Achilles tendinopathy is considered to be one of the most common overuse injuries in elite and recreational athletes and the recommended treatment varies. One factor that has been stressed in the literature is the lack of standardized outcome measures that can be used in all countries. One such standardized outcome measure is the Victorian Institute of Sports Assessment – Achilles (VISA-A) questionnaire, which is designed to evaluate the clinical severity for patients with Achilles tendinopathy. The purpose of this study was to cross-culturally adapt the VISA-A questionnaire to Swedish, and to perform reliability, validity and structure evaluations.Cross-cultural adaptation was performed in several steps including translations, synthesis of translations, back translations, expert committee review and pre-testing. The final Swedish version, the VISA-A Swedish version (VISA-A-S) was tested for reliability on healthy individuals (n = 15), and patients (n = 22). Tests for internal consistency, validity and structure were performed on 51 patients.r = 0.89, ICC = 0.89) and healthy individuals (r = 0.89–0.99, ICC = 0.88–0.99). The internal consistency was 0.77 . The mean [95% confidence interval] VISA-A-S score in the 51 patients (50 [44–56]) was significantly lower than in the healthy individuals (96 [94–99]). The VISA-A-S score correlated significantly (Spearman's r = -0.68) with another tendon grading system. Criterion validity was considered good when comparing the scores of the Swedish version with the English version in both healthy individuals and patients. The factor analysis gave the factors pain/symptoms and physical activityThe VISA-A-S had good reliability for patients (pain/symptoms and physical activity, and can be used in both research and the clinical setting.The VISA-A-S questionnaire is a reliable and valid instrument and comparable to the original version. It measures two factors:  Achilles tendinopathy is a common overuse injury, especially among athletes involved in activities that include running and jumping -4. SeverDespite the high incidence of Achilles tendon disorders there have been few randomized treatment studies in patients with Achilles tendinopathy ,11-16. IRobinson et al.  developeTherefore, the purpose of this study was to translate and culturally adapt the English VISA-A questionnaire to Swedish, to perform reliability and validity evaluations of the Swedish version, and to analyze the factor structure of the questionnaire.To establish good face validity and content validity, the translation and cultural adaptation of the VISA-A questionnaire into Swedish was performed in several steps . The EngThe panel of experts consolidated the various versions into one pre-final version of the VISA-A questionnaire – Swedish version (VISA-A-S). The pre-VISA-A-S was pilot tested on five patients and five healthy individuals. After pilot testing question 1 was made clearer by entering the minutes in the boxes.The final version of the VISA-A-S  with the results of the two patient groups, the non-surgical group (n = 45) and surgical group (n = 14), in the original article by Robinson et al. (2001). The results of the healthy individuals in our study were also compared with the results from the healthy individuals in the original study.The structure of the VISA-A-S questionnaire was evaluated with a factor analysis.All data were analysed by SPSS 11.5 for Windows. Descriptive data are reported as mean, standard deviation and 95% confidence interval.r, as performed for the VISA-A English version [Test-retest data was analysed by Pearson's  version . Inter-CU test. For comparison of the VISA-A-S with the VISA-A, a two sample t-test was used since only means and standard deviations, and no raw data, were available from the results in the original study [Comparison of VISA-A-S with Stanish et al. (2000) tendon grading system was performed by calculating the Spearman's rank correlation coefficient for non-parametric data. VISA-A-S scores for the healthy group and the patient group were compared using the Mann Whitney al study . The levA principal axis factoring with varimax rotation, eigenvalue over 1.0, was applied for evaluation of the structure of the questionnaire.r > 0.88, ICC > 0.88) and for patients (Pearson's r = 0.89, ICC = 0.89). There was no significant difference between the scores on test days 1 and 2. When analyzing each question separately . The patients with Achilles tendinopathy had a significantly lower score (p < 0.0001) compared with the healthy individuals. The mean VISA-A-S score for patients in the present study was significantly (p < 0.01) lower than the mean VISA-A score for the non-surgical group in the original article by Robinson et al. (2001). When comparing the VISA-A-S score for patients in the present study with the VISA-A score of the surgical group in the original study [The VISA-A-S score correlated significantly with the tendon grading system by Stanish et al. (2000)  and physical activity (questions 7 and 8).The factor analysis revealed two factors of importance : A widely-used clinical outcome measure for patients with Achilles tendinopathy would help comparisons between treatments in various clinics and research studies. The VISA-A questionnaire is an easily self-administered questionnaire. Since research is performed in various countries it is important to properly translate, culturally adapt and evaluate instruments like a questionnaire in order to be able to compare the results .face validity and content validity. The test-retest reliability and the internal consistency were considered good. A significant and strong correlation between the VISA-A-S and the tendon grading system by Stanish et al. (2000) indicates good construct validity. The comparison of the results of the patients and healthy individuals in the present study with the results of the non-surgical group, the surgical group and the healthy individuals, as reported in the original article by Robinson et al. (2001), indicates good criterion validity.This study demonstrates that the Swedish version of the VISA-A questionnaire has good reliability and validity. With careful translation and cultural adaptation, we established good pain/symptoms (questions 1–6) and physical activity (questions 7 and 8), strongly confirming that the questionnaire is valid for evaluating the patient's symptoms and its effect on physical activity. The factor analysis and an internal consistency of 0.77 as measured by Cronbach's alpha indicate that no question should be excluded.The factor analysis gave the two factors: We did not include a separate group of pre-surgical patients as in the original study by Robinsson et al. (2001). This is because the advances in the non-surgical rehabilitation of patients with Achilles tendinopathy during recent years have resulted in markedly reduced number of patients awaiting surgery. The patients in the present study can therefore be viewed as representing patients from both groups  of patients used in the original study . This woThe one week duration between the two tests in the test-retest reliability part was somewhat long. This duration was chosen because this is the usual time that lapses between a patient's first and second visit with the physical therapist. This may explain why two of the questions differed significantly between test-days. During this week the patients have met with their physical therapist, which may have caused the patients to change their view on their symptoms and physical ability.Good criterion validity indicates that the Swedish version and the English version of the VISA-A questionnaire evaluate the same aspects of clinical severity in patients with Achilles tendinopathy. It can, thus, be expected that similar scores in the two versions indicate the same index of severity in patients with Achilles tendinopathy.The 11 physical therapy clinics throughout Sweden, which participated in this study, all reported that the questionnaire was easily administered, and required a minimum of communication between the physical therapist and the patient. The physical therapists perceived the questionnaire as a good clinical tool and useful when treating patients with Achilles tendinopathy.A review of the literature in regards to treatment of Achilles tendinopathy yielded only a few randomized treatment trials ,11-16. Tpain/symptoms and physical activity, reinforces that the VISA-A questionnaire can be used as an index of clinical severity. The present study culturally adapts and validates the VISA-A-S questionnaire  declare that they have no competing interests.KGS conceived of the study, participated in its design, performed data acquisition, analyzed and interpreted the data and drafted the manuscriptRT conceived the study, participated in its design, interpreted the data and helped to draft the manuscript.JK participated in the study design, interpreted the data and helped to draft the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:VISA-A-S questionnaire The Swedish version of the VISA-A questionnaire in MICROSOFT WORD format.Click here for file


