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  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.  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 . 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 . 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 .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.


Chronic neck pain is a common complaint in the Netherlands with a point prevalence of 14.3%. Patients with chronic neck pain are often referred to a physiotherapist and, although many treatments are available, it remains unclear which type of treatment is to be preferred.The objective of this article is to present the design of a randomised clinical trial, Ephysion, which examines the clinical and cost effectiveness of behavioural graded activity compared with a physiotherapy treatment for patients with chronic non-specific neck pain.Eligible patients with non-specific neck pain persisting longer than 3 months will be randomly allocated to either the behavioural graded activity programme or to the physiotherapy treatment. The graded activity programme is based on an operant approach, which uses a time-contingent method to increase the patient's activity level. This treatment is compared with physiotherapy treatment using a pain-contingent method.Primary treatment outcome is the patient's global perceived effect concerning recovery from the complaint. Global perceived effect on daily functioning is also explored as primary outcome to establish the impact of treatment on daily activity. Direct and indirect costs will also be assessed. Secondary outcomes include the patient's main complaints, pain intensity, medical consumption, functional status, quality of life, and psychological variables. Recruitment of patients will take place up to the end of the year 2004 and follow-up measurement will continue until end 2005. Neck pain is a common complaint that causes substantial morbidity in western countries with a reported prevalence ranging from 9.5 to 22% ,2. Of alThere are many potential causes of neck pain, but mostly no specific underlying pathology is found so that it is designated as non-specific . AlthougIn the Netherlands, patients with neck pain are often referred for physiotherapy. Moreover, physiotherapy accounted for 84% of the total direct medical neck pain costs in 1996 [Two treatment models have been described in the literature, both of which are applicable within the field of physiotherapy. The first, a biomedical model, considers pain to be a sign of physiological damages and treatment according to this model aims to remove the pathologic condition so that the pain will no longer occur ,10. MoreMany conservative physiotherapeutic treatments are available for treating neck pain, but there is insufficient evidence to allow to conclude that one type of treatment is more effective then others ,20.In a review on chronic pain, operant behavioural therapy was found to be beneficial to waiting list control groups on outcomes such as pain experience, mood effect other than depression, social role, and for the expression of pain behaviour . CompareIn summary, it remains unclear which type of conservative, including behavioural, treatment is to be preferred in the management of chronic neck pain. Therefore, this study, Ephysion (Effectiveness physiotherapy in neck pain), aims to evaluate the clinical and cost effectiveness of an operant behavioural programme  compared with a physiotherapy treatment in patients with chronic non-specific neck pain. In addition, we aim to identify subgroups of patients who benefit most from one of the two treatments, and to identify the most important determinants for recovery from chronic non-specific neck pain.Because a biased study design can produce incorrect conclusions, the design of a trial should be carefully examined before adopting its conclusions . A desigA randomised clinical trial (RCT) has been designed to assess the effectiveness of behavioural graded activity compared with physiotherapy treatment in patients with chronic non-specific neck pain. The study design has been approved by the Medical Ethics Technical Commission of the Erasmus MC, University Medical Centre in Rotterdam and is in compliance with the Helsinki Declaration.Forty general practitioners (GP) in region West Brabant in the Netherlands will select the patients. Patients are eligible if they are aged between 18 and 70 years old, have suffered from neck pain for over three months, and have an adequate knowledge of the Dutch language. Excluded are patients diagnosed with a specific disorder , those who have had physical/manual therapy during the previous six months, those with a chronic disease (e.g. rheumatoid arthritis or coronary artery disease), or those who have to undergo surgery in the near future. Eligible patients will receive an information leaflet from their GP and the GP then informs the research department.Thereafter, the research assistant contacts the patient, provides additional information about the implications of participation, re-checks the eligibility of the patient, and completes the informed consent procedure.The sample size for this study is calculated according to the global perceived effect (GPE). Based on previous studies, a 20% difference in GPE is expected after completion of either treatment (9 weeks) and is considered to be clinically relevant; 160 patients are needed to detect this difference. In this calculation a power (1 - β) of 80% is taken into account. Thus, the inclusion of 80 patients per treatment group is planned.An independent examiner