Wounds may be caused by surgery, trauma, or as a result of diseases such as diabetes. The disruption of the skin/internal tissues and the contact with the external environment may lead to microbial infection. Wound dressings are capable of providing a protective barrier and accelerate the wound healing process . ChitosaIn vitro drug release studies were performed with a Franz diffusion cell system, combined with UV–Vis absorption spectroscopy for drug quantification. The efficiency of HHP was evaluated by sterility tests. Chorioallantoic membrane (HET-CAM) tests were done to study potential irritation of the skin.Two different chitosan-based materials were produced starting from Bioceramed formulations, AbsorKi (suitable for absorbing exudate from the wound) and HemoKi (enhancing haemostasis). The latter was modified by the addition of genipin, a cross-linking agent. The dressings were individually loaded with two different drugs, polyhexamethylene biguanide  and chlorhexidine diacetate (CHX), by soaking in solutions containing each drug . The physical properties  of the materials were studied prior and upon sterilisation by high hydrostatic pressure . Surface morphology was analysed by scanning electron microscopy (SEM). Both dressings present a porous structure and an extremely high swelling capacity (>1700%) before sterilisation. HPP affected the materials in different ways: it increased the swelling capacity of AbsorKi but decreased it for HemoKi. The drug release profiles indicated that the concentrations of PHMB and CHX increased in a sustained way on the first day. HHP increases the amount of drug released, except for AbsorKi with PHMB. Regarding the mechanical properties, sterilisation improved the resistance of both dressings. HET-CAM tests suggest that the produced materials do not lead to irritation.HHP revealed an efficient method to ensure the materials sterilisation making the drug loaded wound dressings potentially efficient devices for the absorption of exudate from the wound bed. The combination of chitosan, a natural antibacterial agent, with the studied disinfectant and antiseptic drugs may lead to promising materials to be used as drug delivery platforms. Further studies in animal models are needed to conclude about the safety and clinical usefulness of the developed dressings.


Introduction Reversal of enamel-only proximal caries by non-invasive treatments is important in preventive dentistry. However, detecting such caries using bitewing radiography is difficult and the subtle patterns are often missed by dental practitioners.Aims To investigate whether the ability of dentists to detect enamel-only proximal caries is enhanced by the use of AssistDent artificial intelligence (AI) software.Materials and methods In the ADEPT  study, 23 dentists were randomly divided into a control arm, without AI assistance, and an experimental arm, in which AI assistance provided on-screen prompts indicating potential enamel-only proximal caries. All participants analysed a set of 24 bitewings in which an expert panel had previously identified 65 enamel-only carious lesions and 241 healthy proximal surfaces.Results The control group found 44.3% of the caries, whereas the experimental group found 75.8%. The experimental group incorrectly identified caries in 14.6% of the healthy surfaces compared to 3.7% in the control group. The increase in sensitivity of 71% and decrease in specificity of 11% are statistically significant (p <0.01).Conclusions AssistDent AI software significantly improves dentists' ability to detect enamel-only proximal caries and could be considered as a tool to support preventive dentistry in general practice. Enamel-only proximal caries is often missed by dentists when examining bitewing radiographs.The use of AssistDent artificial intelligence software results in a 71% increase in ability to detect enamel-only proximal caries accompanied by a 11% decrease in specificity.Artificial intelligence software could be considered as a tool to support preventive dentistry in general practice. Only then can the ideal, personalised caries assessment of adult patients and their preventive care be developed.The early detection and treatment of enamel-only proximal caries can preserve tooth structure and prevent the subsequent cycle of treatment and re-treatment that is involved with more invasive treatment. Recent guidelines from the NHS encourage preventive care in dental practices, especially for young children. Patients differ widely on their willingness to pay for preventive therapies,Selection criteria for dental radiography in determining the frequency of use of bitewing radiography.3 Nevertheless, systematic reviews have consistently shown that detection of proximal caries on bitewing radiography has a low sensitivity.4 A number of studies have reported poor diagnostic sensitivity for radiographic detection of demineralisation by dentists. In a classic study by Mejàre et al.,5 premolar and adjacent teeth surfaces were examined radiographically and visually. The premolar teeth were then extracted for orthodontic reasons. They found that the sensitivity of detection of enamel-only proximal caries was 36.7%. Other studies have found similarly low sensitivity values.6,7Radiographic examinations can increase the number of carious lesions that are detected over those that would be detectable by clinical examination alone. The Department of Health recommends that dentists use the Faculty of General Dental Practice (UK) guideline document 8 It is an aid to the dentist, assisting their clinical decision-making by providing on-screen prompts for potential locations of enamel-only proximal caries. The final judgement about whether enamel-only proximal caries is present, or not, is a decision for the clinician.The introduction of artificial intelligence (AI) methods allows routine tasks to be conducted more quickly and efficiently. AssistDent is an AI software product, developed by Manchester Imaging Limited, and uses machine learning algorithms to search for evidence of enamel-only proximal caries on bitewing radiographs.The null hypothesis of this research was that there is no difference in the performance of dentists in diagnosing the presence of enamel-only proximal caries on bitewing radiographs with and without the use of AssistDent.9 was conducted with dental students to assist in developing the methodology and to provide initial data for a sample size calculation. The final study protocol, participant information sheet and consent forms for the study were approved by the Manchester University Research Ethics Committee (Ref: 2020-9892-15955).A pilot studyParticipants were recruited from two sources: 1) dentists practising as general dental practitioners who, in addition, provide tutorage for dental students within the University of Manchester Dental School; and 2) practising dentists undertaking postgraduate training within the University of Manchester Foundation NHS trust. All signed informed consent forms.The dentists were randomly divided into control and experimental arms by pairing the participants within the recruitment sources according to the order in which they were enrolled. The first of each pair of participants were randomly assigned to either the control or experimental arm, with the second assigned to the other group. This method ensured random assignment while maintaining even arm sizes equally balanced between the recruitment sources as the study progressed.Both arms examined the same images using the same graphical user interface as shown in So that the study would have applicability to general practice, a total of 1,446 bitewing radiographs were collected from a range of different sites . Separate ethical approval had been received from the Integrated Research Application System . As illustrated in Gold standard annotation of all classes of proximal caries was obtained from a panel of five dento-maxillofacial radiologists and one professor of restorative dentistry, each of whom annotated the location and grade of caries on a set of images independently of each other. Each image was annotated by at least three members of the panel. These individual expert annotations were consolidated by retaining, in the gold standard set, any region identified by any expert as enamel-only proximal caries while removing duplicate annotations, resulting in a gold standard set of 1,972 examples of enamel-only proximal caries for algorithm training and evaluation.The caries annotations entered by each participant were collected remotely via a web application and analysed to determine whether they were true positives  or false positives . Annotations corresponding to dentine proximal caries were recorded but excluded from this analysis. The true positive rate or sensitivity of diagnosis is a measure of how well a participant detected the enamel-only proximal caries and was calculated as the sum of true positives divided by the sum of the gold standard caries. True negative rate or specificity is a measure of how well the participant identified healthy surfaces and did not mark them as carious. The probability of a false detection is quoted in terms of a false positive rate, calculated as the sum of false positive detections divided by the sum of healthy surfaces, which is equal to 1-specificity.A per-participant breakdown of the evaluation scores and performance measures, together with the aggregate scores and measures for each arm, is presented in the online supplementary information. The data demonstrate that 23 dentists were recruited, 11 in the control arm and 12 in the experimental arm. These were balanced within the arms between the two recruitment sources of general dental practitioners and practising dentists undertaking postgraduate training. All participants analysed all 24 images.10 In a review of diagnostic studies reported by Keenan and Keenan,11 the authors found lower sensitivities for the detection of proximal lesions in clinical studies than in vitro studies. The mean sensitivity in the clinical studies was 0.24 and for the in vitro studies was 0.43.Small areas of proximal surface enamel demineralisation are difficult to detect visually on radiographs.in vitro studies. In our in vivo study, we mainly used a sample of radiographs from a range of dental practices that were taken in the course of the examination of patients. The low sensitivity and high specificity of our control group in detecting caries on bitewing radiographs is comparable with the above studies, and with that reported in a systematic review.12Other studies that have measured sensitivity and specificity of caries detection using radiographs have found a large variation. This may be due to variation in the range of caries depth or the use of 13 However, increased sensitivity carries with it the likelihood of an increased number of false positive detections. This trade-off is inevitable, but it is clearly desirable that the false positive count should be kept as low as possible. The risk to patients arising from false positive detections is minimal provided clinicians follow guidelines and do not restore teeth with enamel-only caries. In a situation where a false positive detection is made when no caries is present, an individual may, as a result, receive preventive advice without any obvious clinical benefit. The decrease in specificity observed from the use of AssistDent accompanies a much larger increase in sensitivity.Low sensitivity in the detection of enamel-only caries is problematic in the context of preventive dentistry. A significant increase in sensitivity is required over the values reported in these studies, even at the expense of an increased number of false positive detections. Enamel lesions are treated in a conservative manner with dietary and interdental cleaning advice, fluoride treatments and, potentially, resin infiltration. This, in conjunction with cooperation from