Antimicrobial resistance is particularly harmful to infectious disease management in low-income countries since expensive second-line drugs are not readily available. The objective of this study was to implement and evaluate a computerized system for surveillance of antimicrobial resistance at a tertiary hospital in Tanzania.A computerized surveillance system for antimicrobial susceptibility (WHONET) was implemented at the national referral hospital in Tanzania in 1998. The antimicrobial susceptibilities of all clinical bacterial isolates received during an 18 months' period were recorded and analyzed.The surveillance system was successfully implemented at the hospital. This activity increased the focus on antimicrobial resistance issues and on laboratory quality assurance issues. The study identified specific nosocomial problems in the hospital and led to the initiation of other prospective studies on prevalence and antimicrobial susceptibility of bacterial infections. Furthermore, the study provided useful data on antimicrobial patterns in bacterial isolates from the hospital. Gram-negative bacteria displayed high rates of resistance to common inexpensive antibiotics such as ampicillin, tetracycline and trimethoprim-sulfamethoxazole, leaving fluoroquinolones as the only reliable oral drugs against common Gram-negative bacilli. Gentamicin and third generation cephalosporins remain useful for parenteral therapy.The surveillance system is a low-cost tool to generate valuable information on antimicrobial resistance, which can be used to prepare locally applicable recommendations on antimicrobial use. The system pinpoints relevant nosocomial problems and can be used to efficiently plan further research. The surveillance system also functions as a quality assurance tool, bringing attention to methodological issues in identification and susceptibility testing. Exaggerated and irrational use of drugs, availability of antibiotics without prescription, the use of pharmaceuticals of doubtful quality and the HIV epidemic may all contribute to the current worldwide surge in antimicrobial drug resistance. Emerging resistance to antimicrobial drugs increases morbidity and mortality by hampering the provision of effective chemotherapy, and makes treatment more costly -3. The sIt is widely held that surveillance of antimicrobial susceptibility is fundamental to combat the emergence of resistance . SurveilThis paper describes the experience with the implementation of a computerized surveillance system for antimicrobial drug susceptibility at Tanzania's major referral hospital, and its use to analyze the susceptibility patterns of 7621 consecutively recorded clinical bacterial isolates.The study was performed at Muhimbili National Hospital (MNH), Dar es Salaam, Tanzania. With more than 1000 beds, MNH is the largest hospital in the country and serves as a national referral and university teaching hospital, as well as a primary and referral hospital for a population of approximately 3.6 million in the Dar es Salaam area. The Department of Microbiology and Immunology at MNH examines specimens from inpatients and outpatients at MNH, and from a number of nearby hospitals. Bacteriological cultures are performed on more than 23,000 specimens per year.st 1998 to December 31st 1999 were recorded and analyzed. The specimens examined included urine, pus/secretions , blood, cerebrospinal fluid, other body fluids, stools and other specimens. Mycobacteria and anaerobic bacteria were not included in the study. Apart from the WHONET software, we used Stata 8.0 for Macintosh  to evaluate differences of proportions by Fisher's exact test .A free-of-charge software for the surveillance of antimicrobial resistance   was implS. aureus NCTC 6571, E. coli NCTC 10418 or Pseudomonas aeruginosa NCTC 10662. The isolates showing intermediate resistance were few and were grouped together with sensitive isolates for the purpose of data analysis. Either methicillin or oxacillin disks were used to test for methicillin-resistance in S. aureus, the results being considered equivalent and interchangeable in the data analysis. ß-Lactamase testing was not routinely performed. The susceptibility of pneumococci to penicillin was examined by the use of penicillin 2 μg disks. Commercially produced antibiotic disks, mostly obtained from Oxoid Limited, were used, however, in some instances, antibiotic disks, prepared locally were used due to financial constraints. The Department of Microbiology and Immunology participates in an external quality assessment program in bacteriology led by the World Health Organization-collaborating centre, the National Institute for Communicable Diseases (NICD), Johannesburg, South Africa. The Department of Microbiology and Immunology at our hospital receives bacterial strains from NICD, performs species identification and antimicrobial susceptibility testing, and report the results back to NICD.The specimens were cultured and the bacterial isolates identified using standard microbiological methods as described in Mackie & McCartney Practical Medical Microbiology . SusceptWe evaluated the strengths and shortcomings of the surveillance system in our setting, particularly in terms of how well it performed in its main application areas, providing locally applicable data to guide empiric therapy, monitoring antimicrobial susceptibility trends, detecting the emergence and spread of new resistance traits and as a tool for quality assurance. We also assessed the cost-implications of implementing the surveillance program in our setting. We considered direct costs, such as the purchase of equipment, and indirect costs, such as those related to the running of the laboratory, including human resources. We also comment on the benefits of the surveillance system related to both direct patient care and long-term implications of containing antimicrobial resistance.Bacillus spp. were recovered from 22.0% (n = 447) and 6.9% (n = 141) of the blood cultures, respectively. Furthermore, five Candida spp. isolates and one Cryptococcus neoformans were recovered. Among the 49 Salmonella isolates, two were identified as S. Typhi, 16 as S. Typhimurium, 16 as S. Paratyphi B and one each as S. Paratyphi C, S. Enteritidis and S. Arizonae. Twelve Salmonella isolates were not serotyped. Among the 41gonococcal isolates, 28 (68.3%) were from genital swabs. Eleven (26.8%) gonococcal isolates were obtained from the neonatal ward, out of which 4 were specified as from eye discharge.A total of 7617 bacterial isolates were registered during the study period, of which 67.4% (n = 5134) were Gram-negative and 32.6% (n = 2483) Gram-positive. Table Specimens from inpatients and outpatients contributed to 53.2% and 31.9% of the isolates, respectively. A further 6.0% were obtained from specimens from other hospitals in Dar es Salaam, while 8.8% were obtained from other or unknown locations. Among the isolates from inpatients, 36.5% were obtained from the Department of Pediatrics, 28.4% from the neonatal section and 8.1% from the other pediatric wards. The other isolates came from the Departments of Surgery (22.4%), Internal Medicine (16.6%), Obstetrics and Gynecology (9.8%), the Intensive Care Unit (4.9%) and other locations . For 4900 isolates, the age or the estimated age group of the patient was known. Of these, 23.6% (n = 1155) were from neonates (≤ 1 month old), 6.8% (n = 335) from children aged one month to seven years, and 69.6% (n = 3410) from adults or children older than 8 years.Salmonella isolates (data not shown). The majority of Pseudomonas aeruginosa isolates was susceptibility-tested to gentamicin only, to which 4.3% (15/350) were resistant. Among the isolates of Neisseria gonorrhoeae, 70.0% were resistant to penicillin, 45.2% to tetracycline, 59.3% to trimethoprim-sulfamethoxazole, 5.9% to erythromycin and none was resistant to spectinomycin, fluoroquinolones or amoxicillin-clavulanate (data not shown).Tables E. coli from inpatients than in those from outpatients as shown in Table Klebsiella spp. from inpatients were more frequently resistant to gentamicin and trimethoprim-sulfamethoxazole than isolates from outpatients.Comparison of resistance patterns of isolates obtained from inpatients and outpatients at MNH did not show large differences. However, ampicillin resistance was more frequent in urinary isolates of Klebsiella spp. were indeed more frequently resistant to gentamicin than those from other specimen types. A significantly greater proportion of blood culture isolates of S. aureus were resistant to tetracycline than among those from other specimen types, whereas for penicillin the isolates from blood cultures were resistant in a lower proportion than the others.Comparison of resistance patterns in isolates blood cultures with those from other specimen types showed apparent great differences for some drugs, however, in most cases the number of blood culture isolates were few and did not show statistically significant differences. However, as shown in Table A great number of bacterial isolates were recorded in the system. All age groups and both inpatients and outpatients were represented in the study. More than a third of the isolates were from outpatient populations from the Dar es Salaam area, however we cannot exclude the possibility of a selection bias in favour of patients with infections caused by resistant organisms, since many patients get treatment at primary health facilities before reaching MNH. We do not know how well the rural population is represented in this material, but we assume that the outpatients in the study are mostly from the Dar es Salaam area. Ten percent of the isolates represented systemic infections, i.e. isolates from blood cultures and spinal fluid. The susceptibility test results were recorded as interpreted values (i.e. "R" (resistance), "I" (Intermediate) or "S" (susceptible)) and not as inhibition zone diameters. In this study, no molecular techniques were available for the detection of resistance genotypes and evaluation of genetic relatedness of bacterial isolates.The direct cost of implementing the surveillance system was limited to the purchase of a computer at approximately 1000 Euro. However, less expensive second-hand computers would be sufficient. The software was downloaded free of charge from the WHO website. The indirect costs of running this surveillance program are related to human sources for operating the software, including data entry and analysis, and the costs of the susceptibility testing activities. It is difficult to separate these indirect costs from the costs of running the daily laboratory activities. In our setting, a laboratory technologist from the department took on the task of operating the software