Inference of remote homology between proteins is very challenging and remains a prerogative of an expert. Thus a significant drawback to the use of evolutionary-based protein structure classifications is the difficulty in assigning new proteins to unique positions in the classification scheme with automatic methods. To address this issue, we have developed an algorithm to map protein domains to an existing structural classification scheme and have applied it to the SCOP database.The general strategy employed by this algorithm is to combine the results of several existing sequence and structure comparison tools applied to a query protein of known structure in order to find the homologs already classified in SCOP database and thus determine classification assignments. The algorithm is able to map domains within newly solved structures to the appropriate SCOP superfamily level with ~95% accuracy. Examples of correctly mapped remote homologs are discussed. The algorithm is also capable of identifying potential evolutionary relationships not specified in the SCOP database, thus helping to make it better. The strategy of the mapping algorithm is not limited to SCOP and can be applied to any other evolutionary-based classification scheme as well. SCOPmap is available for download.The SCOPmap program is useful for assigning domains in newly solved structures to appropriate superfamilies and for identifying evolutionary links between different superfamilies. Protein structure classifications are commonly used for studying structural and evolutionary relationships between proteins (namely remote homology inference), protein structure and function prediction, identification of potential functional residues and binding sites, understanding sequence/structure/function relationships in proteins, and as an aid in describing protein folds and families.Several structural classification schemes such as SCOP , CATH 22, and DaAvailable tools for assigning proteins to existing classification schemes use either structure-based or sequence-based comparison methods. Classification predictions from structure comparison tools like SSM , GRATH99, and F2In this paper, we describe an algorithm developed to map domains within protein structures with their homologs in an existing classification scheme. The general strategy employed by this algorithm is to combine the results of several existing sequence and structure comparison tools in order to determine classification assignments. The comparison tools incorporated in the algorithm each utilize a different methodology for identifying homologous domains, and consequently, these tools have different advantages and limitations. An approach combining different methods of homology detection is expected to capitalize on the proficiencies of each comparison tool while the limitations of those tools are neutralized by the inclusion of other methods.Our algorithm, named SCOPmap, has been developed to map domains in protein structures to the SCOP database, which is a manually curated hierarchical classification scheme based on the structural and evolutionary relationships between proteins. SCOPmap assigns protein domains at the superfamily level, which is the broadest level of homology in the SCOP database. SCOPmap also performs assignments at the SCOP fold level when confident superfamily level assignments cannot be made. SCOPmap has two general applications. First, domains within newly solved protein structures can be identified and assigned to the appropriate SCOP superfamily. Second, SCOPmap can be used to find new links in SCOP by identifying potential evolutionary relationships between existing SCOP superfamilies. The strategy employed by this algorithm is not limited to SCOP and could be applied to any other similar database or classification scheme as well.We have evaluated the performance of SCOPmap on two test sets, each of which includes over 4500 protein domains. The first set is comprised of the proteins that are included in SCOP v1.63 but not in SCOP v1.61, while the second set contains the proteins that are included in SCOP v1.65 but not in SCOP v1.63. SCOPmap was able to correctly map greater than 94% of both test sets at the SCOP superfamily level. Comparison of SCOPmap results and SUPERFAMILY  results Results of SCOPmap performance on the tweaking set are shown in Table Results of SCOPmap performance on the testing set (see Methods) are shown in Table Because the score cutoffs used by SCOPmap's individual comparison tools were determined while considering domains from the tweaking set, those cutoffs were therefore influenced by the specific collection of domains in that set. Had a different test set been considered when establishing these cutoffs, it is likely that the score cutoffs would be slightly different. Thus, the few false positive assignments observed in the second test set are not unexpected. Furthermore, the number of false positive domain assignments made is higher than the number of incorrect hits between query and library domains that are accepted. Due to redundancy in the test set , the 7 domains mapped incorrectly essentially reflect only 3 different examples of false positive assignments.Each incorrectly assigned domain has less than 10% sequence identity to the nearest library representative from the same SCOP superfamily. Furthermore, all of the false positive assignments are due to scores from the individual comparison tools which barely meet the cutoffs required for acceptance. Such cases reflect the influence that a few specific domains can have in determining the exact values of the minimum score threshold requirements. All incorrect assignments were made due to a hit accepted by one of the comparison tools that includes both sequence and structure components.Escherichia coli  correct assignments; 5.7% (tweaking set) vs 5.3% (testing set) false negative assignments; and 0.0% (tweaking set) vs 0.2% (testing set) false positive assignments. The most significant apparent differences are in the results for the specific types of correct assignments: true positives with ranges accurate within 10 residues, true positives with ranges that are not accurate within 10 residues, and true negatives. These seemingly disparate results are predominantly reflections of inconsistencies in test set composition rather than in SCOPmap performance. More specifically, these variations are primarily due to the number of query domains that belong to new SCOP superfamilies. The most obvious consequence is the fraction of each test set given true negative assignments , which is directly dependent on the fraction of each test set that belongs to new SCOP superfamilies. If domains from new SCOP superfamilies are ignored, the apparent disparity in SCOPmap boundary definition accuracy is reduced. For example, if the entire test sets are considered, there is a 2.3% difference in the number of domains correctly assigned whose ranges are accurate within 10 residues of the SCOP-defined boundaries. However, when considering only domains that can potentially be mapped correctly (i.e. domains that do not belong to new SCOP superfamilies), 86.8% of the tweaking set domains are correct assignments that are accurate within 10 residues, compared to 86.4% of the testing set domains. Similarly, 92.4% of all correctly assigned domains in the tweaking set are accurate within 10 residues, compared to 91.6% for the corresponding domains in the testing set.Table The comparable results are a reliable indication of the consistency of SCOPmap performance because the two test sets are of nearly equivalent difficulty. First, the two test sets include approximately the same fraction of trivial assignments: 73.7% of mappable domains in the tweaking set are assigned by gapped BLAST while 73.6% of mappable domains in the testing set are assigned by gapped BLAST, where "mappable" means the domain is both evolutionarily relevant and is a member of a SCOP superfamily that exists in the version of SCOP used as the library. Of the non-trivial mappable domains (i.e. mappable domains that are not assigned by gapped BLAST), the average sequence identity between the query domain and the closest library representative from the same SCOP superfamily is 29.2% in the tweaking set and 28.6% in the testing set.Fold level assignments are attempted for regions of query chains at least 20 residues in length for which no superfamily assignment was made. Results are shown in Figure Similar to the superfamily level assignments, the apparent disparity in fold level assignments are due primarily to the relative composition of the two test sets rather than inconsistency in performance. There are two principal attributes of test set composition that result in improved fold level results. First, domains from new folds are typically given no fold level assignment by SCOPmap, so a smaller fraction of unmapped domains from new folds will result in a decreased number of domains for which no assignment is made. Second, because the structural similarity between two domains from the same superfamily is likely to be greater than that between two domains from different superfamilies within the same fold, a larger fraction of unmapped domains from existing superfamilies will result in an increased number of correct fold level assignments. Both of these attributes favor the testing set over the tweaking set (results not shown). This indicates that the testing set is less challenging in terms of fold level assignments, which is consistent with the improved results relative to the tweaking set Figure .et al. report that MAMMOTH Z-scores greater than 5.25 are generally reliable for fold predictions ,CSVn is the sum of position-pair conservation scores of the aligned query positions vs. library domain positions , S1 is the sum of position-pair conservation scores of the chosen query positions against themselves (query positions vs. query positions), S2 is the sum of position-pair conservation scores of the chosen library domain positions against themselves (library domain positions vs. library domain positions), and Srand is the sum of position-pair conservation scores of the chosen positions for all-against-all query positions vs. library domain positions normalized over length.where SA COMPASS-based conservation score is also calculated for each query-library domain pair. In this scoring system, a COMPASS-based position-pair score, which describes the similarity between any two given positions, is determined based on the methodology introduced in the COMPASS method . Then, tcompass,D = [CSn - CSrand]/ [(CS1+CS2)/2 - CSrand],CSVn is the sum of COMPASS-based position-pair scores of the aligned query positions vs. library domain positions , CS1 is the sum of COMPASS-based position-pair scores of the chosen query positions against themselves (query positions vs. query positions), CS2 is the sum of COMPASS-based position-pair scores of the chosen library domain positions against themselves (library domain positions vs. library domain positions), and CSrand is the sum of COMPASS-based position-pair scores of the chosen positions for all-against-all query positions vs. library domain positions normalized over length.where CSConservation score hits are accepted if they meet one of the following sets of criteria:cons,D ≥ 0.1 and ZD ≥ 5;1) CSVcons,D ≥ 0.25 and ZD ≥ 2;2) CSVcompass,D ≥ 0.4 and ZD ≥ 5;3) CSVcompass,D ≥ 0.5 and ZD ≥ 2.4) CSVcons,D scores, CSVcompass,D scores, and DaliLite Z-scores of 4000 randomly chosen pairs of SCOP domains from SCOP v1.61.These cutoffs for accepting hits were determined based on the CSVIn cases for which the DaliLite program produces no output, conservation pattern analysis is performed using pairwise alignment produced by MAMMOTH instead of FSSP alignments. The conservation