the patient, will prevent operative intervention and the entry of the tooth into a restorative cycle of increasingly larger restorations and eventual extraction. An increase in sensitivity of detection is important as it provides a better assessment of the full extent of the patient's caries experience. The dentist should create an individually tailored dental care plan with the patient based on a true picture of their oral health. Some aspects of this risk assessment are based on the findings from bitewing radiographs obtained during the process of screening for early, enamel-only caries. In those individuals who suddenly develop many early lesions, this would merit an investigative effort to determine any underlying medical or social causes followed up by more frequent recall intervals. The number of lesions present is an important variable when predicting future caries experience.et al.14 has described the basics of AI and explored its potential use in diagnostics, treatment planning and conduct in dentistry. Computer image analysis is a particular application of AI in dentistry . An earlier, scoping review15 refers particularly to the application in image diagnostics. There is a relatively small number of studies related to caries detection. For example, Lee et al.16 investigated the performance of a convolutional neural network on classification of caries in images of individual teeth isolated from periapical images. The study did not focus specifically on enamel-only caries. Srivastra et al.17 also trained a convolutional neural network for fully automatic detection of caries in bitewing images.There has been a growing interest in general dental publications and online forums in the use of AI in dentistry. A recent review by Schwedicke et al.15 recommend that 'the dental community should appraise [the AI systems] against the rules of evidence-based practice'. This study is an example of such an appraisal.To our knowledge, AssistDent is the only commercially available AI system for use in the clinical diagnosis of enamel-only proximal caries, acting as a prompting system to support dentists' diagnostic decisions. Schwendicke et al.18 investigated the effect of ICDAS training and found a significant increase in over-treatment recommendations. The reproducibility of any diagnostic system depends on the experience of the clinician and this is especially so where the diagnosis is a visual score rather than AI-based.13 How should our profession address the issue of poor sensitivity in detection of enamel-only proximal caries? Audit, reflection with peer review and evaluation of past performance are essential parts of dental practice. An audit of caries diagnosis will rely on identifying opportunities for improvement, comparison with an accepted standard of care and implementing change. The increased sensitivity arising from AI-supported detection may provide a useful standard for audit in caries detection. Testing will also reveal clinicians with unacceptable variation in bitewing analysis competency assessments, with direction towards further training. More efficient caries detection may help in identifying patients with higher caries risk, while the associated display of detected caries can provide a basis for encouraging a detailed discussion with the patient of their oral health and the factors affecting it. The intuitive graphical screen display of AssistDent may be more readily understood by patients in comparison to other algorithm-based tools such as the Cariogram. The latter uses a pie chart of important factors to illustrate the probability that future caries may be prevented.19,20 However, once the high-caries risk individuals are identified, it is important to further investigate all the factors predisposing to caries, such as a high sugar intake and infrequent brushing.21It is important that dentists receive appropriate training with any new diagnostic system. Qudeimat Caries assessment by dentists using AssistDent is compatible with the ICCMS caries management system of the ICDAS Foundation and could be used in conjunction with it. AssistDent provides feedback on the proximal enamel surfaces using radiographs, whereas ICCMS is a mainly visual assessment of the non-proximal surfaces for caries. Both systems aim to maintain tooth structure and encourage preventive care by developing a caries assessment for each patient. At this stage of its development, this AI software is used as an aid to diagnosis, but future developments could include monitoring the progression of caries..AssistDent AI software increases dentists' sensitivity when assessing enamel-only proximal caries. This increased sensitivity is accompanied by a smaller decrease in specificity. The increase in false positive diagnoses may occasionally result in unnecessary preventive treatment and associated use of limited healthcare resourcesThe increase in sensitivity in detecting enamel-only proximal caries should enable better informed targeting of preventive treatments. This would contribute to avoiding the requirement for later restorative treatment, resulting in an overall saving of resources and an improvement in the dentition of patients.


A panel of 37 rabies virus isolates were collected and studied, originating mainly from the northern and central regions of Namibia, between 1980 and 2003.n = 10) sharing 97.2–100% similarity with jackal isolates, and 97–100% similarity with those isolated from domestic dogs. Phylogenetic analysis suggested that these viruses were all of the canid rabies biotype of southern Africa. The viruses from kudu were closely associated with jackal isolates (n = 6), bat-eared fox isolates (n = 2) and domestic dog isolates (n = 2) at the genetic level and identical at the amino acid level, irrespective of the year of isolation.These virus isolates demonstrated a high degree of genetic similarity with respect to a 400 bp region of the nucleoprotein gene, with the virus isolates originating from kudu antelope (These data suggest that jackal and kudu may form part of the same epidemiological cycle of rabies in Namibian wildlife, and might demonstrate the close-relationship between rabies virus strains that circulate within Namibia and those that circulate between Namibia and its neighbouring countries such as Botswana and South Africa. It shares borders with South Africa in the south, Botswana and Zimbabwe in the east, and Angola and Zambia in the north , Mokola virus (genotype 3) and Duvenhage virus (genotype 4) [Canis familiaris), jackal  and bat-eared foxes  [Cynictis penicillata) which maintains the virus, whereas in reality there is no evidence that true viverrids (such as civets and genets) are maintenance hosts of RABV [Classical rabies virus  has a single-stranded, negative-sense RNA genome, and is a member of the genotype , Mokola genotype , Mokola otype 4) . The cangalotis) . The mon of RABV . Mongoos of RABV .Otocyon megalotis) [Rabies is a notifiable disease in Namibia . Here, agalotis) . Canid rgalotis) ,14.By the mid-1970s there was sporadic but endemic rabies throughout most of Namibia, generally with dog and human rabies in the more populous north, jackal and cattle rabies in the central ranching areas and sporadic canid or mongoose rabies in the arid sheep farming areas of the south .Tragelaphus strepsiceros) was first observed in 1975 near Windhoek. However an epizootic of rabies in these antelopes began to the north in Okahandja district in 1977 and was in the same year that rabies was first confirmed in two kudu in the Etosha National Park. The number of confirmed cases rose steadily throughout 1978–1979 [The unusual occurrence of rabies in the kudu antelope  is a wild ruminant with spiralling twisted horns, and is generally found in areas of broken rocky terrain where there is easy access to water [Acacia species, including Acacia hereroensis (Berg thorn), along with a variety of other plants in the savannah woodland [The kudu  were geographically located in the northern districts of Grootfontein and Etosha, and contained one virus isolate from kudu, two from jackal and one from a dog from Grootfontein , along with three isolates from jackals and one hyena sample from the Etosha National Park . However, strains of group A were not restricted to this area. This group also included a single isolate (RV135), which was collected from a kudu in South Africa in 1980.A second group of identical sequences (Group B) originated from more central regions of Namibia and comprised two isolates from kudu from the Okahandja district , two isolates from kudu and one from an eland from the Omaruru district , and one isolate from a kudu from Windhoek (RV1488). The jackal group (Group C) contained three isolates from jackals around Windhoek , one from a jackal in the Khorixas district (RV1498) and one from a jackal in the Okahandja district (RV1497). Also demonstrating 100% similarity with respect to this region of the N-gene were two virus isolates from kudu from the neighbouring regions of Windhoek and Okahandja . Finally, two virus isolates from dogs in the Oshikoto and Oshakati districts  were also found to share identical sequences. With the exception of RV135, sequences represented by multiple isolates were geographically localised , all of these isolates originated from Namibia. With the exception of RV1500 and RV1499, all of the isolates in the upper cluster had identical deduced amino acid sequences in the 400 bp region.Unlike all other species analysed in this study, all of the virus isolates from kudu clustered together  is the most similar of the South Africa Group to the Namibian and Botswanan canid isolates demonstrating again the close relationship between isolates from neighbouring countries due to the movement of such species. The mongoose species responsible for isolate RV1917 has not been identified but it is most likely to have been a yellow mongoose, which is commonly found in the Southern Namibian region of Keetmanshoop. The spillover of canid biotype into mongoose hosts is also demonstrated by the inclusion of the South African water mongoose isolate 2900AFS)  and N-gene analyses were congruent (data not shown). Furthermore, phylogenetic analysis of the concatenated sequence (1000 bp) yielded no significant improvements in terms of either the bootstrap values or topography (data not shown). Phylogenetic analyses of the dataset using either the maximum likelihood (DNAML) or parsimony (DNAPARS) programmes within the PHYLIP package did not improve the resolution of isolates or demonstrate significant differences in topography or bootstrap support (data not shown) which lends support to the co-existence of closely related urban and sylvatic rabies cycles in Southern Africa.The molecular sequence and phylogenetic analysis of the Namibian rabies virus isolates described here, has demonstrated a high degree of similarity between isolates originating from different species, emphasising clearly the ease of transfer of RABV throughout Namibian wildlife, and between wildlife and domesticated species.Viverridae) have been shown to be phylogenetically distinct from the canid biotype [The demonstration of two epidemiological cycles of RABV circulating in southern Africa is well documented. Within the canid biotype of RABV there appears to exist two epidemiological cycles ; an urba biotype . Our dat biotype . This ma biotype . However biotype .A number of clinical signs have been observed in kudu infected with RABV, the most common of which is loss of fear . An infeRABV clearly poses a threat to human and animal health in Namibia and throughout the African continent, as demonstrated by the similarity between viruses from neighbouring countries. The highest population density in Namibia is located in the north of the country, where there are many dogs and little wildlife. In this region, rabies is detected mainly in dogs, and poses a threat to human and bovine health . AccordiDuring the years preceding the 1977–1983 rabies epizootic in kudu, above-average rainfall and abundant growth of vegetation made Namibia a very favourable environment for kudu and the population had greatly increased [Herpestidae such as the yellow mongoose [Our analysis confirms that the RABV strains that infected the kudu population during both epizootics was of the canid biotype. This biotype however, was phylogenetically distinguishable from the group of viruses isolated from mongoose . The earmongoose . Furthermongoose . HoweverThe high degree of similarity among the kudu isolates suggests that once RABV enters this species it spreads rapidly through the population with little interaction with other species. The kudu population then appears to maintain its own cycle of RABV infection. Epizootic observations at the time of the kudu outbreak suggest a horizontal spread of RABV throughout the species, particularly as the only other wild herbivore affected was the eland . The numThis theory is however, not exclusive. An alternative hypothesis for the non-bite route of transmission of RABV throughout the kudu population has been suggested. Kudu live in small herds which remain together, moving and feeding at the time and living in relatively close contact . One of Our data suggest that the isolates studied show a high degree of genetic similarity between a range of species and geographical locations and demonstrates how closely-related the RABV strains circulating in Namibia are to those from neighbouring countries such as Botswana and South Africa.The epidemiological cycles (urban and sylvatic) of RABV circulating throughout the countries of southern Africa are complex, with a large number of vector species and the existence of at least two distinct biotypes of RABV (mongoose and canid). Different species appear to maintain their own cycles of infection, such as that by non-bite transmission seen in the kudu during the epizootics. Our data suggests that jackal and kudu may form part of the same epidemiological cycle of rabies in Namibian wildlife. The canid biotype is now highly established throughout southern Africa. This makes any control programme difficult to implement, due to the large variation in species and habitat. We believe that rabies is highly prevalent and widespread in southern Africa and that the disease poses a continual threat to a large variety of wildlife species, to livestock and to human health.Tragelaphus strepsiceros), black-backed jackal (Canis mesomelas), domestic dog (Canis familiaris), eland (Taurotragus oryx), bat-eared fox , mongoose and hyena (Crocuta crocuta) between 1980 and 2003 were sequenced (Table A panel of thirty-seven viruses isolated from kudu (Previously published RABV nucleotide sequences from Namibia (n = 3) and the neighbouring countries of Botswana, Zambia and South Africa were also included in the analysis Table . A domes® (Invitrogen) following the manufacturer's instructions, and resuspended in HPLC purified water (Sigma Aldrich). RNA was then diluted to 1 μg/μl in HPLC water, and stored at -80C.RNA was extracted using TRIzolReverse transcription and polymerase chain reaction were performed as described previously  using thPCR products were purified using the QIAquick™ PCR Purification Kit, and sequenced in both directions using either the Big Dye Kit (Applied Biosystems), or the Beckman QuickStart kit (Beckman-Coulter), with primers at 3.2 pmole/μl. Forward sequences were produced with primer JW12, whereas reverse sequences were obtained using either JW6 DPL or JW6 UNI (CARTTVGCRCACATYTTRTG).Nucleotide sequences were edited to 400 bp using the DNAstar programme . Multiple sequence alignments were generated using the Clustal W programme  and the Additional sequence analysis was performed with a reduced dataset, using a 600 bp region of the glycoprotein gene (nucleotide co-ordinates 3375 – 3974). Sequences from the glycoprotein gene were derived as previously described . A 1000 The authors' declare that they have no financial or personal relationships with other people or organisations that could inappropriately influence this research study. All authors have access to all data in the study and held final responsibility for the decision to submit for publication.KM performed data analysis and interpretation, RT-PCR and sequencing, phylogenetic tree construction, figure preparation and writing the manuscript. LMcE participated in phylogenetic tree construction, data interpretation and writing the manuscript. OH and FM co-ordinated the study with veterinarians in Namibia and supplied the panel of Namibian isolates for analysis. CS participated in data analysis and in supplying the panel of Namibian isolates and supplied archived Namibian isolates for analysis. LN participated in data interpretation and writing the manuscript. ARF conceived and co-ordinated the study with veterinarians in Namibia and participated in study design, data analysis, data interpretation and writing the manuscript. All authors read and approved the final manuscript.


This indicates that sustained post hoc analyses in regards to efficacy of disruption for every single study group member may be required.The Cre/loxP-system has become the system of choice for the generation of conditional so-called knockout mouse strains, i.e. the tissue-specific disruption of expression of a certain target gene. We here report the loss of expression of Cre recombinase in a transgenic mouse strain with increasing number of generations. This eventually led to the complete abrogation of gene expression of the inserted Cre cDNA while still being detectable at the genomic level. Conversely, loss of Cre expression caused an incomplete or even complete lack of disruption for the protein under investigation. As Cre expression in the tissue of interest in most cases cannot be addressed  In recent years, the Cre/loxP-system has become the system of choice for the generation of conditional so-called knockout mouse strains, i.e. the tissue-specific disruption of expression of a certain target gene Any deliberately chosen DNA sequence can be flanked with loxP-elements. LoxP-sites are inserted in the intronic spacers of exons which encode vital structures of the protein of interest. Cre mediated recombination subsequently leads to the deletion of the sequence between two loxP-elements and a truncated gene product if any at all.Cre recombinase can be expressed in a given tissue or cell type under the control of a defined promoter fragment. Theoretically, expression occurs exclusively in the cell type, where the promoter usually is active The mechanisms causing these undesired effects are widely unknown. In the first place, the activity of many promoters in most cases is not fully understood. Hence, there might be developmental stages or other environmental factors affecting the activity of a promoter that have not yet been characterized. When Cre expression occurs within an early embryonic stage, all cells derived from this lineage will carry the deleted gene.Secondly, Cre transgenic mouse strains are mostly generated by random integration of a DNA construct comprising promoter and Cre cDNA into the host DNA. This may lead to unspecified genetic interactions, i.e. transactivations, at the site of integration which might be concurrently causative for unspecific Cre expression in many cell types.position-effect variegation (PEV), the phenomenon of mosaic expression has originally been described as the genetic cause of heterogeneously coloured eye-discs in mutants of Drosophila melanogasterThirdly, a number of articles on the effects of integration site on the expression pattern of a given transgene have been published. Termed Silencing is assumed to be due to site specific effects such as condensation of chromatin These previous publications altogether suggest that various processes may cause impaired expression of transgenes. We here demonstrated that this might, at least in some cases, precludes reliable prediction of tissue-specific gene disruption by tail-biopsy genotyping.Alb) have been generated Alb promoter driven expression of Cre recombinase, it has been reported, that the maximum level of recombination in hepatocytes occurs at two weeks of age bona fide knock-outs. Subsequently and to generate animals for several follow-up studies we observed inconsistencies between the phenotype initially observed Alb Cre construct.By means of site directed recombination, mouse models with a liver specific inactivation of various proteins employing Cre expression under the control of the Albumin promoter  responsible for these effects. These findings highlight the importance of sustained and comprehensive monitoring of the presence of disruption on a transcriptional or translational level for tissue-specific knock-out models.Animals were generated Isolation of genomic DNA and RNA was performed as previously described Protein samples were prepared as described


One of the major problems related with anticancer chemotherapy is resistance against anticancer drugs. The ATP-binding cassette (ABC) transporters are a family of transporter proteins that are responsible for drug resistance and a low bioavailability of drugs by pumping a variety of drugs out cells at the expense of ATP hydrolysis. One strategy for reversal of the resistance of tumor cells expressing ABC transporters is combined use of anticancer drugs with chemosensitizers. In this review, the physiological functions and structures of ABC transporters, and the development of chemosensitizers are described focusing on well-known proteins including P-glycoprotein, multidrug resistance associated protein, and breast cancer resistance protein. MDR1. The efflux pump Pgp is responsible for transporting various xenobiotics (not limited to anticancer drugs) out of cells by using ATP  is especially problematic in acquired drug resistance. MDR is the phenomenon in which cancer cells exposed to one anticancer drug show resistance to various anticancer drugs that are structurally and functionally different from the initial anticancer drug. The most investigated mechanisms with known clinical significance are: a) activation of transmembrane proteins effluxing different chemical substances from the cells; b) activation of the enzymes of the glutathione detoxification system; c) alterations of the genes and the proteins involved into the control of apoptosis . The cell membrane, cytoplasm, and nuclear protein participate in these resistance mechanisms . The resTP   and breaAlthough the physiologic functions of ABC transporters are not well known, they are expressed constitutively in not only tumor cells but also normal cells in the digestive system including the small intestine, large intestine, liver, and pancreas; epithelial cells in the kidneys, adrenals, brain, and testes; and endothelial cells Table . From thmdr1 knocked out[mdr1 knocked out and in mice administered with the Pgp inhibitor, PSC-833 [Pgp is mainly present in the apical membrane of intestinal mucosal membrane and lowers bioavailability of drugs by preventing the absorption of the drugs. Digoxin, which shows a low bioavailability and is mainly excreted through stool in normal mice due to poor absorption in the mouse intestine, shows a high bioavailability and mainly excreted through urine in mice with ocked out. The bio PSC-833 .MDR1 polymorphisms including more than 20 single nucleotide polymorphism (SNP) have been identified. The mutations at positions 2677(G→T) and 2995(G →A) of MDR1 in normal cells were firstly reported [MDR1 polymorphism could be not only associated with alteration of Pgp expression and/or function, drug disposition and treatment outcome but also increase the risk of diseases such as Parkinson's disease and ulcerative colitis [MDR1 SNP(C3435T and G2677T) on disposition of Pgp substrates or treatment outcome has been examplified in