in addition to her regular duties. In our experience, for a hospital of our size, it is recommendable to allocate approximately 50% of a laboratory technologist position to operating the surveillance software. In our setting, this would translate into a monthly cost of approximately 100 Euro for the department. The surveillance system is dependent on susceptibility testing of acceptable quality. The susceptibility testing incurs costs related to human resources and the purchase of laboratory reagents including antimicrobial disks and agar media. Implementing a surveillance system may increase these costs by focusing on the importance of quality reagents. However, since the susceptibility testing activities are an integral activity of the department, which would have been performed regardless of the surveillance system, we choose not to attribute their costs to the surveillance system in this context. The benefits of a surveillance system are difficult to quantify, but are of potentially great magnitude. Foremost, surveillance data may improve empiric therapy for infections and thus save lives and reduce suffering. It may reduce treatment costs by enabling the use of the least expensive effective drugs. Additionally, surveillance systems may contribute to containing or reducing antimicrobial resistance, which in the long term perspective may have great benefits in reducing morbidity and mortality, and diminish the need for expensive second-line antimicrobial agents.The strengths and weaknesses are elaborated on in the Discussion part.Experience from the World Health Organization's External quality assurance system for antimicrobial susceptibility testing has shown that disk diffusion testing is suitable for routine surveillance . HoweverE. coli, Klebsiella spp., Proteus spp. and Salmonella. Chloramphenicol may fail to cure as much as a quarter of infections caused by Salmonella and half or more of infections caused by E. coli, Klebsiella spp. and Proteus spp. Fluoroquinolones appear to be the only reliable drugs for oral treatment of infections caused by common Gram-negative bacilli, whereas gentamicin and third-generation cephalosporins remain useful for parenteral therapy.Consistent with observations from a number of other countries in the region -15 and eS. aureus, consistent with previous data from the same hospital [The study showed a very low prevalence of methicillin-resistant hospital ,18. Whilhospital . It is rhospital -21. Whilhospital ,23, the hospital  from 199Recommendations for antibiotic treatment of serious bacterial infections such as bloodstream infections and meningitis should preferably be based on knowledge of the prevalence and antimicrobial susceptibility patterns of pathogens isolated from blood and spinal fluid. While a fair number of bacterial isolates were tested in the current study, the number of blood culture isolates was limited . As shown in Table E. coli and gentamicin and trimethoprim-sulfamethoxazole resistance in Klebsiella spp. were more frequent in urinary isolates from inpatients than from outpatients. Apart from that, there were no dramatic differences between isolates from inpatients and outpatients. The data from the study should be representative for both the hospital setting and to some degree the population in Dar es Salaam. However, the majority of the population of Tanzania lives in rural areas, where resistance patterns may be substantially different. Thus one should be cautious to extrapolate the results of the current study to be valid for populations in the countryside.It is important to specify for which population the surveillance data are valid. At our hospital, specimens from both inpatients and outpatients were examined. The hospital is to a great extent used as a primary hospital for the population in the Dar es Salaam area. However, among the cases coming to the hospital, there may be a degree of selection of patients with infections caused by resistant microbes, since many patients rely on health centers and pharmacies to cure simple ailments, and only come to the hospital when primary treatment fails. The study found that a few resistance traits, such as ampicillin resistance in E. coli has increased from zero in 1978–79 [E. coli has increased from 2% in the late seventies [Certain trends in antimicrobial susceptibility could be identified by comparison with data from other studies. While resistance to ampicillin, tetracycline and sulfonamides in Gram-negative bacteria was frequent already in the seventies ,27, it i 1978–79  and 8% ieventies  to 20% aeventies .E. coli and Klebsiella spp. were tested for susceptibility to third-generation cephalosporins and other methods for detection of ESBL  were not available.Resistance to gentamicin is common in Gram-negative bacteria with extended-spectrum beta-lactamases (ESBL), sometimes in as much as 96% of isolates . Such anP. aeruginosa the rate of gentamicin-resistance has increased, from zero in the seventies [S. aureus isolates in this study. However, the rate of tetracycline resistance (49%) was lower than reported from the same hospital in 1979 (57%) and 1982 (74%) [Vibrio cholerae, the use of the drug was subsequently greatly reduced, and this may have contributed to a concurrent decline in the rate of tetracycline-resistance in an unrelated species such as S. aureus.Also in eventies  to 4% in82 (74%) . In the 82 (74%) . Due to For meaningful comparison of data from different studies, whether from the same or different laboratories, the same method of susceptibility testing should preferably be employed. In our laboratory, the same method has been used for a number of years. The WHONET software features a number of sophisticated ways to analyze susceptibility information based on the measurements of inhibition zone diameters. Recording the diameter of the inhibition zones in disk diffusion testing is generally recommended , and mayFurthermore, variations over time in the battery of antibiotics tested makes comparison of data less useful. Laboratories in low-income countries are sometimes vulnerable to this because of unreliable supplies of antibiotic discs.S. aureus and ESBL in Gram-negative bacteria. The current surveillance indicated that methicillin-resistance is rare in S. aureus at the hospital. Ideally this should be confirmed with PCR-based methods to detect the mecA gene. Likewise, the disk diffusion testing showed the presence of resistance to ceftazidime in Gram-negative isolates, albeit at a low rate, which calls for further investigation with regard to the possible presence of ESBL. Our laboratory did not employ molecular methods for detection of resistance genes on a routine basis, but a recent study showed low prevalence of methicillin-resistant S. aureus (MRSA) [Disk diffusion testing may give indications of emerging resistance traits such as methicillin-resistance in s (MRSA) . ResistaKlebsiella spp., which may suggest possible nosocomial spread. The analysis of antibiograms did not produce convincing evidence of clonal patterns spread of bacterial isolates, possibly partly due to the variations in the battery of antibiotics tested. Molecular methods for the evaluation of the genetic relatedness of bacteria were not available in this study.The WHONET software is well suited to analyze antibiograms in order to detect suspicious nosocomial outbreaks. These functions too are dependant on the use of a consistent battery of test drugs, and also works better when results are entered as actual values for MIC or zone diameters, as opposed to the interpreted value . In our hospital, comparison of resistance rates did not show dramatic variation between isolates from inpatients and outpatients. The exception was a trend for more frequent gentamicin-resistance in inpatient isolates of Gram-negative bacteria, particularly in vitro methods are followed, and only in Europe the number is at least ten [In vivo clinical assessment is of great importance in understanding bacterial drug resistance and the gold standard for evaluating resistance in malaria parasite. The multitude of methods employed for antimicrobial susceptibility testing has to some extent hampered the meaningful sharing and comparison of resistance data among countries. Recently, much work has been done in Europe to harmonize resistance surveillance efforts across country borders [In 2002 a total of 880 laboratories in 76 countries across the world used the software, including 41 laboratories in 4 African countries. The WHONET system has been implemented at MNH since 1998. Unfortunately, there is no international consensus on a recommended method for antimicrobial susceptibility testing. Worldwide at least twelve different east ten . Furthereast ten . In addi borders ,34. WhilStreptococcus pyogenes were reported as resistant to penicillin. This was subsequently double-checked, and consulting the laboratory bench-book we found that clerical errors were the explanation for this. The use of the surveillance software enabled the easy detection, investigation and correction of such errors, and consequently may contribute to increase the attention to quality issues and generally improve the performance of the lab. The current surveillance project highlighted some methodological issues, most of which were caused by budgetary limitations, such as the occasional use of locally made antibiotic disks and limitations in the identification of organisms due to lack of reagents.The implementation of the surveillance system brought focus on methodological issues, including microbial identification and susceptibility. The WHONET software has built-in functions to alert the operator if isolates with unexpected resistance patterns are entered. During the surveillance exercise in our laboratory, it was discovered that four isolates of Routine surveillance makes use of available large data sets at little additional cost and may be representative for a greater part of the population. However, often it is necessary to supplement the routine surveillance with ad hoc studies aimed at investigating particular problems. While ad hoc studies generally are more expensive to conduct, they allow for the use of more advanced and expensive laboratory methods and are better at targeting particular populations of interest. The current surveillance study identified a need for more data from bloodstream infections in order to provide reliable guidance for the treatment of serious bacterial infections. As a consequence of this, we started a study of bloodstream infections with the pediatric department at the hospital. Another laboratory-based research was started to ascertain the finding that methicillin-resistance in staphylococci is still relatively infrequent at this hospital.) in