using a computer-generated randomisation schema performs randomisation. To prevent unequal distribution, patients are pre-stratified based on three important prognostic factors: gender, age and the severity of the complaint, which are recorded at baseline . FurtherPatients are told to receive physiotherapy but are blinded to allocation of the two treatments; the content of the treatments is not described in the information leaflet. This enhances the quality of the study, because the patients themselves measure the effect of treatment. GPs are also blinded for allocation to prevent accidentally informing the patients of the allocated treatment. The physiotherapists are not blinded for allocation, but the physiotherapists from each treatment group are kept strictly separate and are not involved in the outcome measurement. Finally, the primary investigator is blinded for patients' allocation but the research assistant is not; neither is involved in the outcome measurement.After receiving written information, 34 physiotherapists in region West Brabant will participate in either the physiotherapy treatment (PT) or the graded activity programme (GAP). To optimise the contrast between the two treatments, both groups are strictly separated throughout the study. The PT group consists of 16 physiotherapists and the GAP group of 18 physiotherapists. The PT physiotherapists participate in a meeting to standardize the physiotherapy treatment. The GAP physiotherapists are instructed on the behavioural graded activity approach during a two-day theoretical and practical training course.Both interventions are performed in an outpatient setting. A maximum of 18 treatments per patient is set and each treatment takes about 30 minutes, which is in accordance with medical insurance policy in the Netherlands. Before treatment starts, physiotherapists receive a completed questionnaire about the patient's main complaints ; this quThe content of the physiotherapy treatment is decided by consensus among the participating PT physiotherapists. Treatment is according to a biomedical model, which implies guidance based on the amount and severity of pain that the patient experiences.By consensus, the physiotherapy treatment is divided into the patient's primary therapy aim, three general treatment goals, and several techniques to attain those goals. The primary therapy aim is defined as the result the patient wants to achieve by the end of therapy. A general treatment goal is a goal for each single treatment and could, therefore, differ per treatment session. Table An operant approach was the basis of the behavioural graded activity programme as used in this study. The treatment is according to a biopsychosocial model, which implies that it is guided by the patients' functional abilities and that time-contingent methods are used to increase the activity level of the patient . The behBefore starting the baseline phase, the treatment vision and the patient's ideas about pain and its causes are discussed. The development and maintenance of pain will be explained and patients are reassured that it is safe to move and to increase their level of activity ,13,30. BDuring the treatment phase, patients systematically increase the time-contingent quotas to enable them to reach their personal aims within a pre-set therapy time period. To ensure a successful experience during the first exercise, the start quota is below the mean baseline score. The pre-set exercise quotas have to be strictly followed; neither over-performance nor under-performance is allowed. During this phase the patient has to practice at home and document every activity or exercise on a performance chart. These charts will be discussed in the following treatment session and achievements will be reinforced while disregarding pain behaviours. Positive reinforcements of healthy behaviour and the patient's experiences of success are considered to be important to enhance the patient's motivations.The generalization phase takes place at the end of the treatment phase. In this phase generalization of learned behaviour and management of relapses will be discussed.Baseline questionnaires are sent after inclusion, which is as soon as possible after patients have consulted their GP. Outcome of intervention will be assessed at 4 and 9 weeks after randomisation; however, if the treatment is not finished at 9 weeks, the patients will receive an additional questionnaire (Ts) after finishing the treatment. Follow-up assessments are planned at 26 and 52 weeks after randomisation.All outcome measures are reported by means of mailed questionnaires. Table Primary treatment outcome of this study is the global perceived effect, which is used to assess recovery from the complaint . In addiCosts are measured using a combination of questionnaires to collect data on direct medical costs , and indirect costs due to sick leave and disability.Secondary outcome measures include main complaints, pain intensity, medical consumption, coping, functional status, quality of life, and psychological variables. Prognostic factors are measured including demographic variables, the baseline variables and the psychological variables table .Descriptive statistics will be used to examine comparability of baseline data between PT and GAP, and to check if randomisation was successful. Before this analysis, decisions about differences considered to be clinically relevant are made and, if necessary, adjustment will be made for these differences in multivariate analysis. Further, all outcome data will be screened for normality and, if