analysis is done for the query-library domain pairs that would have otherwise been submitted to the DaliLite algorithm for structural comparison (see above). Only those residue pairs in which the Cα atoms are located within 4Å, which are indicated by an asterisk (*) by the MAMMOTH algorithm, are considered. Again, a window size of 3 is used in the AL2CO program and only the top scoring 25% of positions are used for calculating the conservation scores. Matrix-based and COMPASS-based conservation scores are calculated as described above. Conservation score hits based on MAMMOTH alignments are accepted if they meet one of the following sets of criteria:cons,M ≥ 0.3 and ZM ≥ 4;1) CSVcompass,M ≥ 0.4 and ZM ≥ 42) CSVcons,M scores, CSVcompass,M scores, and MAMMOTH Z-scores of 2000 randomly chosen pairs of SCOP domains from SCOP v1.61.These cutoffs for accepting hits were determined based on the CSVD > 0 and BLAST, PSI-BLAST, or RPS-BLAST E-value ≤ 100, the number of correctly aligned residues  in the sequence alignment is calculated using the DaliLite alignment as a reference. Hits are accepted for which ZD > 0, E-value ≤ 100, and Nali ≥ 15. These cutoffs were determined based on the DaliLite Z-scores, E-values, and number of equivalently aligned residues from 1000 randomly chosen pairs of SCOP domains from SCOP v1.61. If an error occurs while running DaliLite for the query domain, agreement of the MAMMOTH alignment and BLAST, RPS-BLAST, or PSI-BLAST alignments is instead calculated for the same potential hits. In these cases, hits are accepted for which ZM > 2.0, E-value ≤ 100, and Nali ≥ 15. These cutoffs were determined based on the MAMMOTH Z-scores, E-values, and number of equivalently aligned residues from 1000 randomly chosen pairs of SCOP domains from SCOP v1.61.Remote evolutionary links between protein domains can be gleaned using a combination of sequence and structural information, even when neither of these methods alone is capable of providing convincing evidence for common descent. In this method, the degree of correlation between a pairwise alignment made by DaliLite and alignments made by the sequence comparison methods is determined so that DaliLite can be used to evaluate potential hits from BLAST, RPS-BLAST, or PSI-BLAST. For any query-library domain pair with ZAccepted hits from the sequence and structure comparison methods are mapped onto the query chain and domains within the chain are then assigned to SCOP superfamilies. In cases where accepted hits from multiple SCOP superfamilies mapped to the same region of the query chain, SCOPmap attempts to choose only one correct SCOP superfamily assignment. If the overlap between two different SCOP superfamily representatives covers <50% of both domains, the conflict is resolved by the domain boundary definition (see "Mapping Step 3" below). Otherwise, SCOPmap attempts to determine which SCOP superfamily among the accepted hits is most likely to be the correct assignment.-10 or Z-scores (ZM or ZD) above 14.0, that SCOP superfamily assignment is accepted as correct. If a SCOP superfamily assignment has still not been made, the domain assignments to that query chain are flagged as unresolved. Of the 4580 tweaking set domains (see Results), only 25 domains (0.5%) were unassigned due to unresolved choice between conflicting SCOP superfamilies. The results obtained by inverting the order of these two steps  were also evaluated. There were no cases where the inverted order gave additional correct assignments, and there was a small number of cases that could be resolved by the original strategy but not by the inverted strategy. Thus, the methodology described above is used for choosing between conflicting superfamily assignments.First, for each of two conflicting assignments, all accepted hits that overlap by at least 75% and are from the same SCOP superfamily are identified. For each set of accepted hits (one set corresponding to each of the conflicting SCOP superfamilies), the number of methods that identified accepted hits to that SCOP superfamily is determined. If one SCOP superfamily is found by more methods than the other SCOP superfamily, the assignment with hits from the greater number of methods is accepted as correct. If both SCOP superfamilies are identified by an equal number of methods, the priority of those methods is used to choose the correct SCOP superfamily. The methods are ranked by reliability, which was subjectively determined based primarily on the observed number of false positives accepted by a given method during SCOPmap development. Priority rankings are as follows: BLAST > RPS-BLAST or PSI-BLAST > MAMMOTH or DaliLite > COMPASS > conservation pattern correlation or agreement of DaliLite and sequence method alignments. If both SCOP superfamilies are found by methods with equivalent priorities, the Z-scores and E-values of the hits are evaluated. If only one of the two conflicting SCOP superfamilies has E-values from any sequence comparison method below 10Domain boundary definitions are assigned by identifying the longest non-overlapping domain assignments, with priority given to assignments made by structure comparison methods. First, DaliLite is run for all query-library domain pairs found by MAMMOTH, and the DaliLite range is used in place of the MAMMOTH range unless there is an error in the DaliLite output. Then, ranges of accepted hits are given priority rankings based on which method determined the range of that hit. DaliLite ranges have highest priority, followed by MAMMOTH ranges, and then all sequence comparison method ranges. The longest non-overlapping segments with the highest priority rankings are then identified. A 3-residue cushion for overlap is allowed. Overlapping domains for which boundaries cannot be reconciled within 3 residues are flagged as unresolved. Of 4580 tweaking set domains, only 3 domains (0.1%) were unassigned due to unresolved domain boundary definition.For query chains with a segment at least 20 residues in length which is not assigned to a SCOP superfamily, mapping at the SCOP fold level is attempted. In the SCOPmap algorithm, MAMMOTH is run comprehensively against the library of representative structures. Therefore, no additional comparisons must be made in order for fold level assignments to be determined. For this reason, MAMMOTH is used for fold level assignments rather than DaliLite, which is typically run against less than 5% of the library domains. The single criterion for potential SCOP fold assignment is a MAMMOTH Z-score > 10. Fold level assignments are made by selecting the hit to an unmapped region with the highest MAMMOTH Z-score (>10) that also covers at least 50% of the library domain. The fold level Z-score cutoff was determined based on the MAMMOTH Z-scores of 106,310 randomly chosen pairs of SCOP domains from SCOP v1.61. These same pairs of domains were used for determining the superfamily assignment cutoffs (see above). Approximately 2/3 of these pairs of domains belong to the same SCOP fold while the remaining 1/3 of the pairs belong to different SCOP folds.-3 to 1 × 10-4, and the E-value cutoff for COMPASS was adjusted from 1 × 10-4 to 1 × 10-10. Because some of the domains in this set were considered while establishing the score thresholds, the first test set is more correctly described as a "tweaking" set rather than a testing set. This set was also used for comparison to SUPERFAMILY, for which the score threshold was also chosen specifically for the purpose of precluding false positive assignments. The recommended 0.02 E-value cutoff for SUPERFAMILY, which would allow for the correct assignment of only an additional ~1% of the tweaking set domains, was not chosen due to the 4.3% false positive rate it incurs. Instead, the E-value cutoff was set at 1 × 10-5, the maximum value for which no false positive assignments were observed. For this comparison, the SUPERFAMILY algorithm was used with the library of SAM [SCOPmap performance was evaluated on two separate test sets. The first set is comprised of the proteins that are included in SCOP v1.63 but not in SCOP v1.61. SCOPmap was run using a library based on the previous SCOP release (v1.61), and the SCOPmap domain assignments were compared to the SCOP-defined classification in subsequent SCOP release (v1.63). This set contains 5133 SCOP-defined protein domains, but analysis of SCOPmap performance is based only on the 4580 SCOP-defined domains with evolutionary relevance: 464 low resolution structure domains, 63 peptides, 21 designed proteins, and 5 domains that were later removed from the database are intentionally excluded. The first test set was used to establish whether the score cutoffs for the individual comparison tools used by SCOPmap were strict enough to avoid false positive assignments. After first running SCOPmap for this set of domains, a false positive rate of ~1.5% was observed. The score thresholds for some of the individual comparison tools were subsequently made more strict in order to avoid all false positive assignments in this set. For example, the E-value cutoff for PSI-BLAST was changed from 5 × 10y of SAM  hidden MThe second set of domains used to evaluate SCOPmap performance contains proteins included in SCOP v1.65 but not in SCOP v1.63. The second test set can be considered a true testing set. The testing set contains 5335 SCOP-defined protein domains, but only the 4941 SCOP-defined domains with evolutionary relevance were used for analysis of SCOPmap performance. Low resolution structures, peptides, and designed proteins were ignored. The library of SCOP representative domains used for mapping the queries in this set is based on SCOP v1.63.SCOPmap can also be used to identify potentially homologous proteins that belong to different SCOP superfamilies. Detection of such homologs is accomplished with a slightly altered strategy from the mapping algorithm described above. The modified algorithm evaluates one SCOP superfamily at a time by attempting to detect potential hits to SCOP domains belonging to other superfamilies via the comparison methods described above. A set of query domains is constructed from the domains that are currently included in that SCOP superfamily (based on SCOP v1.63). As in the original mapping algorithm, the query sequences are first clustered at high sequence identity to reduce the computational time. Next, each of the 8 comparison methods described above is employed for each representative query. In the original mapping strategy, queries for which accepted hits are detected via gapped BLAST are not submitted to any of the other comparison methods. However, in this modified strategy, all comparison tools are run for all representative queries, regardless of the results of the gapped BLAST step. The output is a list of all accepted hits from each of the comparison methods to SCOP domains that do not belong to the query superfamily. All hits to SCOP domains within the query superfamily are simply ignored and excluded from the output. Finally, manual analysis of potential hits was performed for selected examples in order to evaluate the significance of those hits and to determine whether an evolutionary link is likely to exist between the two SCOP superfamilies in question.. SCOPmap results for representative PDB structures that are not included in the SCOP database are available here as well.The SCOPmap script and instructions for library construction are available for download at SC developed the code, tested program performance, analyzed the results, and drafted the manuscript. YQ contributed to code development. SSK determined score thresholds for the individual comparison tools used. LNK proposed many additional suggestions for improving algorithm performance. NVG conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.