Tanzania.Resistance surveillance is a platform from which to promote focus on antimicrobial resistance issues, both within the hospital and the medical community, but also among the general population. In conjunction with the surveillance exercise, we have highlighted issues regarding antimicrobials and resistance in local newspaper letters , and theThe study suggests that laboratories, which perform susceptibility testing, can gain useful information on antimicrobial susceptibility with a minimal budget. As appropriate software can be obtained free-of-charge, the main cost of the surveillance system is associated with purchasing a computer. However, there are other, indirect costs, which may be attributed to the surveillance program depending on the situation of the laboratory, such as running costs for microbiologic procedures, including susceptibility testing. Particularly, it is important to ensure availability of antimicrobial discs of satisfactory quality. A susceptibility surveillance system also implies the need for some additional human resources for data entry and analysis. In our experience, it is recommendable to allocate approximately 50% of a laboratory technologist position to this task. While the WHONET program is excellent for entry, analysis and reporting of resistance data, the software is not intended to function as a complete patient management system for the laboratory. Data can be transferred from other databases into WHONET by the use of a complementary software called BacLink . However, in laboratories such as ours, where the management of patients' laboratory tests  is handled manually via register-books, the data must be punched into WHONET by hand. Since the WHONET database is not used directly for patient management, the surveillance activity tends to become less integrated in the clinical routine work than it should. Thus, although the program performs its task very well, in a long-term perspective, a surveillance system that is integrated with a patient management system might be more sustainable. It is difficult to quantify the potential benefits of a well-functioning surveillance system. However, we are fully convinced that the modest costs of the surveillance program are highly justified since the data generated may improve empiric therapy, help contain or prevent the further emergence of antimicrobial resistance, decrease the need for expensive second-line antimicrobial drugs and, ultimately, save lives and reduce suffering.It is imperative to preserve the effectiveness of common antibiotics by promoting rational use of antibiotics based on sound knowledge of local resistance patterns. In a hospital with bacteriology services, the implementation of a computerized surveillance system is a low-cost tool to make use of available resistance data. In our hospital, the resistance surveillance system has generated information on resistance patterns that is useful as guidance for empiric therapy of infections. It can help alert clinicians of trends of antimicrobial resistance, guide drug-policy decisions and facilitate rational use of drugs to prevent the further emergence of antimicrobial resistance. The surveillance system has also served as a quality assurance tool and led to increased focus on antimicrobial resistance and prudent use of drugs. There is need for more data from blood cultures for reliable guidance for the treatment of severe, systemic bacterial infections. For antibiotic policy recommendations to be applicable for the general population, more information is needed from outpatients and rural areas.There is limited information on antimicrobial resistance trends on the African continent. Only four African countries use the WHONET system for antimicrobial resistance surveillance, although some countries may use other similar software. Recently much work has been done to establish consensus and a more standardized approach to resistance surveillance in Europe . SusceptThe authors declare that they have no competing interests.BB was the principal investigator, participated in the planning and execution of the study, performed data entry and data analysis, and was the main responsible author. DSMM, WU, SYM and AD participated in the planning of the study and contributed to the writing process. MM contributed to designing the WHONET database, performed data entry and microbiological work, and contributed to the writing process. SH participated in the writing. NL was the project coordinator and participated in planning, data analysis and writing.The pre-publication history for this paper can be accessed here:


Birds have smaller average genome sizes than other tetrapod classes, and it has been proposed that a relatively low frequency of repeating DNA is one factor in reduction of avian genome sizes.Gallus gallus) autosomes were quantified and compared with those in human autosomes. In the chicken 10.3% of the genome was occupied by DNA repeats, in contrast to 44.9% in human. In the chicken, the percentage of a chromosome occupied by repeats was positively correlated with chromosome length, but even the largest chicken chromosomes had repeat densities much lower than those in human, indicating that avoidance of repeats in the chicken is not confined to minichromosomes. When 294 simple sequence repeat types shared between chicken and human genomes were compared, mean repeat array length and maximum repeat array length were significantly lower in the chicken than in human.DNA repeat arrays in the sequenced portion of the chicken (The fact that the chicken simple sequence repeat arrays were consistently smaller than arrays of the same type in human is evidence that the reduction in repeat array length in the chicken has involved numerous independent evolutionary events. This implies that reduction of DNA repeats in birds is the result of adaptive evolution. Reduction of DNA repeats on minichromosomes may be an adaptation to permit chiasma formation and alignment of small chromosomes. However, the fact that repeat array lengths are consistently reduced on the largest chicken chromosomes supports the hypothesis that other selective factors are at work, presumably related to the reduction of cell size and consequent advantages for the energetic demands of flight. Genomes sizes (as measured by the DNA mass per diploid nucleus) are smaller on average in birds than in other tetrapod classes, and genome sizes within the class Aves show less variation than those of other tetrapod classes ,2. It haAn alternative to the hypothesis that the reduced genome size is adaptive is the hypothesis that it resulted from an event of genomic DNA loss that was fixed in the ancestor of all birds due to genetic drift. The fixation of even a deleterious mutation is possible if the population undergoes an extreme bottleneck . Some auGallus gallus) and human (Homo sapiens) genes. They found that corresponding introns were significantly shorter in chickens, indicating that numerous independent deletions have occurred in the introns of birds. These results support the hypothesis that genome size reduction in birds is adaptive, since it is unlikely that such a large number of independent deletion events were due to chance alone. Additional evidence in support of the adaptive hypothesis is provided by the observation that a secondary increase in genome size has occurred in avian lineages which have become flightless or have reduced flying ability [In order to decide whether genome reduction in birds was adaptive or due to a random event, Hughes and Hughes  compared ability .It has been suggested that an important factor in genome size reduction in birds has been that birds have lower levels of repetitive DNA than other vertebrates . GenomesThe sequencing of a substantial portion of the chicken genome has made it possible to examine quantitatively the distribution of repeating sequences on different chromosomes in the genome. Here we compare the distribution of repeats on 28 sequenced autosomes of chicken with that on the 22 human autosomes in order to test the hypothesis that reduction in repeat density in the avian genome has occurred as a result of adaptive evolution.The characteristics of repeat arrays on the 28 sequenced chicken chromosomes are summarized in Table In the chicken genome, there was a significant positive correlation  between chromosome length and the percentage of the chromosome occupied by repeats (% repeats) based on reassociation kinetics ,12. By c% based oMoreover, when simple sequence repeat array types shared between chicken and human genomes were compared, mean repeat array length and maximum repeat array length were significantly lower in the chicken than in human. The fact that these differences occurred consistently in nearly 300 distinct array types is evidence that the reduction in repeat arrays in the chicken has involved numerous independent evolutionary events. Mutational changes to simple sequence repeat arrays typically involve slippage events that either decrease or increase the number of repeat units . The facThere were substantial differences among chicken chromosomes with respect to the percentage of the chromosome occupied by repeats, and % repeats increased significantly as a function of chromosome length. This trend implies that the avian genome is characterized by an especially pronounced avoidance of longer repeats on the smaller chromosomes. This finding is consistent with the hypothesis of Burt  that theHowever, even the largest chicken chromosomes had repeat densities much lower than human chromosomes of similar length Figure . This imGallus gallus) genome assembly  was downloaded from Ensembl web site at . Only autosomes were used in the analyses; data were available for chromosomes 1 through 24, 26, 27, 28 and 32. We extracted Ensembl annotations of the features of repeat arrays . The human genome assembly ) was downloaded via the UCSC Genome Browser . Repeat information based on the RepeatMasker annotations  was extracted from the UCSC genome annotation database. Only autosomes (chromosomes 1 through 22) were used. For both chicken and human, repeats tallied included simple sequence repeats, class I elements, class II elements, low-complexity regions, and satellite regions. In addition, we compared between genomes a set of 294 simple sequence repeats which were present in both genomes; i.e., repeats of the same short nucleotide motif were present in both genomes. For these 294 repeat types, mean, minimum and maximum length of repeat arrays were compared in pairwise fashion between human and chicken.The chicken (HP gathered and summarized the data. ALH conducted statistical analyses and wrote the manuscript. Both authors read and approved the final manuscript.


