several enzyme/protein ratios (1:50 (w/w), 1:20 (w/w) and 1:2 (w/w)) were tested for each endoprotease. The digestions were performed at room temperature and aliquots were analyzed from 30 sec to 10–240 min. Digested samples were desalted using ZipTipC18 or ZipTipC4 pipette tips (Millipore) according to the protocol specified by the manufacturer and their mass directly evaluated by MALDI-TOF. Eventually, partially proteolyzed mixtures of larger quantities (10 nmol of SSO0551) were fractionated by reverse-phase HPLC using an Aquapore RP-300 column  developed at 200 μL/min with a linear gradient from 5 to 90 % of acetonitrile in TFA 0.1 % over 45 min. The elution was monitored at 220 nm with an Agilent 1100 Series HPLC system equipped with a G1315 diode array detector. Individual fractions were concentrated by evaporation in a SpeedVac (Savant) and directly analyzed by MALDI-TOF.For 10H14N2O2S1) should be 226.293 amu if average mass is considered or 226.078 amu in monoisotopic mode. Modification of lysine residues was carried out by incubating 1.25 nmol of SSO0551 in 20 mM HEPES, pH 7.2, with various amount of freshly prepared NHS-biotin reagent dissolved in anhydrous dimethylsulfoxide. After 30 min of incubation at room temperature, the reagent in excess was removed by a 30 min micro-dialysis against 20 mM HEPES, pH 7.2. Samples were directly desalted by using ZipTipC4 (Millipore) prior MALDI-TOF analysis. They were eventually digested overnight with an endoprotease (trypsin, GluC or ArgC) and desalted by using ZipTipC18 pipette tips (Millipore) prior mass analysis.N-hydroxysuccinimide-biotin (NHS-biotin) was used to label ε-amino groups of SSO0551 lysines. After reaction the biotin labels resulted coupled to the lysines through a stable amide bond. The increase in mass for each label (C6H8O3S2) or 87.998 amu (C3H4O1S1) when DTT treated. The increase in mass for an intramolecular cross-link between two lysines should be 173.981 amu (C6H6O2S2) or 175.997 amu (2 × C3H4O1S1) when DTT treated. Therefore after reduction of the disulfide bridge by DTT, an additional increase of 2.016 amu should be measured. Reaction was carried out by incubating 0.25 nmol of SSO0551 in 20 mM NaH2PO4/Na2HPO4, pH 7.5 containing 150 mM NaCl, with various amount of DTSSP reagent (molar ratio of 20, 35, and 50 mol of DTSSP per mol of polypeptide). After 30 min of incubation at room temperature, the reagent in excess was removed by a 30 min micro-dialysis against 20 mM NaH2PO4/Na2HPO4, pH 7.5 containing 150 mM NaCl. Prior overnight trypsin proteolysis, urea  was added to each sample. Before being desalted by using ZipTipC18 pipette tips (Millipore), the digested peptide mixture was eventually reduced with 50 mM DTT for 30 minutes at 37°C to reduce the thiol linker.3,3'-Dithio-bis [sulfosuccinimidyl-propionate] (DTSSP) was used to cross-link two ε-amino groups of SSO0551 lysines, essentially as described in . The masAb initio modeling was performed using the ROBETTA server [Sequence searching was performed using PSI-BLAST with default parameters. Multiple sequence alignments were performed using VectorNTI software package (Informax Inc). Secondary structure predictions were obtained through the PSIPRED v2.4 web-interfaced facilities  describeA server ,47. EachA server ,48,49. RA server ; PellequA server . Model vA server  and rendA server .amu, atomic mass unit; COG, Cluster of Orthologous Group; DTSSP, 3,3'-dithio-bis [sulfosuccinimidyl-propionate]; IPTG, isopropyl-γ-D-thiogalactopyranoside; HPLC, high performance liquid chromatography; EDTA, Ethylenediaminetetraacetic acid; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; HEPPS, N-(2-hydroxyethyl)piperazine-N'-(3-propanesulfonic acid);IMAC, immobilized metal ion adsorption chromatography; MALDI-TOF, Matrix-assisted Laser Desorption/Ionization Time-of-Flight; NHS-biotin, N-hydroxysuccinimide-biotin; PSI-BLAST, Position-Specific Iterated Blast; Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.ab initio modeling of SSO0551 and drafted the original manuscript. AD actively participated in conception of the mass spectrometry strategies, advised OS on execution and interpretation of mass spectrometry experiments and assisted in figure design. OS performed and interpreted all mass spectrometry experiments. JLP contributed its experience for the modeling aspects of the project. EQ contributed its experience in mass spectrometry-based topology. All authors participated in manuscript preparation, read and approved the final manuscript.JA conceived, coordinated the study and participated in all its experimental aspects. He analyzed the genomic distribution of this family of proteins, designed and engineered the recombinant SSO0551 molecule, conceived the mass spectrometry strategies and interpreted the data. He proposed