necessary, logarithmic transformations or non-parametric methods of analysis will be applied.The first aim is to evaluate the clinical and cost effectiveness of GAP compared to PT. Clinical effectiveness will be examined with a Student's t-test (continuous), a Chi-square test (dichotomised) or a Wilcoxon test  according to the intention-to-treat principle. This means that patients will be analysed in the treatment group to which they are randomly allocated. For missing data, imputation techniques will be used. When the dropout rate is 10% or more, or loss to follow-up is 20% or more, per-protocol analysis will be performed. The results on primary outcome will be dichotomised into improved versus not improved. Improved implies completely recovered and much improved, whereas not recovered implies slightly improved, not changed, slightly worsened, much worsened, and worse than ever .Cost effectiveness will be calculated from a societal perspective. Costs (direct as well as indirect) will be related to the treatment effects, based on the primary outcome measure, by calculating cost-effectiveness ratios.The second aim is to identify subgroups of patients that benefit most from one of the two treatments. The following subgroups will be investigated: duration and severity of the complaint, depression, and fear of movement.The third aim is to identify important variables for recovery. For this purpose multivariate analysis will be performed to investigate the influence of prognostic variables and patient characteristics on the outcome. Separate analyses will be conducted to investigate prognostic factors for short-term (3 months) and long-term (12 months) recovery.This study is designed to evaluate the clinical and cost effectiveness of a behavioural graded activity programme compared with a physiotherapy treatment in patients with chronic non-specific neck pain. Since physiotherapists perform both treatments in this study, contrast between the two treatments is a very important issue. There are contrasts both in the composition of the treatment and the way the physiotherapists approach the patient. With regard to the composition, the graded activity programme (GAP) starts with a systematically performed baseline measurement; this is in contrast to the physiotherapy treatment (PT), where treatment is based on history taking and physical examination. In GAP quotas are set based on the patient's behaviour, whereas in PT they are set based on pain levels and training principles. After quotas are set GAP uses a time-contingent treatment approach, which involves a pre-set systematic increase in activities. In contrast, PT uses a pain-contingent approach, which means that treatment is adapted to the patient's reaction to previous treatment sessions. Furthermore, GAP uses a hands-off approach, whereas PT may contain hands-on techniques, such as massage, traction etc Table .This study addresses an important question because chronic neck pain is a common complaint and it remains unclear which type of physiotherapeutic treatment is most effective. Recruitment of patients will take place until up to the end of 2004; follow-up measurement will continue up to end 2005.The authors declare that they have no competing interests.APV and BWK conceived the study, developed the design of the randomised clinical trial and participated in writing the article. MG is an expert in the field of graded activity and contributed to the content of the article. CJV advised on the content of the article. FV conducts the research, participated in the completion of the study design and wrote the article. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Female mate choice may be adaptive when males exhibit heritable genetic variation at loci encoding resistance to infectious disease. The Hamilton-Zuk hypothesis predicts that females should assess the genetic quality of males by monitoring traits that indicate health and vigor . Alternatively, some females may employ a more direct method of screening and select mates based on the dissimilarity of alleles at the major histocompatibility loci . Empirical studies suggest that both forms of mate choice exist, but little is known about the potential for natural selection to shape the two strategies in nature.We used computer simulation models to examine the evolutionary fates of the two forms of mate choice in populations at risk for infection by debilitating parasites. We found that populations exhibiting random mating (no mate choice) can be invaded and replaced completely by individuals practicing CD type mate choice. We also found that an allele encoding OA choice can increase when rare in randomly mating populations, but that it does not go to fixation under selection. A similar result was obtained when the OA strategy was introduced into populations practicing CD mate choice. As before, we found that the OA choice allele will increase when rare, and that it will not go to fixation under selection. The converse however was not true, as CD individuals gain no rare advantage when introduced into an OA population.Taken together, the results suggest that, when rare, OA is the best strategy for parasite evasion (of those considered here). The consequence of OA increasing in the population, however, is to reduce the parasite driven genotype oscillations and facilitate the breakdown of linkage disequilibrium at the disease-resistance loci. This leads to a neutrally stable situation in which different strategies have equal fitness, and suggests that multiple forms of mate choice may be expected to occur in