digoxin, phenytoin, fexofenadine, nelfinarvir, cyclosporine, talinolol and loperamide [Recently, multiple reported . MDR1 po colitis . The infperamide . Polymorperamide -163.If a substance in food affects Pgp, this substance also could affect the bioavailability of substrate drugs for Pgp. It was reported that substances present in grape juice or orange juice could increase the bioavailability of a drug being the substrate of Pgp by inhibiting it. . These smdr1 gene is knocked out in mice [One of the important physiological functions of efflux pump present in the cell membrane is to provide a pharmacological sanctuary for tissues present in the blood-tissue barriers such as in the case of blood-brain barrier (BBB), blood-placental barrier and blood-testes barrier. Hydrophilic substances present in blood could not go into tissues when they are not small enough to pass through the tight junction with simple diffusion. Nonetheless, various hydrophobic substances could not enter these tissues because they are effluxed out by efflux pumps. Actually, Pgp effluxes neurotransmitters or neuromodulators such as glutamate  and opio in mice -171. Whe in mice . Thus, qPgp is a 170-kDa membrane protein glycosylated at the first extracellular loop Fig. . In the When one of two NBDs of Pgp is inactivated, not only drug transport but also ATP hydrolysis of normal NBD is inhibited. This result indicates that two NBDs would function cooperatively and they could not hydrolyze ATP independently . It was One of the causes for the failure of chemotherapy in the treatment of cancer is the emergence of MDR. Once MDR appears, chemotherapy is not effective even when using high doses of drugs enough to overcome resistance, toxic effects are brought about and the resistance mechanism could be further stimulated. These problems could be resolved by the use of the anticancer drugs that could bypass the resistance mechanism. For example, we could use other anticancer drugs such as alkylating drugs (cyclophosphamide), antimetabolites (5-fluorouracil), and the anthracycline modified drugs (annamycin and doxorubicin-peptide) that would not function as the substrates of ABC transporters -185. TheMany drugs such as the calcium channel blocker verapamil and the immunosuppressant cyclosporin A would inhibit resistance by functioning as competitive substrates of Pgp regardless of their innate pharmacological functions. Different clinical studies also showed that these drugs could reverse resistance to anticancer drugs. Verapamil is the first reported chemosensitizer inhibiting MDR  and its Most chemosensitizers bind with TMD in transporter, but steroid and flavonoid are new recently introduced chemosensitizers, which inhibit transporters by binding with NBD. The binding site of steroid is different from the binding site of ATP but is probably in the vicinity of the ATP binding site . On the in vivo has been limited due to its neurotoxicity [The fungal toxin, fumitremorgin C (FTC), is a strong inhibitor of BCRP but its use toxicity . It was toxicity .Since ABC transporters can be coexpressed in some types of cancer cells, the development of chemosensitizers against MRP and/or BCRP as well as Pgp has been highly demanding. These include VX-710 against Pgp and MRP ,83, GF12One of the major causes of failure in anticancer chemotherapy is resistance against anticancer drugs. Overexpression of ABC transporters such as Pgp, MRP and BCRP has been shown to be responsible for the major portion of MDR. Therefore elucidation of the structure and the function for each ABC transporter is prerequisite for understanding how these transporters work and for reversing MDR. One strategy for reversal of MDR cells expressing ABC transporters is combined use of anticancer drugs with chemosensitizers. Second-generation chemosensitizers have been developed for the purpose of obtaining higher efficacy and lower toxicity than first-generation chemosensitizers. Inhibitors of ABC transporters can be exploited to enhance the oral bioavailablilty or the brain penetration of various drugs.Combination of a conventional anticancer chemotherapy with new strategies such as chemosensitizers, receptor-mediated targeting and nanotechnology will shed light on cancer patients in the near future.


Vector-borne and zoonotic diseases generally display clear spatial patterns due to different space-dependent factors. Land cover and land use influence disease transmission by controlling both the spatial distribution of vectors or hosts, and the probability of contact with susceptible human populations. The objective of this study was to combine environmental and socio-economic factors to explain the spatial distribution of two emerging human diseases in Belgium, Puumala virus (PUUV) and Lyme borreliosis. Municipalities were taken as units of analysis.Negative binomial regressions including a correction for spatial endogeneity show that the spatial distribution of PUUV and Lyme borreliosis infections are associated with a combination of factors linked to the vector and host populations, to human behaviours, and to landscape attributes. Both diseases are associated with the presence of forests, which are the preferred habitat for vector or host populations. The PUUV infection risk is higher in remote forest areas, where the level of urbanisation is low, and among low-income populations. The Lyme borreliosis transmission risk is higher in mixed landscapes with forests and spatially dispersed houses, mostly in wealthy peri-urban areas. The spatial dependence resulting from a combination of endogenous and exogenous processes could be accounted for in the model on PUUV but not for Lyme borreliosis.A large part of the spatial variation in disease risk can be explained by environmental and socio-economic factors. The two diseases not only are most prevalent in different regions but also affect different groups of people. Combining these two criteria may increase the efficiency of information campaigns through appropriate targeting. Numerous human infectious diseases, particularly vector-borne and zoonotic diseases, are thought to emerge or re-emerge in some parts of the world due to environmental changes. The spatial distribution of two emerging human diseases were investigated and compared in Belgium: Puumala virus (PUUV) and Lyme borreliosis. Since the former is a zoonotic rodent-borne infection  and the latter a tick-borne infection, zoonotic and vector-borne at the same time, both strongly depend on the natural environment.Bunyaviridae) are causative agents of human zoonoses called haemorrhagic fever with renal syndrome (HFRS). Hantaviruses are transmitted via rodent excretions, unlike the other viruses from the Bunyaviridae family that are transmitted by arthropod vectors [Clethrionomys glareolus) [In Europe, hantaviruses (family  vectors . In westareolus) -5. EcoloBorrelia burgdorferi. This zoonosis is a tick-borne disease present in the northern hemisphere where the main vector species in European countries is Ixodes ricinus [Lyme borreliosis is an infection caused by a spirochete named  ricinus ,7. The c ricinus . ConsequPUUV and Lyme borreliosis infections followed different evolutions in Belgium over the last decade. PUUV human infections display interannual fluctuations that follow that of their carrier rodent population -11, withThe spatial distributions of both diseases in Belgium are also dissimilar but both are highly clustered in space Figure . HantaviVector-borne and zoonotic diseases generally display clear spatial patterns that can be due to different space-dependent factors: (i) the limited pathogen dispersal, (ii) the spatial distribution of vectors, hosts and reservoirs, and (iii) the human exposure to the infectious agent. Ostfeld et al. (2005) define spatial epidemiology as "the study of spatial variation in disease risk or incidence" . Land coGeographical information systems (GIS) and remote sensing are appropriate tools to describe the spatial distribution of infectious diseases and predict disease risk -20. ThesThe objective of this study was to combine environmental and socio-economic factors to explain the geographic distribution of disease incidence of Puumala virus and Lyme borreliosis in Belgium. We used multivariate statistical analysis to explain the number of PUUV and Lyme borreliosis infections per municipality according to variables related to land cover, land use, settlement characteristics, socio-economic variables, risk behaviours, and host population. We used the disease incidence as proxy for disease risk because: (i) data on human infections are better inventoried than data on hosts (in the case of PUUV) or vectors (in the case of Lyme borreliosis), and (ii) human infection results from the presence of vectors/hosts and the pathogen but also from people's exposure to vectors/hosts . Human eThe surveillance of Lyme borreliosis and hantavirus infections is carried out through the Scientific Institute of Public Health (IPH) sentinel laboratory network. As the diagnosis of these infections is complex and needs to be confirmed by a specialised laboratory, sentinel laboratories are asked to send positive samples to the Reference Laboratories for confirmation. These Reference Laboratories  send monthly data on confirmed cases to the IPH and duplicated records are eliminated. The IPH collects data per postal code, which is a subdivision of the Belgian municipality. As socio-economic census data are only publicly available at the level of municipalities, we simply aggregated data from postal codes to municipalities and conducted the study at that level.The number of human infections was used as dependent variable and the population number was added as offset variable in the model. Therefore, the model explains the incidence rate per municipality, as it was done in , under tThe data collection method was slightly different for the two diseases. While PUUV patients were georeferenced according to their residential municipality in the IPH database, Lyme borreliosis patients were georeferenced according to the municipality where they thought the contamination took place. Since, in most cases, Lyme contamination by a tick took place close to one's house, we assumed that the municipality of contamination that was recorded is the municipality of residence. We thus considered municipalities of residence for both diseases. This assumption allows combining socio-economic and environmental characteristics of municipalities in explaining disease occurrence.Independent variables included several factors that have been related to the spatial variation in disease risk, including proxies for human behaviours that facilitate contacts with pathogenic agents  Table .Land cover determines the spatial distribution of vectors, hosts and animal reservoirs according to their habitat preferences. Broad-leaved forests, being considered as the favourite habitat for bank voles and ticks alike are expected to have an impact on disease risk. The CORINE Land Cover 2000 database, with a spatial resolution of 100 meters, was produced between 1999 and 2001 on the basis of satellite image interpretation . The proLand use determines human presence in a given place  at particular times of the year and of the day. In built-up areas, the presence of vectors and hosts is very constrained ecologically and disease risk is expected to be low. A higher disease risk is expected in landscape mosaics of human-dominated and natural covers, where contacts between humans and vectors or hosts are more frequent . To testSettlement density and types are expected to have an impact on the likelihood of contacts between humans and vectors or hosts. People living at the vicinity of forests are more likely to be infected because they are more often in direct contact with soil and wood potentially infected by PUUV. For some PUUV human cases in France, the only risk factor found was the proximity between habitation and