Emerging Themes in Epidemiology (ETE) is a new, online, Open Access peer-reviewed journal. The Journal is unique in that it was conceived and is managed by research degree students in epidemiology and related public health fields. The Journal's management is overseen by its Editor-in-Chief and Associate Faculty Editors, all of whom are senior members of faculty. ETE aims to encourage debate and discussion on the theoretical, methodological and practical aspects of epidemiologic research and practice. In addition, ETE is dedicated to the promotion of Open Access publication and the training of research students in the scientific publishing process. This editorial, to coincide with the launch of ETE, sets out the Journal's philosophy and aims. Epidemiology is a rich and innovative science that has much to gain from broader discussion of the causal frameworks that underpin it. ETE aims to be a major forum for such discussion. Causality. There is no escape from it, we are forever slaves to it. Our only hope, our only peace is to understand it, to understand the 'why'["the 'why'."- The MerovingianAs far as we know, the Wachowski brothers' Merovingian was not an epidemiologist, but his sentiments should resonate well with those in the field. Probably more than those in any other profession, epidemiologists are slaves to causality. Our professional lives are dedicated to the pursuit of pumphandles, spiderwebs and causal pies ,3. AddinDictionary of Epidemiology does not include the term 'cause', opting instead to give a definition of 'causality' that involves a brief discussion of necessity and sufficiency [The epidemiologic literature on causality certainly makes for stimulating reading, but it would be interesting to know how many of us have causal pies and fractals on our minds as we reach for that red folder labelled "Logistic regression 101". Discursive articles on the usefulness of such metaphors are widely regarded as philosophical flights of fancy that we might eventually get around to reading after clearing that backlog of papers waiting to be written in the next two months. Yet what are our alternatives? The newly-qualified epidemiologist leaves their degree with a solid grasp of error, bias, confounding and statistical methodology, but with perhaps a single lecture on Koch-Henlé postulates and Bradford-Hill 'criteria' as the extent of their training into causal thinking. It is interesting to note that Last's ficiency . Any reaficiency , or admiFinal Destination [Rocky Mountain High ironically playing in the background, she tries to calm herself by making some tea. Moving to the kitchen sink, she fills the kettle with water, spilling some down the side. She wipes the kettle, turns towards the gas stove and tosses the towel carelessly behind her, which catches onto a knife block. With the kettle whistling, she picks up a school coffee mug, drops two tea bags inside and fills it with boiling water. Picking up the mug, she suddenly recognizes the school logo and, in shock, reflexively throws the mug's contents into the sink. Opting now for something stronger to calm her nerves, she takes some ice cubes from the freezer, drops them into the still-warm mug and re-fills it, this time with vodka. Oblivious to the crack that has appeared in the mug, she walks towards the living room, leaving a trail of vodka behind. As she stands by her computer monitor, vodka drips into the circuitry. An electrical surge creates a spark that ignites the alcohol, causing the monitor to explode and sending out flying shards of glass that slash her throat. Shocked and bleeding, she stumbles towards the kitchen sink, chased by a trail of burning alcohol. Reaching the stove, the trail of flames ignites the gas burners, lighting up her clothes and hair. Falling to the ground and still bleeding, she rolls around violently trying to put out the flames. In an act of desperation, she reaches up and grabs the dangling towel, tilting the knife block and sending a half dozen blades cascading into her stomach while flames catch the curtains and set the house on fire.James Wong's 2000 teen horror movie tination  is unlikSuppose you were the investigator arriving on the scene. There is a half burnt-down house, a blown-up computer, a broken mug and a corpse with third-degree burns, stab wounds and a cut throat, but no signs of struggle. What would you determine was the cause of this tragedy? The severe burns, the protruding knives and the neck wound would be pretty obvious choices. But perhaps it is more complicated than that. Perhaps there were extenuating circumstances without which