An online educational module introduces students to concepts of quantitation and numerical simulations in developmental biology Driven by the massive datasets that are generated by “omics” research, the molecular life sciences are entering a new phase. This phase is characterised by a shift in focus from individual genes and their products to networks and whole systems ,2,3. ForThe successful application of quantitative tools in the molecular life sciences requires a good understanding of these tools and sufficient knowledge of the biological system under study. This can be achieved by collaboration between quantitatively trained scientists such as physicists on the one hand and biologists on the other. However, cultural differences hamper such collaboration : even atA more productive approach is therefore to prepare students better for the quantitative nature of the molecular life sciences by integrating quantitative thinking and biology in the life science curriculum. This can be achieved in various ways. For example, a curriculum could be developed in which mathematics, the physical sciences, and biology are introduced together . Howeverhttp://mbedu.fbt.eitn.wau.nl/demo_plos/). Some of the features of this module and the thinking that led to its development are quite general, and so we present the module here as a case study in the hope that this might inspire and guide others to create similar resources.As an example of the integration of quantitative teaching and cutting edge biology, we have implemented an educational module in which numerical simulations are used in an existing course on developmental biology  early during ogenesis . The genSecond, we designed the simulation environment in such a way that biology students with their existing mathematical background can build quantitative models and run numerical simulations themselves. In this environment , studentThird, we wanted to make sure that students would use the simulation environment effectively. Therefore, a clear goal is formulated when students enter the simulation environment. For example, they are asked to make a model that generates a Dpp gradient that fulfills a number of specific criteria, or simulates certain mutants. After running a simulation, students can view feedback that helps them draw conclusions or consider the next step to be taken. If a student's model, for example, generates a gradient that is too shallow, the student has to indicate which change in the model he expects to be useful for generating a steeper gradient. The student then receives an intuitive explanation of the usefulness of the given suggestion. If an increase in the synthesis of one of the proteins, Short gastrulation, is proposed, for example, feedback is given that this could indeed be useful, since there would then be more Short gastrulation available to transport Dpp, such that the gradient can become steeper. In this way, the student is stimulated to carefully consider each step and is provided with sufficient support to decide which is a useful step to follow. In addition, with this type of feedback, explanations are given that relate quantitative changes in the model to qualitative changes in its behaviour, which should increase the student's understanding of the behaviour of the biological model.We consider it important that students, while using the module, are not distracted too much by quantitative issues from the actual biological principles and facts. These have to be mastered in order to obtain a strong biological background. If students want to learn more advanced quantitative skills, they can still follow courses that are specifically aimed at this aspect.Quantitative analysis is already gaining importance in molecular life sciences. Therefore, it is desirable that curriculum changes are implemented in the short term. This poses challenges to faculties, especially to those whose members do not have much, if any, experience with the application of quantitative tools in their own research. Therefore, it may be useful to initially focus on the development of learning materials that are rather self-contained, such that their application requires relatively little competence in quantitative analysis from the teaching staff. If these materials are openly available they can be incorporated rapidly into existing courses, such that even the current generation of students may be better prepared to integrate quantitative thinking and biology in their future research.


There has long been a recognition that place matters in health, from recognition of clusters of yellow fever and cholera in the 1800s to modern day analyses of regional and neighborhood effects on cancer patterns. Here we provide a summary of discussions about current practices in the spatial analysis of georeferenced cancer data by a panel of experts recently convened at the National Cancer Institute. Recently, the North American Association of Central Cancer Registries (NAACCR) formed a Geographic Information Systems (GIS) Task Force that prepared a handbook to aid cancer registry staff in using GIS for the collection, analysis, and presentation of cancer registry data . The firThe purpose of the meeting was to provide guidance from experts in this field who have experience applying these methods to health data, acknowledging that opinion will change as the field continues to evolve. Consensus of recommendations for any technical field is difficult to achieve, but we have attempted to include contributors with a wide-ranging set of backgrounds and experiences in the hope that what is presented represents, if not clear "best practices", at least sound principles for the analysis of spatial health data. This paper introduces motivating ideas and provides a broad overview of an upcoming series of reports by subgroups of the attendees. A listing of initial reports appears in Table Interest in and use of GIS for health data has grown tremendously during the past decade. The recognition of local geographic influences on health date back at least to the development of spot maps of yellow fever and cholera in the earlier-to-mid 1800's . While wIn addition to the impact of the computer revolution is the increasing recognition that all health data are spatial, i.e., referenced to place. A recent call for more widespread use of GIS in the U.K. National Health Service points out that GIS could "act as powerful evidence-based practice tools for early problem detection and solving" . Many heWith the increasing interest in and availability of georeferenced health data comes the need for methods to properly analyze them, taking into account the spatial correlation of outcomes in nearby places. Recognition of spatial influences in statistical inference date back to some of the earliest developments of modern statistical methods leading, for example, to notions of randomized plot designs for agricultural field trials .Development of theoretical methods for spatially referenced data includes point process models ,14, spatApplication of spatial statistical methods is more common now that both GIS and spatial statistical software packages are widely available. While there are several texts focused on statistical methods for spatial health data -25, and We specifically focus on spatial and spatio-temporal statistical methods appropriate for observational human health data, not clinical trials or data from other types of designed experiments. A challenging but common problem with this type of data is the difficulty in obtaining accurate exposure and disease outcome data for the time and place most relevant to that disease. Health consequences are the result of a continuum of multiple and varied exposures which often occur over a long period of time and in various places. How can we capture this complex pattern of exposure over decades and, most important for the topic at hand, to what geographic location do we assign the exposure? A subgroup of meeting attendees discussed the problems of defining "place" and locating appropriate data in detail , offering no information on previous residences or workplace location(s). Accumulating and validating data required for analysis from these multiple sources usually takes longer than the analysis itself, where data validation plays an essential but time-consuming and often overlooked role.Of increasing concern in this field is protecting the privacy and confidentiality of the study subjects. While all researchers agree that this is important, it is often difficult to reconcile these needs with data needs for a proper analysis. In particular, spatial data analysis and mapping of results are often hampered by the lack of specific addresses. Data collection agencies and medical facilities are imposing increasingly strict requirements for data release and often only identify a place  to a broad administrative unit. For example, the recently enacted Health Insurance Portability and Accountability Act (HIPAA) often requires removal of geographic subdivisions smaller than the state, and the National Center for Health Statistics only releases death certificate data aggregated to the county level or for places with large populations. The reportable specificity of location is often not good enough to allow the analysis to answer research questions about the spatial patterns of the disease. Methods are currently being explored that would allow use of specific individual information in the analysis but would mask identifying characteristics in the results reported only at an aggregated level. In addition to such federal reporting restrictions, state and local governments may add additional regulatory requirements.).This said, such regulations need not prohibit spatial analysis of health data completely, rather they change the context within which such analyses may occur. For instance, mutually agreeable memoranda of understanding between analysts and agencies holding data often allow analysis of data with individual-level identifiers provided all reports include only aggregate results. Such memoranda also specify when, if ever, detailed maps of locations may be reported. As an example, the National Cancer Institute's Long Island Breast Cancer Study Project provides a protected environment (on site or remotely accessible) within which registered users may analyze (but not remove) sensitive georeferenced health data (for more information, see In addition to general concerns regarding the analysis of health data, the spatial data analysis of cancer data poses unique challenges. Most cancers develop over a period of 20 to 30 years and are a result of multiple exposures interacting with the individual's genetic susceptibility. Few Americans live in a single place for decades – migration presents the problem of which residential address to use for a case's location. Because latencies differ by cancer type and most likely by an individual's susceptibility, little guidance is available for this question. The rarity of cancer also causes a sparse data problem for analysis, both for detecting clusters in data with high spatial variability and for communication of results without violating confidentiality. John Snow's illustration of his theorized cause of cholera in London via a map of case residences was possible because of the large number of cases in a small geographic area with a single, precisely located exposure . The detDespite such concerns, important discoveries in cancer research do result from spatial data analysis. Although U.S. mortality data had been published in tabular form for many years, it wasn't until mortality rates were mapped in 1975 that spatial patterns emerged, such as the cluster of high oral cancer rates in southeastern states, later found to be due to smokeless tobacco use ,31. LateFinally, in order to ultimately improve public health, the results of the complex analyses of georeferenced cancer data must be disseminated to those in a position to take action, such as state epidemiologists and local cancer control specialists. This audience often needs to obtain statistical data quickly for rapid response to health problems and cannot be expected to have the technical expertise to understand the statistical detail underlying the methods. Statisticians must consider this audience and design maps and reports in such a way as to be easily accessible by them. A forthcoming article in the International Journal of Health Geographics by Bell, Hoskins, and Wartenberg Table  addresseIn closing, participants in the NCI workshop addressed a wide range of topics summarized in Tables