Within the peer-reviewed literature, associations between two things are not always recognized until commonalities between them become apparent. These commonalities can provide justification for the inference of a new relationship where none was previously known, and are the basis of most observation-based hypothesis formation. It has been shown that the crux of the problem is not finding inferable associations, which are extraordinarily abundant given the scale-free networks that arise from literature-based associations, but determining which ones are informative. The Mutual Information Measure (MIM) is a well-established method to measure how informative an association is, but is limited to direct (i.e. observable) associations.Herein, we attempt to extend the calculation of mutual information to indirect (i.e. inferable) associations by using the MIM of shared associations. Objects of general research interest (e.g. genes, diseases, phenotypes, drugs, ontology categories) found within MEDLINE are used to create a network of associations for evaluation.Mutual information calculations can be effectively extended into implied relationships and a significance cutoff estimated from analysis of random word networks. Of the models tested, the shared minimum MIM (MMIM) model is found to correlate best with the observed strength and frequency of known associations. Using three test cases, the MMIM method tends to rank more specific relationships higher than counting the number of shared relationships within a network. This is reflected by the growth in the number of scholarly journals published every year as well as the number of total records indexed in biomedical literature reference databases such as MEDLINE[Most scientific fields are data-intensive, but perhaps even more so for biology and medicine. Sequencing efforts have generated billions of base pairs of genetic information across hundreds of thousands of species, and ushered in the relatively recent completion of the Human Genome Project. Microars MEDLINE. In any transitive", "implicit", "indirect" or "inferable" relationships. Deciding that no relationship exists when no co-mentions exist is somewhat of an over-simplification, but a necessary one. Realistically, several co-mentions between terms could be observed without a definitive relationship present. However, if one uses a greater-than-zero cutoff to define when a relationship exists, false-negatives become a problem: Some co-mentions below the cutoff will constitute a real relationship. Using zero co-mentions as a cutoff is a convenience to avoid this problem even if the end result is that some relationships are declared "known" when they really are not.Most scientific knowledge comes from peer-reviewed articles and is written in free-form text, which is difficult to analyze algorithmically. However, the idea that novel relationships within text could be computationally identified based upon existing relationships has its roots in an approach developed by a researcher named Don Swanson, who used software to identify words shared between article titles . Using tWhile pioneering, a keyword-based method such as Swanson and Smalheiser's is both limiting and highly burdensome, especially where a large body of literature is concerned, because the number of unique keywords grows quickly per record analyzed. Neither is the method amenable to open-ended querying – that is, telling a user what is implicitly related to a query term. Rather, one must essentially begin by postulating a relationship between a query term, A, and another term, C, where a set of intermediate terms, B, can be found that connect the two. Even improvements in visualizing or exploring records that share commonalities and/or define entities of interest ,9 are liIt was previously demonstrated that, because the number of associations between terms follows a scale-free, or inverse power-law, distribution, the number of inferable associations with any given term rapidly approaches the maximum number of possible associations as the number of direct associations grows. That isimplied mutual information content of two unrelated terms.Previous work demonstrated that it was feasible to identify pertinent implicit relationships by ranking inferred relationships and preferentially examining those at the top of the list. One of The general approach to associating objects by searching for their co-occurrence within text has been used in many fields as a simple, yet comprehensive way to identify potential associations. In biology and medicine, co-occurrence has been used to identify potential relationships between genes ,14, protimplicitly associated with each other, and new associations can be potentially inferred by evaluation of their shared associations. Since there are many implicitly associated objects, the relevance of each one is also evaluated using the MIM. However, a MIM can be calculated to evaluate the relevance of an association between A and B and between B and C, but it is not clear how each of these individual scores extends to the inference of an association between A and C. Therefore, we explore and evaluate different methods.To begin a search for novel, inferable associations within the literature, relevant "objects" of interest in scientific research were first defined by assimilating database entries from relevant databases into one central database. By doing this, both words and phrases can be identified within text, and it permits synonymous terms to be mapped to primary terms. All electronically available literature was then analyzed for associations between objects of interest by searching for their co-occurrence within MEDLINE records (titles & abstracts), summing the total number found. The significance of this collective set of co-occurrences is evaluated using the mutual information measure (MIM), which was originally based upon Shannon's Entropy theory , but hasCode was written in Visual Basic 6.0 (SP5) using ODBC extensions to interface with an SQL-based database, with database queries written in SQL. Programs were executed on a Pentium 4 3.06 GHz machine with 1 GB of RAM and two ultra-fast SCSI hard drives. The National Library of Medicine graciously provided an electronic archive of MEDLINE records in XML format. To obtain a set of common words for analysis, the Merriam-Webster dictionary was parsed into individual words and each word summed by the number of times it was observed within the dictionary. 10,000 words were chosen with dictionary frequencies ranging from 322 to 28. This range was selected so that no extremely common or rare words would be within the list. To create a database of random word associations, only 100,000 titles/abstracts were used. This was done to avoid network saturation (i.e. having a significant number of objects related to every other object) and to ensure that the distribution in the number of associations between words resembled the same power-law distribution observed for biomedical objects.The occurrence of such objects within scientific text is identified by comparing phrases within MEDLINE records to entries in the object recognition database (ORD). This ORD is built by inputting terms found in several different biomedical databases, all freely available for download. Objects classified as diseases, disorders, syndromes or phenotypes were obtained from Online Mendelian Inheritance in Man (OMIM) ; chemicaAcronyms for entries, if not explicitly stated within the assimilated database, were obtained from an acronym database. SimilarA scoring scheme based upon the Mutual Information Measure (MIM)  is used AB is the measured probability that A and B will be observed together in the same abstract, while PA and PB are the probabilities of observing A or B, respectively, in a given abstract. Furthermore, because scientific research and discovery is a time-dependant process, prior information can be incorporated to refine the probabilities in Equation (1). The describing of a disease or discovery of a gene, for example, will occur at a given point in time  represents the larger of the two values between Af and Bf. And At is the total number of records processed.Where TMIM in Equation 1 then evaluates to 1 and the log value to zero – the information gained on one object by observing the other. If the probability of observing A increases when B is mentioned, then MIM > 0. If A and B are rarely mentioned together, then MIM < 0. When considering scientific writing style with reference to biomedical objects such as genes, diseases and chemical compounds, there is a probability that two of them might be mentioned together in the same record without having an established association. For example, one of the objects may be very commonly used in many studies (e.g. the gene LacZ is used for staining assays, luciferase is used for luminescence, etc), or one of the objects may be of great scientific/medical interest and authors may make an extra effort to speculate how their results might relate to such objects (e.g. cancer, diabetes, heart disease, apoptosis). The MIM provides a way of quantifying literature-based object dependencies. However, taking the log value can provide a negative weighting to an association when two frequent terms are mentioned together. Optimally, irrelevant or uninformative associations  would be ignored entirely rather than penalized. Therefore, the log function is removed and the equation becomes:As an example of how the MIM score is used, assume that the probability A will appear in any given record within a database of records is 10% and the probability of B appearing is the same. If the appearance of A is completely independent of the appearance of B then no information about one can be gained by observing the appearance of the other. The probability both A and B will be observed in the same record is thus 0.1*0.1 = 0.01. The value of The possibility remains that rare associations might receive a very high MIM score , but it t objects in this set, each individual object can be given the symbol "Bn", where 0 <n <t. For each Bn, there is another set of objects related to it by literature co-occurrence, called "C". Each object in the set C may or may not be connected to the primary object, A. That is, an association may consist of A↔B↔C where an object in the set C also belongs to the set B. The symbol "↔" is used here to represent the existence of a non-directional association between two objects.Figure n and Bn-C associations. Two models are thus proposed and evaluated, the numeric score obtained by any one of them will only be relevant in terms of how well it assigns a relative importance to each A-C connection within a list.Objects in the set C having no literature-based association with the primary object, A, represent associations that have not been previously made, or at least documented, by others. These represent new associations that can potentially be inferred by virtue of their shared associations. Because the number of implicit associations rapidly increases with each established association, the goal here is to provide a quantitative measure of the strength of an implicit association based solely upon