forest . OutdoorCertain professional or leisure activities that are dominant in certain municipalities could be reflected in the socio-professional level. For example, municipalities with many farmers and forest workers, such as loggers or foresters, which are more exposed to disease risk, generally correspond to lower mean incomes. By contrast, peri-urban zones, characterized by large gardens surrounded by woods generally correspond to higher incomes. A higher income may also enhance the propensity to consult a doctor and therefore the probability to detect the disease. Since 2000, the Directorate-general Statistics Belgium performs an annual survey on Belgian household budgets. We used data on average taxable income per tax return form in each municipality from 2002, the most recent publicly available data . Data onSpecific human behaviours are expected to increase risk for the two diseases. Risk activities for PUUV include working in forest, farming, animal trapping, woodcutting or reopening rodent infested buildings ,10,32, aIxodes ricinus – although not a reservoir – plays an important role of maintenance and co-feeding for ticks [The roe deer, the main host of or ticks ,36. Roe or ticks . The absor ticks . Censuseor ticks . Data onor ticks . Using tCount data such as the number of human infections often follow a Poisson distribution. This distribution requires that the mean is equal to the variance, a condition commonly not fulfilled. Our data follow such an extra-Poisson model. The dependent variables  for both diseases have highly asymmetrical statistical distributions Figure . VariancAs we are interested in inference of regression parameters, the use of a model that allows for the possibility of extra-Poisson variation is preferable . The negWhen k = 0, the negative binomial distribution is reduced to a Poisson distribution.Likelihood ratio tests and Dean's tests for overdispersion were performed with the DCluster package of the R software. The three tests were very significant and the null hypothesis of overdispersion was not rejected Table .i of municipalities and the logarithm of Ni was added as an offset variable in the models. The distribution parameter k is estimated by the maximum likelihood method, as the other parameters of the negative binomial regression model. The Akaike's information criterion (AIC), which takes the number of estimated parameters into account, was used to select the covariates to retain in the model. Variables were tested for collinearity and confounding factors.Generalized linear regression models were computed using the R statistical software. All variables were relative to the population size NSpatial autocorrelation appears when a spatial dependence due to neighbouring effects  exists between spatial entities. Spatial dependence usually results from a combination of exogenous and endogenous processes. In an exogenous spatial process, the identified spatial pattern is generated by independent factors, whereas endogenous spatial dependence is an inherent property of the variable of interest . SpatialSince spatial dependence between observations skews parameter estimation, the presence of residual spatial dependence in the error term needs to be examined . EmpiricFor linear regression models, one can distinguish between endogenous and exogenous spatial processes by comparing spatial lag and spatial error models . This api is the expected value of the dependent variable y for the municipality i, xin are the dependent variables with their associated regression coefficients βn, σεi is the disturbance term and YEj in each municipality. Covariates included the same variables as for the non-spatial model computed for both the central and its neighbouring municipalities. Interaction variables were also included to maximize the performance of the model. In the second stage, these linear predictions where Yr (1977) . FirstlyBoth non-spatial regression models have a high explanatory power, with all parameters being very significant and a residual deviance value close to the number of degrees of freedom (DF) (with a ratio of 0.81 for PUUV and 1.1 for Lyme), indicating a good model fit. For PUUV, we reduced the deviance from 1,167.8 for the null model to 474.9 (with 584 DF). For Lyme, it was reduced from 857.9 to 642.4 (with 584 DF) Tables  and 4. EThe variograms of residuals for these non-spatial regressions lead to a different diagnostic for the two diseases Figure . For PUUTo test for spatial endogeneity in the Lyme borreliosis model, a linear predictor of the dependent variable in neighbouring municipalities was estimated. For comparative purpose, the same approach was applied to the PUUV model, even though the spatial dependency was already reduced in the non-spatial model for this disease. Both spatial models have a high explanatory power and very significant parameters Tables  and 4. FThe variograms of the residuals from the spatial regressions show that the spatial dependence is further reduced for PUUV Figure . SpatialThe spatial model on PUUV is composed of four variables, each with the expected sign, in addition to the spatial dependency term that is also significant. Two variables are related to the landscape , one to a socio-economic characteristic of the population (income), and another one to a risk behaviour of humans (hunting). The PUUV incidence rate is higher where the proportion of broad-leaved forests is high and where urbanisation is low. Broad-leaved forests are the favourite habitat for bank voles and are thus positively related to the disease. Moreover, bank voles use small areas and only rarely leave forests. Therefore, humans generally catch the disease in the ecological habitat of bank voles, in broad-leaved forests. A high level of urbanisation thus limits PUUV transmission. Income is negatively correlated with the disease incidence because PUUV particularly affects forest workers and farmers who are in frequent contact with bank voles or infected soil in forests or at their edge. These socio-professional categories are generally characterised by a low income. Hunters also spend time in forests and are thus particularly vulnerable to PUUV infection.The spatial model for Lyme borreliosis infections has four explanatory variables, each of them with a positive sign, in addition to the spatial dependency term that is also highly significant. One variable is related to the host population (roe deer), two variables represent landscape attributes that favour Lyme borreliosis transmission , and one characterises the human population by its socio-professional level. The model confirms the hypothesis that roe deer density has an impact on spatial variations in Lyme disease transmission risk in Belgium, probably by favouring tick reproduction and co-feeding. Lyme borreliosis incidence rate is positively linked to forest cover but also to the proportion of people living in separated houses. This suggests that heterogeneous landscapes with a fragmented land use mixing forests and houses are more at risk. In areas with a high proportion of separated houses, people are more likely to have gardens and thus spend time outdoors. The presence of forests nearby further favours human-vector contacts. Peri-urbanisation could thus be one of the major causes of the recent increase in Lyme borreliosis infections. In Belgium, it largely affects Walloon Brabant and the province of Antwerp, two wealthy peri-urban areas. The positive relationship between Lyme borreliosis incidence rate and mean income supports this hypothesis since higher incomes are likely to be associated with large gardens in wooded areas and thus a larger interface between settlements and forests in peri-urban areas. Higher incomes – especially when they are associated with education level – could also increase the probability of being aware of the disease and of consulting a doctor, thus increasing the likelihood of disease detection.The spatial distribution of PUUV and Lyme borreliosis infections are thus associated with similar factors, in particular the presence of forests. For both diseases, it is linked to the vector or host habitat preferences. Other variables influence both diseases but in opposite directions, such as mean income and urbanisation. This reflects the fact that PUUV incidence is more associated with professional outdoor activities , whereas Lyme borreliosis is rather associated with recreational and peridomestic outdoor activities .Most importantly, both diseases are associated with a combination of factors linked to the vector and host populations, to human behaviours, and to landscape attributes. The latter includes both land cover (forests), as a function of vector/host ecology, and land use that determines human presence in critical habitats, the frequency of contacts between vectors and human populations, and thus human exposure to infection.Residuals of the spatial regression model of Lyme disease still display strong spatial dependence, which suggests that one or more external factors with spatial structure are missing. However, several factors could influence and skew the spatial effect in the Lyme model. Firstly, the roe deer density data were only available at the province level in Flanders and at the forest district level in the Walloon region and Brussels. This variable was thus spatially disaggregated at the level of municipalities. Removing this variable did not reduce the spatial autocorrelation. However, representing more ecologically-relevant boundaries that separate independent deer populations  could reduce spatial autocorrelation compared to administrative boundaries . SecondlThe objective of this study was to explain the spatial distribution of a zoonotic  and a vector-borne (Lyme borreliosis) disease in Belgium. The combination of environmental and socio-economic factors allowed explaining the major part of the spatial variation in disease risk. Besides the influence of land cover on the spatial distribution of vectors and hosts, land use also determines the prevalence of vector-borne and zoonotic diseases by controlling the degree of exposure of people to contact with vectors or hosts. PUUV infection risk is more prevalent in remote, low-income areas with large forests whereas the probability of Lyme borreliosis infection is higher in areas with a large interface between settlements and forests in wealthy peri-urban areas. This study showed the importance of adding variables related to human behaviour and land use in addition to land cover and habitat variables in epidemiological models. Spatial dependence diagnostics in error terms revealed a combination of endogenous and exogenous processes causing spatial clustering of incidence for both diseases. The spatial dependence could be accounted for in the model of PUUV incidence but not for Lyme borreliosis.The results of this study have important implications to define priority areas for public health policies aimed at preventing diseases by informing the public and promoting the use of protective measures. For example, the Institute for Public Health produces information sheets on the diseases for distribution to the public. Each disease is not only most prevalent in different regions but also affects different groups of people. Combining these two criteria may increase the efficiency of information campaigns through appropriate targeting.The author(s) declare that they have no competing interests.CL collected data, performed the statistical analyses, interpreted the results and wrote the first draft of the manuscript. PL participated in data acquisition, analysis and interpretation. PH, GD and VL collected data on human infections. KT helped in data acquisition. SOV helped in statistical analysis and interpretation of results. EFL had the initial idea and led the design of the study, advised CL and PL, and helped to write the manuscript. All authors read and approved the final manuscript.