In the UK the health service is investing more than ever before in information technology (IT) and primary care nurses will have to work with computers. Information about patients will be almost exclusively held in electronic patient records; and much of the information about best practice is most readily accessible via computer terminals.To examine the influence of age and nursing profession on the level of computer use.A questionnaire was developed to examine: access, training received, confidence and use of IT. The survey was carried out in a Sussex Primary Care Trust, in the UK.The questionnaire was sent to 109 nurses with a 64% response rate. Most primary care nurses (89%) use their computer regularly at work: 100% of practice nurses daily, compared with 60% of district nurses and 59% of health visitors (p < 0.01). Access to IT was not significantly different between different age groups; but 91% of practice nurses had their own computer while many district nurses and health visitors had to share (p < 0.01). Nurses over 50 had received more training that their younger colleagues (p < 0.01); yet despite this, they lacked confidence and used computers less (p < 0.001). 96% of practice nurses were confident at in using computerised medical records, compared with 53% of district nurses and 44% of health visitors (p < 0.01.) One-to-one training and workshops were the preferred formats for training, with Internet based learning and printed manuals the least popular (p < 0.001).Using computers in the surgery has become the norm for primary care nurses. However, nurses over 50, working out in the community, lack the confidence and skill of their younger and practice based colleagues. In the UK its National Health Service (NHS) is investing in computerisation. Large contracts have been set that will run until 2013 to provide electronic patient records ,5) In adIn this environment primary care professionals will need the skills to read and enter data in computerised medical records, access guidelines and other information on-line, receive alerts about changes in practice via email and use computer as a learning resource. Nurses: practice nurse, district nurses, health visitors, and others, make up a substantial proportion of the primary care workforce. Previous studies have suggested that they have limited access and low levels of confidence and competence in using IT (Information Technology) -11. HoweA literature review was carried out of standard bibliographic data bases: CINAHL, British Nursing Index and Medline to identify any recent publications about use of IT or computerised medical record by practice or district nurses and health visitors. In addition the primary care electronic library (PCEL) , and infThe study took place in a single Primary Care Trust in Sussex, a mixed urban and rural county in southeast England. A Primary Care Trust is a geographically defined locality of approximately 1 – 200,000 people that commissions hospitals, general practitioners and other health providers to deliver comprehensive local NHS healthcare. A PCT can also provide care itself. Most community nurses are employed directly by their PCT, and this PCT is no exception.All the practices in this PCT are computerised with computers linked to the patient database located in all consulting and treatment rooms; and most offices. In common with nearly all UK general practice the practice is networked and the network is connected to the internet via a fixed ISDN-2  link. The ISDN (64 K link) actually connects the practices to the NHS own intranet (NHSnet) and via secure gateways to the internet . The NHSThis PCT has a predominantly female (97%) workforce with one-and-a-half times as many nurses in the over 40 age group compared with the national average for nursing . The Nur.A questionnaire was considered the most appropriate way to gather the information required. Two previous studies indicated that primary care nurses appeared willing to complete the questionnaires and provide rich data ,11. The The questions were based on those used in two earlier studies; with the clarity of questions was improved. Especially distinguishing between a clinical computer system (i.e. one that is used to enter data into an electronic patient record) from other "work" computer systems; and, between home and work use. The questionnaire was tested by a pilot group of nurses located in another PCT in a neighbouring county. The pilot group of six nurses were asked to complete the questionnaire on their own. They were then invited to join a discussion group – where the purpose of the study was explained. Then the questions were gone through one by one to identify any ambiguity. The nurses were also invited to suggested alternative or additional questions. They were given SB's contact details should anything come to mind later.Ethical approval was obtained from the local research ethics committee to conduct the study.Primary care nurses were identified using a list provided by the PCT. The questionnaire was sent out with a covering letter explaining the nature of the study. A courier addressed envelope was provided so the questionnaire could be returned without cost. A reminder and second questionnaire was sent out to non-responders.The questionnaire responses were coded and entered into SPSS  for analysis. The results were analysed by age bands and professional grouping as we wished to test whether either of these factors predetermined the level of computer use. Additional comments were entered into Microsoft Word, and the SPSS data from the questionnaire was placed alongside the relevant comments – they were flagged with the professional group and age group of their originator. Microsoft Excel was used to allow them to be sorted by age group and profession.To simplify the analysis only three age-groups were considered: age under 30, 30 to 39, 40 to 49, and 50 and over. There were no nurses under 30, and only one over 60. The nursing professions were divided into: District nurses, health visitors and midwives, practice nurses, and others. In this study qualified nurses who work in the community primarily supporting district nurses or health visitors were analysed as part of that nursing specialist group. The health visitors and community midwives were combined into the "health visitors" group. The term "primary care nurse" is used to embrace all qualified nurses included in this survey; "community nurse" refers to nurses who still do a proportion of their work in patients' homes, Figure There was a 67% (67/105) response rate to the questionnaires. There was some variation between nursing groups: the response from practice nurses was 73% (n = 22/30), for district nurses 51% (n = 17/33), for health visitors and midwives 67% (n = 19/28), and other nurses, community psychiatric nurses and managers 64% (9/14).Computers were readily available to primary care nurses. Over 90% of nurses had access to a personal computer (PC) at work, though for nearly half (45%) this was shared access; home access was even higher – 96%, with very few (12%) describing their access as shared. The remainder had access elsewhere: library, college etc. Not all the computers were connected to the Internet: 11% of those provided a PC at work and 6% at home could not go on line. After taking into account "other" access to the Internet only 3% of primary care nurses lacked access.The pattern of access to computers, the Internet, and receiving training changed with age. The younger nurses had more access to PCs and the Internet at work, while older nurses had higher levels of access at home, and had received more training. These trends are shown in Table 62% of nurses had received some training, although it was often superficial and run in house to provide basic knowledge of how to use the clinical computer system. Some learned from family members, colleagues or were self-taught, in the use of Microsoft Word and Excel. A few respondents received more formal training on the use of Internet for literature searching from a library, evening classes or as part of high education. Open question data shows that the training received are very diverse both in subjects and level of skills:"...two fifteen minute sessions in 1991 when we first got computers, a second when system was updated""...only one day. All other courses since have been at difficult times or locations""...very basic""...not sufficient though""...no formal courses, only shown by colleague when I was new in post""Protected time for PC training would be most valuable – so often this is hurriedly given during the odd 5-minute break rather than in a training session.""...adequate training is really necessary and protected time; 10–15 minutes between patients is not adequate."Respondents were asked to rate their preferred format for ICT training on a 4-point scale: from 0 for least desirable to 3 for highly desirable. The result showed that 'one-to-one' training and workshops were the preferred formats for training, printed manual the least Table .The preferred location for the training reflected the format of training requested Table . Most waConfidence with computers appeared to be age related, with younger nurses having higher levels of confidence across all the areas of competence than their more senior colleagues. However, only two of these trends, use of spread sheets and electronic patient records (EPR) were found to be statistically significant, see Table The raw data also suggested that there was a discernable trend in computer usage with age. 90% of nurses age 30 to 39 years used their workplace computer daily, 70% of those 40 to 49, and only 59.3% of those over 50. This trend was not significant, see Table Colleagues were the most used source of information across all ages and nursing professions. Older nurses tended to use books and journals from their personal collections, and to use libraries. However, the use of libraries, books and journals is quite low.Younger nurses seem to use journals at work or electronic resources more. Neither of these trends, shown in Table Nurses summarised their experiences in access information in a number of free text comments:"don't know where to look", "don't know website address","vast amounts of irrelevant articles under same headings","I am not as good as I should be with CIHNAL, Medline","information not found or not relevant some difficulty sometimes refining the search for specific information""lack of training/unable to access information I am looking for quickly enough – quicker to look in a book""like trying to get a glass of water from Niagara Falls"This study demonstrated that primary care nurses have high levels of access to IT and only a small minority have no access at all. Practice nurses, working within the surgery, all use the computerised medical record and nearly all feel confident to use it; over three-quarters have access to the Internet. However, community bases nurses in the second half of their careers despite receiving more training; have less access and are less confident about computers, especially use of the EPR, and prefer to use paper-based information resources. Across all ages and professions using libraries, reading journals and accessing on-line resources are minority activities. One to one learning in the workplace, and