the associations shared by two objects. After all, if no known relationship is documented, then these shared associations will be the only way to understand the nature of an relationship between A and C. Since directly associated objects also share associations with other objects, it is reasoned that the strength of known associations can be used to benchmark how well the scores from implicit associations correlate with the relative importance of an association. However, it is not clear how A-C relationships are best evaluated given a set of component A-Bn and Bn-C MIM association is averaged over a total of t shared connections and then finally divided by t to normalize the total score by the total number of connections. The function for the normalized averaged MIM (AMIM) model is:The first model to be tested assumes that the total information content of an implied A-C association can be approximated by the mutual information measure of each component connection. Thus, the MIM scores for each A-BAs model by which A-B and B-C values were summed was also considered, but it would be functionally indistinguishable from the AMIM model in terms of ranking implicit relationships, so it was not included.n association as well as the Bn-C connection, and the information potential an A-C connection will be no greater than the least mutual information given by A-Bn or Bn-C. This is equivalent to assuming that a chain can be no stronger than its weakest link. The equation for the normalized minimum MIM (MMIM) model is:The second model views the process of inferring an A-C connection as function of each of its component processes, limited in its potential by the mutual information in each step of the inference process. That is, inferring an A-C connection depends upon how much information is in the A-BA total of 12,899,016 MEDLINE records recorded from 1967 to May 2003 were processed in chronological order to create a network of 10,873,926 associations between a total of 112,805 unique objects assimilated from the databases mentioned. When including synonyms, the total number of recognizable phrases for these unique objects was 223,540 .The distribution of objects found in MEDLINE ranges from more general categories (e.g. "blood", "tumor", "stress", "lesions") that are found in a higher percentage of records  to the more specific. The frequency of objects when plotted follows a power-law distribution and resembles that of a scale-free network, which is reasonable given that new objects are typically studied in terms of their relationship to known objects .Records were chosen for analysis due to their electronic availability and are also because they are a good source of pertinent information due to their brief, focused nature that presumably contains a summary of the most important findings in each report. Several objects were examined to see if associated objects with high MIM scores correlated with the relative importance of the association. This was done by obtaining summary descriptions of an object from various authoritative sources such as review articles, glossaries or biomedical databases. Table Table known, relevant associations are ranked within the set of all inferable associations, the better the ranking method is.When ranking inferred associations, the goal is for the score assigned to an inference to correlate well with the amount of mutual information gained from any given association. The only reference basis for this is the mutual information contained within established associations. Since they too will frequently contain shared associations, they can be evaluated independently using only their shared associations Figure . Severalknown relationships is added together, in order from highest MIM score to lowest, to reflect the fastest rate by which they could be discovered. When plotted, this curve is what would be observed were mutual information preserved exactly (the "exact" curve). Because it's neither expected that all possible relationships are known, nor that mutual information is static as the scientific discovery progresses, it is not anticipated that this curve would or even should be followed exactly . However, it is reasonable to expect established relationships with high mutual information content to retain a relatively high mutual information content when evaluated on the basis of its shared relationships. Thus, it is expected that the implicit MIM curve follow the "exact" MIM curve.To evaluate this, a graph is drawn to reflect the rate that established relationships are discovered within the set of all objects analyzed. The total of all MIM scores for Figure To gain a better quantitative estimate of performance, 50 objects were chosen at random from both the MEDLINE and random word databases. Each object was analyzed to identify and rank other objects that shared relationships with it as described and the area under the curve (AUC) was taken for each of the ranking methods shown in Figure A peculiar effect was noted with the average MIM-based scoring model: Some implicitly associated objects received higher MIM scores than the primary object itself, which is also analyzed as a control. There tend to be relatively few, sometimes none, such instances per analysis, but it occurs when a relatively rare object shares several or more associations with the primary object. This effect was not present in the minimum MIM model.The scores assigned by inference methods so far have no meaning by themselves, but only as a means of ranking the potential relevance of an inference. Because the majority of database objects will be present in the list of implicit connections, the question naturally arises as to where a significance cutoff can be drawn. A range of significance for a given MIM score can be estimated by analysis of a random word network in which we would expect that meaningful relationships are only encountered by chance.t random associations and dividing by t would also be expected to have an average  MIM score of 1. This is true for any set of A-B associations as well as a corresponding set of B-C associations, thus an average value of 1 should still be obtained when calculating the average minimum MIM score. Figure Since the MMIM model performed slightly better than the AMIM model, we evaluated it using the random words database. Words in the random network were effectively chosen at random from the Merriam-Webster dictionary , and so relevant associations between these words co-occurring within MEDLINE records should occur predominantly by chance. An uninformative association  would have an average MIM score of 1 (e.g. see Equation 5). Thus, summing a set of ileum is frequently used to test capsaicin effects because of its contractile response. Tachykinins Substance P and Neurokinin A as well as the neurotransmitter acetylcholine[tetrodotoxin[atropine[Using the example of capsaicin brought up earlier, we analyzed it using the methods described to identify and rank objects sharing relationships with it Table . When raylcholine are resprodotoxin or atrop[atropine. These iper se. To do so presumes that Swanson's initial study was the "correct" way of finding relevant implicit relationships and Swanson did not employ the open-discovery model in these examples anyway. It would be useful to know, however, where Swanson's predictions rank among others using models in which implicit relevance is judged by counting the number of shared relationships and where it is evaluated by the MMIM. Both Raynaud's and Migraine headaches were analyzed as starting objects (A), the goal being to find all C objects that share relationships and rank them by their relevance. Both known and implicit relationships were displayed. The top 10 results are summarized in Table Finally, we also sought to re-evaluate some of Swanson's original hypotheses as has been done in other text-mining studies ,11,30. IWhen ranking implicit relationships by the number of shared relationships, fish oil scored #1025 in the Raynaud's list and magnesium (the link Swanson found with migraines ) scored sumatriptan is a drug used to treat migraines and other items ranking highly on the list such as nausea, vomiting, and dizziness typically accompany migraines. Notably, one of the important links that Swanson used to surmise the role of magnesium is also on this list: Seizures, which cause migraines.Examining the relationships that tend to rank highly in both models it is apparent that, when ranking by the number of shared relationships, the higher-scoring entries tend to be more general and vague in nature . When ranked by the MMIM, their relevance to the object in question is more readily apparent. For example, Information retrieval (IR) methods are limited to querying what is known; yet often the most valuable information is what is not directly known. Mutual information measures have been used successfully in many IR applications, and a method has been presented here to extend it to inferable associations. We find that the normalized MMIM method of ranking inferences based upon their shared associations correlates best the level of currently established mutual information. A good correlation is suggestive that mutual information is being captured even though evaluation proceeds indirectly, through intermediates. For simplicity, we have used a cutoff of zero co-occurrences to suggest that no association between objects has been made, but it is quite possible that a number of co-occurrences could be noted between two objects yet no specific relationship between them documented. Or additionally, a certain relationship may be known between the two, but other important relationships still remain to be inferred. At this point, however, it is not clear how this would effectively and quantitatively be taken into account.The method reported was applied to biomedical research, but could ostensibly be applied to any domain in which the goal is to identify undiscovered relationships. Importantly, this method of automated inference ranking provides a quantitative way of prioritizing inferred associations when available literature is growing rapidly in size and scope.