Chronic headaches from head trauma and whiplash injury are well-known and common, but chronic headaches from other sorts of physical traumas are not recognized.Specific information was obtained from the medical records of 15 consecutive patients with chronic headaches related to physically injurious traumatic events that did not include either head trauma or whiplash injury. The events and the physical injuries produced by them were noted. The headaches' development, characteristics, duration, frequency, and accompaniments were recorded, as were the patients' use of pain-alleviative drugs. From this latter information, the headaches were classified by the diagnostic criteria of the International Headache Society as though they were naturally-occurring headaches. The presence of other post-traumatic symptoms and litigation were also recorded.The intervals between the events and the onset of the headaches resembled those between head traumas or whiplash injuries and their subsequent headaches. The headaches themselves were, as a group, similar to those after head trauma and whiplash injury. Thirteen of the patients had chronic tension-type headache, two had migraine. The sustained bodily injuries were trivial or unidentifiable in nine patients. Fabrication of symptoms for financial remuneration was not evident in these patients of whom seven were not even seeking payments of any kind.per se can be discounted as the cause of the headaches. So can fabrication of symptoms for financial remuneration. Altered mental states, not systematically evaluated here, were a possible cause of the headaches. The overall resemblance of these headaches to the headaches after head or whiplash traumas implies that these latter two headache types may likewise not be products of structural injuries.This study suggests that these hitherto unrecognized post-traumatic headaches constitute a class of headaches characterized by a relation to traumatic events affecting the body but not including head or whiplash traumas. The bodily injuries  Chronic headaches from head trauma -4 and whThe 15 patients in this series were all those seen by the author from February 1997 through December 2001 for chronic headaches apparently related to traumatic events that involved the body but did not cause either head trauma or whiplash injury  ,13. The Detailed information had been systematically collected on the headaches' characteristics , duration and frequency (when episodic), and accompaniments , and on the patients' use of pain-alleviative drugs. With this information, the headaches were classified by the 1988 diagnostic criteria of the International Headache Society (IHS) , extant identifiable damage to a part of the body . All but two of the events  were sudden and unexpected.The physical traumas were very diverse Table . None ocwithin minutes , within hours , within days , and within weeks . The interval for the one patient who did not have an abrupt traumatic event (patient 8) was arbitrarily set as the interval between his hospital discharge and headache onset.The reported intervals between the traumatic events and the headaches' onsets are listed in Table chronic tension-type headache [migraine without aura, and one of them also had headaches fitting the criteria for migraine with aura [Thirteen of the 15 patients had serious continuous headaches of varying intensity. Among these, 11 patients had headaches that met the IHS's diagnostic criteria for headache  fully, with aura . Table 1new daily-persistent headache, which has the same features as chronic tension-type headache, but is distinguished from it by becoming daily and unremitting within three days of its onset [chronic tension-type headache is used herein.After the completion of this study, the IHS added a new primary headache class called ts onset . Many, iThe IHS's criteria for chronic tension-type headache and migraine without aura  are listHeadache frequency more than 15 days/month.At least 2 of the following pain characteristics:1. Pressing quality2. Mild or moderate severity3. Bilateral location4. No aggravation by routine physical activityBoth of the following:1. No vomiting2. No more than one of the following: Nausea, photophobia or phonophobiaHeadaches last 4 to 72 hours.Headache has at least 2 of the following characteristics:1. Unilateral location2. Pulsating quality3. Moderate or severe intensity4. Aggravation by routine physical activityDuring headache at least one of the following:1. Nausea and/or vomiting2. Photophobia and phonophobiaThis 46-year-old man was seen in 1999 for headache that began five days after an "injury" at work five months earlier. Ten minutes after forcefully yanking a wrench to loosen a rusted bolt, he felt pain in his neck and right shoulder. An urgent-care facility prescribed analgesics after taking (unremarkable) cervical radiographs. This pain disappeared before I saw him. The headache was a continuous dull to moderate ache mostly in the right cranium. It was unaffected by physical activities or neck movements, and was not nauseating. Neurological examinations were normal. Pressure on his posterolateral neck was not painful. A cranial CT was normal. His only other symptom was insomnia. Analgesics, taken just a few days per week, had little effect. Amitriptyline lessened the headache's intensity enough for him to return to work.This 48-year-old man with a neurologic impairment of gait was seen in 1999 for continuous headache that began a few hours after he fell while walking two months earlier and cut his face on the edge of a metal sign. His head was not struck and he was not stunned. He bled profusely from the laceration, which was closed with 26 stitches in the emergency room. His neurologist detected no new neurologic findings and a cranial CT was normal. His headache was a non-nauseating steady ache of mild to moderate intensity in his forehead, unaffected by exercises, brightness, or noise. Non-prescription analgesics and opioids had been ineffective and discontinued.This 40-year-old man was seen in 2000 for a continuous headache that began 10 months earlier soon after he awoke from anesthesia for a cystoscopy. When the urologist did not report the (negative) result of the procedure to him in the recovery suite, the patient became visibly angry and soon complained about his treatment to the health-care facility. His anger persisted and was expressed at his consultation. The headache was a steady non-nauseating pain that fluctuated from dull to moderate intensity at the vertex, temples, and posterior neck, and was unaffected by physical activities, brightness, or noise. He worked despite it and no longer took analgesics. A cranial MRI had been normal. A trial of amitriptyline had been unsuccessful. At his last report a month later he reported improvement on buspirone.This 47-year-old woman was seen in 2001 for symptoms that she attributed to a snowmobile accident seven weeks earlier. She was seated behind the driver when he mistakenly shifted into reverse sending the machine backwards five feet into a tree. At impact, he fell on top of her without hurting her or himself. She recalled no impact of her helmet against the tree or the snowmobile. She felt no pain, but was upset and asked to be taken home. On the next day, her neck ached. Cervical radiographs taken eight days after the accident were normal. Two days later, she developed severe headache, nausea, dizziness, and confusion. A cranial CT taken later that day was normal. Subsequent MRIs of her head and neck were normal.Headache soon became her most prominent pain. It was a continuous pressing ache of mild to moderate intensity in her temples and orbits, unaffected by physical activities. It was sometimes nauseating, without emesis. Brightness and noise bothered her. She also complained of dizziness and impaired thinking and memory. Infrequent doses of analgesics were not beneficial. She was unable to work. Neurologic examinations were normal. Preventive medications were refused. When seen next, by a colleague, two months after her visit with me, the headache and dizziness had lessened considerably, but her thinking difficulties remained disabling. Four months later, she reported continuing improvement.This 53-year-old man was seen in 2001 for a headache of six-months duration that began two days after he struck the right side of his chest against a rock without striking his head when he toppled over in a beach chair. His chest pain was extreme. He obtained a prescription for hydrocodone/acetaminophen tablets that day, but discontinued taking them after several doses because of side effects. Coughing, sneezing, and lying on his right side were excruciating. His physician diagnosed a fractured rib. Two days after the injury, he returned to work despite his chest pain and new headache. The chest pain disappeared in a few weeks, but the headache persisted. It was a continuous, non-nauseating, bifrontal "tightness" of dull to moderate intensity, unaffected by mild physical activities, brightness or noise. Amitriptyline and propranolol had been ineffective and produced side effects. When I saw him, he was taking only occasional doses of non-prescription analgesics. Neurological examinations and cranial MRI were normal. He declined other medications. Seven months later, he reported that his headache persisted.This 43-year-old man was seen in 2001 for a continuous headache that he first became aware of soon after discharge from hospital, in 1997, where he had undergone 25 days of intensive treatment for a high-voltage electrical injury that had necessitated amputation of his arms. Unconsciousness had been instantaneous, but brief, and post-traumatic amnesia lasted about ten minutes.His headache was a bi-occipital, non-pressing ache, usually of mild to moderate intensity, and only occasionally severe enough to force him to cease physical activities. Then it was nauseating, without emesis. Some loud noises, but not brightness, seemed to intensify it. He had been getting slight relief from a few doses of ibuprofen per week, but had received no preventive medications.His cognitive and emotional states and cranial MRI were unremarkable. He had been provided with prosthetic upper limbs with grasping hands. Amitriptyline decreased the headache slightly. The addition of progressively larger doses of dextroamphetamine limited the headache to only a few days per month.Thirteen of the fifteen patients had, besides headache, at least one other post-traumatic symptom of the type commonly seen after head trauma ,17,18. SSeven patients  were not seeking and could not seek financial compensation for their headaches. Three patients  were receiving Workers' Compensation (WC) payments for their headaches and other symptoms. One patient (number 15) was receiving both WC and Social Security (SS) disability payments. One other receiving WC payments was also seeking SS disability payments (number 1). One patient (number 8) was seeking SS payments only. One patient (number 2) was litigating for payment of medical bills. Only one patient (number 4) had pursued a lawsuit for monetary compensation for the symptoms, but he continued to experience headaches even after receiving the award.This report has presented evidence suggesting that traumatic accidents and other events without head trauma or whiplash injury but with other physical effects can induce chronic headaches. Such headaches have barely been alluded to before (see Background). The 15 patients presented here blamed their headaches and other symptoms on their traumas and are supported in this by the juxtaposition of their headaches to the traumas. Twelve of the 15 patients developed their headaches minutes, hours, or a few days afterwards  and thereby foster their investigation.Both head-trauma and whiplash headaches form clinical classes based on their preceding traumas, but the headaches of the present series have no single trauma to join them. They were, however, all related to acute and unexpected (with one exception) traumatic events that affected the patients physically. Hence, they could be designated The headaches themselves were serious chronic headaches. They had the features of continuous chronic tension-type headaches in 13 patients and frequent migraines in the other two ,16. ThisThe apparent overall likeness of the traumatic-event headaches to those after head and whiplash traumas suggests a pathogenic link between them. Such a link would be discredited if, as many believe, headaches after head and whiplash traumas are due to structural injuries to the brain and neck, respectively ,24,27-29This study indicates that chronic headaches similar to those after head trauma and whiplash injury can follow other types of acute and unexpected physically injurious traumatic events. The evidence suggests that these traumatic-event headaches are psychogenic. The occurrence of such headaches supports the concept that the chronic headaches after head trauma or whiplash injury may likewise not be products of structural injuries.The author(s) declare that they have no competing interests.DCH was the sole investigator and author.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/4/17/prepub