workshops were believed to be the best way to learn, with printed manuals and on-line tutorials of least value.The implication of this study is that community nursing needs more silver surfers. Silver surfers are those over 50 years who enjoy using the Internet. The primary care nurses over 50, despite more training, are still looking for information on paper; using their computers less at work; and less confident in computer use. Market strategies are making the over 50 s the fastest expanding group of Internet users this decade ,18; but The small sample size and single locality of this study are its principal weaknesses. The former prevented any more detailed sub-group analysis, as the groups became too small. A further reminder may have improved the response rate.This study shows that nurses have made enormous strides in acquiring IT skills and access to computers since earlier surveys -12; althFurther research is needed with a larger sample to see if some of the non-statistically significant trends seen in this data become significant in a larger sample drawn from a national sample. It is important for the NHS to understand what input is needed to raise the level of use of the EPR and online information; and how quickly this change can be achieved. What nurses believe to be most effective ways of changing behaviour, and promoting computer use needs to be tested.There is a consensus among primary care nurses of the sort of training they require to improve their computer use. A significant proportion of community nurses in the second half of their careers need this additional support if they are to achieve silver surfer status; and, have the skills needed to work in the new computerised NHS. Studies need to test whether providing the training wanted overcomes the barriers reported. Although primary care nurses have acquired more computer skills than previously reported there is no room for complacency – community nurses need the opportunity to develop into silver surfers.CPHVA Community Practitioners and Health Visitors Association, a representative body of community nurses.EPR Electronic patient record, synonymous with Computerised Medical Record.IT Information TechnologyNHS National Health Service. The UK's state funded health service.PCEL Primary Care electronic Library, an on-line information resource for primary care.PC personal computerPCT Primary Care Trust – organises and commissions care for a locality of about 100,000 population.RCN Royal College of NursingSNOMED CT Systematized Nomenclature of Medicine – Clinical TermsSPSS Statistical Package for Social Sciences – a computerised statistical package.The author(s) declare that they have no competing interests.All authors conceived the study, and contributed to all aspects of the paper. The major contributions of each author is as follows: TC Analysed the data for the study, SB Recruited the nurses, sent out and collated the questionnaire, SdeL helped develop the questionnaire used in a earlier study and wrote the initial draft of the paper.The pre-publication history for this paper can be accessed here:Community nurses access to and use of computers: questionnaireClick here for file


Microarray experiments are becoming a powerful tool for clinical diagnosis, as they have the potential to discover gene expression patterns that are characteristic for a particular disease. To date, this problem has received most attention in the context of cancer research, especially in tumor classification. Various feature selection methods and classifier design strategies also have been generally used and compared. However, most published articles on tumor classification have applied a certain technique to a certain dataset, and recently several researchers compared these techniques based on several public datasets. But, it has been verified that differently selected features reflect different aspects of the dataset and some selected features can obtain better solutions on some certain problems. At the same time, faced with a large amount of microarray data with little knowledge, it is difficult to find the intrinsic characteristics using traditional methods. In this paper, we attempt to introduce a combinational feature selection method in conjunction with ensemble neural networks to generally improve the accuracy and robustness of sample classification.We validate our new method on several recent publicly available datasets both with predictive accuracy of testing samples and through cross validation. Compared with the best performance of other current methods, remarkably improved results can be obtained using our new strategy on a wide range of different datasets.Thus, we conclude that our methods can obtain more information in microarray data to get more accurate classification and also can help to extract the latent marker genes of the diseases for better diagnosis and treatment. With the successful completion of the Human Genome Project (HGP), we are entering the post genomic era. Facing mass amounts of data, traditional biological experiments and data analysis techniques encounter great challenges. In this situation, cDNA microarrays and high-density oligonucleotide chips are novel biotechnologies as global (genome-wide or system-wide) experimental approaches that are effectively used in systematical analysis of large-scale genome data. In recent years, with its ability to measure simultaneously the activities and interactions of thousands of genes, microarray promises new insights into the mechanisms of living systems and is attracting more and more interest for solving scientific problems and in industrial applications. Meanwhile, further biological and medical research also promoted the development and application of microarray.Typical issues addressed by microarray experiments include two main aspects: finding co-regulated genes for classification based on different cell-type , stage-sMicroarray experiments usually generate large datasets with expression values for thousands of genes 2000~20 000) but not more than a few dozens samples (20~80). Thus, very accurate classification of tissue samples in such high-dimensional problems is difficult, but often crucial, for successful diagnosis and treatment. Several comprehensively comparative and improved methods have been proposed recently 00~20 000. In this, so we simplify this step and only use all datasets exactly as we found them in their transformed data.The flowchart of our method can be seen in Figure Due to the characteristic of small sample numbers in microarray data, in order to improve the accuracy, robustness and generalization of issue classification, we apply bootstrap mechanism to resample 100 iterations. During each iteration, we input the resample training data into three cooperative and competitive neural networks, and then by averaging their decisions, the neural network set can output their discrimination. From Figure After completing the 100 iterations, we can get 100 individual answers about the problem. In this situation, how to combine these answers into one more precise result is still a problem. Here, we simply use majority voting to combine the result and then give the ultimate solution about this classification problem. As noted above, here we adopt the soft-voting mechanism, that is, we can combine the confidence of each net. All the implementations of our framework were written in Matlab, using the hardware platform of a PC running 2.4 GHz.. We adopted their transformed data format for further research. All datasets we used can be reduced to three categories: binary class with testing samples, binary class without testing samples and multiple class problem. Here we classify samples into binary class with testing samples and without testing samples just according to the reference authors for each dataset. One important reason is that in this way we can easily compare our result with others based on the same training and testing sets. These datasets are shown in Table In this section, simple illustrations of the datasets we used in this paper for exploring the performance of our classification are given. The datasets in our paper have been downloaded from the following website: We use the three datasets below as the example of the first category, for which performance of our classification can be tested using the error ratio of testing samples.The training dataset consists of 38 bone marrow samples (27 ALL and 11 AML), with 7129 probes from 6817 human genes. Also, 34 samples testing data is provided, with 20 ALL and 14 AML.More information and raw data can be found in Golub et al. .The dataset can be reduced to the problem of classification between malignant pleural mesothelioma (MPM) and adenocarcinoma (ADCA) of the lung. The training set contains 32 tissue samples, which consists of 16 MPM and 16 ADCA and the testing samples are constitutive of 15 MPM and 134 ADCA. Each sample is described by 12533 genes. More information about this dataset can be found in Gordon et al. .For the prostate cancer dataset, detailed explanation and raw data is available in Singh et al. . This daAnother three recently popular datasets have been used as the representative of the second category. Using these kinds of datasets, we apply cross-validation to validate our classification performance.This dataset is used for discriminating distinct types of diffuse large B-cell lymphoma (DLBCL) using gene expression data. There are 47 samples, 24 of them are from "germinal canter B-like" group while the rest 23 are form "activated B-like" group and each sample can be described by 4026 genes. More detailed explanation can be found in Alizadeh et al. .. More information can be found in Petricoin et al. [The goal of this significant experiment is to identify proteomic patterns in serum that distinguish ovarian cancer from non-cancer. The proteomic spectra were generated by mass spectroscopy and the dataset provided here is 6-19-02, which includes 91 controls  and 162 ovarian cancers with 15154 molecular mass / charge (M/Z) identities. Here we use the transformed normalization data in The dataset Contains 62 samples collected from colon-cancer patients. Among them, 40 tumor biopsies are from tumors (labelled as "negative") and 22 normal (labelled as "positive") biopsies are from healthy parts of the colons of the same patients. Two thousand out of around 6500 genes were selected based on the confidence in the measured expression levels. Raw data and more information can be found in Alon et al. .Finally, we can generalize our method from binary class to multi-class problems. In this paper, we evaluate the performance using the dataset below.. More information can be seen in Armstrong et al. [This dataset contains training data consisting of 57 leukemia samples (20 ALL, 17 MLL and 20 AML) and testing data consisting of 4 ALL, 3 MLL and 8 AML samples. We adopted the transformed data from g et al. .First we primarily focus on the binary class problem. Because most of problems can be reduced to binary class problems, such as diseased vs. normal, survival vs. lethal, two opposite subtypes of some diseases and so on. Finally we generalize our classifier to multi-class application. In this paper, we evaluate the performance of different classification methods using predictive accuracy, which can be defined as:TN1,TN2,…,TNn respectively denote the correct classification numbers of the samples belonging to a corresponding class; totalnum represents total sample numbers.Here, For the first category of the datasets, we evaluate the performance of our classifiers using predictive accuracy of testing samples compared with the best performance of the current available methods. In this paper we use bagging to resample just as Tan and Gilbert , and we For the most popularly used AML-ALL leukemia dataset, to our knowledge, the best classifiers of this dataset can be found in -27, whicIn the same way, we trained our ensemble of