Several problems exist with current methods used to align DNA sequences for comparative sequence analysis. Most dynamic programming algorithms assume that conserved sequence elements are collinear. This assumption appears valid when comparing orthologous protein coding sequences. Functional constraints on proteins provide strong selective pressure against sequence inversions, and minimize sequence duplications and feature shuffling. For non-coding sequences this collinearity assumption is often invalid. For example, enhancers contain clusters of transcription factor binding sites that change in number, orientation, and spacing during evolution yet the enhancer retains its activity. Dot plot analysis is often used to estimate non-coding sequence relatedness. Yet dot plots do not actually align sequences and thus cannot account well for base insertions or deletions. Moreover, they lack an adequate statistical framework for comparing sequence relatedness and are limited to pairwise comparisons. Lastly, dot plots and dynamic programming text outputs fail to provide an intuitive means for visualizing DNA alignments.). GATA uses the NCBI-BLASTN program and extensive post-processing to identify all small sub-alignments above a low cut-off score. These are graphed as two shaded boxes, one for each sequence, connected by a line using the coordinate system of their parent sequence. Shading and colour are used to indicate score and orientation. A variety of options exist for querying, modifying and retrieving conserved sequence elements. Extensive gene annotation can be added to both sequences using a standardized General Feature Format (GFF) file.To address some of these issues, we created a stand alone, platform independent, graphic alignment tool for comparative sequence analysis (GATA GATA uses the NCBI-BLASTN program in conjunction with post-processing to exhaustively align two DNA sequences. It provides researchers with a fine-grained alignment and visualization tool aptly suited for non-coding, 0–200 kb, pairwise, sequence analysis. It functions independent of sequence feature ordering or orientation, and readily visualizes both large and small sequence inversions, duplications, and segment shuffling. Since the alignment is visual and does not contain gaps, gene annotation can be added to both sequences to create a thoroughly descriptive picture of DNA conservation that is well suited for comparative sequence analysis. The most widely used methods for aligning DNA sequences rely on dynamic programming algorithms initially developed by Smith-Waterman and Needleman-Wunsch ,2. TheseWhen aligning protein coding sequences, dynamic programming works quite well. Evolution exerts significant functional constraint on protein coding sequences. When an inversion, duplication or segment-shuffling event occurs, the protein is often compromised by truncation due to the introduction of frame shifts and stop codons. These deleterious mutations are typically lost and not observed in the surviving population. When aligning this type of constrained sequence element, dynamic programming works quite well.Functional non-coding sequences do not appear to be as constrained in the ordering of elements as protein coding sequences -6. CompaGATA employs a two tiered architecture in aligning DNA sequences. GATAligner executes and processed BLASTN output. GATAPlotter displays the processed alignments and annotation from GATAligner.The GATAligner application figure  uses theOur initial goal was to create a high resolution sequence alignment and visualization tool to use in identifying small sequence rearrangements, like those associated with evolving non-coding regulatory DNA. We initially divided the first sequence into overlapping windows offset by one base pair. A Smith-Waterman dynamic programming algorithm was then used to align each window against the entire second sequence. Windows were scored, merged, and saved as above. Although this method is more rigorous than using BLASTN, it took 20–50 times as long, and did not produce significantly different results (data not shown). It should be noted that BLASTN requires seven consecutive identical bases to align two sequences. Thus in rare cases, some windows will be missed, for example, GGGGGGcTTTTTTaCCCCCCgAAAAAA versus GGGGGGaTTTTTTgCCCCCCtAAAAAA.The GATAPlotter application figure  takes suGATAPlotter also has the capability to display extensive gene annotation for one or both input sequences. The principle component of gene annotation rendering by GATA is the "GeneGroup" figure . Each GeDrosophila melanogaster and D. pseudoobscura. Figure To illustrate the types of rearrangements GATA can distinguish, examine figures Several related alignment and visualization tools have proven useful in comparative sequence analysis. Dot plot analysis can be used to identify duplications and inversions. Programs such as Dotter, JDotter, Dotlet, and Family Relations -16 generOne program that is proving quite useful in avoiding the collinearity problem while still using a dynamic programming algorithm is Shuffle LAGAN . AlignmeOne of the major challenges facing bioinformaticians is the development of alignment and visualization tools for multi-species comparative sequence analysis. Within the fly community alone, 12 divergent species of diptera and hymenoptera will be sequenced within 3 years. A variety of higher eukaryotes including human, mouse, rat, dog, chimp, cow, chicken, opossum, and platypus have or are in the process of being completely sequenced. How can one visualize the alignment and species-specific annotation for 12 orthologs of a particular gene or a genomic segment? The GATA alignment paradigm is well suited to this challenge and will play a prominent role in GATA's development.As comparative sequence analysis accelerates, scientists need more sophisticated alignment and visualization tools to define the evolutionary relationships and functional significance between particular orthologous sequences. This is especially true for regulatory, non-coding DNA that can show significant small-scale rearrangements. These new tools must incorporate detailed annotation alongside views of sequence conservation while providing easy access to the underlying sequence information. GATA provides one such solution.Project name:GATA, graphic alignment tool for comparative sequence analysis.Project home pages: and Operating system(s):Platform independentProgramming language:JavaOther requirements:Java 1.4 or higherLicense:GNU GPLAny restrictions to use by non-academics:NoneDAN designed and constructed the GATA programs with advice and supervision from MBE.


Dyslipidemia has been associated with hypertension. The present study explored if polymorphisms in genes encoding proteins in lipid metabolism could be used as predictors for the individual response to antihypertensive treatment.1-adrenergic receptor blocker atenolol for twelve weeks.Ten single nucleotide polymorphisms (SNP) in genes related to lipid metabolism were analysed by a microarray based minisequencing system in DNA samples from ninety-seven hypertensive subjects randomised to treatment with either 150 mg of the angiotensin II type 1 receptor blocker irbesartan or 50 mg of the βThe reduction in blood pressure was similar in both treatment groups. The SNP C711T in the apolipoprotein B gene was associated with the blood pressure response to irbesartan with an average reduction of 19 mmHg in the individuals carrying the C-allele, but not to atenolol. The C16730T polymorphism in the low density lipoprotein receptor gene predicted the change in systolic blood pressure in the atenolol group with an average reduction of 14 mmHg in the individuals carrying the C-allele.Polymorphisms in genes encoding proteins in the lipid metabolism are associated with the response to antihypertensive treatment in a drug specific pattern. These results highlight the potential use of pharmacogenetics as a guide for individualised antihypertensive treatment, and also the role of lipids in blood pressure control. Hypertension is a complex trait caused by multiple environmental and genetic factors interacting through the cardiac, vascular and endothelial systems. Several drug classes with different mechanisms of action, including inhibitors of the renin-angiotensin-aldosterone system (RAAS), calcium channel blockers, adrenergic receptor blockers and diuretics, are available for treatment of hypertension. However, the response to antihypertensive treatment is highly variable between individuals, which makes it difficult to predict the efficacy of a specific drug in the individual patient -3. CurreTwin studies have estimated that as much as half of the variability in blood pressure levels between individuals is due to genetic factors ,8. Based1-adrenergic receptor blocker atenolol [The RAAS and the sympathetic nervous system play key roles in blood pressure regulation. We have earlier shown that polymorphisms in the angiotensin converting enzyme gene  and a SNatenolol . DyslipiWe have recently developed a microarray based minisequencing system for parallel genotyping of multiple SNPs in blood pressure regulating candidate genes . Here we1-adrenergic receptor blocker atenolol once daily as monotherapy. The dose was doubled after six weeks if the diastolic blood pressure was ≥ 90 mmHg. Blood pressure was measured by trained nurses using a mercury sphygmomanometer, after the patients had rested for at least 10 min in the seated position. Left ventricular hypertrophy was defined as left ventricular mass index of > 131 g/m2 for men and > 100 g/m2 for women, assessed by echocardiography. The data presented relates to the change in blood pressure after 12 weeks of treatment. For details on the SILVHIA trial, see Malmqvist etal [DNA extracted from blood samples from 97 hypertensive patients from the double blind parallel group "Swedish Irbesartan Left Ventricular Hypertrophy Investigation versus Atenolol" (SILVHIA) trial  were anaist etal . Baselin) and the SNP Consortium  databases and validated in a pooled DNA sample representing the Swedish population. A subset of these SNPs located in genes involved in lipid metabolism and that were polymorphic in the Swedish population were included in the study presented here, together with one additional SNP in the apolipoprotein A-V gene. See Table In our previous study , 98 SNPsFragments comprising the SNPs were amplified in multiplex PCR described previously . A microAnalysis of covariance (ANCOVA) with each SNP as factor, baseline blood pressure as covariate and the change in blood pressure as response, was performed. The analyses were performed by