Macrophages play an integral role in the host immune system, bridging innate and adaptive immunity. As such, they are finely attuned to extracellular and intracellular stimuli and respond by rapidly initiating multiple signalling cascades with diverse effector functions. The macrophage cell is therefore an experimentally and clinically amenable biological system for the mapping of biological pathways. The goal of the macrophage expression atlas is to systematically investigate the pathway biology and interaction network of macrophages challenged with a variety of insults, in particular via infection and activation with key inflammatory mediators. As an important first step towards this we present a single searchable database resource containing high-throughput macrophage gene expression studies.The GPX Macrophage Expression Atlas (GPX-MEA) is an online resource for gene expression based studies of a range of macrophage cell types following treatment with pathogens and immune modulators. GPX-MEA follows the MIAME standard and includes an objective quality score with each experiment. It places special emphasis on rigorously capturing the experimental design and enables the searching of expression data from different microarray experiments. Studies may be queried on the basis of experimental parameters, sample information and quality assessment score. The ability to compare the expression values of individual genes across multiple experiments is provided. In addition, the database offers access to experimental annotation and analysis files and includes experiments and raw data previously unavailable to the research community..GPX-MEA is the first example of a quality scored gene expression database focussed on a macrophage cellular system that allows efficient identification of transcriptional patterns. The resource will provide novel insights into the phenotypic response of macrophages to a variety of benign, inflammatory, and pathogen insults. GPX-MEA is available through the GPX website at  They have been shown to play a key role in both the innate and adaptive immune response and are capable of responding to a range of signals leading to classical or alternative activation of immune responses which may be classified as innate, cellular, or humoral, . Their fo emerge . ClinicaGPX-MEA is a gene expression database that stores MIAME-compliant  microarrA major challenge when using microarray data to elucidate molecular pathways is the development of methods for the reliable comparison of results from different studies. This issue is further compounded by the fact that microarray assays are highly sensitive to technical perturbations and the lack of standard units of gene expression. It has been shown that microarray experiments, even using the same platform, can vary greatly between groups and laboratories . In an aHere we report on the data curation process, quality assessment, content, search facilities and integration tools that are components of the GPX-MEA.). The GPX system comprises two MIAME-compliant microarray databases, GPX Repository and GPX Discovery. The data is first curated into GPX Repository, a biochip Laboratory Information Management System developed at the GTI. GPX Repository is designed around a set of web enabled data entry forms that assist in the data capture process ensuring that MIAME-compliant information is obtained.GPX-MEA is part of the GPX database system at the Scottish Centre for Genomic Technology and Informatics . Enhancement to the maxdSQL schema include extensions to store image analysis files and annotations from the NCBI UniGene database [The underlying database schema of GPX Discovery, maxdSQL-Plus, is an enhanced version of maxdSQL 1.2 , and any MIAME information that were not reported in the original publication or database entry. Responses were received from eleven of the initial eighteen investigators contacted to supply data and information to GPX-MEA.In addition to experimental information and data obtained from the public domain, GPX-MEA also contains original unpublished microarray experiments conducted at the GTI. These experiments characterise the gene expression profile of murine bone marrow derived macrophages grown under standard and serum free growth conditions. Information and associated data files for these and other experiments may be retrieved from GPX-MEA using the GPX Discovery web interface.Data submitted to the GPX-MEA has followed a manual curation process. At present, subjective evaluation criteria are employed during the quality assessment of microarray data. As a result, expert manual curation was undertaken to ensure the value and biological relevance of information held within the GPX-MEA. Efforts in the future will be directed to methods for the automation of this process.The curation process follows standard operating procedures that include the assessment of the experiment data against defined criteria and validation of experiment information against MIAME standards.® platform have been preferentially selected since they have widely recognised quality control parameters, use of standardized manufacturing and experimental protocols and familiarity of the platform with the biological and computational users. Future releases of GPX-MEA will contain experiments hybridized using spotted custom array platforms and other proprietary platforms, such as those from Agilent and GE Healthcare.For this first public release of GPX-MEA, studies conducted using the Affymetrix GeneChipBefore submission to the database, analysis files were all processed using the same software program and normalisation algorithm. Affymetrix DAT files were processed using Affymetrix-Gene Chip Operating System (GCOS). The CHP files were generated from CEL files using Affymetrix Global scaling normalisation to a Target Intensity Value of 100 (TGT-100). In some instances DAT files were unavailable and the original CEL files were used to generate the CHP files. The use of this global normalization algorithm provides a uniform measure which allows a preliminary comparison of expression values across experiments within the databases. Other values that are stored within the database from the CHP files are the detection call  and the p-value of detection.GPX-MEA contains experiments of varying complexity and experimental design collated from a variety of resources. To take into account the variations in quality and statistical power of experiments a GTI-derived ordinal scale quality assessment score is assigned to each experiment added to the database. The score enables the user to make an informed judgement on the reliability of results obtained from experiments within GPX-MEA and allows for experiment selection based on quality requirements. It also functions to provide a metric for systems biologist interested in integrating data sets from the database.The quality assessment score ranges from 0 (low) to 28 (high) and is derived from six defined categories each with a 3-point 0, 1, 2) scale. The score is also stored as a percentage of the maximum attainable value within the database. Categories are also weighted by relative importance of the assessment on a 3-point  scale. The scoring categories are as follows: use of consistent standard operating procedures (SOPs) , sample , 2 scaleIt is worth noting that given the complexity and variety of experimental designs and data structures, it is not feasible to develop fully objective criteria based on non-arbitrary threshold values, instead we have sought to provide the user with a graded indication of the underlying quality of an experiment with a scoring system that is reproducible.The first step in scoring an experiment is to obtain MIAME compliant information about an experiment's design and implementation. Using this information the score for SOPs, sample size and sample quality control can be assigned. The next criterion assesses image files and includes analysis of the background noise (RAWQ) and variation in mean signal intensity  for the arrays. The final criterion seeks to measure sample level variation and gene level variation from visualizations output using R and Bioconductor softwareTo illustrate the assessment method the scoring of the category "sample size" is described. In the category "sample size", the number of biological replicates per group is determined and scored using the criteria in table A record detailing the score in each category for an individual experiment can be viewed using GPX-MEA. Further, the combined overall percentage score can be used to select and retrieve experiments using the GPX Discovery interface.Future versions of GPX-MEA will incorporate non-Affymetrix experiments. This will require modifications to the scoring system which are likely to include; more stringent requirements for the numbers of biological replicates used in custom arrays based experiments, due to the larger noise variation, the use of different visualisation techniques to assess quality and an additional criterion to address the suitability of the reference sample.® platforms (Table The current release of GPX-MEA May 2005) consists of 11 public microarray experiments with a total of 152 hybridizations from 3 Affymetrix GeneChip005 consiAt present, GPX-MEA has a further 12 MIAME-compliant experiments with a total of 246 hybridizations stored in a restricted access site. This allows the accommodation of unpublished microarray experiments. Experiments within this access controlled site will be made publicly accessible following scientific publication. Further macrophage gene expression studies have been identified for deposition in the GPX-MEA database and routine updates to the database are scheduled to include emerging research findings.. Searches can be performed in three different sections of the interface; experiments, arrays and probes. A search is made using a number of separate criteria such as accession number, technology type, target organism, quality assessment score or keywords. For example, all microarray experiments conducted using humans as the source organism can be retrieved by performing a search selecting Homo sapiens from the organism search field. The results of a query are displayed in a table with hyperlinks to the corresponding experiment, array or probe record.Users can browse and retrieve data from GPX-MEA using the user oriented GPX Discovery web based graphical interface Experiments are defined in GPX-MEA as the complete set of bioassays (hybridizations) and their descriptions for a common purpose, following Microarray Gene Expression Data Society (MGED) ontology specifications. Once an experiment has been selected the corresponding database entry is displayed. An experiment record shows the provenance, experimental design, protocols, sample information, array details, quality assessment and a list of array measurements. Expression