neural networks using 32 training sets of lung cancer and then predicted the 149 separate testing sets still with 0 error number. And three (1:2) testing error numbers can be reached using methods by Li et al. , which iFor the third prostate cancer dataset, after training the classifier using 102 training sets, only one wrong classification can happen using our ensemble neural networks to predict the 34 separate testing samples. We did not find a more accurate classification result except for the bagged decision trees in  based onIn order to further validate the effectiveness of predictive accuracy, we also performed the leave-one-out cross-validation (LOOCV) respectively only on the above three training samples. We also obtained the 100% accuracy both on the AML-ALL leukemia dataset and the lung cancer dataset, which are the same results as using individual testing samples. At the same time, 96.08% accuracy can be got based on the prostate cancer dataset, which is a little lower than using individual testing samples. For the purpose of comparison, we also list these results in Table From the results of the above three testing datasets, we can also see that many different classifiers obtain the best results when they concern some certain dataset, but there is still no general best strategy for tumor classification problems based on a wide range of different datasets. Furthermore, from Figure Without separate testing samples, we cannot evaluate the performance of our classifiers with the predictive accuracy of testing samples in the same way as above. Many performance evaluation methods have been proposed, of which various cross validations are most popularly used, such as 3-fold cross validation, 10-fold cross validation, leave-one-out cross-validation (LOOCV), and others. Here, we used the leave-one-out cross validation (LOOCV) to evaluate the performance of ours based on these available datasets. For further comparison with recent published methods based on the same datasets, we also perform 10-fold cross validation just as they used in their research. In Table In the first data column of Table 2 feature selection heuristic and support vector machine (SVM). In our method, only 75 features in total were used, and 99.21% and 98.82% accuracy was obtained respectively by LOOCV and 10-fold cross validation. Thus, we think that our method is comparable to theirs to some extent.For the ovarian dataset we used, Liu et al.  reportedFor the colon tumor dataset, we found that 85.48% predictive accuracy is the best classification result obtained in Dettling et al. , where tFrom the results of the above three datasets, we can see that our method is better, or at least comparable to current other best methods. Also, we need to note that these named best methods can get the best results based on certain datasets but may get worse results based on other datasets ; however, general performance improvement can be obtained using our method.Finally, we generalize our method from binary class to multi-class problems. After minor adjustments to the corresponding parameters of our framework, we obtained 100% classification result of the above-mentioned multi-class dataset – MLL_Leukemia dataset, that is, 4 ALL, 3 MLL, 8 AML of 15 test data can be predicted correctly. Similarly, 100% accuracy also was obtained by Li et al. . In thisIn this paper, we introduce a combinational feature selection and ensemble neural network method for the classification of gene expression data. On a wide range of recently published datasets, our method performs better, or is at least comparable to, the current best methods of our knowledge. As a further test, we randomly selected genes of the same amount as the feature instead of any of the three individual selected features in our research and then used the ensemble neural networks based on these features for classification again. The apparently worse discrimination power can be seen in this strategy. Moreover, we also used the output of a unitary network based on all the same features as the ultimate classification result and the result was also worse than ours. Thus, we believe such remarkable performance improvements of our method are due to the fact that our combinational feature selection mechanism induced more useful information for discrimination, and the ensemble neural network framework improved the stability, robustness and generalization of learning.We performed simple majority voting mechanism to combine the individual networks produced by bagging and got a more accurate solution. The advantage of the ensemble is to reduce the variance, or instability of the neural network, and avoid the error surface of neural network training being trapped into local minima. The ensemble model tends to cancel the noise part as it varies among the ensemble members, and it tends to retain the fitting to the regularities of the data. In this paper, our ensemble neural network model has 100 members; However, further research is needed to determine how many members working together can reach the best performance.In this paper, we focused on classification problem, so we didn't give a detailed analysis about how the importance of each different gene we select and the interaction between them influenced the diseases, which is a very important issue for application and will be researched in our future work.Note that the only drawback of our approach is the problem of increasing computational complexity and the fact that it consumes a little more time than others. However, considering the lost caused by wrong prognosis or diagnosis of disease, we believe that the remarkable improvement in corresponding accuracy deserve these costs.By aggregating various information and ensemble neural networks, we reached a more accurate classification decision based on several datasets. We think that making full use of all available information will more clearly elucidate the latent mechanisms of many diseases. For example, we can combine various imaging techniques, such as CT, MRI, PET and others, which can detect the change of phenotype for the corresponding disease, with microarray data for further research. In this way, we can recognize the nature of various life phenomena both from macro and micro viewpoints. Also, we can retrieve the information of genes that are used in microarray, such as gene functions and gene locations. In this way, we can make use of prior knowledge combined with the microarray data for further research.Feature selection is one of the most important issues in classification, which is a transformation process of observations in order to obtain the best pathway for getting to the optimal solution. At the same time, it can reduce the complexity of the data to make it more comprehensible. It is particularly relevant for microarray datasets with thousands of features because it has been reported that many diseases, especially tumors, have never been caused by a single gene mutation but are the result of a series of gene changes. Such genes are highly relevant to the studied phenomena of diseases. On the other hand, the expression levels of many other genes may be irrelevant to the distinction between tissue classes. We can say that the extraction and selection of features determine the ultimate performance of classifiers. Both for cost and for biological insight, making full use of the most informative genes and finding small feature sets with high classification accuracy are very essential. At the same time, highly informative genes that are part of known biochemical pathways give insights into the processes that underlie the differences between classes, and those of unknown function suggest new research directions.Some classifiers, such as trees, perform automatic feature selection and are relatively insensitive to the variable selection scheme, but most classifiers need to perform feature selection first. So far, various feature selection schemes have been used in microarray data analysis, such as the most popular method of selecting the top-ranked genes based on various different scores  ,22,25,29Just as we alleged above, in such a high-dimension space, finding accurate and significant features (genes) is very essential for classification, for cost savings and for biological insights. However, it has been verified that differently selected features reflect different aspects of the dataset and some selected features can obtain better solutions on certain problems. This is because one feature selection mechanism corresponds to one different artificial hypothesis, but which hypothesis is most near to the true hypothesis on a special problem is unknown to us. Here, we propose combining the above mentioned several feature selection methods to reflect different profiles of samples in order to obtain more useful information for classification and to produce a good approximation to true hypothesis by averaging the different hypotheses. In this paper, we select features using wilcxon's ranksum test  to get tAn Artificial Neural Network (ANN) is an information-processing paradigm that is modeled on biological nervous system, which is composed of a large number of highly interconnected processing elements (neurons) working in unison to solve specific problems. In fact, since the basic model was proposed, various improved algorithms and theories have already been successfully applied in many fields. Because neural networks are best at identifying patterns or trends in a large amount of data with little theory, they are well suited for prediction or forecasting needs. That's just the case for microarray data. However, instability and little intrinsic knowledge of neural networks are obstacles to its further generalized application in some specific problems. Here we ensemble multiple networks in an attempt to solve this problem to some extent.Since multi-net systems were introduced by Sharkey in 1996 , the comGenerally speaking, in neural networks ensemble, two problems need to be resolved: how to generate the individual network and how to combine them together. Using bootstrap or boosting resample mechanism to obtain the individual network is the most popular method to solve the first problem. Bootstrap is the most popular resample mechanisam of sampling with replacement, therefore some observations are duplicated and some are omitted. Boosting means to boost a "weak" learning algorithm into a "strong" learning algorithm. Their differences are that using bootstrap can resample uniformly and can get the individual network immediately, but boosting weights every sample in each iteration and must generate the individuals in sequence. Several recent published papers claimed that adaboost-the basic boosting algorithm is not fit to microarray data analysis ,26, and Considering the complexity and noise of microarray data, it's very difficult to get a perfect solution using a unitary neural network based on some certain selected features. At the same time, accurately extracting and selecting the most informative genes is also very difficult using individual available methods. Thus, in this paper we attempt to combine multiple modes of information available from gene expression data using neural networks ensemble to get a better solution. We presented a cooperative and competitive neural network system that each of nets has the same architecture and topology, and each can respectively learn to classify a set of patterns based on partial information of the patterns and then by combining their classification results we can get a more precise result. The detailed framework of combing various features and neural networks ensemble and its implementation methods are discussed below.BL carried out the design of the method and performed the related analysis. QC participated in discussions of algorithms and manuscript preparation. TJ and SM instructed the whole study. All authors read and approved the final manuscript.