this tragic outcome might not have occurred. The exploding computer maybe, or what about the vodka that burned leaving no trace, or the cracked mug? No, maybe it was the towel, complicitly catching onto the knife block. Or maybe we should blame the teacher's drinking habit.The point of this rather unsavoury story is that without the benefit of such extrinsic observation, detailed reconstructions of causal processes are unattainable. A reasoned observer might conclude that all of the above factors were in some way responsible. They all contributed to the process in their own small way, and had any one of them not been involved things may not have turned out the way they did. They were all what one might call 'component causes'. But is this enough to convince us of what the real cause was? Clearly not.Suppose now that you were an audience member and were somehow able to communicate directly with the characters in the movie. You might wish to warn Valerie of her impending ill fate. At which point in the whole sequence would you alert her so that her death could be prevented? Clearly you would not deny her a mug of tea and you would most likely have no way of knowing that the towel would land on the kitchen block with dire consequences just a few moments later. You might, of course, realize this at the last moment and warn her against reaching up and grabbing the towel, but she might still have died from her neck wound. You might have shouted for her to grab a fire extinguisher and put out the trail of flames, but it is unlikely she would have listened to you as she tried hopelessly to stop the bleeding from her neck. You might, with better foresight, have pointed out to her that vodka was dripping from her mug. Or perhaps with hindsight, you might have recognized that the best thing would have been to provide some moral support and consolation in her time of grief, with which the whole sorry incident might have been avoided altogether.One thing is now clear. There are steps within causal processes on which we can act to try and alter their course- the trail of flames might have been extinguished, and the consequences of the dripping vodka might have been avoided. There are other steps on which we can have no influence, eg. the tossed towel landing on the knife block. Another important thing is also apparent: causal processes have hierarchies. Depending on what happens at one stage, a number of alternative events may result at the next. Had Valerie been able to stop the trail of flames, she might have reached the kitchen sink, realized she could not stop the bleeding and called an ambulance. Or she might have run out of the house shouting for help and her neighbour, trained in first aid, might have saved her life. Saving victims of horror movies, however, is not an easy job. Knowing when best to step in is not necessarily that simple, as we have seen with Valerie. In some cases we may be given a number of opportunities and intervening at any of them might lead to a positive outcome. But in other cases, once certain factors are in place there will be an air of inevitability in everything that follows, and all our attempts to intervene thereafter may prove to be little more than an exercise in futility.Perhaps it is now time to admit that I have extended this fanciful analogy far beyond what is appropriate. I make no apology if in so doing I have in some way managed to convey the idea that epidemiology thrives on causal processes, on elucidating their complexities and identifying the most effective points for intervention no matter at what level of their elaborate hierarchies. If this is a worthwhile venture, then the discussion of how we conceptualize and study these causal processes surely is so too.Emerging Themes in Epidemiology (ETE) was born out of this ideal- that contrary to common belief, epidemiology is not merely a collection of standardized tools to be applied at will to any health-related situation, but that it is a rich and innovative science that aims to describe reality in all its complexity, spanning the molecular to the global, with the ultimate goal of improving the health of individuals and populations. And that in order for this to be achieved, we need to improve our understanding of how factors, at any level of biological or social organization, eventually lead to ill health.ETE is founded on three core principles:• That epidemiology and epidemiologists have much to gain from a broader and more fundamental discussion of the concepts and theoretical frameworks that underpin the practice of epidemiologic research• That Open Access publication has a crucial role to play in reducing the current inequities in access to scientific