treatment group and blood pressure measurement (systolic and diastolic blood pressures). Correction for multiple testing was performed by calculation of critical p-values corresponding to a nominal type I error of 5% using a permutation test . Two taiWe explored possible associations between individual genotypes of ten SNPs and reduction in systolic and diastolic blood pressure as response to treatment with atenolol or irbesartan Figure  in samplCorrection for multiple testing using a permutation test  yielded The SNP C711T in the apolipoprotein B gene is located in the coding region of the gene, and alters a threonine residue to an isoleucine residue in the protein. This SNP is located in the amino-terminal part of the enzyme and has been suggested to affect the dimerisation of apolipoprotein B and low density lipoprotein during cholesterol transport . The C16We have recently found circulating apolipoprotein B to be the most powerful predictor of endothelium-dependent vasodilation of the commonly used markers of cholesterol metabolism . It is nIn our earlier exploratory study, 74 SNPs with a minor allele frequency over 5%, including nine of the SNPs analysed here were tested as predictors of blood pressure regulation in the SILVHIA study samples using a multiple regression model . The maiA remaining weakness in our study is the small number of samples available for analysis, which does not allow detection of small to medium size gene effects, and results in uncertain estimation of the the magnitude of the effects detected. Moreover, in a small study there is the risk of a non-representative group of patients with respect to gender, age, and genotype distribution. Despite these limitations, we detected a significant effect of the SNP C711T in the apolipoprotein B gene and the SNP C16730T in the low density lipoprotein receptor after correction for multiple testing. The pharmacogenetically interesting results from our study need to be replicated in other studies.As the C711T SNP in the apolipoprotein B gene predicted response to treatment with irbesartan, and the C16730T SNP in the low density lipoprotein receptor gene appeared to predict response to atenolol treatment, our results point at possible use of SNPs in genes encoding components of lipid metabolism in pharmacogenetic panels for selecting the optimal drug for each patient. To our knowledge our study is the first one to investigate the relationship between polymorphisms in genes involved in lipid metabolism and the response to antihypertensive treatment.The authors declare that they have no competing interests.UL performed the development of genotyping technology, genotyping lab work, interpretation of data and had a substantial role in writing the manuscript. LL provided clinical expertise, participated in selection of candidate genes and contributed to writing. LK provided clinical expertise, established a database of the SILVHIA phenotypes, and in writing. LB performed the statistical analysis. TK provided the SILVHIA samples and contributed to writing the manuscript. A-CS contributed by planning and supervision of the project, and to writing the manuscript.The pre-publication history for this paper can be accessed here:


Hirudo medicinalis, has been used in plastic and reconstructive surgery, to relieve venous congestion and to improve the microrevascularization of flaps. In many countries, wild leeches are still provided from local markets and utilised with antibiotic prophylaxies. In this research, results of identification of bacteria in the transport fluid is reported, oral and intestinal floras and the antibiograms of the identified microorganisms are investigated. Also, to avoid possible infections, the ability of hypochloric acid, a disinfectant, to suppress the relevant microorganisms without changing the life style and behavior of leeches in terms of sucking function, is investigated.Medicinal leech, ppm concentrations in different groups of 25 leeches. Finally, 20 leeches were applied atraumatically to the bleeding areas of rats, the duration of suction was determined and compared statistically between the leeches treated and not treated with hypochloric acid solution.Bacterial identifications and antibiograms of oral and intestinal flora and transport medium were performed for 10 leeches. The optimum concentration of hypochloric acid which eliminated microorganisms without affecting the viability and sucking function of the leeches were determined by dilution of hypochloric acid to 100, 50, 25, 12.5, 6.25 Aeromonas hydrophilia was the most commonly identified microorganism and found to be resistant to first generation cephalosporins, frequently used in prophylaxis at surgical wards. In the next stages of the study, the leeches were subjected to a series of diluted hypochloric acid solutions. Although disinfection of the transport material and suppression of the oral flora of hirudo medicinalis were successful in 100, 50, 25, 12.5, 6.25 ppm concentrations; 12.5 ppm solution was the greatest concentration in which hirudo medicinalis could survive and sucking function was not affected significantly.External decontamination of wild leeches with 12.5 ppm hypochloric acid enables bacterial suppression without causing negative effects on leech sucking function and life. Hirudo medicinalis, has been used with increasing frequency during the past few decades for salvage of venous compromised pedicled flaps, microvascular free-tissue transfers and replantations. Although the therapeutic use of leeches in medicine dates back 50 BC; for centuries they were collected from various water supplies and utilised under septic conditions with the risk of wound infection and infestation. The supply of leeches was modernized by medicinal leech farm set up in the 1970s [The medicinal leech, Aeromonas is the most common microorganism in leech infections and may cause a wide spectrum of deseases such as cellulitis, ocular infections, arthritis, myocarditis, peritonitis, meningitis, bacteremia and sepsis [Today, leech therapy is indicated in plastic and reconstructive surgery, to relieve venous congestion and to improve the microrevascularization of flaps Fig ,2,3,4 ord sepsis . In manyIn the first stage of the study, bacterial content of the transport fluid of leeches that were bought from local markets were studied, also oral and intestinal flora cultures and antibiograms of the identified microorganisms were performed subsequently.In the next stages hypochloric acid, a disinfectant, was tested on animals that were not bred in specific laboratories, to suppress the relevant microorganisms in order to avoid possible infections, without changing the life style and behavior of leeches in terms of sucking function.This study was approved by the Animal Care and Ethics Committee of our institution.A. hydro/caviae and A. sobria for definite identification. Since bacterial growth was observed in specimens taken from oral and intestinal flora or transport fluid of all animals except one ; antibiograms for aeromonas isolates were performed.Ten leeches were obtained with their 100 cc original water from different stores. Their sizes varied between 6–10 cm in length and 0, 5 – 1, 5 cm in width. They were transported in sterile boxes immersed in the water which the animals were kept at the market. For 2 days they were observed at room temperature. Specimens were taken and smears from transport fluids were prepared. All leeches were held with sterile gloves and prepared in sterile conditions. Specimens were obtained from their mouth  with the help of sterile cotton – swab. After traction was applied between the mouth and posterior muscular organ, the animal was cleaned with alcoholic solution of povidone-iodine, as described by Hokelek et al. [ppm solutions were prepared by adding apropriate amounts of hypochloric acid into the transport fluids of 25 leeches seperately with five animals in each concentration group. After 10 minutes of contact with hypochloric acid solutions at 20°C, smears were taken from mouths and transport fluids of each leech under sterile conditions with cotton-swabs. Cotton-swabs were put into a neutralizing media (lecithinized agar media) because they could also contain disinfectant fluid which would prevent microorganism reproduction. Then the neutralized smears were planted into MacConkey and blood agar [ppm solutions lost their muscular activity and died ;the leeches which were treated with 12.5 and 6.25 ppm hypochloric acid solutions were alive. Bacterial growth was not observed in either concentration group for the specimens taken from oral flora and transport fluid, however bacterial cultures were positive for in specimens taken from the intestine or crop. Of these microorganisms, bacterial colonies for Aeromonas spp were counted. To compare the changes in the number of Aeromonas colonies in leech intestinal flora induced with the application of hypochloric acid, the intestine of five new leeches were exposed as described for Stage 1. Based on the findings of Stage 2, a concentration of 12.5 ppm was chosen for Stage 3 studies.30 leeches from different stores were obtained and transported in different sterile boxes with their 100 cc original water as in stage 1. On the following day, samples were taken from transport fluids and oral region and planted in MacConkey and blood agar. Twenty five leeches, which had bacterial growth in either oral flora or transport water were used for the second stage. 100, 50, 25, 12.5, 6.25 ood agar . The leeppm hypochloric acid solution was prepared with the transport fluids. After waiting for 10 min at 20°C temperature, smears were taken from the oral floras and fluids again. All leeches were put into boxes containing distilled water and watched for survival and blood suction function on sedated rats (ketamine 30 mg/kg and xylazine 10 mg/kg). After shaving dorsal region of 20 rats, 2 mm long skin incissions were done in order to produce bleeding. The leeches were applied to the bleeding areas atraumatically and the duration of suction was determined and compared statistically (ANOVA) between the leeches treated and not treated with hypochloric acid solution. Statistical significance was presumed at p < 0.05.20 leeches were obtained with their 100 cc original water from different stores and equally divided into two groups. One group was treated with hypochloric acid while the others had no treatment at all. After taking specimens from the transport fluids and oral floras, for 10 leeches 12.5 aeromonas species, peudomonas species, acinetobacter species, sphingobacterium species) was observed. In one media, Gram (+) growth was seen :meticilline susceptible coagulase (-) staphylococcus. Dominant microorganisms were aeromonas species, Table Aeromonas spp. was investigated by using conventional methods and a commercial kit ID 32 GN ATB System (BioMérieux). In this study, with this system the results were obtained as A. hydro/caviae, A. sobria, A. salmonicidia and A. spp, with the latter nomenclature designating all other Aeromonas species. The most commonly encountered agents, A. hydro/caviae and A. sobria underwent further biochemical studies, as suggested by Abbott, et al.