Cancer gene therapy will benefit from vectors that are able to replicate in tumor tissue and cause a bystander effect. Replication-competent murine leukemia virus (MLV) has been described to have potential as cancer therapeutics, however, MLV infection does not cause a cytopathic effect in the infected cell and viral replication can only be studied by immunostaining or measurement of reverse transcriptase activity.env gene with the red fluorescent protein (RFP) and separately cloning GFP-Env into a retroviral vector. Co-transfection of both plasmids into target cells resulted in the generation of semi-replicative vectors, and the two color labeling allowed to determine the distribution of the individual genomes in the target cells and was indicative for the occurrence of recombination events.We inserted the coding sequences for green fluorescent protein (GFP) into the proline-rich region (PRR) of the ecotropic envelope protein (Env) and were able to fluorescently label MLV. This allowed us to directly monitor viral replication and attachment to target cells by flow cytometry. We used this method to study viral replication of recombinant MLVs and split viral genomes, which were generated by replacement of the MLV Fluorescently labeled MLVs are excellent tools for the study of factors that influence viral replication and can be used to optimize MLV-based replication-competent viruses or vectors for gene therapy. Replication-competent retroviruses (RCRs) encoding suicide genes linked via an internal ribosome entry site (IRES) offer a significant advantage over replication-deficient vectors in cancer gene therapy, since they are able to spread efficiently in vivo -4. Unconin vivo ,6. The fInvestigating murine leukemia virus (MLV) replication is technically inconvenient because MLV infection does not cause a cytopathic effect in the infected cell. Viral replication can only be studied by immunostaining, measurement of reverse transcriptase activity or syncytia formation. We have developed a tool to simplify these analyses. We generated an MLV tagged with a fluorescent envelope protein, which allows viral replication and Env attachment to target cells to be followed by flow cytometry. This method will be useful for optimizing RCRs or retroviral vectors for gene therapy.Aequoria victoria in its envelope. A replication competent ecotropic MLV variant was generated (GFP-EMO) that had the 53 aas of the epidermal growth factor (EGF) fused to the N-terminus of Env and the GFP sequences inserted into the proline-rich region (PRR) [We previously constructed a modified ecotropic murine leukemia virus (Mo-MLV) bearing the green fluorescent protein (GFP) from on (PRR) . We deleSequestering of EGF-Env-containing viral particles has been described before ,9. ViralViral entry is initiated by the binding of the envelope protein (Env) to the retrovirus receptor at the target cell surface. To test whether labeling of Env with GFP allows viral attachment to be monitored, we incubated supernatants of NIH3T3 cells producing GFP-EMO or GFP-MOV with cells that either express mCAT, the receptor for ecotropic MLV  (NIH3T3)i-MLV). These cells have only negligible numbers of mCAT molecules on the cell surface, because Env expression leads to their retention within the cell (receptor interference). As expected, NIH3T3i-MLV cells produced no shift when incubated with GFP-MOV supernatants , where the env gene and replacing it with the red fluorescent protein (RFP)  Fig. . RFP is FP) Fig. . The GFPFP) Fig. . This veFP) Fig. . Co-tranIn addition, we examined the stability of the GFP-tagged Env in the split genome approach. As shown in Figure in vitro and in vivo. The biodistribution of replication-competent viruses in animal models and their safety for cancer treatment could, thereby, be assessed.Our data demonstrate that labeling the MLV Env with a fluorescent protein is an easy method of monitoring MLV replication and the attachment of Env to target cells. This is especially useful for the development of novel cancer gene therapies that use replication-competent MLV encoding a cytotoxic gene . LabelinA further improvement of replication-competent viruses would be tumor cell-specific entry. The inclusion of tumor-specific ligands into Env is one option to potentially expand the ecotropic host range of MLV to human tumor cells ,5. EcotrIn our experiments using semi-replicative retroviral vectors, we found that a rapid increase in GFP-positive cells correlated with the appearance of recombinations and the formation of full-length MLV genomes. This indicates that semi-replicative vectors have to be improved to avoid intergenomic recombination before they can be considered to be used for gene therapy. The recombinants did contain the GFP-Env gene, providing further proof that insertion of GFP into the proline-rich region of Env did not interfere with viral fitness.Fluorescently labeled MLVs are excellent tools for the study of factors that influence viral replication and can be used to optimize MLV-based vectors or viruses for gene therapy. This method is not limited to ecotropic Env, but can be extended to amphotropic MLV, since it has been shown recently that the amphotropic MLV Env can also be tagged with GFP .2.NIH3T3, A431, 293T and COS-7 cells were grown in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal calf serum, 4 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in 10% COenv (position 5777) was deleted to allow translation to start at the inserted GFP sequence [The construction of GFP-EMO has been described previously . GFP-MOVsequence . We replPlasmids encoding the MLV genomes or soluble receptor binding fragments (sRBDs)  were traSupernatants of tissue culture cells were collected, filtered through a 0.45 μm pore filter (Millipore, Eschborn, Germany) and added to target cells. After 5 min. at room temperature, the cells were spun down, redispersed in PBS and immediately monitored by fluorescence-activated cell sorting (FACScan, Becton Dickinson, Heidelberg) using the Cellquest software.Green fluorescence protein (GFP) expression was monitored by a shift to green fluorescence (FL-1) and red fluorescent protein (RFP) by a shift to red (FL-2). FACS analysis was performed with FACScan (Becton Dickinson, Heidelberg) using the Cellquest software.Genomic DNA was isolated after proteinase K digestion and phenol/chloroform extraction. PCR was performed using the manufacturers protocol (Qiagen, Hilden, Germany).N-terminal EGF-Env sequences were analyzed using the primers BS-5: 5'-TCT GAG TCG GAT CCC AAA TGT AAG and MLV-5'-Env: 5'-TAA CCC GCG AGG CCC CCT AAT CC, which amplified a 899 bp fragment from GFP-EMO and a 726 bp fragment from wt MLV. The generation of full-length genomes was analyzed using the primers p1: 5'-GAA TAG AAC CAT CAA GGA GAC and p2: 5'-CTC GAG AAG CTT AGT ACT GA, which amplify a 600 bp fragment from full-length MLV. No fragment should be amplified from the semi-replicative vectors, because the primers bind to genes on separate constructs. The stability of the GFP-Env fusion gene was analyzed using the primers p3: 5'-GTC AGT AAG CTT CTC GA and p4: 5'-GGT TTT GTC AGG ACT GGT GAG, which amplify a 1.5 kb fragment from gfp-env and an 800 bp fragment form wt env.The author(s) declare that they have no competing interests.Katja Sliva and Alexandra Bittner performed the experiments. Katja Sliva, Otto Erlwein and Barbara Schnierle participated in the design of experiments, oversight of the conduction of the experiments, and in the interpretation of the results.


Flexible video bronchoscopes, in particular the Olympus BF Type 3C160, are commonly used in pediatric respiratory medicine. There is no data on the magnification and distortion effects of these bronchoscopes yet important clinical decisions are made from the images. The aim of this study was to systematically describe the magnification and distortion of flexible bronchoscope images taken at various distances from the object.Using images of known objects and processing these by digital video and computer programs both magnification and distortion scales were derived.Magnification changes as a linear function between 100 mm (×1) and 10 mm (×9.55) and then as an exponential function between 10 mm and 3 mm (×40) from the object. Magnification depends on the axis of orientation of the object to the optic axis or geometrical axis of the bronchoscope. Magnification also varies across the field of view with the central magnification being 39% greater than at the periphery of the field of view at 15 mm from the object. However, in the paediatric situation the diameter of the orifices is usually less than 10 mm and thus this limits the exposure to these peripheral limits of magnification reduction. Intraclass correlations for measurements and repeatability studies between instruments are very high, r = 0.96. Distortion occurs as both barrel and geometric types but both types are heterogeneous across the field of view. Distortion of geometric type ranges up to 30% at 3 mm from the object but may be as low as 5% depending on the position of the object in relation to the optic axis.We conclude that the optimal working distance range is between 40 and 10 mm from the object. However the clinician should be cognisant of both variations in magnification and distortion in clinical judgements. The flexible bronchoscope has been used in pediatrics for more than 20 years  yet therMagnification of an object refers to the virtual or real visualized and measured increase in size of that object after rays of light from that object have passed through a lens system. Magnification varies across the field of view and with the distance of the lens from the object . DistortA flexible bronchoscope (Olympus BF Type 3C160, Olympus, Tokyo, Japan) and light source (Olympus Evis Exera CLV-160, Olympus, Tokyo, Japan) and processor (Olympus Evis Exera CV-160, Olympus, Tokyo, Japan) were connected to a TV monitor (Sony HR Trinitron, Sony Corporation, Shinagawa-ku, Tokyo, Japan) and a colour video printer (Sony Mavigraph, Sony Corporation, Shinagawa-ku, Tokyo, Japan) as supplied by the Olympus company. The image signals from the Evis Exera CV-160 were co-recorded by a digital camera . These digital video images were viewed and stored as uncompressed 640 × 480 pixel, millions of colours as TIFF files. These images were then analysed by an image processing program  and a computer (MAC Power Book G4. Apple Inc,, Cupertino, Ca., USA).A precisely measured object consisting of 4 concentric circles with emanating radii on 1 mm × 1 mm graph paper was created and drawn to precision using design software (Auto CAD, Autodesk, San Rafael, Ca, USA) Fig  would alMagnification was calculated by repeatedly measuring the image diameters AE, BF, CG, DH across the 4 concentric circles Fig.  at distaGeometrical distortion was defined as the radial length ratios from each interlocking circle with radii A taken as the denominator for all subsequent radial measurements within the outer circle and then other elements of radius A for the other circles. The uniformity of the ratios was then tested in quadrant or sectors: a sector or quadrant being an area defined in accordance with mathematical convention whereby The changes in magnification across the field of view was further defined as the differences in calculated measurements for a known distance of 2 mm across the field of view at a fixed distance (15 mm) from the object. These measurements were made in the horizontal plane. As with the magnification calculations, the mean distortion value from the entire set of 3 experiments was taken as the defined distortion at the particular distance from which the measurements were made. All of the aforementioned experiments were then repeated on 3 separate Olympus BF Type 3C160 bronchoscopes. The experiments were performed separately with the central geometrical axis of the bronchoscope and then the optic axis of the bronchoscope .2 value) for each of the curves for circles A,B,C,D was 0.999, 0.999, 0.999 and 0.999 respectively. When the bronchoscope was aligned