information, which should be a universal and freely-available resource for the benefit of the whole of society• That students of epidemiology and related fields can make substantial contributions to the introduction of new concepts and ideas into mainstream epidemiologic research, not only through writing, but also through having a direct influence over what is publishedETE will not generally publish research reports, although exceptions may be made in cases where the results can be placed within a broader public health context to present a new concept or theoretical framework.In keeping with this philosophy, we recognize that epidemiology has much to gain from developments in other fields and we welcome contributions from all public health professionals. We will consider articles that comment critically on current epidemiologic theory and practice, either generally or within a specific specialty, including articles from other fields that have implications for the conduct of epidemiologic research. Emerging Themes in Epidemiology a global forum for the discussion of new developments in epidemiologic thinking and practice that will benefit the global public health community. In doing so, we recognize that there is much that the scientific community can do to support Open Access publication. The health consequences of inequitable access to scientific information remain largely ignored, yet for years we have adhered to a system of publication that is restrictive and largely subsidized by institutions and libraries at great expense. Open Access is an important step towards making the publication process, and its associated costs, more transparent. We thus call on individuals, institutions, funding bodies and governments to engage in the Open Access movement by promoting and supporting Open Access publication. This will involve a major shift not just in publishing costs, but also in thinking. Our current measures for assessing the impact of academic research are inherently intertwined with our inequitable publication tradition. Developing new ways of assessing the quality of scientific research that are independent of journals' perceived 'impact' are imperative for the wider recognition of Open Access.By making all of this material freely available online under the auspices of the Open Access publisher, BioMed Central, we aim to make ETE to be a training ground for postgraduate students, providing them with an opportunity to be involved at every stage of the publication process, including commissioning, reviewing and writing articles. Our editorial board is formed principally of research students, who operate the Journal with support from an international group of associate faculty. Our collaborations extend to a growing number of students from diverse institutions serving as article referees. We welcome suggestions for extending these collaborations in the future.In promoting the role of students in the publication process, we intend for Emerging Themes in Epidemiology will be a tool for epidemiologists confronting that challenge.The third millennium has brought with it exciting challenges for epidemiologists. An explosion in the availability of genetic and molecular information, the development of bioinformatics, the increasing application of sophisticated statistical analyses to model complex systems and the gradual incorporation of sociological approaches to understanding health inequalities have arrived together with sobering statistics on the state of global public health. Knowing when and how to apply these and other developments at a time of rapid political, social and biological change while maintaining clear sight of our public health goals will be the major challenge for current and future generations. It is our hope that


The 156 breeds of dog recognized by the American Kennel Club offer a unique opportunity to map genes important in genetic variation. Each breed features a defining constellation of morphological and behavioral traits, often generated by deliberate crossing of closely related individuals, leading to a high rate of genetic disease in many breeds. Understanding the genetic basis of both phenotypic variation and disease susceptibility in the dog provides new ways in which to dissect the genetics of human health and biology.in situ hybridization (FISH). The two data sets show excellent concordance. Excluding the Y chromosome, the map features an RH/FISH mapped BAC every 3.5 Mb and an RH mapped BAC-end, on average, every 2 Mb. For 2233 markers, the orthologous human genes have been established, allowing the identification of 79 conserved segments (CS) between the dog and human genomes, dramatically extending the length of most