[A. hydro/caviae was in fact A. hydrophila, and A. sobria was in fact A. veronii biovar sobria , intestinal flora in 9 animals (90%) and transport media in 5 animals (50%). In one leech, no growth was observed in either oral, intestinal and transport fluid cultures. In all culture media, 4 different gram (-) bacterial growth  however the difference between control group and 6.25 ppm group was not significant.Fluid and oral flora cultures that were prepared before hypochloric acid application showed multiple microorganism growth, as it was in stage 1. After adding hypochloric acid there was no growth in transport fluid and oral media cultures in either concentration so that no identification and antibiogram study could be performed. After 10 minutes in hypochloric acid solution, leeches were taken to sterile containers which contained distilled water. The muscle activity of all leeches that had been treated in 100 All the leeches survived while hyperactivity followed by hypoactivity was seen in the group of leeches treated with hypochloric acid. In this group, two of the leeches never made suction but the other eight leeches sucked the bleeding area for 5–16 min (mean 12.2 ± 3.8). Non hypochloric acid treated group sucked for 9–22 min (mean 15.5 ± 4.4) and the difference between two groups was not significant .Aeromonas spp) were observed. After hypochloric acid application no growth was observed in any of the specimens taken from animals, therefore no identifications or antibiograms could be performed.In 70 % of cultures that were prepared from transport fluids of leeches and in 50% of cultures that were prepared from oral floras of leeches before hypochloric acid application, bacterial growth  rods, Acinetobacter Iwofii and A sobria were isolated. All of the isolates were sensitive to ciprofloxacine, cefotaxime, ceftazidime, gentamycine and trimetoprim-sulfamethoxasole [Aeromonas hydrophylia colonies which produced inducable betalactamase. In case of infections with these species, beta lactam antibiotics except carbapenems should not be used.In countries, where special farms for medicinal leeches do not exist, animals obtained from markets should be studied for their floras and antibiotic susceptibilities. Eroğlu et al. investigated the floras and the antibiotic susceptibility of leeches in Black Sea region. Most commonly hoxasole . LikewisIn addition, we aimed to supress possible bacterial contamination of the transport media and oral floras of leeches but not intestinal flora because of the endosymbiotic relationship of the aeromonas and leech, with hypochloric acid solutions at disinfectant concentrations that does not affect the life style of leeches.Aeromonas spp. are sensitive for 12 hours but could not eradicate aeromonas from the intestines [Investigators have attempted to disinfect the guts of leeches before they are placed on patients by placing the ectoparasites in % 0.02 chlorhexidine for 15 seconds or in antibiotic solutions (tetracycline or cefoperazone solutions) for 12 hours, but these attempts were unsuccessful [testines .We preferred hypochloric acid which is an ideal disinfectant and has a wide antibacterial spectrum for desired purpose. Chlorine in the form of hypochloric acid exhibits rapid microbicidal activity by inhibiting of key enzymatic reactions within the cell and eventual protein denaturation. It has a rapid bactericidal effect and can dissolve in water. However, it has some disadvantages as it irritates mucosal membranes, has interactions with some chemicals and metals .ppm concentrations on pseudomonas was achieved within 10 min exposure [Aeromonas spp. cause similar nosocomial infections and has similar antibiotic susceptibility with pseudomonas spp. Therefore, in the second stage of our study, beginning with 100 ppm, we applied decreasing concentrations of hypochloric acid in transportation fluids, observed bacterial growth by taking specimens from transport material and oral flora of hirudo medicinalis. We tried to find the dilution ratio that was closest to 100 ppm, suppresing oral flora and transfer liquid and also allowing the leech to survive with normal function. As a result of the second stage of the study we found out that concentration to be 12.5 ppm.It was showed that bactericidal effect of hypochloric acid of 100 exposure . AeromonIn the third stage, we observed that mean sucking duration of the hypochloric acid treated group was shorter than the other group but the difference was not significant (12.2 min & 15.5 min respectively). However hypochloric acid had successful disinfection effect for transport liquid and oral flora. In our opinion, such a decrease in duration of sucking function can be preferable to infection possibility. Since leeches are much cheaper than antibiotics, it is logical to use them once, after treating with hypochloric acid, and never utilise them again.We do not advise to use "full-up" leeches over and over again by putting them in hypertonic solutions to force them vomitting because if they regurgitate, the intestinal flora can readly contaminate the environment where the leeches are kept and cause infection eventually. Although the oral flora and transport enviroment studies do not really address the problem since excretory contamination is probably the major factor in leech infections ; we believe that to take any measure in order to decrease the infection risk during utilisation of ordinary leeches is valuable.ppm hypochloric acid solution with transport fluids of ordinary leeches obtained from the local market for 10 minutes and then taking the leeches gently from water before application can prevent possible infections caused by contamination from leech oral flora and transport medium.We can comment that preparation of 12.5 None declared.AA and SVK participated in design of the study, involved in the dissections of leeches. EH and ST participated in the design of the study and writing of the manuscript.HN, NG and BO involved in the antibiogram tests. SNK participated in design of the study.The pre-publication history for this paper can be accessed here:


Stevie Wonder and Ray Charles are often cited as evidence that blindness confers superior musical ability. Wonder lost his sight after an incubator-related oxygen overdose during infancy; Charles lost his as a boy to glaucoma. It's impossible to know whether sight would have compromised their success, but many gifted musicians, from Jose Feliciano to Rahsaan Roland Kirk, lost their sight at an early age.A number of human studies show that blind persons perform nonvisual tasks better than those with sight. Neuroimaging studies of blind persons performing nonvisual tasks, including hearing, show activity in brain areas normally associated with vision. But much remains to be learned about the nature and extent of this phenomenon: how these “visual areas” are used, the mechanisms that generate individual differences , and the neural processes that underlie it.The task of localizing sound—which requires integrating information available to one ear only  or information derived from comparing sounds binaurally—is particularly suited to investigating the neural remapping that seems to follow vision loss. In a previous study, Franco Lepore and colleagues showed that people who lost their sight at an early age could localize sound, particularly from monaural cues, better than those who could see. These findings suggested that areas of the brain normally dedicated to processing visual stimuli  might play a role in processing sound in these individuals. In a new report, Lepore and colleagues use functional imaging studies to investigate the functional relationship between neural activity and enhanced hearing abilities in the blind, and find a strong correlation between superior sound localization skills and increased activity in the brain's visual center.The authors hypothesized that if visual cortex recruitment bolstered auditory function in some individuals, then visual cortex activity would correlate with individual differences in performance, and the degree of activity should predict such differences. Nineteen people—seven sighted and twelve who lost their sight at an early age—were placed in an echo-free chamber and asked to indicate where a sound was coming from, using either one  or both  ears. The participants then performed the same tasks within a positron emission tomography (PET) machine, which measures brain activity through changes in cerebral blood flow (CBF).Five of the blind participants could accurately localize sounds monaurally; most of the sighted could not.  Only the blind individuals with superior localization skills showed increased CBF in the visual cortex while performing monaural localization tasks. Interestingly, during binaural localization, the sighted participants showed decreased CBF in visual cortical areas. This decrease comports with previous studies showing that engaging one brain center—say, the temporal lobe, which processes sound—inhibits activation of others—such as the occipital lobe, which processes visual cues. These inhibitions appear to be absent in blind persons, though it's not clear why. It could be that blind persons don't need such inhibitions, the authors speculate, or maybe unrestricted access to the visual center serves to compensate for vision loss by boosting nonvisual senses.Whether the enhanced auditory performance reported here simply reflects increased efficiency of auditory processing or indicates “supranormal” powers, Lepore and colleagues argue that their results show that the visual cortex is “specifically recruited to process subtle monaural cues more effectively.” It will be interesting to learn whether blind persons can recruit visual centers for other auditory tasks or to help them navigate the world without sight. Such studies would be vital for tailoring sensory support to suit individual needs and maybe even suggest ways to facilitate the neural cross talk that enhances auditory performance. But don't expect such innovations to recreate the likes of Rahsaan Kirk or Ray Charles anytime soon.