populations at risk from infectious disease. Since Darwin published his classic work on sexual selection , biologiIn a previous study, we investigated the potential for active mate choice to indirectly favor the maintenance of sexual reproduction . We founThe results from our study show that when populations are confronted with infectious disease, alleles encoding OA and CD mate choice both increase when rare in randomly mating populations Figs.  and 2A. As the allele for OA increased in the population, the oscillations in both host and parasite resistance genotypes underwent a dramatic reduction in amplitude Fig . In addiIn contrast, as the allele for CD choice increased from its rare starting point, the oscillations in genotype frequency increased, rather than decreased Fig. . This reUnder the conditions studied here, both OA and CD alleles increase when rare in randomly mating populations, but only the CD allele goes to fixation due to sexual selection. How does CD fare against an OA population? We found that when introduced into a population of 20,000 OA individuals, a rare allele encoding CD choice was not favored by selection, but that it could become established and increase in frequency as a result of genetic drift Fig. . This reIn contrast, OA did increase when rare due to selection in a population of CD individuals; hence CD is not an ESS Fig. . The oveAmong the three strategies of mate choice for parasite avoidance considered in this study, only OA is evolutionary stable. However, OA will not eliminate either RM or CD-choice through the action of sexual selection, since OA becomes selectively neutral at intermediate frequencies in both situations by causing reductions in the fitness variance among resistance genotypes. Thus, OA and RM, OA and CD, or all three could coexist in the short term in large populations (until OA goes to fixation by drift). As such, in populations at risk from disease, females might practice more than a single type of mate choice.The methods employed in this investigation are similar to those used in our previous studies of host parasite coevolution ,25. We uT. For the two locus version of the model, T was set to a value of 0.8, and parasites underwent two generations for each host generation. In the three-locus, five-allele version of the model, higher rates of parasite transmission coupled with a greater asymmetry of host and parasite generation times were required to sustain infection in the host population. For this version of the model, T was set to a Poisson distributed mean of eight, and parasites underwent ten generations for each host generation. In all cases, if a parasite matched a host exactly at all interaction loci, the host was marked as infected and the parasite was placed in a pool of potential reproductives. Once infected, individual hosts were protected against further attack by parasites. Reproduction in hosts and parasites was accomplished by drawing individuals from their respective populations at random with replacement. The parasite life cycle included a "free-living" stage in which reproductive adults emerged from infected hosts to mate. When an individual (host or parasite) was selected for reproduction, a second individual was randomly selected for cross-fertilization. Gametes from the two haploid "parents" were then brought together to form a diploid zygote stage, where free recombination between the interaction loci made possible the production of a genetically diverse brood of haploid offspring. All else equal, sexual individuals (hosts and parasites) produced a lifetime average of 10 haploid offspring. The number of offspring produced by hosts, however, was reduced according to the status of parasitic infection for each of the parents. The reproductive output of each host parent was discounted according to 10(1-E), where E simulates the detrimental effect of parasitism on host reproduction (virulence). In the present study, E was set to a value of 0.8 to simulate the effect of a moderately virulent parasite. Following reproduction, a maximum of 20,000 parasite and 20,000 host offspring were selected at random to become the next generation of adults.During each parasite generation, hosts were drawn sequentially and exposed to a randomly drawn parasite with a probability of n, where n was the total number of alleles present at each locus. Prior to data collection, the simulation was allowed to run for 200 host generations; this allowed for establishment of long-term coevolutionary dynamics arising from the host-parasite interaction. Next, we tracked the fates of alleles encoding different types of mate choice in the host population. Mating preferences in hosts were controlled by a single locus encoding one of three strategies: random mating (RM), condition-dependent mate choice (CD), or opposites attract (OA) mate choice. Random mating was simulated by pairing two randomly drawn adults. Condition-dependent choice was implemented by allowing the first reproductive drawn  to identify and discriminate against parasitized "males". For the opposites attract strategy, females preferred "males" with alleles different from their own at each of the two interaction loci. For both OA and CD choice, "females" were allowed to choose the "best" available male from a sequence of 20 randomly drawn "males".At the beginning of each run, the interaction alleles at each locus in the host and parasite populations were initialized to a frequency of 1/RSH wrote the computer code and implemented the simulations. Both authors participated equally in all other phases of the work.