previously described CS.To facilitate both genetic mapping and cloning efforts, we have constructed an integrated canine genome map that is both dense and accurate. The resulting resource encompasses 4249 markers, and was constructed using the RHDF5000-2 whole genome radiation hybrid panel. The radiation hybrid (RH) map features a density of one marker every 900 Kb and contains 1760 bacterial artificial chromosome clones (BACs) localized to 1423 unique positions, 851 of which have also been mapped by fluorescence These results provide a necessary resource for the canine genome mapping community to undertake positional cloning experiments and provide new insights into the comparative canine-human genome maps. Three major advances in the development of resources for mapping canine disease genes have been: 1) the development of a radiation hybrid (RH) map composed of large numbers of microsatellite markers and genes that link the canine and human genomes -5; and 3The above mapping efforts are complemented by the recent release of a 1.5x sequence of a Standard Poodle genome . The seqUsing the above resources, canine researchers have undertaken genome-wide screens for linkage to a variety of disease loci as well as morphological traits defining differences between breeds. Many of these studies have met with success. Disease loci have been genetically mapped or otherwise localized in the dog for several disorders including: vision-associated disease such as progressive rod cone degeneration, early retinal degeneration, cone degeneration, and collie eye anomaly -11, kidnTo facilitate positional cloning efforts in the canine community, we have localized a large set of randomly selected canine-specific BACs onto the canine 5000-rad RH map, a subset of which we have also localized cytogenetically. The resulting map of 4249 markers includes 1760 mapped BAC-ends, 851 of which have been FISH mapped, to generate a dense, accurate and highly integrated map of the canine genome.This novel RH map of the dog genome contains 4249 markers of three different types: 900 genes, 1589 microsatellites and 1760 BACs. The map was generated by genotyping 1092 new BAC-end markers on the RHDF5000-2 panel, and recomputing the new vectors with those from the previous map  and TSP/Analysis of the entire dataset of 4249 RH vectors by MULTIMAP  at a Lod. Again, the chromosome with the fewest is CFA 38, with 16 BACs localized to 14 unique positions. This resource provides a dense scaffold on which to build regional physical maps and search for new genes. Of particular interest to disease gene mappers will be the 26 BACs that are RH mapped to the X chromosome, 13 of which are also localized by FISH. Only two BACs were localized to the Y chromosome, one of which is also FISH mapped. See Table The resulting 4106 linked markers defined 3090 unique positions regularly spaced across all autosomes. The largest chromosome, canine chromosome 1 (CFA 1), contains 220 markers, while CFA 38, the smallest chromosome at 38 Mb, has only 32 markers  between the human and the dog genomes is 79. CS were identified by both FISH localization and RH mapping with four chromosomes showing four human/dog CS, and the remainder showing one to three. The number of markers and the resolving power of the 5000 rad panel allowed us to detect several instances in which CS appear to be split in sub-segments. In several such cases, the order of markers in the two adjacent sub-fragments is conserved, although the two sub-fragments are oriented in opposing directions. Following the previously agreed definitions, these sub-fragments are termed CSO for "conserved segment order" . Such a As shown in Table Canidae. Finally, with the imminent release of the 7x canine genome assembly, the resources described in this paper will provide a long-awaited means to translate canine cytogenetics data into canine DNA sequence data, thus advancing our knowledge of canine and comparative genomics.Of interest to many researchers will be the multiple applications that a cytogenetically defined and RH verified set of canine BAC clones can provide. For example, the ordered set of 981 FISH mapped canine BAC clones with a unique cytogenetic location will be welcomed by cancer geneticists as a means to characterize chromosome aberrations in canine tumors. The dense cytogenetic coverage available for most chromosomes allows the use of serial differential labeling of some or all clones in chromosome-specific panels designed to 'tile' structurally aberrant chromosomes. This approach will allow a much more accurate assessment of chromosome rearrangements than is possible with single color whole chromosome paint probes. In addition, the ordered