A systematic search among the latter for a fusogenic activity had led to the identification of two bona fide genes, named syncytin-1 and syncytin-2, most probably co-opted by primate genomes for a placental function related to the formation of the syncytiotrophoblast by cell-cell fusion. Here, we show that one of the newly identified envelope gene, named envP(b), is fusogenic in an ex vivo assay, but that its expression – as quantified by real-time RT-PCR on a large panel of human tissues – is ubiquitous, albeit with a rather low value in most tissues. Conversely, the second envelope gene, named envV, discloses a placenta-specific expression, but is not fusogenic in any of the cells tested. Altogether, these results suggest that at least one of these env genes may play a role in placentation, but most probably through a process different from that of the two previously identified syncytins.A recent  These sequences  share strong similarities with present-day retroviruses, and are the proviral remnants of ancestral germ-line infections by active retroviruses, which have thereafter been transmitted in a Mendelian manner (reviewed in -3). The ectively ,8. The sh genome . The seqenv gene, namely on the alignment of a conserved domain of the transmembrane (TM) subunit [env gene is closely related to that of MER66, MER84 and Z69907 families. This gene seems to be part of a very degenerate proviral structure, with only the LTR being identifiable . 3' LTRs are also found just downstream of the envelope genes . The analysis of the PBS (Primer Binding Site) region located downstream of the two 5' LTRs of this family reveals a high degree of homology to the PBS for Val-tRNA , are clribed in ).env genes with an open reading frame would be amplified among all the envelope genes of a given family, by positioning them within domains of maximal divergence between the coding and the non-coding copies. For the HERV-V coding envelope, the primer pair was designed in the 3' part of the gene, where the two envV genes are the most divergent (79% identity in the last 200 nt). An additional primer pair was also designed to monitore the expression of the truncated HERV-V env gene. To assess the specificity of each primer pair for the corresponding env gene, the PCR products obtained upon amplification of genomic DNA were cloned into a pGEM-T vector and 6 clones per amplicon were sequenced. In each case, the 6 sequences corresponded to the expected env gene. Analysis of the expression level of the coding envP(b) and envV genes was achieved on a series of 19 healthy human tissues, and the results are represented in Figure envV was found to be placenta-specific. Interestingly, the truncated envelope of the HERV-V family is highly expressed in the placenta as well, but poorly in other tissues (data not shown). EnvP(b) expression, on the other hand, was observed at a rather low level in almost all the tissues tested, without any specificity for the placenta.To determine whether these two genes could play a role in human placentation, we then characterized their expression pattern and fusogenic properties, as previously performed for the 16 coding envelope genes already identified . To get env genes of the human genome tested in [envW (syncytin-1) and envFRD (syncytin-2), had been found to be fusogenic in an ex vivo assay. As these two env genes were highly and specifically expressed in the placenta, it was suggested that they are involved in a major physiological process within this organ, namely fusion of the cytotrophoblast cells to form the syncytiotrophoblast layer. The two newly identified env genes were therefore similarly tested. To do so, they were first cloned and introduced into a eukaryotic expression vector. The envP(b) gene was PCR-amplified from the DNA of BAC RP11-828K24 by using a proofreading DNA polymerase and running a 15-cycle PCR reaction, whereas the envV gene -not available as BAC DNA- was PCR amplified from the genomic DNA of a Caucasian individual using the Expand long template enzyme mix (Roche Applied Science). Both env genes were then assayed for cell-cell fusion on a large panel of mammalian cells  using a transient transfection assay and two clones from each construct. As shown in Figure envP(b), and in none of them for envV. The truncated envelope protein member of the HERV-V family was also tested and, as expected, was not fusogenic (data not shown). In some respect, these results are surprising. Indeed, the putative protein encoded by envP(b) is fusogenic despite the absence of a canonical fusion peptide, i.e. of a hydrophobic region located at the N-terminus of the putative TM subunit, just downstream of the SU-TM cleavage site . To check that the lack of fusogenicity of the latter gene is not due to a fortuitous gene polymorphism of the envV gene from the selected individual, we PCR-amplified, cloned and assayed the envV gene from two other individuals (for both the complete and the truncated envV genes): no cell-cell fusion was observed either (data not shown). Finally, we identified and cloned the chimpanzee orthologous envV gene (which is fully coding as well): neither did it display any fusogenic activity in our assay (data not shown).Among the 16 coding ested in , only twenvV and envP(b) genes are most probably not "syncytin-like" genes, sensu stricto. Additional experiments should now be devised  to assess their role -if any- in human physiology.In conclusion, the present analysis shows, rather paradoxically, that the envelope protein with fusogenic properties is not placenta-specific, whereas the one which is exclusively expressed in the placenta -a characteristic pattern of the two previously described fusogenic syncytin-1 and syncytin-2 gene products- is not fusogenic. In this respect, these results suggest that the two newly identified HERV, human endogenous retrovirus; TM, transmembrane; LTR, Long Terminal Repeat; PBS, Primer Binding Site.The author(s) declare that they have no competing interests.env genes and the cell-cell fusion assays.SB carried out the cloning of the NdP analyzed the sequences, constructed the phylogenetic tree, designed and carried out the Real-Time RT-PCR experiments, and drafted the manuscript.TH conceived the study.


Doctors and researchers often look for the rapid proliferation of T cell populations, key defensive players in the immune system, as a telltale sign that the body is working hard to fend off a foreign threat. Every one of these circulating white blood cells carries a T cell receptor (TCR) that binds to a specific protein, or antigen, when displayed on the surface of a cell. A match between TCR and displayed antigen results in the cell's death and the subsequent expansion of T cell clones, all programmed to recognize the original offending protein. Some TCRs bind and expand in response to pathogenic antigens, such as viral or bacterial proteins. But T cells can also react and proliferate inappropriately in response to the body's own proteins, leading to destructive autoimmune diseases such as multiple sclerosis, which is characterized by immune system attacks on nervous tissue. Self-recognizing TCRs, however, can also target and destroy tumors—though full activation of these T cells is inconsistent and poorly understood.PLoS Biology, Frances Crawford and colleagues have developed a novel method for rapidly identifying TCR mimotopes—peptide sequences similar or identical to epitopes that also elicit the immune response—which can be used to determine the antigen of a given T cell population.Identifying the particular antigen behind an exploding population of T cells is invaluable for finding the source of autoimmune diseases and studying immune responses to cancer. But it's a laborious and time-consuming process, as researchers are faced with the prospect of sifting through millions upon millions of possible matches between TCRs and their prospective antigen epitopes—the part of the antigenic molecule to which the receptor binds. Now, as they report in this issue of Working backwards, the team started off with two different T cell clones that had been previously selected for with a known antigen—a peptide called p3K. One clone was derived from mice genetically engineered to have broadly reactive T cells; the other, a conventional clone, was much more sensitive to the precise molecular structure of p3K.Crawford and colleagues then created a “peptide library” comprising more than 30,000 baculoviruses (viruses that selectively target insect cells), each one carrying a slightly different version of the p3K gene, varied in regions of the peptide known to be important for TCR binding. These p3K genes were embedded within a major histocompatibility complex (MHC) gene—a type of cell surface protein that holds displayed antigens and is also important for proper TCR recognition. The team then unleashed their virus library onto insect cells that, once infected, began to produce the specific peptide–MHC complexes encoded on the viral DNA. The insect cells then shuttled these proteins to their surfaces, resulting in a vast array of cells that each displayed a unique variant of the p3K–MHC complex. This “display library” was then incubated with fluorescently labeled TCRs from the two different clones. By observing and isolating the insect cells that lit up, the researchers could see which of the thousands of cells displaying peptide–MHC possessed a mimotope capable of binding a TCR. Because the genetic information about the displayed complex was still stored within the virus-infected cell, the researchers could determine the full peptide sequence responsible for the identified mimotopes.Confirming the effectiveness of their method, the results of the fluorescence experiments echoed the authors' original characterizations about the two populations of T cells. The broadly reactive TCR bound to several different uniquely displayed complexes; it had 20 mimotopes. The conventional TCR, however, bound only to one peptide–MHC complex, an almost perfect match to the original p3K peptide. Though this study was based on a known antigen and epitope , the baculovirus display library technique described here could easily be used on T cell populations with unknown antigens. With such a tool, researchers could, for example, identify the antigens connected with tumor-fighting T cells and, through inoculation, possibly induce the production of similar T cells in cancer patients who lack them.