to the object through the optical axis, the magnification was reduced generally but by as much as 22% at 5 mm and 14.5% at 10 mm from the object  to 0.9811 (95%CI: 0.8827 to 1.000). These intraclass correlation data indicate and support the goodness of fit of the regression curves. The variability in magnification measurement between the bronchoscopes was extremely small and there are virtually no differences between the bronchoscopes  and the object is not aligned through the optical axis. This could be corrected by movement of the object closer to the line of the optical axis of the lens. However there are no markers on the bronchoscope to indicate that this position has been gained. At close proximity to this point there is separation of the light beams, however while the use of this is possible in laboratory settings, it is not currently possible in the clinical context where there are differences in reflectance and absorption of light. The second issue is that barrel distortion is generally regarded as homogeneous and independent of working distance however when the lens is offset as it is in this type of instrument it cannot be, given the differences between the geometrical and optical axes of the bronchoscope. In the clinical context these issues indicate that clinicians must be aware that their judgements always contain "distortion effects" but particularly when working very close to the object eventhough the image appears clear and is centred on the screen.The limitations of this study are the fact that the object positioning could only be moved with micrometer precision in the longitudinal plane. However an instrument that allowed for lateral movement of the object with micrometer precision may have produced less distortion but in the clinical context such precision could only ever be transient and thus these methods have allowed for a greater appreciation of the extent and complexity of distortion. Even though computer based systems have been used to correct for some of the elements of distortion,5,6 noneThe clinical relevance of this work may not be obvious. However, we argue that it has a number of important clinical applications and that all bronchoscopists should be cognisant of these factors, which may influence their judgement of the size of lesions. Firstly, the distance of the tip of the scope to the lesions being assessed must be measured and not approximated by judgement as distances under 5 mm may result in very considerable differences in magnification and thus the estimates of size or even presence or absence of a lesion. The most obvious example of this is in assessments of a curvaceous left main stem bronchus that may initially appear as malacia but appears to become "normal" as it is viewed at a closer distance. Indeed in defining malacia a viewing or operating distance must be stated otherwise these perspective difficulties will prevail and comparative statements and judgements become meaningless. Another example occurs when assessing malacia or airway cross sectional area changes across the respiratory cycle. In that scenario the movement of the object is away from the lens during inspiration making the image relatively smaller than is real for the increase in lung volume. These optic effects combined with the venturi effects from the bronchoscope itself on reducing intraluminal pressure again compound the issue and reduce the image size or its relative changes. Bronchoscopists need to be aware of these apparent paradoxical effects, that the lumen is greatest at the end of expiration. Indeed at this point in time and until the distance of airway movement can be measured in vivo, measurements can only be made precisely at one point in the respiratory cycle, and that generally is the end expiratory point. If end inspiration is used there is likely to be intrusion of the mucosa into the field of view during the subsequent expiration. The most recent published example of most of these effects can be seen in the images provided in that paper by Okasaki et al  where thSecondly, paradoxical concepts also apply to the use of the flexible bronchoscope in assessment and or removal of foreign bodies situated peripherally in the airways. When dealing with peripheral or markedly angulated bronchial branches it is not always possible to position the image in the centre of the screen. Here estimates of size may be misconstrued by the interactions of variable magnification across the lens, curvilinear distortion and potentially geometric distortion. These effects and the "iceberg effects" of the presented foreign body size usually mean that the foreign body appears smaller than it really is and the inexperienced bronchoscopists might tend to dismiss the object as trivial or inconsequential. To the contrary attempts to remove it should be maximized. Finally, in terms of research, defining the position of the bronchoscope in terms of the lesion being assessed and photographed is vital if realistic comparisons are to be made across time or between groups. In this regard techniques that allow for distance and lesions measurement and assessment need to be developed and integrated into routine clinical use. Using our technique described in this paper, we are quantifying airway size in a variety of airway lesions  associated with significant respiratory morbidity.The fact that performance of each bronchoscope was remarkably similar in terms of magnification and distortion measurements obviously reflects company's production quality. In a clinical session where a number of bronchoscopes might be used and in research where inter-group comparisons might be desired, this information suggests that a significant level of confidence can be maintained in perception terms for the bronchoscopists. This does not negate the need for individual bronchoscope calibration and for objective and accurate measurements in bronchoscopic work. Detailed data from the Olympus Company was not available for comparison; however our own validation experiments show these data to be correct and accurate. Despite the latter, as there are now many instruments available to the clinician, we suggest that companies and manufacturing regulators make readily available the magnification and distortion characteristics of each instrument type or size so that more effective clinical appraisals can occur.Although this study has shown that the Olympus BF Type 3C160 video-bronchoscopes produce remarkably consistent magnification across the working ranges of 100 mm to 3 mm from the object, an understanding of the influence of distance on magnification and distortion of the image obtained by a flexible bronchoscope is an essential step in the development of an invivo technique of measuring airway sizes. We have provided graphic appreciation of these effects and the importance of optical versus geometric axis orientation. The optimal working distances for this bronchoscope are between 40 mm and 10 mm from the object. The study also provides reasonable working magnification factors for this type of bronchoscope and as such could allow for a better appreciation of real or actual airway sizes.Mean magnification and the mean whole of field magnification at defined distances.Click here for fileComparison of bronchoscope axis and optical axis aligned magnification measurements.Click here for file


After apprenticing at her mother's side for some eight years—the first three clinging to her body—an orangutan is ready to make her own way in the forest canopy. The only great ape specializing in arboreal living, orangutans forage the treetops mostly for fruit, nuts, insects, leaves, and tree bark. They can recognize hundreds of species of edible fruit from trees and woody climbers and remember their location and fruiting season. Malay legend says orangutans  derived from humans who sought refuge from their species in the wilds of the forest. Today, with the last orangutan refuges shrinking drastically, the man of the woods has nowhere else to go.Hundreds of thousands of orangutans once ranged throughout southeast Asia. Now just two orangutan species inhabit just two countries: Indonesia  and Malaysia (Sabah and Sarawak, in northern Borneo). The Sumatran orangutan is listed as critically endangered; the Bornean, endangered. In Indonesian Borneo and Sumatra, logging operations clear an estimated 5 to 6 million forest acres a year, leaving the apes stranded in isolated stands of trees and the normally fire-resistant rainforest at sudden risk. Another force driving orangutan extinction in Indonesia is the poaching and illegal killing (mothers do not give up their babies without a fight) that secures orangutan babies for the exotic pet trade. In Sabah, Malaysia, the primary threat comes from clearing the forest for agriculture.Conservation efforts depend, among other things, on having reliable data on population size, density, and distribution, but estimates of orangutan numbers in Sabah—which range from 2,000 to 20,000—are outdated. In a new study, Marc Ancrenaz and colleagues report an innovative method of directly estimating orangutan numbers from the number of nests detected during aerial surveys. (Orangutans are tough to spot directly, so researchers count the nests they sleep in at night.) Their survey, which covered the entire orangutan range throughout Sabah, estimates a total population of 11,000 orangutans—a drop of 35% in the past 20 years, based on a 1988 World Wildlife Federation report.Counting orangutans from the ground can be very time-consuming, difficult work, especially when faced with the hip-deep muck and steep slopes of the rainforest floor. Though helicopters obviously cover greater distance and more remote territory than is possible by foot, they're generally used to survey animals in more open landscapes. By using ground survey data to refine their aerial survey results, Ancrenaz and colleagues could directly assess the distribution and size of orangutan populations throughout Sabah. (Sabah covers roughly 72,000 square kilometers.)Over the course of two years, ground surveys—requiring 1,100 hours of field work—and aerial surveys—requiring just 72 hours—were conducted throughout all the major forests of Sabah. Commercial logging occurs in about 76% of all Sabah forests in commercial forest reserves. During the overflights, information was recorded on altitude, forest type, forest disturbance , and signs of human activity.The authors attribute the 35% decline in Sabah orangutan numbers primarily to habitat loss from agricultural development. The surveys revealed that lowland forests harbored the greatest density of nests and orangutans, with the densest populations found in several highly disturbed, fragmented forests along newly created palm oil plantations. These high orangutan densities could reflect an influx of refugees from recently destroyed forest habitat into areas that are still forested. In logged forests, higher population densities were found in old exploited or sustainably logged forests than in conventionally logged reserves.While the authors acknowledge the density estimates could be more precise—better measures of nest decay and construction rates are needed—their survey reveals crucial information on orangutan numbers and distribution. Most orangutans in Sabah, including those making up one of the largest unfragmented populations in Borneo, live outside protected areas, in commercially exploited forests. These results suggest that orangutans may adapt better to degraded forests than previously thought—provided illegal hunting and agricultural conversion are controlled.More field research will help quantify the impacts of human activity—from logging to stealing babies—on great ape ecology and survival, and determine whether exploited forests can support conservation. It may be, for example, that integrating agricultural fields with forested corridors could sustain orangutan populations over the long term. With time of the essence, these aerial surveys will speed that work, and help sustain orangutans' refuge in the treetops, above their human relatives.