clones will allow expansion of the current canine BAC microarrays, which in turn may be used for a variety of applications including array-based Comparative Genome Hybridization (CGH) analysis of canine tumors , array-pThe presentation of a map containing 900 canine specific genes, 1589 microsatellites and 1760 BACs provides the canine genetics community with nearly all the resources it needs to undertake experiments aimed at both mapping and cloning traits of interest. A dense set of microsatellite markers (MSS-2) for undertaking genome wide scans was provided in the previous iteration of the map [Escherichia coli (E. coli) and repetitive DNA sequences. BAC sequences were also compared using Cross_Match with the complete E. coli genome to remove contaminating sequences of bacterial host origin. Finally, sequences were examined for interspersed repeats and regions of low sequence complexity using RepeatMasker . Primers defining each BAC-end were designed to regions of high quality sequence data using Primer3 software . Primers of 25 bp lengths were preferentially designed in order to minimize problems associated with non-specific amplification, to generate amplicons of 200 to 500 bp, and to work under a single optimal set of PCR conditions.Canine BACs were randomly selected from the RPCI-81 canine BAC library  and wereGenotyping was performed using the RHDF5000-2 panel, which is comprised of 118 cell lines. The panel was constructed by fusing dog fibroblasts irradiated at 5000 rads with TK-HTK3 hamster cells and has an experimental retention frequency of 22% with a theoretical resolution limit of 600 Kb .PCR reactions were carried out at the University of Rennes and the Fred Hutchinson Cancer Research Center (FHCRC) as described previously in 15 μl volumes -35 usingDuplicate genotypes were obtained for the 1092 new BACs added to the map. Data were considered consistent when the number of discrepancies between data sets was ≤ 16%, a value determined to correspond to a distance lower than the resolution limit of the RHDF5000-2 panel. In the rare cases where two independent typings yielded >16% discrepancies, a third typing was done and the resulting vector was either integrated into the map construction, or the marker was discarded if no agreement was observed between two of three genotypes.RH vectors corresponding to the BAC-end markers and marker vectors of the previous map  were comIndividual linkage groups were analyzed using the multipoint approach of the rh_tsp_map version 2.0 of TSP/CONCORDE, as described previously ,29. Inte.A total of 851 BAC clones from the RH map were also localized by multi-color FISH analysis. DNA from each clone was prepared from 2.5 ml cultures using a BAC RealPrep  protocol. Two hundred nanograms from each sample were labeled using nick translation to incorporate one of five fluorochromes, Spectrum Red/Orange/Green dUTP (Vysis, Downers Grove, IL), diethylaminomethylcoumarin (DEAC)-5-dUTP (NEN/Perkin Elmer Life Sciences, Boston, MA), or Cy5-dUTP (Amersham Biosciences, Piscataway, NJ). Typically, 25 ng of each of five differentially labeled probes were pooled and precipitated in the presence of 15 μg of sonicated genomic dog DNA as competitor. Chromosome preparation, probe hybridization and post hybridization washes were performed as described previously ,36. Chro). For 648 of the BACs (34%), each of the paired end-sequences gave a best hit to human genomic locations that are separated by 50–500 Kb, and aligned head-to-head (mean span of human genomic DNA = 191 Kb). The remaining 1262 BAC-end sequences were searched against scaffolds of the 1.5x assembly [Nineteen 384-well plates of BAC clones from the RPCI-81 library  were sel and .Accession numbers, PCR conditions, primers and BAC-end sequences are available for all markers at: BAC-Bacterial Artificial Chromosomein situ HybridizationFISH-Fluorescence PCR-Polymerase Chain ReactionRH-Radiation HybridCGH-Comparative Genomic HybridizationcR-centiRaysMb-megabasesKb-kilobasesBp-base pairMin.-minutesE. Coli-Escherichia coliSOM-Supporting Online MaterialDAPI-Diamidino-2-phenylindoleCS-Conserved SegmentsCSO-Conserved and Ordered SegmentsTIGR-The Institute for Genomics ResearchFHCRC-Fred Hutchinson Cancer Research Centerin situ hybridization, and microscopy. MB performed all the cytogenetic analysis to generate detailed probe ordering. MB and RT completed the merging of RH and FISH data, and prepared the drawing of Figure MB, RT, AS, and RH all contributed to the FISH mapping aspects of the manuscript, including isolation of BAC DNA samples, generation of canine chromosome preparations, probe labeling and purification, fluorescence FISH Information: RH Map Information:  and .