This article traces the career of Celso-Ramon Garcia (1922–2004), noted physician, educator, and internationally renowned pioneer in the field of reproductive endocrinology. His work helped to formulate oral contraceptives used by millions of women throughout the world. Garcia's research collaborators included Gregory Pincus and John Rock, who together finalized the landmark clinical data needed to secure initial FDA approval for "the pill" in 1960. In addition to Garcia's monumental work in contraceptive endocrinology, his scholarly interests encompassed physiology of the menopause, minimally invasive reproductive surgery, as well as psychological aspects of infertility. Closely identified with the University of Pennsylvania, Garcia was instrumental in establishing the first formal clinical program in reproductive biology and influenced countless young scientists whose training he supervised and mentored. His distinguished career was emblematic of the best of the medical profession, characterized by compassion, intellect, and a sincere desire to help others. Our manuscript outlines Garcia's wide range of interests, acknowledges his superior fund of knowledge, and honors his humanitarian spirit – all of which contributed to an impressive legacy of medical discoveries. The impact of Prof. Garcia's work will continue to be felt for many years. Celso-Ramon Garcia Figure , who depGarcia was raised speaking Spanish, but his English diction and elocution were so honed by his teachers in grammar and high school that hardly a trace of accent was left to be detected by the Boston Brahmans he would later encounter. Along the way his interests took seemingly improbable turns, yet there was a clear rationale that was beyond the imagination of the average physician. Indeed, his work presaged several important developments in the field of reproductive medicine. Garcia began his career with an interest in obstetrics, but ended up conducting clinical research on contraception that on more than one occasion was nominated for the Nobel Prize in Medicine or Physiology. He was revered as a meticulous abdominal infertility surgeon, yet he embraced the fledgling field of endoscopy and endoscopic surgery. While being celebrated for his surgical technique, he promoted research into the psychological aspects of infertility. Towards the end of his career he became interested in sexuality in the menopause, bringing his career through the full cycle from obstetrics, to family planning, to infertility to reproductive aging. His accomplishments in each of these spheres were well recognized by his peers who chose him President of the American Association of Planned Parenthood Physicians, Chairman of the Board of Directors of Planned Parenthood of America, Founding President of the Society of Reproductive Surgeons, and President of the American Society of Reproductive Medicine and bestowed upon him the Scientific Leadership Award from the Global Alliance for Women's Health, a non-governmental organization of the United Nations.Garcia's course to greatness was based on a diverse set of skills that he used creatively throughout his career. He majored in chemistry at Queens College where he revealed a remarkable aptitude for analysis of organic compounds. During his undergraduate studies, Garcia served as a teaching fellow in chemistry at New York University and authored two papers on qualitative and quantitative analysis. These are the earliest public manifestations of his passion for rigor and precision, which subsequently guided his clinical practice and his scholarship. After receiving his baccalaureate degree he entered medical school at the Long Island College of Medicine (now SUNY-Downstate) and graduated in 1945. He completed a rotating internship at the Norwegian Hospital in Brooklyn and then served two years in the U.S. Army Medical Corps in Fairbanks Alaska and then the Phoenixville General Army Hospital outside of Philadelphia. Garcia started a residency in pathology at Cumberland Hospital in Brooklyn where he was introduced to Sam Lubin, who became his friend and mentor. Lubin's work focused on uterine pathology and contractility and this sparked Garcia's interest in obstetrics and gynecology. With Lubin, Garcia would later publish papers on the effects of meperidine on labor.After a year of pathology residency and a year as a research fellow, Garcia transferred to the obstetrics and gynecology program at Cumberland Hospital. With a pathologist's keen eye and an appreciation of tissue biology/healing, Garcia was well-equipped to excel in the art of reproductive surgery. In the operating rooms at Cumberland Hospital Garcia met Shirley Stoddard, the newly appointed Director of Surgical Nursing; she was to become his wife and lifelong companion. After completing residency, Garcia explored openings in academia but few opportunities were available. One exception was an advertisement for assistant professor of obstetrics and gynecology at the newly established medical school at University of Puerto Rico, for which Garcia applied and was accepted in 1953.Garcia's research during residency, mentored by Lubin, had focused on obstetrical issues – something that he could have easily developed as a junior faculty member at the University of Puerto Rico. However, Garcia was about to embark on the first of several sharp turns that would lead him into seemingly improbable arenas. His arrival in Puerto Rico fortuitously coincided with the time for the first large scale clinical trials of the oral contraceptive pill. Within a year of Garcia's arrival, Gregory Pincus  was in Puerto Rico looking for sites to conduct these trials. It was there that Pincus was first introduced to Garcia. Pincus had an uncanny ability to sense talent; he always surrounded himself with the brightest people. Pincus, undoubtedly impressed by Garcia's fund of knowledge in organic chemistry, training in pathology, and of course, the fact that he was bilingual in addition to his training in obstetrics and gynecology, promptly recruited Garcia to oversee his important research program in Puerto Rico.Pincus would subsequently introduce Garcia to John Rock, who arranged for Garcia to be named Sydney Graves Fellow in infertility and gynecology at the Free Hospital for Women in Brookline, Massachusetts. There, Garcia continued to work on oral contraceptives but also pursued clinical work in infertility. Although the Graves fellowship no longer exists, contemporary reproductive medicine was developed by its incumbents. Rock had an interesting manner of selecting these fellows. He usually interviewed candidates over breakfast in his hotel room at the annual meeting of the American College of Obstetricians and Gynecologists. He could identify the smart ones within a minute, and had little patience for individuals who were not well-spoken. The diction and elocution drilling Garcia had endured and his insistence on precision brought him through with flying colors.Garcia quickly became indispensable to Rock. Garcia moved to Boston, where he held assistant and instructor faculty posts at Harvard Medical School. During this time, Garcia also commuted to Puerto Rico to manage the oral contraceptive trials. Rock had a thriving infertility practice and Garcia did much of the surgery. Garcia's contributions to Rock's clinical practice were, however, underappreciated and when it came time for Garcia to be proposed for a full-time position at the Free Hospital for Women, the job did not materialize. This scenario is not unfamiliar to practitioners in our field, and it elicited the obvious reaction: Rock and Garcia pulled up stakes and took their hugely lucrative practice to the Faulkner Hospital in Boston. But after a short interval, the Rock/Garcia partnership was persuaded to return to the Free Hospital where an appropriate appointment was arranged for Garcia. Mrs. Stanley McCormick, a benefactor of remarkable vision who had provided initial funds to Pincus and colleagues at the Worcester Foundation to begin studies on oral contraceptive steroids, helped establish the Rock Reproduction Study Center at the Free Hospital campus, greatly facilitating research and training.By 1960, Pincus had recruited Garcia to the Worcester Foundation as a senior scientist and director of the training program in Physiology of Reproduction. Sponsored by grants from the Ford Foundation and National Institutes of Health, this was the first training program of its kind. The discipline of reproductive biology as we know it today had its roots in the Worcester program. Although Garcia was appointed chief of the infertility clinic at Massachusetts General Hospital in 1962, he continued to visit Puerto Rico to perform follow-up examinations on women who had participated in the oral contraceptive trials in gratitude for their role and with genuine concern for potential untoward long-term effects. That was a personal mission, not a mandated component of the study protocol. This post-trial surveillance helped identify rare side effects that ultimately led to product reformulation and lowering of the steroid content of oral contraceptives. Garcia subsequently carried out groundbreaking studies on patient acceptance of oral contraceptives, recognizing that future developments in family planning must address the social and demographic aspects, including cultural differences in preferences for methods.th century. This is in good part Garcia's legacy [The technologies that gave men and women the ability to control their own reproduction through contraception and infertility treatments are arguably among the most important medical advances of the 20s legacy -3. WhileImportant medical advances like "the pill" or IVF do not come without controversy, societal baggage, and rhetoric. The pill's creators fully understood these implications, and knew such peripheral distractions would take away from the significance of their work. Consequently, there was no phone call from Sweden despite several nominations to the Nobel committee. But it is best to look back on the history of the development of the oral contraceptive with awe and wonder. Indeed, the commercial availability of oral contraceptives was also important because it established a formal approval and regulatory framework for pharmacologic agents of an entirely new class: drugs designed to be used by healthy people for long periods of time. It provided a clear understanding of how post-marketing surveillance can lead to product reformulations that reduce side effects without reducing efficacy. It yielded an understanding of how sociology and demography must be incorporated into the development of pharmaceuticals and the practice of medicine.Garcia maintained his connections with Rock and Rock's family of protégés, one of whom (L.M.) later lured him and other Rock/Pincus trainees, Edward Wallach and John V. Kelly, to the Department of Obstetrics and Gynecology at the University of Pennsylvania. Garcia spent the rest of his career at Penn developing the specialty of reproductive surgery, particularly microsurgical approaches. Here again came the totally un-expected. Garcia was at his best when confronted with the most challenging open abdominal procedures, which always ended with the pelvis in a pristine state, not a drop of blood, all surfaces meticulously re-peritonealized, no risk of inflammation or adhesion. Garcia's novel regimen of intraperitoneal corticoids and antihistamines (an early anti-adhesion therapy) was merely icing on the cake. The two-stage tuboplasty procedure was celebrated both locally and internationally. Yet this master of the open abdomen quickly recognized the potential of minimally invasive surgery, establishing one of the first endoscopic surgery programs in USA after a brief sabbatical with his colleague, Professor Kurt Semm in Kiel, Germany.During his career, Garcia authored numerous textbook chapters on surgery of the ovaries, tubes and uterus that guided a generation of gynecologists in reconstructive surgery. He was also an early advocate of laser and electrosurgical methods, as well as microsurgical techniques designed to minimize tissue trauma. When Garcia's residents and fellows under-performed, he could not mask his displeasure, yet when they excelled he could not hide his joy. His concern for the outcomes of a rapidly evolving medical specialty led him to develop one of the first electronic infertility data registries .In 1968, Garcia made yet another change in tack, developing an interest in psychological aspects of infertility. While this may have puzzled colleagues who knew Garcia only by reputation as a surgeon, those of us who worked with him knew well his holistic commitment to medicine and his devotion – unprecedented even in the current era – to his patients and their families. He had a profound understanding of the human frailties that emerge during the rigors of infertility treatment. One of us (J.F.S.) assisted Garcia in the care of an attorney's wife who underwent a two-stage tuboplasty. The husband demanded to be present during all medical examinations of his wife, he tape recorded all sessions, and refused to pay for Garcia's services, despite the fact that she conceived within three months after the hoods were removed from her reconstructed tubes. Garcia did not press the attorney for payment – he knew that the treatment course was exceptionally arduous with considerable post-surgery discomfort. When this same lawyer called eight months later asking if Garcia would serve as an expert witness in a plaintiff's medical malpractice case, Garcia thanked him for his call and politely said his schedule would not permit it, never mentioning the unpaid bill. He forgave, but he did not forget.Later in his career, Garcia and his colleagues undertook studies on the menopause that presaged the current interest in female sexual dysfunction. In retrospect, this was a logical extension of his long-standing interest in the social and psychological aspects first of contraception and family planning, and later of infertility. Garcia published on perimenopausal sexuality, and reported the first studies the relationship between sex steroid levels and sexual behavior. Garcia also recognized the need for multidisciplinary collaboration in the care of women, particularly in the post-reproductive years, and co-authored an early self-help manual for menopausal women . The WomGarcia gracefully retired from practice to devote time to his wife and family. This life transition, seamlessly executed, surprised many who were familiar with Garcia's intensity. But there should have been no surprise. Garcia demanded the highest technical standards from residents and fellows in the operating room, the same standards that he held for himself. He would not carry on if he could not perform to those standards, and he would not neglect his wife who needed increasing medical attention.Throughout his professional life, Celso-Ramon Garcia was a major force driving innovation in clinical care, and ultimately the evolution of the field of reproductive medicine. Let all who follow in the arena of reproductive endocrinology honor his contributions, appreciate the unique attributes that made him a great physician and educator, and gain insight from a truly remarkable career.The author(s) declare that they have no competing interests.JLS and LM contributed equally to this work and its revisions.