The PTDS yields both a PTSD diagnosis according to the Diagnostic and Statistical Manual of Mental Disorders 4812 people living in Sarajevo or in Banja Luka in Bosnia-Herzegovina, of whom the majority had experienced a high number of traumatic war events, were administered the PTDS and other measures of trauma-related psychopathology. The psychometric properties of the instrument were assessed using Cronbach's alpha and principal components analysis, and its construct validity was assessed via Spearman correlation coefficients with the other instruments.The PTDS and its subscales demonstrated high internal consistency. The principal components revealed by an exploratory analysis are broadly consistent with the DSM-IV subscales except that they reproduce some previously reported difficulties with the "numbing" items from the avoidance subscale. The construct validity of the PTDS was supported by appropriate correlations with other relevant measures of trauma related psychopathology.The Bosnian version of the PTDS thus appears to be a time-economic and psychometrically sound measure for screening and assessing current PTSD. This self-report measure awaits further validation by interview methods. To obtain a diagnosis of PTSD and an estimation of PTSD severity a wide range of measures either relying on interviews or self-report exist in many languages. However, most of the relevant validation studies for these instruments were carried out for English-language versions . For manSelf-report instruments have several advantages as compared to interview measures. They are relatively economic in terms of administration and demand minimal clinician time. If clinicians are not familiar with psychiatric diagnostic procedures and especially the clinical diagnosis of PTSD, it is more advisable to use a psychometrically sound self-report measure which is less prone to mistakes than interview measures.A good self-report measure for PTSD should allow a diagnosis of PTSD as well as an estimation of PTSD severity and should conform to the DSM-IV criteria for PTSD . The EngIn terms of convergent validity, Foa, Riggs, Dancu, and Rothbaum  comparedThe symptom items of the PTDS, which reflect more or less verbatim the corresponding items in the DSM-IV criteria, in empirical studies do not necessarily fall into the three groups explicit in DSM-III-R and DSM-IV. The results of a number of factor-analytic studies suggest that the avoidance symptoms load on two separate factors -13. One Because of the many advantages of the PTDS we decided to use it for estimating rates of PTSD in a series of studies in different samples of war-traumatized inhabitants of Sarajevo and Banja Luka, Bosnia and Herzegovina. The results of these studies have been published elsewhere or are still in the process of being published -16.The PTDS had to our knowledge never been used before in the area of former Yugoslavia; instead, many studies have used similar but more or less ad-hoc constructed checklist versions of the DSM-IV criteria. The introduction of the PTDS would therefore mean providing clinicians and researchers with a sound Bosnian version of an internationally accepted PTSD self-rating instrument. The goal of this paper is to report first results of the psychometric evaluation of the Bosnian PTDS.Although all applied measures are questionnaires, not all subjects proved literate enough to complete them on their own. Therefore in some cases the interviewers had to read some of the questions to them and sometimes to reread or reformulate the questions. Thus the administration deviated slightly from the standard procedures.Posttraumatic Stress Diagnostic Scale  is a qule  and hasle  is a 90CL-90-R; ]. The SCThe Beck Depression Inventory  is probThe following data was collected between February 1998 and October 1999 in Sarajevo, Banja Luka and Prijedor, which are all in Bosnia-Herzegovina. Sarajevo is in the Federation of Bosnia and Herzegovina, namely that part of Bosnia and Herzegovina which has a predominantly Muslim and Catholic population, and Banja Luka and Prijedor are in the other part, the Republika Srpska, which is predominantly Serbian Orthodox. The samples were stratified by age and sex. The number of years of schooling was also recorded. All subjects participated voluntarily and gave fully informed consent. Table In total 812 persons participated. Inclusion criteria for all were a) age between 16 and 65, b) not suffering from a psychotic disorder and c) literate enough to answer the questionnaires with help. All subjects completed the PTDS and the SCL-90-R; therefore correlations for these subscales are based on the data of all the subjects. However for reasons of economy, in 1999 the full package of questionnaires including the BDI and IES were only administered to a random selection of participants in only the two Sarajevo sub-samples. All other participants in 1999 only answered a smaller package of questionnaires including the PTDS. Correlations between the PTDS and BDI and IES are therefore based on a smaller dataset.In 20 cases an entire instrument was missing, as detailed in table The medical and psychological samples were assessed through a total of 15 experienced counsellors/therapists, who were working at a variety of clinics and counselling centres in Sarajevo. All other samples were assessed by pairs of final year and third year students of Psychology at Sarajevo University and Banja Luka University. All interviewers were trained in the use of the questionnaires. Two pilot studies were performed to insure the appropriate use of the assessment. During the studies constant supervision for all interviewers was provided.To obtain an estimation of internal consistency Cronbach's alpha was calculated for the total scores and the subscales of the PTDS. Convergent and divergent validity were estimated by using Spearman correlations between the scales. Spearman correlations were used because most of the distributions were not normal. For the principal components analysis, oblimin oblique rotation was used.The standardised Cronbach's alphas for the Bosnian PTDS were .93 for the total symptom score, .89 for the reexperiencing subscale, .84 for the avoidance subscale and .84 for the arousal subscale. The results correspond well with other published results.The Spearman's correlations between the total scale and the subscales were all quite high at .89, .93 and .87 for re-experiencing, avoidance and hyperarousal respectively; re-experiencing correlated .74 and .67 with avoidance and hyperarousal; and the correlation between avoidance and hyperarousal was .72.The item characteristics for the symptom items and subscale totals are shown in table The items from the symptom subscales were submitted to a principal components analysis with oblimin oblique rotation. Factors with eigenvalues greater than 1 were retained. Items were considered as belonging to a factor if their loadings on that factor were above 0.4.  found ourselves in, namely that very few world-standard instruments existed. If one does find, as we did, inter-instrument correlations similar to those for the corresponding instruments in other languages then that provides at least some provisional evidence for the psychometric quality and construct validity of all of those instruments.One of the main uses of the PTDS is to provide a PTSD diagnosis in an economical way. As the PTDS assesses in questionnaire form all the information necessary for the diagnosis according to DSM-IV, the PTDS prevalences can be easily calculated and are in fact 24.72% for the whole sample, 31.37% for women and 17.40% for men.The most important factor which restricts the interpretation of these results is that the PTDS was not compared with clinical interview, which would have been standard procedure in this kind of study. However, when we began the study there was no suitable validated interview available in the Bosnian language, which meant that we would have had to translate and extensively validate such an interview ourselves, and again we would have run into the problem of validating the interview against instruments which had also not been validated at that time. It also should be stressed that this study says very little about the cultural or contextual validity of the instrument or the construct PTSD which it is intended to measure.On the other hand, the samples are quite large and taken together quite heterogeneous, and the selection methodologies in each case provided a reasonable approximation to randomness, so that all in all the data can be considered to be of good quality.In conclusion it can be said that the psychometric properties of the Bosnian version of the PTDS are as good as those published for other languages. The internal consistencies are at least as good and the Bosnian version appears even to distinguish a little better than the American and German versions between PTSD as measured by the IES and depression as measured by the BDI. The principal components revealed by an exploratory analysis are broadly consistent with the DSM-IV subscales except that they reproduce some previously reported difficulties with the "numbing" items from the avoidance subscale; this issue might explain the poor specificity of the avoidance scale with respect to the IES subscales. None of the analyses revealed anything unusual or indicated problems either with the translation or with the application of the concepts inherent in the instrument to the post-war Bosnian population, all of which indicates that the Bosnian PTDS can be given the green light for further application in the future. Yet our results are only a necessary first step in the validation of the applied measures; a comparison with a validated translation of a Bosnian interview measure for PTSD still needs to be done.The author(s) declare that they have no competing interests.RR participated in the design of the study, and drafted the manuscript.SP carried out the actual study and performed the statistical analysis.Both authors worked on and approved the final manuscript.group 0: injury to selfWere you severely injured during the war?group 1: sexual violence to selfWere you raped or sexually assaulted during the war?During the war, were you sexually assaulted by a member of your close family who had been forced to do that?During the war, were you sexually