Is biodiversity decreasing or is it being replaced by a splendid profusion of microbes -- and does this matter? There is an old Chinese curse: ‘May you live in interesting times.’ According to those who know about such things, we live in a momentous time, the time of the Sixth Mass Extinction! But most of us do not feel at all cursed. Because, in fact, the Sixth is quite different to the previous Big Five—no-one would notice this one if we were not repeatedly reminded of it by ecologists. Previous mass extinctions were not so bashful, so discreet. The fossil record reveals the disappearance of pollen during previous ones, replaced by an abundance of fungus spores, telling us of a world of devastated forests rotting away. The earliest sediments after the mass extinction that did away with the dinosaurs are barren of fossils: so it is not just that species were going extinct, conditions for life itself were bad. Not only did species diversity drop, the abundance of life did as well.But conditions for life itself have never been better than today. In the history of the planet, there has never been anything as productive of life as a wheat field in Kansas. It may not have a large diversity of species, but that is a different matter. In fact, one of the reasons for the ongoing loss of plant diversity from grasslands is the very reason the wheat field is so productive—fertilisation. We are pouring nitrogen fertiliser into the environment and, through the wellstudied ‘paradox of enrichment’, this reduces species diversity while increasing actual biomass. Now, there is no question that if current trends of habitat alteration and climate change continue then we will ultimately lose large numbers of species—diversity will drop—but this does not necessarily translate into a loss of abundance of life, and that is a big difference between now and previous mass extinctions. Looking at specific groups of organisms tells the same story. So, for example, many island bird species are threatened, like the kagu of New Caledonia, but British seabird populations, like puffins, are booming. Worldwide amphibian diversity is threatened, but cane toads are a pest in Australia. Introduced species pose a threat to diversity—the ‘McDonald-isation’ of nature—precisely because they achieve enormous abundances.Actually, all six mass extinctions may have one very important thing in common: from the point of view of the vast bulk of life on the planet they are probably not mass extinctions at all. By any criterion—number of individuals or total biomass—the vast majority of life on earth is invisible—microbial. So, for example, at least 10% of the living biomass on earth consists of bacteria living deep in the oceans' sediments: it would take more than an asteroid impact to disturb them. And microbial life is extraordinarily robust: microbes can be found living happily in pressurised water hotter than your boiling kettle, in concentrated acid, and in rock, and their spores can survive for years in the rigours of outer space.In talks and lectures, the renowned oceanographer and paleontologist Jeremy Jackson paints a vivid picture of what is currently happening to coastal ecosystems, talking about a wall of slime emanating from populated areas and growing outwards inexorably towards the open oceans, replacing beloved ecologies like coral reef systems. What he means is that the visible life that we find attractive and useful—pretty fish, turtles, and so on—is being replaced by microbes in splendid profusion. It is taken completely for granted that this is disastrous. From a utilitarian point of view indeed it is disastrous, since we like eating fish and turtles, and don't like snorkling in slime. But from the point of view of life per se, again things have never been better. Life is so abundant that in some places all the oxygen in the water is completely used up. These are called ‘dead zones’, but they are no more ‘dead’ than the Dead Sea, which is actually teeming with life—just not fish. But, nonetheless, we consider what is occurring to be a disaster not just from a utilitarian point of view, but at some deeper level giving us an emotional reaction to the word ‘slime’—somehow it is just plain wrong.But this reflects nothing other than our evolutionary origins. Evolution has programmed us to be positively interested in plants and animals, our food, and to be repelled by slimes and oozes, teeming with potentially harmful microbes. These emotional responses colour our view of ecology, for example, in a way that has no parallel in other sciences: physicists do not just study particles that they find pretty. No ecologist wants to study the rich ecosystem that each of us carries around inside our gut, because evolution has programmed our brains to find bottom-related matters disgusting. I think it likely that naturalists from a different planet, silicon entities evolved under very different circumstances, would find tropical forests uninteresting  and animal guts fascinating, with their complex metabolic networks in which each node is manned by different species with wildly varying means of energy production.Our guts should be an ecological scientist's dream come true, ecological theatres that are replicated billions of times, which operate on a fast time scale and are easy to get to!  Many natural experiments are going on all the time as antibiotics and probiotics are administered and people find all sorts of different ways, voluntary or otherwise, to establish migration links between their gut ecologies. Microbiologists are increasingly interested in our guts from an ecological point of view but, unlike ecologists, they are used to faeces from their work in sewage plants.Two thousand years ago the Roman senator Cicero noted the creation of barren desert-like land in North Africa after the forests were felled for their timber, providing the earliest record of an ecosystem ‘service’ provided by forests—the stabilisation of soils. Other services provided by biodiversity readily come to mind, like pollination and carrion clean-up, and there may be many more. But perhaps the clearest example of an ecosystem service provided by biodiversity comes from our gut. Throughout our history, until very recently, we all had worms. In rich countries we have quite happily eradicated them from our inner ecosystems with none of the handwringing we expend on rhinos. But it is increasingly believed that the loss of worms from our internal ecology is responsible for the upsurge in inflammatory bowel disorders such as Crohn's disease and colitis. In fact, there are clinical trials underway in the United States testing the efficacy of worms as treatment for these diseases. The mechanism is clear: worms trigger one arm of the immune system which down-regulates another, inflammatory arm. Our immune system has evolved to expect a certain constellation of species in our gut: in that context, worms provide an ecosystem service of balancing the immune system.Our perception of our impact on the planet as equivalent to a mass extinction simply reflects the evolutionary prism through which we view life. Of course, we may yet live up to our own publicity and pull off something apocalyptic like a runaway greenhouse that sterilises the Earth. But it is at least as likely that the microbial world, resentful at being either ignored or exterminated, will come up with something to consign us to a footnote in the history of life when it is ultimately written by the silicon entities. The Spanish flu, SARS, and HIV have just been early experiments.