Disturbances in the immune system has been described in Turner syndrome, with an association to low levels of IgG and IgM and decreased levels of T- and B-lymphocytes. Also different autoimmune diseases have been connected to Turner syndrome , thyroiditis being the most common.Besides the typical features of Turner syndrome  ear problems are common .Levels of IgG, IgA, IgM, IgD and the four IgG subclasses as well as T- and B-lymphocyte subpopulations were investigated in 15 girls with Turners syndrome to examine whether an immunodeficiency may be the cause of their high incidence of otitis media. No major immunological deficiency was found that could explain the increased incidence of otitis media in the young Turner girls. Recurrent otitis media is often a problem in children with Turner syndrome (TS) ,2. More TS is caused by the presence of only one normally functioning X-chromosome. The other sex chromosome can be missing  or abnormal and mosaicism is often present. Occurring in one of every 2000 female births, TS is one of our most common sex chromosome abnormalities . TS is cImmunological disturbances have previously been described in TS, with an association to reduced levels of serum IgG and IgM, increased IgA and decreased levels of circulating T- and B-lymphocytes. However, the results have not been conclusive -12.2 deficiency commonly develop recurrent acute otitis media. It is believed that these infections are secondary to impaired antibody response, rather than Eustachian tube dysfunction [In the normal population children with IgGfunction . As immuThe aim of this study was to investigate immunoglobulin and lymphocyte subpopulations in girls with Turners syndrome to examine whether an immunodeficiency may be the cause of their high incidence of otitis media. Immunotherapy would then be a possible treatment.Blood samples from patients with the diagnosis TS, genetically confirmed, were investigated according to the Swedish ethical record no 88–265.Analyses regarding immunoglobulin- and lymhpocyte subpopulations were performed in 15 girls, aged 5–17 years (median age 11 years), randomly selected from all girls in this age group with TS attending the Karolinska Hospital, Stockholm . Of these 53% (n = 8) had suffered from repeated attacks of otitis media. All TS girls had been treated with growth hormones and their karyotypes were: 45, X (n = 8); 45, X/46, XX (n = 4); 45, X/46, X, i(Xq) (n = 2); and 45, X/46, X, r(X) (n = 1) (r = ring chromosome).A medical history was attained, focusing on autoimmune diseases, previous and current ear diseases and other infectious diseases, ear operations, and hearing problems.9/L) were analysed in a Coulter MicroDiff II (Beckman-Coulter). The differential leukocyte  counts and percentages were obtained by 2-color FACS-analysis with CD14/CD45 markers. The number and percentage of lymphocyte subpopulations were obtained by standardized 2- or 3-color FACS-analysis on Epics XL or Elite flowcytometer (Beckman-Coulter) using commercial reagents. CD19+ was marker for B-cells and CD3+ for T-cells, CD3+CD4+ for helper T-cells, CD3+CD8+ for cytotoxic T-cells, CD56+CD3- for NK-cells and HLA-DR+ for activated T-cell subsets. The ratio of CD4+/CD8+ was also calculated. The monoclonal antibody clones used were: UCHT1 (CD3+), SFCI12T4D11/T4 (CD4+), SFCI21Thy2D3/T8 (CD8+), 116/Mo2 (CD14+), 89B/B4 (CD19+), KC56 (CD45+), NKH1 (CD56+) and 9-49/I3 (HLA-DR+), all from Cytostat, Beckman-Coulter. All FACS-analyses were performed at the routine laboratory, Department of Clinical Immunology, Karolinska Hospital and the results were compared to age-related in-house and published reference ranges (5 to 95 percentiles) [+CD3- for which an adult reference was used (10–90 percentile).Leukocyte counts -allotyping of IgG2 were analysed by standard methods and compared to age related reference ranges used at the routine laboratory, Department of Clinical Immunology, Karolinska Hospital, Stockholm.Hemolytic complement , IgA antibodies to gliadin and endomysium, IgG antibodies to pneumococcal polysaccharide and tetanus toxoid antigen, the serum concentrations (g/L) of circulating IgA, IgG, IgM, IgD, IgGMedians of continuous parameters were compared between groups by Mann-Whitney U-test and correlations were performed by Spearman rank analysis. A two-tailed p < 0.05 was considered significant.+ and CD8+ T-cells (HLA-DR+). However, the CD4+/CD8+ ratio was in the lower range (girls aged ≥10), with one girl having a very low ratio (0.6).The leukocyte counts as well as the absolute counts and percentages of lymphocytes, monocytes, and granulocytes were within normal limits for all 15 Turner girls. Likewise most girls had normal counts and percentages of lymphocyte subpopulations as compared to the 5 to 95% percentiles age-related reference ranges Fig.  and 1b iHemolytic complement  was within normal limits for all 15 Turner girls.1 (10.2 and 10.8 g/L), one with low IgG2 (0.4 g/L) and two girls with low IgG4 (<0.01 g/L).The serum concentrations of IgG, IgA, IgM, IgD and the four IgG subclasses were for most Turner girls within the age-related 95% confidence intervals Fig. . The excThe frequency of homozygous G2m(23)-negative Turner girls was 33% (5/15).Normal levels of IgG antibodies to tetanus toxoid and polysaccharide antigen were detected among most Turner girls, except for two respectively one, having too low levels. Slightly elevated IgA antibodies to gliadin were observed in 3 (20%) girls, whereas no IgA antibodies to endomysium could be detected in any of the 15 girls.+ (p = 0.0053) and CD4+HLA-DR+ (p = 0.035), as well as the percentage of CD19+ (p = 0.023). Also IgG2 increased with age (p = 0.05). These findings are in line with the reference literature for the normal population [When comparing girls aged <10 years (n = 4) and ≥10 years (n = 11) the following parameters were found to be influenced by age with decreased values among the older girls: total counts of leukocytes (p = 0.0093), lymphocytes (p < 0.05), monocytes (p = 0.0093), granulocytes (p = 0.015), CD19pulation .+ T-cells (p = 0.0087), CD4+ T-cells (p = 0.012) and CD4+HLA-DR+ (p = 0.05) as well as in the percentage of CD3+ T-cells (p = 0.05) in otitis prone (n = 5) compared to otitis free (n = 6) Turner girls was shown. No such differences were noticed for any immunoglobulin levels, antibody titers, CD4+/CD8+-ratio or CD8+, CD19+, CD56+CD3- lymphocyte subpopulations.The girls with TS were divided into two groups according to their history of recurrent otitis media. As age influenced some of the parameters we only considered girls ≥10 years old (n = 11). Significant increases in absolute counts of lymphocytes (p = 0.004), CD3Any apparent influence, of the different karyotypes, on any of the parameters studied was not observed within the group.In this study no major derangement in the immune status was found among the girls with TS. Normal levels of most lymphocyte- and immunoglobulin subpopulations were registered. The few outliers noted must be considered as a normal individual variation.+/CD8+ ratio in the lower range [+ population. Although, the patients were few, we noticed some differences between the otitis prone and otitis free Turner girls. The elevated counts of lymphocytes, CD3+, CD4+ cells and CD4+HLA-DR+ cells seen among the otitis prone girls, probably reflects a secondary effect of an activated immune system involving T-helper cells, rather than any immune deficient state. Moreover, the levels of IgG antibodies to pneumococcal polysaccharide antigen, which are important in the defense of bacteria, were normal. A homozygous lack of the IgG2m(23) allotype was seen in 33% of the girls, which is the same frequency as in the normal population [2. In the study group a negative IgG2m(23) allotype was not correlated to a positive history of recurrent otitis media, neither could the different karyotypes be associated to the levels of immunoglobulin- or lymphocyte subpopulations. Perhaps the cause of the repeated attacks of otitis media in Turners syndrome is not to be found in the periphery, but rather more locally. Even if earlier computed tomography scans of the temporal bone have not shown any abnormalities [However, as described in an earlier study of Turner girls, the present study confirmed a CD4er range , supposepulation . A negatmalities . The SHOmalities . As the Our findings of normal immunoglobulin- and lymphocyte subpopulations are not entirely in concordance with some earlier studies, where a reduction of circulating IgM and IgG as well as T- and B-lymphocytes has been observed ,10. HoweIn conclusion, we did not find any major immunological deficiency in immunoglobulins or lymphocyte subpopulations that could explain the increased incidence of otitis media observed in girls with TS. Therefore, treatment with immunotherapy is not an option in this patient group. Further studies are warranted to elucidate local pathology, both from an immunological and anatomical point of view.AES participated in the design of the study, performed the statistical analysis and drafted the manuscript. LS participated in the design of the study and collected the blood samples. CGMM performed the statistical analysis. MH participated in the design and coordination of the study and collected the blood samples.All authors read and approved the final manuscript.