assaulted by a member of your close family who was not forced to do that?group 2: torture to selfWere you tortured during the war?group 3: other threat to selfDuring the war, were you in a situation in which you strongly believed you would be severely injured or killed?During the war, did a bullet come so close to you that you could have been severely injured or killed?During the war, did a bomb or grenade explode so close to you that you could have been severely injured or killed?During the war, did anyone threaten to kill you or severely injure you?Were you captured or held in a detention camp during the war?During the war, were you without food or water for so long that you strongly believed you would die?During the war, were you so cold that you strongly believed you would die?During the war, did you stay in a cellar longer than 3 weeks without a break?During the war, were you assaulted in a non-sexual way by a member of your close family who had been forced to do that?During the war, were you assaulted in a non-sexual way by a member of your close family who had not been forced to do?Were you in the army during the war?During the war, were you seriously ill because of the war (e.g. heart attack)group 4: witnessed: loved onesDid you eyewitness a loved one being killed during the war?Did you see dead body of a loved one who had been killed in the war? Did you see a loved one being tortured or physically assaulted during the war?Did you see a loved one being sexually assaulted during the war?Did you touch a loved one who had been killed or wounded in the war?During the war, did you see a loved one who was severely injured before he/she received medical help?group 5: witnessed: othersDid you eyewitness somebody being killed (not a loved one) in the war?Did you see the body of a person (but not a loved one) who had been killed in the war? Did you see someone being tortured or physically assaulted during the war (but not a loved one)?Did you see someone being sexually assaulted during the war (but not a loved one)?Did you touch someone (but not a loved one) who had been killed or wounded in the war?During the war, did you see a severely injured person (not a loved one) before they received medical help?group 6: losses, nuclear familyWas your father killed in the war?Was your mother killed in the war?Was your spouse killed in the war?Was a child of yours killed in the war?Was a brother or sister of yours killed in the war?group 7: losses, other loved onesWas a close relative of yours killed in the war?Was a close friend of yours killed in the war?group 8: threat, violence, injury to loved onesWas a loved one in the army during the war?Was a loved one severely injured in the war?Was a loved one raped or sexually assaulted in the war?Was a loved one tortured in the war?Was a loved one captured or held in a concentration camp during the war?During the war, was a loved one seriously ill (e.g. cancer or heart attack) or had some chronic health problem?group 9: other war eventsOther traumatic event since 1991 due to war: 1Other traumatic event since 1991 due to war: 2Other traumatic event since 1991 due to war: 3group 10: other events since 1991 not related to warDid a loved one die in the war for reasons unrelated with the war?(Other stressful and traumatic events since 1991 and unrelated to the war)The pre-publication history for this paper can be accessed here:


Cryptococcus revealed sequential evolutionary events that fashioned this large, highly unusual region. We hypothesize that MAT evolved via four main steps, beginning with acquisition of genes into two unlinked sex-determining regions, forming independent gene clusters that then fused via chromosomal translocation. A transitional tripolar intermediate state then converted to a bipolar system via gene conversion or recombination between the linked and unlinked sex-determining regions. MAT was subsequently subjected to intra- and interallelic gene conversion and inversions that suppress recombination. These events resemble those that shaped mammalian sex chromosomes, illustrating convergent evolution in sex-determining structures in the animal and fungal kingdoms.Sexual identity is governed by sex chromosomes in plants and animals, and by mating type (MAT) loci in fungi. Comparative analysis of the MAT locus from a species cluster of the human fungal pathogen  A comparative genomic analysis of the sex determining region in fungi reveals a remarkable similarity between its evolution and the events which shaped mammalian sex chromosomes Saccharomyces cerevisiae, the a and α mating types are established by the mating type (MAT) locus, which spans only 642 bp or 747 bp, respectively, and encodes only one or two cell type factors  and contains more than 20 genes , and the MAT locus has been characterized in both . a, the only MAT homeodomain proteins) with the locus composed almost entirely of divergent alleles of a common gene set. Fourth, the MAT gene cohort has been dramatically rearranged during evolution (Cn. var. neoformans at Stanford and The Institute for Genomic Research (TIGR), Cn. var. grubii at Duke and the Broad Institute, and C. gattii at the University of British Columbia and the Broad Institute, reveal that these rearrangements at MAT are highly atypical compared to the non-MAT regions of the genome in all three species . In addition to the original set of shared genes . In total, genic sequences comprise approximately 50% of MAT  pathway. The second and third classes  represent intermediate genes that have progressively higher nucleotide identity between the a and α alleles, and less discrete, although still MAT-specific, phylogenetic patterns  began diverging after this time. The tree topologies of STE20, ZNF1, SPO14, and RPO41 were shown to be statistically different (p < 0.0001) by the Shimodaira-Hasegawa test , but is a flanking gene in the third (Cn. var. neoformans), providing a unique opportunity to test this model ,, intermediate class I (2.00 > sK > 0.70), intermediate class II (0.60 > sK > 0.35), and most recently acquired genes (sK < 0.17)  see . These gCryptococcus MAT locus are no longer stratified by age, because the members of the four identified classes have been heavily shuffled during speciation and mating type divergence, and this rearrangement appears ongoing. However, an analysis of relative gene locations in MAT reveals two alleles in which higher levels of synteny are evident: Cn. var. grubii MATα and C. gattii MATa , followed by more recent acquisition of the intermediate class I genes, whose role in mating has not yet been studied (2.00 > sK > 0.70). In constrast, the intermediate class II genes (0.60 > sK > 0.35) were recruited by expansion of the ancient tetrapolar homeodomain-containing locus. In this model, the expanding ancestral loci are separated by the five genes that exhibit species-specific phylogenies  the LPD1 gene exhibits this hybrid phylogeny, but in C. gattii the 5′ region in both mating types resembles the α alleles, and a sK value of zero for this region is indicative of recent gene conversion . Second, the number of pheromone genes varies between alleles. The a alleles contain three unlinked 130-bp aMF pheromone genes embedded in 900–5,000 bp amplicons identical within an allele but not between species. In contrast, the alleles contain three or four MFα pheromone genes embedded in approximately 500-bp conserved repeats, usually flanking the syntenic PRT1/ZNF1 gene pair in inverted orientation. These repeats are likely maintained by intra-allelic gene conversion, ensuring maintenance of these important fertility genes in the absence of meiotic recombination. This is a striking difference to the MAT locus of S. cerevisiae, where gene conversion plays the unrelated and very distinct role of driving mating type switching  into the ancestral pheromone/pheromone receptor locus (ancient class). This was followed by a second round of acquisition of genes with an unknown role in mating (intermediate class I). Next, the ancestral homeodomain locus acquired genes that we hypothesize function in the dikaryon or meiosis  based on their known roles in S. cerevisiae and Ustilago maydis . This created an intermediate tripolar mating stage in which one mating type had one large contiguous MAT locus and the partner retained the ancestral gene clusters formed from the unlinked tetrapolar loci. During this transitional tripolar intermediate stage, only half of the meiotic progeny would be fertile. The incipient alleles of the opposite mating type were then either gene converted onto the newly-formed bipolar chromosome or, more likely, were linked via dual recombination events , collapse system .SXI1α is now linked to the aSTE3 pheromone receptor gene, and in which aSXI2 is linked to the STE3α pheromone receptor gene. These recombinants could mate with each other, but would not complete the sexual cycle with the original MATa and MATα members of the population, because while cell-cell fusion would occur, two copies of only one of the homeodomain-encoding genes would be present, and both are required for completion of the sexual cycle . Thus, whereas the parental strains could mate with 50% of the population, the recombinants could not and would have a disadvantage. This selective pressure for fecundity could have driven the inversions that we hypothesize occurred to prevent recombination between the two linked sex-determining regions.Thus, we hypothesize that a chromosomal translocation juxtaposed the two previously unlinked sex-determining regions and produced a bipolar mating type system in which the two distinct regions are linked by a common block of sequence information. Subsequently, inversions occurred that obscured these boundaries and, as a consequence, suppressed recombination between the two regions. What was the evolutionary pressure to suppress recombination between the two linked loci, leading to their rearrangement in not only one but all three lineages? If the ancestral tetrapolar system was not multiallelic, and the four mating types in the population were in equal proportions, then any given individual in the population could mate with only 25% of the other members. However, in a population in which the two loci are linked, any individual can mate with 50% of the other population members. A recombination event in the common spacer region would result in two new mating types in which Cryptococcus, the events that led to the initial formation of the homeodomain- and pheromone/pheromone receptor-based gene clusters involved in sexual processes are less clear. How can recombinationally suppressed sites such as the original tetrapolar loci initiate expansion to create a larger nonhomologous region? One likely hypothesis involves the presence of the large number of transposable elements in the genome. It has been suggested that the spread of a mobile element through a population can be facilitated by increasing the probability of sex in its host  may play related roles. Finally, Y-specific genes are maintained by intrachromosomal recombination and repair of genes embedded within palindromes in an inverted orientation  during the mammalian life cycle. By contrast, in fungi such as Cryptococcus for both the a and α MAT alleles, and therefore for their host chromosome. The MAT locus occupies 6% of the 1.8 MB chromosome on which it resides, and thus has not expanded to occupy nearly the entire chromosome, in contrast to the mammalian sex chromosomes. A more similar analogy to the fungal MAT locus is the sex chromosomes of the plant papaya (Carica papaya), in which the sex-determining region occupies only about 10% of the 41-MB primitive Y chromosome  and E566 (a) from the Australian environment. E. coli DH5α was used as the library host strain. LB or FB media supplemented with the appropriate antibiotic was used for E. coli culture  libraries and analysis of the mating type alleles were  culture .C. gattii serotype B strains WM276 and E566 were constructed in pBACwich (Cn. var. neoformans to identify overlapping sequences that span the MAT locus (Large-insert libraries (insert sizes 100–120 kb) for the candidate pBACwich , a derivpBACwich , using HpBACwich . Clones pBACwich  and hybrAT locus . BAC DNAAT locus  that werAT locus . 