Anyone who's endured their share of childhood scrapes has probably heard some version of the motherly admonishment, “Don't pick that scab, you'll just make it worse!” It turns out, Mom was on to something. Tissue damage in humans triggers a well-characterized response marked by rapid blood clotting and a recruitment of epidermal cells to the injury. When you remove a scab, you're also removing some of the newly regenerated tissues growing underneath, thereby interfering with the healing process.Drosophila melanogaster, to create a novel system for studying wound healing. After stabbing fruitfly larvae with a needle to create a nonfatal puncture wound, Galko and Krasnow then analyzed significant morphological, cellular, and molecular events at various stages of healing.Many different cell types and proteins have been linked to the repair process, but the complexity of the mammalian wound response has challenged efforts to determine their individual roles. Only the first step—the signaling cascade that promotes blood clotting—is understood at the molecular level. Since dissecting the wound response pathway at the molecular level requires an organism that lends itself to relatively easy genetic manipulations, Michael Galko and Mark Krasnow of Stanford University turned to the quintessential genetics organism, Immediately after wounding, the larvae begin to bleed, and about 15 minutes after wounding, the wound darkens and a “plug” of cellular debris forms in the wound. The plug staunches the bleeding and provides a protective layer. Within two hours the plug's outer layer forms a dark, tough scab that presumably serves as an effective barrier to further blood loss. Two or three days later, the healing process is complete.Galko and Krasnow observed the activity of epidermal cells during healing by labeling their nuclei with a fluorescent protein and staining their membranes. Soon after wounding, cells along the wound margin elongated and aligned themselves toward the wound, then fused together to form a large, multinucleate (multiple nuclei) epidermal cell surrounding the scab. Over the next six hours, these cells also spread along and through the plug, re-establishing a continuous epidermis.Since this epidermal spreading resembles that seen during a developmental stage of the fruitfly, where the process depends on the JNK signaling pathway, the authors investigated JNK signaling to shed light on the genetics and cellular events of healing. Sure enough, they found that the JNK pathway was activated during the peak hours of wound healing. Inhibiting the pathway in fly mutants—the classic approach in fly genetics—had dramatic effects on the wound-healing process. The early stages of healing—including plug and scab formation—weren't affected, but epidermal spreading to regenerate the intact epidermis was either blocked or defective. In contrast, larvae with defects in a gene required for the generation of crystal cells—a type of blood cell implicated in processes linked to scab formation—could not properly form scabs. In these scabless wounds, the JNK pathway was hyperactive, epidermal cells at the wound's margin started to spread to close the wound but often failed, and the wound did not heal. Score one for Mom.But apparently Mom wasn't totally right: As Galko and Krasnow discovered, a scab isn't always necessary. When the authors pinched larvae without puncturing the overlying cuticle, the wounds did not form scabs. However, they still saw many of the same processes at work around this “pinch” wound that they saw around the puncture wound, and the pinch wounds healed efficiently. This indicates, the authors argue, that the scab functions primarily to provide stability to the wound site and help restore tissue integrity when both the epidermis and overlying cuticle are damaged during wounding.While the stages of wound healing outlined here occur at a particular time and place, these results suggest that each stage is controlled by distinct genetic programs and signaling pathways triggered by the wound. Since many aspects of the fly wound response resemble those in mammals, it's likely that the molecular components are also shared. That makes identifying the molecular underpinnings of wound healing a high research priority. And thanks to the powerful system presented here, this task should be all the easier.


Hemin, the oxidized form of heme, accumulates in intracranial hematomas and is a potent oxidant. Growing evidence suggests that it contributes to delayed injury to surrounding tissue, and that this process is affected by the heme oxygenase enzymes. In a prior study, heme oxygenase-2 gene deletion increased the vulnerability of cultured cortical astrocytes to hemin. The present study tested the effect of HO-2 gene deletion on protein oxidation, reactive oxygen species formation, and cell viability after mixed cortical neuron/astrocyte cultures were incubated with neurotoxic concentrations of hemin.50 of 1–2 μM; astrocytes were not injured by these low hemin concentrations. Cell death was consistently reduced by at least 60% in knockout cultures. Exposure to hemin for 4 hours, a time point that preceded cell lysis, increased protein oxidation in wild-type cultures, as detected by staining of immunoblots for protein carbonyl groups. At 10 μM hemin, carbonylation was increased 2.3-fold compared with control sister cultures subjected to medium exchanges only; this effect was reduced by about two-thirds in knockout cultures. Cellular reactive oxygen species, detected by fluorescence intensity after dihydrorhodamine 123 (DHR) staining, was markedly increased by hemin in wild-type cultures and was localized to neuronal cell bodies and processes. In contrast, DHR fluorescence intensity in knockout cultures did not differ from that of sham-washed controls. Neuronal death in wild-type cultures was almost completely prevented by the lipid-soluble iron chelator phenanthroline; deferoxamine had a weaker but significant effect.Continuous exposure of wild-type cultures to 1–10 μM hemin for 14 h produced concentration-dependent neuronal death, as detected by both LDH release and fluorescence intensity after propidium iodide staining, with an ECThese results suggest that HO-2 gene deletion protects neurons in mixed neuron-astrocyte cultures from heme-mediated oxidative injury. Selective inhibition of neuronal HO-2 may have a beneficial effect after CNS hemorrhage. Hemin is a potent oxidant that accumulates in intracranial hematomas and may contribute to neural cell injury ,2. It isAll HO inhibitors that are currently available have numerous non-specific actions that may complicate the interpretation of experimental results, including inhibition of nitric oxide synthase and guanyl cyclase, and modification of voltage-gated calcium currents -14. SomeThe disparate effects of HO-2 gene deletion on hemin toxicity to astrocytes and hemoglobin toxicity to neurons may reflect the inability of neurons to tolerate the products of heme metabolism, i.e. iron, carbon monoxide, and bilirubin. Alternatively, it may reflect the different oxidant properties of hemin and hemoglobin. Although the oxidant effect of hemoglobin may be due in part to hemin release to membrane lipids , other mThe present study was designed to test the effect of HO-2 gene deletion on the oxidative neuronal injury produced by extracellular hemin. We specifically tested the hypothesis that targeted deletion of the HO-2 gene attenuated oxidative cell injury in a primary cell culture model of hemin toxicity.50 was 1.85 μM. LDH release was significantly reduced in knockout cultures subjected to the same treatment. At 3 μM hemin, only 12.6 ± 4.1% of LDH had been released at this time point. Control experiments demonstrated that these low hemin concentrations do not interfere with the LDH assay.In preliminary experiments, we observed that overnight 14 h) exposure to low micromolar concentrations of hemin consistently produced morphologic evidence of neuronal injury in wild-type cultures , which are markers of oxidation, were then derivatized and detected with a dinitrophenylhydrazone antibody. Increased immunoreactivity was apparent in lysates of wild type cultures treated with hemin . Experiments were conducted at 12–16 days in vitro. Serum was washed out of cultures with MEM10 (> 1000X dilution) prior to addition of hemin. Cultures were incubated at 37°C in a 5% CO2 atmosphere for the entire exposure interval.ROS formation was quantified by staining with dihydrorhodamine 123 (DHR, Molecular Probes, Eugene, OR), which is a cell-permeable, non-fluorescent compound that is oxidized by cellular peroxides to fluorescent rhodamine . FluoresProtein oxidation was assessed using the Oxyblot™ kit (Chemicon, Inc., Temecula, CA). At the end of the hemin exposure interval, culture medium was aspirated, and cells were washed and then harvested in 100 μl of lysis buffer containing 210 mM mannitol, 70 mM sucrose, 5 mM HEPES, 1 mM EDTA, and 0.1% sodium dodecyl sulfate. Carbonyl groups were derivatized to 2, 4-dintrophenylhydrazone (DNP-hydrazone) by reaction with 2, 4-dinitrophenylhydrazine, following the manufacturer's instructions. Proteins were then separated on a 12% polyacrylamide gel and were transferred onto a polyvinylidene difluoride Imobilon-P transfer membrane filter (Millipore, Billerica, MA) using a semidry transfer apparatus (Bio-Rad, Hercules, CA). Carbonylated proteins were detected with rabbit anti-DNP (1:150) followed by goat anti-rabbit IgG (1:300). Immunoreactive proteins were visualized using Super Signal West Femto Reagent (Pierce Biotechnology, Rockford, IL) and Kodak ImageStation 400.After examination of cultures using phase contrast microscopy, cell death was quantified by measurement of LDH activity in the culture medium, as previously described . To faciCell death was also quantified by staining with propidium iodide (13 μg/ml for 15 min). When viewed with a rhodamine filter, the nuclei of cells with disrupted membranes stain red, while cells with intact membranes exclude propidium. Random 100X fields were captured with a Nikon Diaphot epifluorescence microscope and were analyzed with IPLab image analysis software. As with LDH data, fluorescence intensity was scaled to that in sister cultures treated with NMDA 300 μM for 40 h, which kills all neurons. Propidium iodide fluorescence was not observed in cells that had an astrocyte phenotype after treatment with NMDA or hemin at the concentrations used in this study.DHR: dihydrorhodamine; DNP: dinitrophenylhydrazone; HO: heme oxygenase; LDH: lactate dehydrogenase; MEM10: minimal essential medium containing 10 mM glucose; NMDA: N-methyl-D-aspartate; PI: propidium iodide; ROS: reactive oxygen species.RFR designed the study, collected and analyzed data, and wrote the manuscript. JC also participated in data collection and analysis, and edited the manuscript. LBZ participated in genotyping and edited the manuscript. All authors reviewed and approved the final manuscript.