values can be selected from the experiment record by selecting the desired measurement records. Measurement data are presented to the users as a comma separated value (CSV) file. Significantly, the user can also obtain original image analysis files, e.g. the Affymetrix CEL files, from the database providing additional flexibility for an extended analysis. Access to the original image analysis files is an especially valuable resource for those interested in computational analysis and data integration.The array type section allows the user to browse the list of array types stored within the database. An array type record displays the description of the array type and the manufacturer. The array layout with probe annotation obtained from a local copy of the UniGene database can also be downloaded as a CSV file from a link within a record.The probe section allows the user to search and retrieve individual probe details from the database based on criteria such as Entrez Gene ID, Gene Symbol and UniGene ID. Once a probe record is selected the user can view probe annotation within the record and by following links to external databases, Adapt, NetAffx® MG U74Av2 and MOE430A version 2 can be compared using BioLink MatchMaker to find probes that share equivalent genes. The results show that there are 7454 unique probe matches between the two platforms. The user can then view the results in a separate webpage or download the results for further analysis.The Probe section provides a link to the BioLink MatchMaker integration tool which enables the comparison of different array types. BioLink MatchMaker uses NCBI's UniGene Cluster IDs to identify probes which represent equivalent genes on two different microarray platforms of the same species stored within GPX-MEA. Users can also submit a list of up to 1000 GenBank or UniGene ID to identify probe matches with any array type in the database using the Batch query function. Results can be displayed either as HTML tables containing hyperlinks to the corresponding UniGene entry or can be downloaded as a CSV file for further analysis. For example, the Affymetrix GeneChipsGPX-MEA is under active development and will continue to collate macrophage expression studies through online database searches and collaboration with principle investigators. This data will be assessed and manually curated within the GPX-MEA and released in routine database updates. In addition, experiments that profile the macrophage response to infection and immune regulators conducted within GTI will also be included in the database. There will also be continual development of curation and quality assessment protocols to maintain the high standard of experimental description and data within the database.New analytical tools in development for the GPX-MEA include the ability to query gene expression using interactive immunological pathway maps and an automated statistical analysis tool to guide the user through normalisation and analysis processes. The export of MAGE-ML (Microarray Gene Expression-Markup language) from the database is also being developed so that data may be shared between other public repositories and users of microarray data. In addition, GPX is part of the Edinburgh National Translational Cancer Research Network Centre resources (NTRAC) and relevant work emerging from this project will be included in GPX-MEA.Here we present GPX-MEA, a MIAME compliant online database that exploits and captures the wealth of expression studies related to macrophage biology data in a centralised resource. Experiments are assessed and submitted to the database by expert curators and includes some information and data, such as image analysis files, that were not previously publicly available.This is the first example of a focussed macrophage gene expression database that allows efficient identification of gene expression patterns in this key player in the host immune system.GPX-MEA also provides highly reliable datasets with associated contextual information that can be used by the computational biologist for systems level analysis. In addition, it also utilises a unique quality assessment scoring system that enables the user to select experiments based on their quality requirements. using web browsers. The GPX Discovery software is available for download at .GPX-MEA is publicly available and can be accessed at GG carried out the data collation, curation, and quality assessment and drafted the manuscript. SM and GG carried out the database development, design and data migration. TF, KR, PD, MC participated in the design of the quality control criteria. GS, AL, MC, AR contributed data to the database. PG and JB conceived of the database, and participated in its design and coordination. PG, JB, TF, KR, PD, SM and MM helped to draft the manuscript. All authors read and approved the final manuscript.


C. elegans and C. briggsae. It has been implemented according to the following project specifications:NemaFootPrinter (Nematode Transcription Factor Scan Through Philogenetic Footprinting) is a web-based software for interactive identification of conserved, non-exonic DNA segments in the genomes of a) Automated identification of orthologous gene pairs.b) Interactive selection of the boundaries of the genes to be compared.c) Pairwise sequence comparison with a range of different methods.d) Identification of putative transcription factor binding sites on conserved, non-exonic DNA segments.C. elegans or C. briggsae gene name or identifier, the software identifies the putative ortholog (if any), based on information derived from public nematode genome annotation databases. The investigator can then retrieve the genome DNA sequences of the two orthologous genes; visualize graphically the genes' intron/exon structure and the surrounding DNA regions; select, through an interactive graphical user interface, subsequences of the two gene regions. Using a bioinformatics toolbox  the investigator is able at the end of the procedure to identify and analyze significant sequences similarities, detecting the presence of transcription factor binding sites corresponding to the conserved segments. The software automatically masks exons.Starting from a C. elegans and C. briggsae. These sequences may contain regulatory transcriptional elements since they are conserved between two related, but rapidly evolving genomes. This software also highlights the power of genome annotation databases when they are conceived as an open resource and the possibilities offered by seamless integration of different web services via the http protocol.This software is intended as a practical and intuitive tool for the researchers interested in the identification of non-exonic conserved sequence segments between Availability: the program is freely available at  Comparative genomics is a powerful bioinformatics methodology for the identification of conserved genomic DNA segments between two related organisms . AlignmeC. elegans and C. briggsae are almost identical in morphology and development [Although elopment , their gelopment . Conservelopment .C. elegans and C. briggsae, starting from a bio-computational project focused on identification of conserved segments in a single pair of orthologous genes.We developed an interactive web-based and user friendly software to help the researchers in the identification of conserved non-coding sequence regions between the genomes of the two nematodes The program developed here is a research tool; hence the design has been sometimes bound to the functionality, in order to optimize the speed and easiness of interaction between all the components. A careful planning of all the required modules has been achieved, however we have used system analysis and design techniques for the most complex parts of this development project., and to the programmer/maintainer of the software, with internal documentation on the scripts. We always relied on feedback from the interested scientist in designing the user interface and ameliorating the software functionality. From the programming language point of view, we adopted standard solutions that were fit to the problem (bioinformatics development).Documentation has been targeted both at the user with a web page The following components have been used to develop the web application:1) A local mirror of Wormbase  database.2) An EnsMart table to search orthologs. EnsMart  is a dat3) A Perl web interface used to retrieve gene and sequences, mask exons, launch analysis tools, and generate gene images (GD libraries). Other web based technologies are implemented inside the Perl code: cursor coordinates capture and client-side image maps are implemented in html and JavaScript.4) A graphical user interface implemented in Java (Swing Applet) for interactive selection of sub-sequences. Using HTTP POST and GET method, the Applet is able to communicate with other elements of the system.dotmatcher and extractseq (EMBOSS package), blast two sequences (NCBI), ssearch (part of the FASTA program suite written by William Pearson), blastz.5) A collection of locally compiled C++ software: rVista)6) An User Agent LWP connection (Perl library) to send subsequences and blastz alignment to a transcription factor database web server , based on information derived from one of the EnsMart datamart tables [CGC (Caenorhabditis Genetics Center) genetic nomenclature [The main functional flow of the software can be summarized as follows Figure : Startint tables . In thisnclature .The user can select a display mode:TEXTMODE: html output only1. SLIDER MODE: graphical visualization with a Java Applet  wherever there is equality between the sequences a dot is placed on the image. Where the two sequences have substantial regions of similarity, many dots align to form diagonal lines. It is therefore possible to glance at local regions of similarity, as these will show diagonal lines . Even if the software selects a default strand configuration, the user can manually choose another strand mode. On the web page, text boxes give the exact position (relative to the effective sequence length) of the cursor on the X and Y-axis; this helps the user in choosing the sequence stretch of interest.•s Figure . In thisSsearch [: Ssearch uses Pearson's implementation of the method of Smith and Waterman [• Waterman  to searcWaterman . The disBlastZ[and rVista [: BlastZ computes local alignments for sequences of any length based on the assumption that the input sequences are related and share blocks of high conservation that are separated by regions that lack similarity and vary in length. Regions of homology are displayed collinear only to the reference sequence, while the order and orientation of the conserved elements is not necessarily the same in the second sequence.