1,100 cSequencing reactions were performed with an MJ Research  thermal cycler using standard BigDye chemistry  and analyzed on an Applied Biosystems PE3700 96-capillary sequencer. Sequence reads were assembled using the PHRED/PHRAP/CONSED package . AdditioC. gattii sequences based on homology to the existing annotation in C. neoformans; in some cases, this led to revision of the C. neoformans annotation. Additional genes that did not have previously defined homologs  were found by comparing the six available MAT allele sequences and by identifying large regions of identity that were unassigned to genes. Based on ClustalW v1.4 .Genes were annotated in the alW v1.4  alignmensK values for comparison of the a and α alleles of all MAT protein-coding genes in each lineage were calculated using DnaSP 3.51  and those in the central portion correspond to those genes hypothesised to have entered the MAT locus most recently. The additional region of identity present in Cn. var. neoformans due to the fixation of the α alleles of the NCM1, BSP3 and IKS1 genes in the MATa allele is circled in blue. Scale is given in kb.The entire MAT(64 KB EPS).Click here for additional data file.Figure S2Phylograms were generated for each protein-coding gene in MAT under the maximum parsimony criterion in an exhaustive search. Numbers next to branches indicate statistical support as calculated in 1,000 bootstrap replicates. Shaded trees indicate genes with an unusual phylogenetic pattern, due to either gene conversion or a hybrid phylogeny pattern.(218 KB EPS).Click here for additional data file.Figure S3The phylograms represent the single most likely trees for each protein-coding gene in the MAT locus. Trees were generated under the best-fitting evolutionary model in an exhaustive search. Numbers besides branches indicate statistical support as calculated in 1,000 maximum likelihood bootstrap replicates. Shaded trees indicate genes with an unusual phylogenetic pattern, due to either gene conversion or a hybrid phylogeny pattern.(225 KB EPS).Click here for additional data file.http://www.ncbi.nlm.nih.gov/) accession numbers for the MAT locus alleles discussed in this paper are WM276 (AY10430) and E566 (AY10429). GenBank accession numbers for other genes discussed in this paper are Cn. var. grubii MATa (AF542528), Cn. var. grubii MATα (AF542529), Cn. var. neoformans MATa (AF542530), and Cn. var. neoformans MATα (AF542531).GenBank (


Drosophila, we present the first high-resolution genetic study of variation within species for female mating discrimination that is enhanced by natural selection. We show that reinforced mating discrimination is inherited as a dominant trait, exhibits variability within species, and may be influenced by a known set of candidate genes involved in olfaction. Our results show that the genetics of reinforced mating discrimination is different from the genetics of mating discrimination between species, suggesting that overall mating discrimination might be a composite phenomenon, which in Drosophila could involve both auditory and olfactory cues. Examining the genetics of reinforcement provides a unique opportunity for both understanding the origin of new species in the face of gene flow and identifying the genetic basis of adaptive female species preferences, two major gaps in our understanding of speciation.Reinforcement occurs when natural selection strengthens behavioral discrimination to prevent costly interspecies matings, such as when matings produce sterile hybrids. This evolutionary process can complete speciation, thereby providing a direct link between Darwin's theory of natural selection and the origin of new species. Here, by examining a case of speciation by reinforcement in  Mating discrimination between two species of Drosophila is more pronounced where they hybridize and genes related to odor appear responsible for this "reinforcement," thus providing insights into the genetics of speciation During reinforcement, mating discrimination is strengthened by natural selection in response to maladaptive hybridization between closely related taxa . Althoug-allele models, alleles conferring mating discrimination spread if they become genetically correlated with alleles reducing hybrid fitness. However, the evolution of such a correlation is opposed by recombination because alleles conferring discrimination in a given species do not confer discrimination in the other species. Consequently, two-allele models require either very strong selection, or tight linkage  between alleles conferring mating discrimination and alleles reducing hybrid fitness  or different (two) alleles conferring mating discrimination spread in the emerging species (Felsenstein 1981). In two fitness . In cont fitness  and so m fitness .Drosophila and present for the first time a high-resolution genetic study of variation within species in female mating discrimination, including a set of candidate reinforcement genes and a discussion of the evolutionary implications of our findings.In addition to providing fundamental information for theoretical models, discerning the genetics of reinforcement will also develop our understanding of both the physiological basis of and forces governing changes in female preference. Furthermore, because the strengthening of female preference is driven by natural selection, the genetics of reinforcement will provide unique insights into the genetics of adaptation, another unsettled issue in evolutionary biology. Finally, high-resolution genetic studies of reinforcement can identify candidate speciation genes with effects on mating discrimination, information almost nonexistent in speciation studies but see, . Here, wDrosophila pseudoobscura and D. persimilis hybridize in nature and produce sterile male hybrids. While D. pseudoobscura occurs alone in non-coastal western United States and Central America, the two species co-occur in California and the Pacific Northwest. Males court females from both species indiscriminately  while females derived from populations where D. persimilis is present exhibited strong mating discrimination (hereafter “reinforced mating discrimination”). This difference in mating discrimination is likely the evolutionary consequence of maladaptive hybridization where the two species coexist: reinforcement has strengthened mating discrimination in the D. pseudoobscura populations co-occurring with D. persimilis. These observations and the recent completion of the genome sequence of D. pseudoobscura (BCM-HGSC 2004) make these species an ideal system to genetically dissect the enhancement of mating discrimination in sympatry.The North American fruitflies minately , but femD. pseudoobscura, or in any system, the genetic basis of other traits contributing to the species' reproductive isolation  is known in detail. All traits contributing to reproductive isolation between D. pseudoobscura and D. persimilis, including traits for basal discrimination, map primarily or exclusively to regions bearing fixed chromosomal inversion differences between the species  and mating discrimination creates the necessary genetic correlations to advance divergence in the presence of gene flow. However, we do not know whether the genetic basis of reinforced mating discrimination corresponds to this picture, and specifically, whether chromosomal inversions are fundamental to this process. Comparing these genetic architectures will provide the most comprehensive view yet on the genetics of mating discrimination contributing to the formation of new species in the face of interspecies gene flow.Although the genetics of reinforcement has not been studied in  species , 2001b. D. pseudoobscura females derived from sympatry (with D. persimilis) exhibited stronger mating discrimination against D. persimilis males than did D. pseudoobscura females derived from allopatry. This pattern holds for both inbred and outbred lines. Also, our data show that both sympatric-derived lines and allopatric-derived lines vary considerably in their degree of discrimination , suggesting some within-population variation in female mating discrimination, both basal and reinforced.D. persimilis males when exposed to sympatric or allopatric D. pseudoobscura females. To exclude this possibility, we measured the copulation latency and number of attempted copulations by D. persimilis males towards D. pseudoobscura females derived from sympatry or allopatry, and found no significant differences between groups . Finally, we investigated the mode of inheritance of the phenotype and observed that F1 females from crosses between sympatric and allopatric flies discriminated as strongly as their sympatric parent, suggesting that reinforced female mating discrimination is inherited as a dominant trait in both inbred and outbred lines . Taken together, these results suggest that reinforced mating discrimination in D. pseudoobscura is exclusive to females derived from areas of sympatry with D. persimilis, is inherited as a dominant trait, and is not markedly affected by inbreeding.Apparent reinforced mating discrimination could result from behavioral differences in ines see . This F1D. pseudoobscura populations. In each case, we performed a male-parent backcross in which a mixture of whole chromosomes from sympatry and allopatry (F1 genome) was substituted into an allopatric background (F2 backcross genome) . For example, sympatric X and fourth chromosomes derived from Mather, California , contribute significantly to reinforced mating discrimination , while the same chromosomes show no detectable effect on reinforced mating discrimination when derived from Mt. St. Helena, California   see A and 2B.D. pseudoobscura despite extensive gene flow within and between populations , and a line derived from Flagstaff, Arizona , and genotyped 275 to 1,500 individuals for 70 markers dispersed along the four major chromosomes in D. pseudoobscura. Our initial single-marker analyses revealed significant associations between reinforced mating discrimination and three regions defined by markers located on the right and left arms of the X chromosome   and the fourth chromosome . We were not able to detect an effect of any single region on the third chromosome even though nine markers were surveyed. Effects identified on XR and Chromosome 4 reinforced mating discrimination when the sympatric allele was present (positive), while the effect from XL was negative. After our initial scan, we used composite interval mapping (CIM) to account for any inflated estimates in the absence of background correction. In addition, several markers were genotyped around the X021 and 4034 regions with the goal of refining the segments containing the quantitative trait loci (QTLs). We measured female mating discrimination against Coy-1, is estimated to lie between two additional markers, X021-A1 and X021-A4 . According to the recently obtained genome sequence of D. pseudoobscura, there are seven genes between these two markers, one of which, bru-3, accounts for one-third of the sequence length of this region. In addition, CIM also identified another QTL, hereafter referred to as Coy-3, located between markers X021 and X021-B2, although with a weaker effect (CIM LOD score = 2. 