A circadian rhythm of symptoms has been reported in allergic rhinitis and some studies have shown the dosing time of antihistamines to be of importance for optimizing symptom relief in this disease. The objective of this study was to examine the efficacy of morning vs. evening dosing of the antihistamine desloratadine at different time points during the day.Patients ≥ 18 years, with seasonal allergic rhinitis received desloratadine 5 mg orally once daily in the morning (AM-group) or evening (PM-group) for two weeks. Rhinorrhea, nasal congestion, sneezing and eye symptoms were scored morning and evening. Wilcoxon rank sum and 2-way ANOVA test were used.Six-hundred and sixty-three patients were randomized; 336 in the AM-group; 327 in the PM-group. No statistically significant differences were seen between the AM and PM group at any time points. In the sub-groups with higher morning or evening total symptom score no difference in treatment efficacy was seen whether the dose was taken 12 or 24 hours before the higher score time. There was a circadian variation in baseline total symptom score; highest during daytime and lowest at night. The circadian variation in symptoms was reduced during treatment. This reduction was highest for daytime symptoms.A circadian rhythm was seen for most symptoms being more pronounced during daytime. This was less apparent after treatment with desloratadine. No statistically significant difference in efficacy was seen whether desloratadine was given in the morning or in the evening. This gives the patients more flexibility in choosing dosing time. Allergic rhinitis is a common illness, which affects approximately 15 % of the population .Antihistamines are important medications in the treatment of allergic rhinitis. One should expect that the effect of an antihistamine is best near or shortly after peak serum level is attained. If this also coincides with the peak in allergy symptoms, an optimal treatment effect should be expected. In one study evening dosing of the antihistamine mequitazine  gave better symptom relief than morning dosing on morning symptoms . DesloraThe aim of this study was to examine the efficacy of the antihistamine desloratadine at different time points during the day and to evaluate whether the time of dosing of desloratadine has any impact on the treatment efficacy in seasonal allergic rhinitis (SAR).This was a randomized, open label, parallel group, multicenter study of two weeks duration in patients with SAR during the birch or grass pollen season. Eighty one medical centers in the Nordic countries participated. The inclusion criteria were: patients 18 years or above with a minimum of two years history of SAR confirmed by either a positive skin prick test or a positive serologic allergen test to the relevant seasonal allergen; clinically symptomatic with SAR at baseline/inclusion with a minimum total nasal symptom score (rhinorrhea, congestion, itching and sneezing) of at least 6 and rhinorrhea being minimum 2 (moderate); willingness to adhere to dosing and visit schedule. Females of childbearing potential had to use medically accepted methods of birth control and written informed consent had to be obtained from all patients.The exclusion criteria were: pulmonary disease, perennial rhinitis, sinusitis, rhinitis medicamentosa, pollen desensitization during the last 6 months, respiratory tract infection within the last two weeks, structural nasal abnormalities (including polyps), use of oral, nasal, ocular decongestants, corticosteroids in any form , other antihistamines , any investigational drug during the last 30 days, pregnant or nursing females.The patients were randomized into one of two treatment groups with dosing of 5 mg desloratadine tablets either in the morning between 07 – 09 (AM-group) or evening between 19 – 21 (PM-group) in a 1:1 ratio. Randomizing was computer generated for the whole study population using SAS version 6.12 and performed in blocks of eight. Each subject unit (bottle with medication) was labelled with randomization number. Physicians in the different Nordic countries recruited the patients. They assigned the medication in consecutive order. The study was monitored by Schering-Plough.The following symptoms were assessed using a scale from 0 to 3 (0=none, 1=mild, 2=moderate, 3=severe): rhinorrhea, nasal congestion, sneezing, itching nose and eye symptoms (itching, burning, tearing, redness). These symptoms were recorded in a patient diary every morning (AM 12 hours reflective and AM last hour) and evening (PM 12 hours reflective and PM last hour) both at baseline and during the 2 weeks treatment period. Interference with sleep and interference with daily activity were also assessed by the patients every day using the same scale from 0 to 3. In addition, the number of hours spent outdoors was recorded.th 2001 to September 2nd 2002. Pollen counts were not recorded.Visit 1 was at day 0 at the start of baseline, visit 2 after one week and visit 3 after two weeks. A wash-out period prior to Visit 1 was necessary if the patient had been on any drugs which could interfere with the study results . Baseline symptoms were recorded in the evening at day 0 and the following morning (day 1) after which the patients started taking the study medication as randomized. A physical examination was performed at visit 1 and 3. All adverse events were recorded. The study period was from April 11The primary objective was to evaluate the efficacy of 5 mg desloratadine taken orally once daily in the morning versus evening. The primary efficacy variable was the mean change from baseline for the AM last hour Total Symptom Score (TSS) over the 2 weeks treatment period. TSS is the sum of the individual symptom scores for the following symptoms that in prior studies  have shoIn a study on morning vs. evening dosing of the antihistamine mequitazine the differences in dosing-time-related efficacy increased in the sub-group of patients having predominantly morning symptoms . A sub-gAll patients receiving at least one dose of study drug and having at least one post dose registration were included in the efficacy analysis (intention-to-treat, ITT), and confirmatory analysis were based on evaluable patients with no protocol violations. Statistical analyses were made with 2-way ANOVA. For evaluation of response of therapy Wilcoxon rank sum test was used. Adverse events were tabulated.The study protocol and the patient informed consent form were approved by Ethics Committees and Health Authorities in each of the participating countries.Six hundred and sixty-three patients were randomized at baseline; 336 to the AM-group and 327 to the PM-group. The two groups were comparable with respect to demographics and baseline characteristics Tab 1). To assess the primary parameter 310 in the AM and 294 in the PM group fulfilled the criteria for ITT. Of the AM group 259 and of the PM group 254 patients completed the study without any violation. Mean baseline TSS varied between 4.64 and 6.10, a difference of 31%; highest during daytime (PM 12 hours reflective) and lowest at night (AM 12 hours reflective). The circadian variation at baseline was more evident for sneezing (around 60% difference between night and day), rhinorrhea and eye symptoms, less so for nasal itching and hardly noticeable for nasal congestion. Fig. . To asseDuring the two weeks period the mean reduction in TSS ± SE for AM last hour (primary efficacy variable) was 1.63 ± 0.17 (30 %) for the AM-group and 1.80 ± 0.17 (35 %) for the PM-group. There was no statistically significant difference  between the groups at this time point (p = 0.456) or at any other time points. The reduction in TSS was highest (2.5 – 41%) for day time symptoms (PM previous 12 hours) and lowest at night. This was evident for all individual symptoms except for nasal congestion.In the subgroup analysis comparing TSS AM last hour and PM last hour at baseline, 32 % of the patients had more severe symptoms in the morning (≥ 1 point difference in TSS) than in the evening, and 37 % had more severe symptoms in the evening. Looking at these two sub-groups, no difference in treatment efficacy on TSS was seen 12 or 24 hours post dosing  compared to morning dosing (24 hours before peak of symptoms). In our study no difference in treatment efficacy was seen 12 or 24 hours after dosing in the sub-group analysis of patients with higher baseline morning or evening TSS. Whatever the cause for this discrepancy between these two antihistamines, other antihistamines may show a variation in effect during the day not only on dermal symptoms ,12 but aThe adverse events recorded were of a magnitude and nature as seen in other studies of desloratadine and other antihistamines -17.Many patients have circadian variations in symptoms. The peak of symptoms can be at different time points from patient to patient. Individual dosing time of medication may improve symptom relief. Desloratadine, however, apparently shows no circadian variation in effect.A circadian rhythm was seen for most SAR symptoms at baseline, being most distressing during daytime, possibly due to long outdoor exposure. This circadian variation is less apparent after treatment with desloratadine. No statistically significant difference in efficacy was seen whether desloratadine was given in the morning or in the evening. This gives the patients more flexibility in choosing dosing time.The study was funded by Schering-Plough in the Nordic countries.None of the authors will gain financially from the publication. There are no patents pending. There are no other competing interests.RH participated in the design of the protocol and is the main author of the article.KH participated in the design of the protocol and as investigator.OB participated as principal investigator in Sweden and enrolled most patients in the study.SF participated in the statistical analysis, drafted the tables and figures and participated in drafting the manuscript.TØ was project leader and participated in the design of the protocol, the statistical analysis and drafting of the manuscript.