Increasing evidence elucidating the pathogenic mechanisms of ulcerative colitis (UC) has accumulated and the disease is widely assumed to be the consequence of genetic susceptibility and an abnormal immune response to commensal bacteria. However evidence regarding an infectious etiology in UC remains elusive.Clostridium difficile infection and accompanying fever, vomiting, bloody diarrhea, and arthritis. Colonic biopsy revealed a histopathology suggestive of UC. Antibiotic treatment eliminated detectable levels of enteric pathogens but did not abate symptoms. Resolution of symptoms was procurable with oral prednisone, but tapering of corticosteroids was only achievable in combination therapy with vancomycin and metronidazole.We report a provocative case of UC with profound rheumatologic involvement directly preceded by C. difficile and the like in patients with UC.An infectious pathogen may have both precipitated and exacerbated autoimmune disease attributes in UC, symptoms of which could be resolved only with a combination of corticosteroids, vancomycin and metronidazole. This may warrant the need for more perceptive scrutiny of  Clostridium difficile infection may be a compounding factor in ulcerative colitis (UC) with data suggestive of a role in disease exacerbation or initiation by the organism in a subset of patients [Clostridium difficile. Herein, we report a case of steroid-resistant UC with profound rheumatologic involvement immediately preceded by C. difficile infection in which remission of UC symptoms was twice achieved in response to adjunct metronidazole and vancomycin therapy. The patient remained symptom-free without supportive therapy for a four-year period.patients . Adjunctpatients ,3. Howevpatients -6 suggespatients -9, thereA 32-year-old white male presented with nausea, vomiting, crampy lower abdominal pain and diarrhea. His initial lab values were hemoglobin 15.6 g/dl (14–18 g/dl), hematocrit 45.8% (40–54%), sodium 131 mEq/l (135–146 mEq/l), potassium 3.9 mEq/l (3.5–5.5 mEq/l), bicarbonate 24 mEq/l (22–24 mEq/l), chloride 91 mEq/l (95–112 mEq/l), BUN 37 mg/dl (7–25 mg/dl), serum creatinine 1.6 mg/dl (0.7–1.4 mg/dl), and normal liver function tests. He was treated with intravenous fluids, Ciprofloxacin and anti-emetics, to which the diarrhea resolved in 2–3 days. After one month he had recurrent watery diarrhea accompanied with rectal pain, bleeding and vomiting. He also developed fever of 101.6 F.Clostridium difficile cytotoxin, and depicted many WBCs (neutrophils 99% and eosinophils 1%), suggesting an infectious etiology. Stool sample was negative for giardia lamblia, salmonella, shigella, campylobacter, aeromonas and plesiomonas. There was no predominance of staphylococcus, yeast, or pseudomonas.The physical exam was remarkable for decreased bowel sounds and moderate lower abdominal tenderness. There were no masses or organomegalies palpable. The rectal exam was remarkable for tenderness and heme-positive mucus. The remainder of the physical exam was uneventful. Stool samples from the initial visit were positive for The patient was initially started on Ciprofloxacin (500 mg bid.) for 3 days without symptom resolution. Metronidazole was added (250 mg tid.), but intolerance of the patient to metronidazole with exacerbations of nausea prompted the replacement to Vancomycin (250 mg qid) in conjunction with Ciprofloxacin for 7 days. During this initial treatment period, the patient characteristically developed migratory polyarticular joint pain and swelling involving the shoulders, elbows, fingers, hips and knees. Lack of response to antibiotics suggested an autoimmune disease as a contributing factor.Clostridium difficile antigen, confirming an autoimmune component to disease pathology in this patient. Prednisone was increased to 30 mg per day at which point gastrointestinal symptoms resolved but joint swelling and pain continued. Over the course of 3 months prednisone tapering was attempted three additional times, with each resulting in symptom recurrence.At this time a sigmoidoscopy and pinch biopsy revealed diffuse acute and chronic inflammation with cryptitis, mucin depletion, and glandular foreshortening and branching suggestive of UC Fig. . The patAfter 3 months of treatment, the patient experienced an acute dental abscess and was prescribed a combination vancomycin (250 mg tid) and metronidazole (250 mg tid). During this period, the patient was able to gradually reduce prednisone dosage without symptom recurrence. Antibiotics were continued for 30 days during which time prednisone was tapered completely. Symptoms did not recur and the patient remained symptom-free without further therapeutic intervention for 4.5 years.Clostridium difficile positive by culture and cytotoxin. Once again the patient was treated with 60 mg daily of prednisone and metronidazole (250 mg tid). Symptoms did not resolve, and vancomycin (125 mg tid) was added, resulting in a significant improvement in symptoms. After 2 weeks of combined therapy metronidazole treatment was stopped and symptoms recurred within a week. Metronidazole was once again added, leading to resolution of symptoms. Combined antibiotic and steroid therapy continued for 2 months during which time prednisone was tapered completely and a second remission of UC symptoms was induced.After this prolonged period of remission of UC symptoms, a second episode similar to the first occurred. The patient once again presented with bloody diarrhea, nausea, vomiting, and joint pain. Stool cultures were C. difficile infection. While various therapeutic regimens of prednisone, vancomycin and metronidazole preceded a negative C. difficile toxin test, remission of UC symptoms could only be induced by the combination of these three medications. C. difficile toxin tests have shown varying efficacy in diagnosing pseudomembranous colitis. It has been demonstrated that toxin-negative patients with C. difficile positive stool culture may still have symptomatic pseudomembranous colitis [C. difficile infection cannot be detected by the cytotoxin assay and suggested that the organism has the ability to vary toxin production [The patient was remarkable in several regards. The onset of UC and spondyloarthropathy coincided with  colitis . Lynch e colitis . Likewisoduction -16. The C. difficile related diarrhea, it is reasonable to assume that the organism may exist undetectably in a symptomatic patient, but especially in a patient with inflammatory bowel disease (IBD). The elusiveness of the bacterium in diagnosis of C. difficile infected IBD patients was confirmed in 2001 by Markowitz et al. They demonstrated that single-toxin assays failed to identify C. difficile infection in approximately 40% of pediatric IBD patients and in a case report where a toxin A positive, toxin B negative variant was detected [C. difficile infection than in healthy controls [C. difficile infection.Since toxin-negative patients have presented with detected ,17. It hcontrols . When coIBD has been shown to be more prevalent in industrialized nations, where antibiotic treatment has its longest history . The incH. pylori and its recently discovered tendency to adhere to glycoconjugates expressed in inflamed gastric mucosa [C. difficile could similarly bind to inflamed colonic tissue in this subclass of UC patients. Further research in this area could yield valuable new data.This case is especially interesting considering c mucosa . In lighThe potential utility of combined therapy is further suggested by the fact that antimicrobial resistance among C. difficile strains to metronidazole and with intermediate resistance to vancomycin is emerging in countries like Hong Kong (where one of 100 C. difficile isolates was resistant to metronidazole) and Spain  -24 It isC. difficile toxin-positive refractory autoimmune symptomatologic UC as opposed to the use of a single agent.Recurrent relapses could only be suppressed in this patient by the combination therapy of corticosteroids, metronidazole and vancomycin. Our observations suggest that this combination therapy may be effective to confront and induce remissions of UC symptoms in patients with C. difficile infection commonly results from the use of broad-spectrum antibiotics like quinolones. The frequent use of these antibiotics in treating IBD suggests that these patients could develop C. difficile infection during treatment, thereby exacerbating symptoms and preventing remission of UC symptoms. It may therefore be practical to probe for C. difficile infections more meticulously in patients with IBD.An opportunistic The author(s) declare they have no competing interests.All authors contributed equally to this work. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Anopheles sp. mosquitoes are a promising strategy for urban areas. However, a precise knowledge of the geographic location and potentially of ecological characteristics of breeding sites is of major importance for such interventions.By 2030, more than 50% of the African population will live in urban areas. Controlling malaria reduces the disease burden and further improves economic development. As a complement to treated nets and prompt access to treatment, measures targeted against the larval stage of 2 of central Dar es Salaam, the biggest city of Tanzania, were systematically searched for open mosquito breeding sites. Ecologic parameters, mosquito larvae density and geographic location were recorded for each site. Logistic regression analysis was used to determine the key ecological factors explaining the different densities of mosquito larvae.In total 151 kmAnopheles sp. larvae density. The probability of Anopheles sp. larvae to be present was reduced when water was identified as "turbid". Small breeding sites were more commonly colonized by Anopheles sp. larvae. Further, Anopheles gambiae s.l. larvae were found in highly organically polluted habitats.A total of 405 potential open breeding sites were examined. Large drains, swamps and puddles were associated with no or low Anopheles sp. larvae could not be identified in this setting. Hence, every stagnant open water body, including very polluted ones, have to be considered as potential malaria vector breeding sites.Clear ecological characteristics of the breeding requirements of  Anopheles sp. mosquito larvae, but it also raises the economic status of the population, allowing improved malaria protection [Urbanization is progressing fast worldwide. By the year 2030, more than 50% of the African population will live in urban areas . It is aotection .Malaria is not only a personal health problem, but also an intolerable economic burden . InvestmIn urban areas, relatively few breeding sites are found per unit population . In addiAnopheles sp. breeding sites are of major importance. Computerized maps have proven to be useful for this purpose, helping to understand malarial epidemiology and guiding interventions [Every campaign to reduce malaria transmission aims to be as effective as possible given existing resources. Malaria control methods are affected by many setting-specific factors such as endemicity, vector species and behaviour, seasonality, disease patterns, health service factors and more. Since these factors are not distributed equally in space, accurate and timely information is required before malaria control can be planned and resources allocated properly. With regard to transmission reduction, attention must be paid to the areas of greatest vector abundance and precise risk maps of ventions -18.Anopheles sp. mosquitoes adapt to new breeding sites created by urbanization, and hence their ecology might differ from rural settings [on Anopheles sp. mosquito larvae ecology have been conducted in rural settings [Besides geographic location, knowledge of ecological features of mosquito breeding sites is a potential key element for implementing efficient and effective larvae control measures. Such measures have been shown to be an important tool to reduce malaria endemicity ,20. In asettings . Most Afsettings ,22, and Anopheles sp. and Culicine larvae in open breeding sites in Dar es Salaam, Tanzania, were examined during an extended dry period and correlations between biological and physicochemical environmental factors and the abundance of mosquito larvae were determined. This work was carried out in order to collect baseline information for a larval control-based mosquito abatement programme implemented by the three municipalities of Dar es Salaam.The spatial distribution of 2. The present study area covers 151 km2. in the central area of the city  was less than half the long-term average from 1961–1990 (576 mm) . FurtherA. gambiae s.l. (gambiae s. s., arabiensis, merus) and A. funestus, larvae collected from the productive breeding sites were raised and hatched in the laboratory so that the adults could be identified morphologically by light microscopy using the key of Gillies and De Meillon [To distinguish between  Meillon .Anopheles gambiae s.l. were not further identified to sibling species by PCR because such a methodology is not practical, affordable or particularly useful in the context of routine operations of a sustainable mosquito abatement program in Dar es Salaam.©, Garmin International Inc., Olathe, US). In addition, 10 year-old breeding site maps from the Japan International Cooperation Agency (JICA) were consulted [Each ward within the study area was searched for open water bodies. To locate areas of potential breeding sites, the search was lead by municipal malaria experts with a good knowledge of the area. Furthermore, one-year-old geo-referenced digital aerial pictures  were examined visually on the computer and all suspected open water areas were checked on the ground by extracting coordinates from the digital picture and locating the area in the field using a global positioning system (GPS)  density in the same sites.The same definition was also used to characterize Culicine mosquitoes  two logistic regression models were fitted: one model was used to determine the key factors influencing whether larvae were present or absent, and one model was used to determine the key factors influencing whether larvae were present at low or high density. For the second model only data from sites that contained at least one larva were used. All measured parameters were included in the logistic models. The three categories "stream", "foot/hoofprint" and "tyre track" were included in the baseline category "other" as they had only four or less records. No "artificial containers" or "rockpools" were found, consequently these categories are not presented in the analysis. Salinity, temperature and pH were measured with an electronic device (HANNA© HI 98130 Combo pH&EC, Hanna Instruments Inc., Kehl am Rhein, Germany). Conductivity measured in milliSiemens/cm was the indicator to measure salinity. The continuous variables "salinity" "temperature" and "pH" were categorized into quartiles for ease of interpretation. As this study is focused on Anopheles sp. larvae, only the significant parameters are presented for the Culicine models. To reduce the risk of α errors given the large number of variables that where investigated, only variables with p-values below 0.01, or those showing a consistent trend in both regression models with p-values below 0.05, were considered in the discussion.Statistical analysis was carried out with SPSS V 11.0 (SPSS Inc., Chicago, US). Logistic regression analysis was used to determine the importance of key factors for explaining the different mosquito larvae densities. For each group of mosquitoes This study did not involve human subjects and permission was obtained from the National Institute of Medical Research in Tanzania (NIMR/HQ/R.8a/Vol.lX/l 12, No. 2003-110-CC-2003-63).Anopheles sp. and 57% were productive for Culicine mosquitoes. From all 150 adult mosquitoes reared from the larvae samples, only A. gambiae s.l. could be found. In one productive breeding site salinity was above 1%, suggesting that these larvae were Anopheles merus [Anopheles sp. were made up of 69% low larvae density sites and 31% high larvae density sites. Of the 186 sites productive for Culicine larvae 23% had low larvae density and 77% had high larvae density. Breeding sites of Anopheles sp. and Culicines and their productivity are shown in Figures A total of 405 potential mosquito breeding sites were examined and mapped. 19% were dry at the time of visit. Of the 327 sites that contained water, 62% were productive for es merus . The 202The frequencies of the different parameters are shown in Table Presence/absence of Anopheles sp. larvae . As Darg Figure . FurtherThis study represents a snapshot of a highly dynamic system and presents the first results of a larger malaria control program. More intensive longitudinal studies are currently being undertaken to complement these results.A. gambiae s.s., A. arabiensis and A. funestus survive during the dry period in discreet habitats, making them an easier target for control interventions [The study was implemented during an exceptionally dry year and, although the study took place during the rainy season, it actually rained very little . As a reventions . Dry seaventions .Very small breeding sites could not be detected by aerial pictures and were, therefore, largely excluded from the study. This could result in the omission of factors potentially important for mosquito control interventions. Further, mosquitoes do not necessarily lay eggs every day in each potential breeding site, thus the reason why a site did not contain larvae could simply be because no eggs were deposited within the last week.Anopheles sp. larvae productivity. But within a city, the influence of humans can never be neglected. The pollution from waste such as oil, soap or industrial by-products is important in potential breeding sites in Dar es Salaam. Larvae may have been absent due to pollution by elements that could not be detected in the frame of this study.One of the key objectives of the study was to detect ecological factors determining Anopheles sp. mosquitoes were not classified down to species level. The goal of the study was to characterize important breeding sites of Anopheles sp. mosquitoes and, consequently, potential foci of malaria transmission, regardless of the species. This is because in the context of sustainable operations in a routine mosquito abatement programme, municipal staff cannot be expected to identify all Anopheles larvae samples to species level without rendering sampling procedures prohibitively laborious and expensive. To achieve a satisfactory impact, exhaustive targeting of all potential vector species is necessary anyway. Furthermore, community acceptance of vector control programmes in Dar es Salaam has been shown to require suppression of all mosquito species, rather than only malaria vectors [ vectors .Anopheles sp. and Culicine larvae seems to be influenced differentially by ecological parameters, as none of them had a significant effect in all four regression models. Only one factor influenced the presence or absence of both Anopheles sp. and Culicine larvae. In turbid breeding sites Culicine larvae were much more likely to be present, whereas Anopheles sp. larvae were much more likely to be absent. Bates [Anopheles sp. breeding in rather clear water, but Gimnig et al. [A. gambiae s.l. larvae densities with increasing turbidity. Robert