84). There are approximately 200 kb and 30 genes between these markers and a recombination fraction of 3.5 cM. Finally, a third QTL was found near the XL telomere and, in contrast to the X021 region, showed a negative and weak additive effect (CIM LOD score = 2. 45). We tested this model for the X chromosome using multiple interval mapping and found that the strongest support is for Coy-1, followed by Coy-3. We were unable to recover any support for the QTL on XL. No epistatic interactions were detected among any QTLs.A more careful examination of the X021 region showed that the QTL location (CIM LOD score = 5. 16), hereafter referred to as Coy-2 and Coy-4, respectively. These QTLs show the strongest effects . As with Coy-1 and Coy-3, these QTL are additive and contribute positively to the degree of mating discrimination of F2 females. Coy-2 is located next to marker 4034-A8. This marker is within a 300-kb region homologous to a D. melanogaster region containing a p-element insertion disrupting normal olfactory behavior (see Discussion for details) . However, a more careful examination of the genes shows that their sequence overlap results from the inability of BLAST homology searches to distinguish the members of this gene family, suggesting that there may be only one or few UDP-glycosyltransferase genes here. Consequently, the number of candidate genes in the region may be reduced from nine to five genes, at least one of which is involved in olfaction. As before, we tested this model using multiple interval mapping and recovered significant support for Coy-2 under stringent conditions and no evidence of significant epistasis among previously identified QTLs.Dissection of the 4034 region using CIM split the effect into two QTLs for reinforced mating discrimination; we refer to them as details) , 2003. TCoy-1 and Coy-2, and that Coy-3 and Coy-4 are suggestive QTLs.Based on these results and those for the X chromosome, we suggest that the strongest evidence for QTLs contributing to reinforced discrimination in sympatry lies with Drosophila pseudoobscura. The resolution of our approach is novel to genetic studies of behavioral discrimination in that we have surveyed the genome with 70 microsatellite markers for chromosomal regions contributing to increased mating discrimination and have narrowed some of these regions to intervals containing as few as five genes. The role of these genes in reinforcing mating discrimination is supported by indirect evidence from D. melanogaster mutants: two of the major QTLs identified in our mapping experiments bear genes identified in smell impairment screenings of p-element mutants (CG13982 (D. melanogaster Chromosome 2L), appears to up-regulate a second gene located in the other interval, bru-3 (D. melanogaster X chromosome). Furthermore, we have shown that the chromosomal contributions to reinforced mating discrimination vary among strains of D. pseudoobscura. Finally, the chromosomal effects on mating discrimination are inherited in a dominant fashion, consistent with general theories on the evolution of adaptive characters  show that all QTLs for this trait map unequivocally to two inverted chromosomal regions separating it from D. persimilis ((DPS4003) just 400 kb away from the QTL identified on the fourth chromosome seems to have introgressed recently between D. pseudoobscura and D. persimilis  was striking in this regard, supporting a potential role of olfactory response in reinforcement in these species.Our results suggest, albeit not conclusively, that reinforced mating discrimination is related to differences in response to olfactory cues. We have shown here that candidate regions on the fourth chromosome bear an unusual excess of olfactory genes, and some of these have been associated with specific olfactory responses in other D. melanogaster variation.We also observed differences among strains in the genetic architecture of reinforced mating discrimination. Such variation in genetic control may be common when populations exchanging genes differ in phenotype because of selection. Multiple alleles from different loci may have increased in frequency because of selection for discrimination, and these alleles sometimes spread into allopatry or are replaced by the allopatric alleles in sympatry. When sampling from single lines, we capture only a fraction of the genetic variation in mating discrimination, and sometimes a high-discrimination allele is even sampled from allopatry (as we observed in the QTL on XL). This observation should be typical in many QTL mapping studies that utilize strains within species with extensive gene flow among populations, as in the many studies of D. pseudoobscura is a composite trait, and there is genetic variation within species in reinforced mating discrimination.In brief, these results show that basal and reinforced discrimination are different, species discrimination in 1 female offspring of crosses between allopatric and sympatric populations of D. pseudoobscura are as reluctant to mate with D. persimilis males as are D. pseudoobscura females from sympatric populations. This result implies that high discrimination can be expressed in heterozygous individuals, suggesting a dominant basis for the phenotype. In contrast, “basal” female mating discrimination seems to be a recessive trait . Taken D. pseudoobscura, (1) high discrimination in sympatry is inherited in a dominant fashion, (2) there is within-species variability for high female mating discrimination as evidenced by the different genetic architectures recovered in the male-parent backcross experiments, (3) there are multiple genes, possibly involved in olfaction, contributing to enhanced female mating discrimination, (4) some candidate genes for reinforcement identified here have been previously identified in p-element mutant screenings for smell impairment in D. melanogaster, (5) the genetic architecture of basal female mating discrimination is different from that of reinforced mating discrimination, and (6) inversions seem to play no direct role in creating or maintaining the genetic differences directly responsible for increased female mating discrimination in sympatry. However, these inversions seem to play a crucial role, as evidenced by previous studies , (2) identify chromosomal regions contributing to reinforced mating discrimination  and compare them to regions conferring basal mating discrimination, and (3) provide a set of candidate genes for increased mating discrimination.Our approach is based on measuring in one species the effects of substituting chromosomal segments from a highly discriminant genome into a less discriminant genome. In particular, we (1) test for within-species variation in the genetic architecture of female mating discrimination , Mt. St. Helena, California (1997), Flagstaff, Arizona (1993 and 1997), and Mesa Verde National Park, Colorado (2001). Isofemale lines were established by rearing the offspring of individual females previously mated in the wild. All lines were maintained under a constant regime of temperature (20 °C) and humidity (85%) in diurnal/nocturnal cycles of 12 h and reared on a mixture of agar, dextrose, and yeast.D. pseudoobscura isofemale lines from each of two populations were crossed: Mather (1997) 52 × 10  and Flagstaff (1997) 16 × 17 , respectively. Virgin F1 females from these crosses as well as D. persimilis males were routinely collected during afternoons and confined for 8 d. On the morning of the eighth day, individual females were confined with individual D. persimilis males. The rationale of this no-choice design is based on behavioral observations suggesting that females tend to copulate more often in the presence of single males than when multiple males approach them . Therefore, no-choice experiments should provide a more conducive setting for mating. The flies were observed for 10 min. If the male attempted fewer than three copulations, the pair was not scored, and the data were discarded. Otherwise, the pair was scored for successful copulation versus not . These protocols are the same used in D. pseudoobscura lines sympatric versus allopatric to D. persimilis. Comparisons between allopatric and sympatric populations were performed both for outbred lines and inbred lines and only between lines that were tested for the phenotype at the same time, thus controlling for environmental error. Our comparisons between the two allopatric lines and between the two sympatric lines were not temporally controlled, and therefore may have been subject to some environmental heterogeneity. We used pairs of D. pseudoobscura inbred lines that significantly differed in their degree of female mating discrimination against D. persimilis in our mapping experiments (see below).Pairs of D. persimilis males of F1 females resulting from crosses between sympatric and allopatric D. pseudoobscura lines. If F1 females discriminated as strongly as the parent derived from sympatry, then we concluded that higher (reinforced) mating discrimination was inherited as a dominant trait. Fisher exact tests where performed to evaluate this hypothesis  and the multipoint-linkage approach implemented in MapMaker version 3.0 (Microsatellite markers include those reported previously  and 100  project . Microsasion 3.0 .n1 = 900 and n2 = 600 flies) were used to determine the chromosomal basis of reinforced mating discrimination and its natural within-species variation. Crossing over does not occur in male Drosophila, and they thus transfer whole chromosomes to their offspring , and its DNA was subsequently extracted. Lines used in each backcross were: for backcross 1, Mather  17 and Flagstaff (Arizona) 1993, and for backcross 2, Mt. St. Helena  7 and Mesa Verde (Colorado) 17. We consider strains derived from California as sympatric and strains derived from Arizona or Colorado as allopatric to D. persimilis.Two male-parent backcrosses  accession numbers for the genes discussed in this paper are bru-3 (FBgn0036379) and CG13982 (FBgn0031811).The Flybase (


The researchers isolated lung lymphocytes from the samples and used two-color flow cytometry to phenotypically characterize the cells. They found that lymphocytes taken from patients with emphysema expressed more CCR5 and CXCR3 receptors, which are associated with a particular type of T cell called T helper 1 (Th1), than did those from control individuals. By contrast, expression of CCR4 receptors, which are found on T helper 2 (Th2) cells, was very low in both control and emphysema groups.In a separate experiment, Kheradmand's team showed that lung lymphocytes taken from patients with emphysema secreted more of three other proteins—interferon gamma, monokine induced by interferon (MIG), and interferon-inducible protein 10 (IP-10)—than control patients. MIG and IP-10 are known to be produced by injured epithelial cells and are ligands for CXCR3 receptors, which are expressed by Th1 cells. Importantly, the researchers were also able to show that isolated peripheral lung macrophages secreted matrix metalloproteinase-12 (MMP12), an enzyme that degrades elastin—a protein important for lung elasticity—in the lungs, in response to IP-10 and MIG. Together these findings, say the authors, indicate that Th1 cells, but not Th2 cells, are required for producing the elastin-destroying lung environment of emphysema.The researchers now intend to investigate the antigens that drive the Th1-based inflammation that underlies emphysema. “Ultimately, we seek to understand the biochemistry of tobacco smoke that triggers inflammation in the first place, and whether such insight might explain other environmentally triggered lung diseases,” explains Kheradmand. “To understand such detailed immune mechanisms, we really need an improved experimental model of disease, and this we are currently working on.”