• rVista. rVista is aimed at high-throughput discovery of cis-regulatory elements, combining clustering of predicted transcription factor binding sites (TFBSs) and maximizing the identification of functional sites.Identifying transcriptional regulatory elements represents a significant challenge in annotating genomes. Our bioinformatics procedure has been transparently connected trough the http protocol to a computational tool, A continuous exchange of ideas and information about this software and its interface occurred between the software developers and the nematode researchers. This user feedback has hence been fundamental in tailoring the graphical interface in functions of his needs.blast2seq (blast two sequences), a heuristic algorithm (BLAST), is used for fast comparison of two sequences.A PAIRWISE alignment performed with Dotmatcher uses a simple but slow algorithm; for this reason we have chosen this as a tool to verify the alignments identified in the first step. When two regions of similarity have been found, this similarity can be quantified with the Smith and Waterman algorithm . This is the most sensitive method available for pairwise sequence comparison, but it works slowly, therefore it is more appropriate for in depth analyses than primary searches.rVista) focused on the identification of transcription factor binding sites related to conserved sequence elements. Continuous update of the transcription factors for vertebrates and, in particular, for nematodes is thus guaranteed. Local execution of blastz algorithm permits a fast genome sequence alignment, while the remote server (rVista) performs transcription factor binding site analysis.The last component of the toolbox is a transparent connection through the http layer to a database search tool  they were able to identify two different Cis acting elements [C. briggsae. By simply using our software we were able to confirm all the elements described in this work without any molecular biology experimentation needed (data not shown). With the help of NemaFootPrinter, from now on, researchers will "first" use our software and "then" test the results by creating just a few transgenic strains in order to just "test" the results and not to "identify" the regions by tedious in vivo experiments.Finally, in order to test the quality of the software, we performed the following experiment: Natarajan and colleagues  have recelements . In a seCisHorto, another tool for transcription-factor identification , uses PoCSTminer  is an usC. elegans and C. briggsae [. We have also demonstrated that usability and functionality in bioinformatics development can be achieved only through a strong and continued feedback from the scientist/user.Genome annotation databases such as Wormbase  or EnsEMbriggsae . With thbriggsae . Where sProject name: NemaFootPrinterProject home page: server side: UNIX type platformsclient side: Any operating systemProgramming language: SQL, Perl, JavaOther requirements:text-only browsers like 'lynx' are compatibles with the software (obviously text-only browser must use the TEXT MODE display). For more compatibility information check the help page: The web-based application was tested and is compatible with the more common Internet browser. For the Slider Applet the user must have a Java Virtual Machine installed and configured on the client. User without Java can use the TEXT MODE to analyze genes. Even the older All authors contributed to the development of NemaFootPrinter.DR wrote most parts of the NemaFootPrinter core, the CGI scripts, the Java applet and the database handlers. AG was responsible of the main bioinformatics programming strategy.PM was respnsible of biological applied aspect.GC made the scientific supervision and interface design. All authors drafted the manuscript and approved the final version.


Biliary papillomatosis (BP) is a rare disease entity with a strong malignant potential. It is characterized by multiple papillary adenomas involving both the intrahepatic and extrahepatic biliary tree. BP was considered in the past to be a disease with low malignant potential. However, a current review of the English literature revealed a high rate of malignant occurrence of approximately 41% and histological analysis along with the expression pattern of mucin core proteins (MUC) and mucin carbohydrate antigens suggests that BP is a borderline or low grade malignant neoplasm with a high malignant potential.A 68 year-old male patient was referred to our hospital due to the presence of sudden right upper quadrant abdominal pain, nausea and dark urine. Imaging workup demonstrated dilatation of the left hepatic duct without the presence of a space-occupying lesion. A left hepatectomy and cholecystectomy were carried out and histological analysis revealed a moderately to poorly differentiated carcinoma of the left hepatic duct in the background of biliary papillomatosis. Postoperative course was uneventful. Unfortunately, two years after initial diagnosis the patient rapidly deteriorated and died from multiple pulmonary secondary deposits.BP should not be considered to be a benign disease. The clinical behavior, the high recurrence rate and the even higher malignant transformation occurrence, as well as the presence of carcinogenetic indicators  strongly support that BP is a low-grade neoplasm with high malignant potential. Biliary papillomatosis (BP) is defined as papillary proliferation of the lining epithelium of the bile duct tree, further classified into 5 classes according to the degree of cytological and structural atypia ; thus, class 1 is defined as BP with low-grade atypia, class 2 as BP with high-grade atypia, class 3 as BP with in situ carcinoma, class 4 as BP with microscopic foci of stromal invasion and class 5 as BP with definite invasion into the hepatic parenchyma or fibromuscular layer of the bile duct wall [A 68 year-old male patient was referred to our surgical department from another hospital's medical department with a presenting clinical picture of sudden right upper quadrant abdominal pain, nausea and dark urine. With an initial diagnosis of obstructive jaundice possibly due to choledocholithiasis the patient was admitted to our department for further investigation. The patient's past medical history included upper gastrointestinal bleeding and chronic pulmonary obstructive disease.Tumor markers  were normal, while a mild elevation of the cholestatic enzymes  were demonstrated with return to normal of bilirubin and transaminases.Abdominal ultrasound, computed tomography and magnetic resonance imaging Fig.  demonstrEndoscopic retrograde and magnetic resonance cholangio-pancreatographies showed anomalous dilatation of the left intrahepatic bile ducts with a concomitant milder dilatation of the pancreatic duct, as well as mucus discharge from the papilla of Vater during endoscopy.Colonoscopy was performed to rule out primary bowel neoplasm and revealed the presence of large bowel polyps. Snare polypectomies were performed and the histological analysis demonstrated the presence of tubulous and tubulovillous adenomas of the colon with mild to moderate degree of epithelial dysplasia.Total bone scan with Tc99 m MDP, thoracic computed tomography and brain magnetic resonance imaging were negative for secondary deposits.With a diagnosis of a cholangiocarcinoma a left hepatectomy with inflow occlusion (Pringle's maneuver) and selective hepatic vascular exclusion and cholecystectomy were carried out. The histology report describes the presence of foci of papillary adenomas with a fibrovascular core connecting each of them with the ductal wall, the cuboidal or columnar cells lining the bile duct epithelium and the presence of excessive intraductal mucus, as well as foci of a moderately to poorly differentiated carcinoma, with sporadic necrotic areas and invasion of the fibrously thickened intrahepatic bile ducts Fig , 3.th postoperative day. Despite an uncomplicated 2 year follow-up period, the patient rapidly deteriorated and died from multiple pulmonary metastatic deposits.The postoperative course was uneventful and the patient was discharged on the 9Since the first case of biliary papillomatosis reported by Chappet in 1894  approximBiliary papillomatosis is an unusual disease entity commonly affecting adults older than 60 years-old , with a The clinical picture of BP consists of recurrent colicky abdominal pain, repeated episodes of acute cholangitis with fever and jaundice, due to partial or intermittent obstruction of the bile duct by mucus, enlarging adenomas or tumor fragmentations ,5-7. TheSeveral pathogenetic mechanisms have been proposed, but a definite one remains to be elucidated. Recurrent pyogenic cholangitis, congenital choledochal cysts and chronic stimulation from lithiasis, infection or pancreatic juice are some of the mechanisms reported to be associated with the papillary appearance of the bile duct epithelium .An enlarged intrahepatic and/or common bile duct with concomitant ill-defined filling defects consist the primary imaging features of BP. Ultrasound can demonstrate non-specific bile duct dilatation and intraductal solid masses with no distal acoustic shadowing. Endoscopic and magnetic resonance cholangiopancreatograms usually show multiple irregular filling defects. In ERCP, direct visualization of excessive mucus discharge from the papilla of Vater, as well as lack of motility on irrigation are typical endoscopic features, while the presence of intraductal masses connecting with a pedicle to the bile duct wall demonstrated in MRCP consist the key radiological features of BP ,8. HowevTsui et al  have defBiliary papillomatosis was considered in the past to be a disease with low malignant potential. However, a current review of the English literature revealed a high rate of malignant occurrence of approximately 41%  report tResection is the treatment of choice when BP is localized according to preoperative imaging workup and with the support of intraoperative ultrasound or cholangioscopy ,17-19. IBP should not be considered to be a benign disease. The clinical behavior, the high recurrence rate and the even higher malignant transformation occurrence, as well as the presence of carcinogenetic indicators  strongly support that BP is a low-grade neoplasm with high malignant potential. Radical surgery and liver transplantation should be considered as the only curative treatment options in order to prolong survival.The author(s) declare that they have no competing interests.IV and VS carried out the surgical procedures and contributed to the design of the study; AM gathered the data form the literature search, equally contributed in the preparation of the manuscript and critically revised it; EK performed the histological analysis of all surgical specimens and provided histological sections as figures for the manuscript; TT, NA and VS revised and finally approved the manuscript for been published. All authors approved the final manuscript.