et al. [A. ambiensis breeding in wells in urban Dakar. A study by Ye-Ebiyo et al. [A. arabiensis was favoured in moderately turbid water, while excessive turbidity limited the production of larvae. The proximity to flowering maize with pollen as food source compensated for the development failure induced by excessive turbidity. Clearly, the simple definition of "turbidity" might not be precise enough. Water which is turbid from particles not edible for Anopheles sp. larvae could disfavour the production of larvae, while water turbid from food particles represents a very suitable habitat. The preference of Culicine mosquitoes for turbid water is coherent with their known breeding site preferences, as they breed successfully in rather polluted environments such as blocked drains and septic tanks [The abundance of t. Bates  supportsg et al.  found int et al.  found a o et al.  found thic tanks ,37.Anopheles sp. were more likely to be present than in larger habitats. This is consistent with the general description of breeding sites for A. gambiae s.l., but not with those for A. funestus [In small breeding sites with diameters less than 1 m, funestus ,36. HoweAnopheles larvae were less likely to be present in swamps, and when so, they were found in low densities. This finding matches the known preference of A. gambiae s.l. for temporary sites [Anopheles sp., the importance of these habitats should not be underestimated because of their great size and their role in supplying water for irrigation ditches, rice fields and various agricultural activities.ry sites ,38. EvenA. gambiae s.l. larvae, and when Anopheles sp. larvae were present there, they were likely to be present in low densities. This finding is not consistent with established habitat descriptions for Anopheles sp. larvae ["Puddle" as a type of breeding site was not favoured by . larvae ,39.Anopheles sp. densities. This result agrees with findings by Yamagata [Anopheles sp. breeding [Large drains were likely to have low Yamagata . Large dbreeding ,28,38.A pH value of less than 7.3 favoured high Culicine densities and a value of less than 7.6 favoured their presence. These results showed clearly that Culicine larvae favoured a pH-neutral environment.A. gambiae s.l. was found in a sewage pond (Figure Anopheles sp. breeding requirements in urban settings. In Lahore, Pakistan, Anopheles sp. mosquitoes were found in the waste water system [A. gambiae s.l. adapted to breeding in organically polluted water habitats [Anopheles sp. larval ecology have been conducted in rural settings, it is likely that unpolluted breeding sites are found and described far more often [Anopheles sp. larvae can breed in nearly every kind of water accumulation. For every ecological factor identified as enhancing or reducing Anopheles sp. larvae productivity, at least one breeding site was found that contradicted these findings. Hence, all water bodies in an urban environment should be considered as potential breeding places and a target for larval control.The findings of this study revealed that d Figure  and 4 anr system . In Accrhabitats . As mostre often ,22, giviet al. [Anopheles sp. breeding [Anopheles sp. mosquitoes within a distance of one kilometer [et al. [et al. [Slopes to riverbeds, riverbeds, borders of swamps, stagnant drains and rivers, areas with restricted access and areas along railway lines represented environments where most breeding sites were found in Dar es Salaam. These sites where mostly associated with agriculture activities. Afrane et al.  stated tbreeding . In Dakailometer . Similar [et al. . Ponds c [et al. .The increasing urbanization of Dar es Salaam will probably help to reduce risk areas in the long-term, as open spaces get rare and swampy areas are filled to regain building land, but unplanned city growth along the city edges could increase the number of breeding sites . In BrazAnopheles sp. on urban larval ecology, showed that Anopheles sp. mosquito larvae are not restricted to clearly defined habitats. Therefore, malaria control interventions, such as environmental measures or insecticide application, have to consider all open water bodies as potential breeding sites. Specific malaria control interventions are currently developed and tested by the three municipalities of Dar es Salaam.Mapping of malaria risk on the basis of breeding sites plays an important role for urban malaria control programs. Also, initial risk mapping of breeding sites, combined with improved knowledge of mosquito ecology and their interactions with humans, is crucial to understand the epidemiology of urban malaria. The present study, in accordance with other studies of MS designed and implemented the study, analyzed the data and drafted the manuscript. DM, MK and ZP were involved in designing and implementing the study and analyzing its data. MT participated in the study design, data analysis and drafting of the manuscript. GK and CL assisted in the study design and implementation, data analysis and writing of the manuscript. All authors read and approved the final manuscript.


H. M. S. Beagle “struck with certain facts” that “seemed to throw some light on the origin of species,” Linnaeus published the first systematic taxonomy of life. In Systema Naturae, the Swedish botanist divided organisms into plants, animals, and minerals, eventually assigning scientific names to 7,700 plant and 4,400 animal species, and popularizing the binomial system—as in Homo sapiens—of naming species.One hundred years before Darwin returned from his voyage on the In the 1700s and 1800s, naturalists classified organisms based on morphology, devoting their careers to naming newfound plants and animals. Today biologists still use Linnaean taxonomy as the foundation of scientific classification. But with just a fraction of the estimated 5–30 million species on the planet already named and too few specialists to do the job, biologists are looking for high-throughput tools that can rapidly and accurately identify both individuals of a species and entirely new species. That's what some scientists say the DNA barcode will do. The DNA barcode, as the name implies, uses genes to identify species much like supermarket barcodes identify products. The idea is that a short stretch of genetic code from a reference gene is unique enough to one species to distinguish it from every other species, and that comparisons of sequence variations in that stretch of gene can reveal evolutionary relationships among species.c oxidase I, or COI) in a study of 260 North American bird species.Such technology could radically advance biologists' attempts to achieve the long-standing goal of cataloging life on earth, but the approach is controversial, with critics questioning both the method and its applications.  Paul Hebert and colleagues offer a proof of the utility of the DNA barcoding concept, using a 648-basepair region of a mitochondrial gene  evolves much faster than nuclear DNA. It evolves so quickly, in fact, that mtDNA sequence variation has been found not just between closely related, or sister, species but also within species. Still, the variation is much greater among than within species, which is why mtDNA divergences have become a tool for identifying species.Hebert and colleagues tested the effectiveness of the mtDNA COI barcode by matching bird species flagged by the COI barcode against those already established by taxonomic methods. The litmus test for DNA barcoding is absence of COI sequence overlap between species. Beyond that, differences within species should be significantly fewer than those between species. And that's what the researchers found. All 260 species had unique COI barcodes, with differences between species for the most part much more frequent—on average, 18 times more common—than those within species. In the 130 species represented by two or more individuals, COI sequences were either identical or closest to other sequences within that species. For these 260 bird species (of the 667 bird species that breed in North America), the authors report, the COI barcodes “separate individuals into the categories that taxonomists call species.”The COI barcode, the authors propose, could help resolve problematic classifications based on morphology, as arise when populations of a single species acquire distinct characteristics after geographic barriers prevent their interbreeding. For example, the similar COI sequences found in American and black oystercatchers here support taxonomic studies suggesting that they are actually color morphs of one species. And conversely, highly divergent COI sequences might bolster taxonomic studies indicating that lineages of uncertain status are indeed distinct species.Future studies will have to determine whether these results can be generalized to animals in other climes and ecosystems, but the authors argue that constructing a comprehensive library of barcodes will facilitate such efforts. Hebert and colleagues conclude that the success of DNA barcoding depends not only on such a repository—with sequences pegged to well-characterized species exemplars—but also on the expertise of trained taxonomists. The hope is that large-scale, standardized testing based on a uniform barcode sequence could go a long way toward finishing what Linnaeus started: a full classification of all plant and animal life. To E. O. Wilson, every species is “a masterpiece of evolution, offering a vast source of useful scientific knowledge because it is so thoroughly adapted to the environment in which it lives.” Faced with what Wilson calls the “worst wave of extinction since the dinosaurs died,” the need for a fast and easy way to identify species has never been greater.