The neuroprotective effect of xenon has been demonstrated for glutamatergic neurons. In the present study it is investigated if dopaminergic neurons, i.e. nerve-growth-factor differentiated PC-12 cells, are protected as well against hypoxia-induced cell damage in the presence of xenon.2-saturated atmosphere, a treatment that induced release of dopamine, reaching a maximum after 30 min. By determining extracellular lactate dehydrogenase concentration as marker for concomitant cellular damage, a substantial increase of enzymatic activity was found for N2-treated cells. Replacement of N2 by xenon in such a hypoxic atmosphere resulted in complete protection against cellular damage and prevention of hypoxia-induced dopamine release. Intracellular buffering of Ca2+ using the Ca-chelator 1, 2-bis(2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester (BAPTA) reduced the neuroprotective effect of xenon indicating the essential participation of intracellular Ca2+-ions in the process of xenon-induced neuroprotection.Pheochromocytoma cells differentiated by addition of nerve growth factor were placed in a NThe results presented demonstrate the outstanding property of xenon to protect neuron-like cells in a hypoxic situation. Many studies have been performed on the mechanisms of glutamate-induced neuronal damage ,2 but real cells  possessial cells . In thesal cells ,11.Numerous approaches have been undertaken to reduce hypoxia-induced neurotoxicity ,12. The Cells kept under normoxic conditions did not release dopamine during the time period studied. If, however, they were kept in an atmosphere consisting of 100% nitrogene, considerable amounts of dopamine were found in the extracellular space reaching a maximum at 30 min of incubation, followed by a subsequent decrease. If under the same conditions nitrogen was replaced by xenon, no such increase in dopamine concentration was found Fig. . The levIn order to test if such hypoxia damaged the cells, extracellular LDH was determined after a two-hour period of treatment. A low level of LDH was found in cells kept under normoxic conditions whereas cells kept under nitrogen showed a significant release of LDH indicating severe cellular damage Fig. . If instHypoxia-induced extracellular increase of dopamine could be caused either by elevated release of dopamine or by a reduced, or even inhibited, dopamine uptake. If hypoxia caused faster release but did not interfere with uptake, uptake-inhibitors would cause a higher concentration of dopamine in the extracellular space. On the other hand, if the release was constant but the re-uptake inhibited by hypoxia, additional inhibition of uptake by inhibitors would have no or little effect. In the presence of 5 nM of the dopamine reuptake inhibitor GBR 1209 the extracellular dopamine concentration did not change in a normoxic or xenon environment. However, in nitrogen the extracellular dopamine concentration did not reach exactly the same value as in pure nitrogen, the dopamine level was slightly but significantly reduced, thus supporting the view that hypoxia-induced extracellular dopamine increase was caused by an enhanced release of dopamine and – to a lesser extent – an interference with the uptake mechanism . Surprisingly, such chelation reduces significantly the neuroprotective effect of xenon, indicating an essential role for intracellular Ca2+ for this effect to occur. A slight but significant reduction in cellular damage is observed when BAPTA-treated cells are incubated in nitrogen-saturated buffer.In order to test if changes in intracellular CaTo exclude that the results obtained were limited to the PC-12-system itself, hypoxia-induced dopamine release and cell damage was investigated in rat embryonic primary mesencephalic cell cultures that are known to contain 0.5 – 1.5% of dopaminergic cells . As show2+-regulated events was obtained when it was shown that xenon blocked cells in metaphase and that the block could be lifted by artificial small intracellular Ca2+- increases [2+-dependent regulatory events [2+-dependent systems. One of those may be the Ca2+/calmodulin-activated calcineurin system that has been implicated in the regulation of monoamine release [2+-dependent events required to occur in hypoxia-induced cell damage. Such a scenario is suggested by our demonstration that the neuroprotective effect of xenon is strongly reduced if PC-12 cells are loaded with BAPTA. Thus, at present all evidence obtained by us [2+-regulating and -regulated systems up to the activaton of transcription factors whereby such targets are probably not essentially and sequentially linked to each other.In hypoxia/ischemia a key feature of secondary damage after the primary neuron-damaging event is the over-release of neurotransmitters . Consequncreases . Since tncreases . It is wncreases -22 as wey events . Thus, i release . Alternaed by us ,14,18,19ed by us ,16 estabTo summarize briefly our main findings: (1) The presence of xenon blocks hypoxia-induced dopamine release in dopaminergic cells. (2) Hypoxia-induced dopamine increase is caused by an enhanced release of dopamine rather than a reduced uptake of dopamine. (3) When measuring LDH release as a marker of cellular damage, xenon was found to block such release, which suggests that xenon reduces hypoxia-induced cellular damage. (4) Increased extracellular dopamine can damage dopaminergic cells directly. This is mainly mediated by D1 receptor agonism rather than D2. (5) Such direct extracellular dopamine-induced damage can be reduced by the presence of xenon, even when the increase in extracellular dopamine has not been caused by an episode of cellular hypoxia. (6) The above described protective effects of xenon depend on the presence of calcium ions.Further studies will show if indeed in the hypoxic cell multiple intracellular targets exist for xenon and how they are orchestrated together to result in cellular protection.Based on the present results obtained with NGF-differentiated PC-12 cells and on the literature cited in this paper, xenon appears to be a neuroprotectant for a broad spectrum of neuronal cells; given its proven non-toxicity based on its long clinical use, it may come close to fulfilling the requirements for an ideal or "gold standard" neuroprotectant.2. For experiments, cells were seeded in 24-well plates at a density of 1 × 105 cells/well and nerve-growth-factor  was added (0.4 μg/ml) whereupon cells entered differentiation. They were used on day five after the addition of growth factor [Rat pheochromocytoma cells (PC-12) were maintained in RPMI 1640 medium containing 5% fetal calf serum, 10% horse serum, at 37°C, 5% COh factor . PrimaryHypoxia-treatment was performed as described . Samplesbis(2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester (BAPTA-AM) was purchased from Molecular Probes, , and all standard chemical products were obtained from Merck .Gases were supplied by AGA-Linde . 1,2-p values less than 0.05 were considered statistically significant.All experiments were repeated at least five times, i.e. in five different plates on five different days. The data were presented as means ± SEM. The results of multiple groups were analyzed using one-way ANOVA with Dunnett's multiple comparison post test or two-way ANOVA with Bonferroni posttests using GraphPad Prism version 3.00 for Windows, GraphPad Software, San Diego California USA. Differences with CP conceived the study, participated in its design and coordination, carried out the cellular studies involving the various gas treatments, participated in the preparation of the cells, and drafted the manuscript. PB and WS participated in the design of the experiments and the gas applications, JM performed neurotransmitter analysis and LDH determinations, and WK participated in the design of the study and performed the statistical analysis. All authors read and approved the final manuscript.