Is evolutionary theory is incomplete and are we failing to understand phenomena as disparate as ecosystem development and the interplay of genes and culture in shaping human evolution? This book, the latest in the excellent Monographs in Population Biology series from Princeton University Press, is a work of advocacy in which the authors argue that evolutionary theory is incomplete and that, in consequence, we are failing fully to understand phenomena as disparate as ecosystem development and the interplay of genes and culture in shaping human evolution. What we are missing, they argue, is an appreciation of niche construction, the process by which an organism modifies the abiotic and biotic environment in which it is subject to natural selection. The authors' major assertion is that the importance of niche construction is so great that it should be regarded “after natural selection, as a second major participant in evolution” and that it is “not just an important addition to evolutionary theory” but “requires a reformulation of evolutionary theory”. Bold claims indeed.After introducing the conceptual framework Odling-Smee et al. set out a series of arguments to support this position, the first of these being the empirical case for the existence of niche construction. Niche construction, as broadly interpreted here, is everywhere. Animals build nests, burrows, and protective cases and so alter the environment they experience in a way that may select for further adaptations. The changes caused by some animal species, such as beavers and earthworms, are of a sufficient magnitude that the environment experienced by a host of other species is affected. Many plant species also modify the environment they experience by generating organic litter; influencing hydrological and biogeochemical cycles; affecting temperature, humidity, and light regimes; and, over the longer term, determining the make up of the atmosphere. Decomposer and chemoautotrophic microorganisms similarly influence biogeochemical transformations, while parasitic species can manipulate the behaviour and internal environment of the hosts they infect. Perhaps less obvious examples of niche construction are the many types of migration and cultural evolution that, like physical transformations, cause the organism's descendants to experience a different selective environment.After this broad, accessible survey, the authors change key rather abruptly and explore two-locus, frequency-dependent population genetics. The novelty here is that selection on one locus depends on the history of gene frequencies at the other, “niche construction”, locus. In an extension, gene frequencies at one locus affect an environmental variable with its own dynamics that in turn influences the second locus. As one would guess, the models display a range of potentially interesting dynamics, though generalisations and broad conclusions are sparse. We guess the aim of the chapter is to illustrate that environmental feedbacks can be potent and general agents of evolutionary change, but the restriction of the theory to such a narrow model, with very technical explanation, risks losing the few readers who we suspect will stay the course .Perhaps aware of the dangers of getting bogged down in detail, the argument then moves to proving a case for the universality of niche construction. Invoking the second law of thermodynamics and Maxwell's Demon, the authors lead us through a challenging thesis that concludes that the persistence of life on earth requires both natural selection and niche construction, thereby justifying some of the bold claims for their new theory. We think they are technically correct, but we are concerned that the demonstration of the inescapability of niche construction, as defined here, does not guarantee that it will actually tell us new and important things about the world, as the theory of natural selection has.The remainder of the book explores the implications of niche-construction thinking for evolutionary biology, ecology, and the human sciences, and in our view is the most successful part. Though it is rare for the authors to offer new analysis and insight, their sideways look at many issues from the niche-construction viewpoint often offers interesting new angles on old problems, and suggests new avenues of enquiry that may be the book's greatest legacy. A good example of this is their convincing and timely argument that a more explicit recognition of evolution's role in environmental feedbacks will help to unify population/community ecology and ecosystem science.avant la lettre, has given way to a more pluralistic theory of succession. It is a great pity that the authors give so little space to plants and plant ecology, as it here that some of the finest examples of niche construction are found, as well as the best-developed conceptual framework for studying the roles of environmental feedback.The chief argument for the prosecution is that niche construction is common but not pervasive, and that wherever ecologists and evolutionists have found interesting examples of it, they have developed appropriate theory and concepts to understand its ramifications. For example, some of the clearest examples of niche construction occur in plant succession where, as F.E. Clements realised nearly a hundred years ago, early-succession plants frequently modify their environment in ways that allow other species to replace the pioneers. Interestingly, the strict Clementsian theory of facilitation, niche construction Other areas where biologists have well-developed theories of the influences and impacts of niche construction include co-evolutionary theory, where the environmental feedbacks are largely biotic, and Dawkins' theory of the extended phenotype. Very close to some of the arguments discussed here, and generously acknowledged, is the idea developed by Jones, Lawton, and colleagues of ecosystem engineers, species that have a major impact on the abiotic environment experienced by a large number of species.A major strength of the book is that it reveals common processes and patterns underlying disparate biology in consistently interesting ways. Its chief contribution is thus not to tell us new things about how nature works but to link together many different aspects of ecology under an umbrella of theory that may in the future lead to new insights. Do they deliver on their grand claims? Time will tell, but our view is that they don't. They engagingly admit that for their project to succeed the new theory must earn its keep by producing significant new biology—something which has yet to occur. However, the great need for the biological and human sciences to integrate across subdisciplines, as the authors bravely attempt here, makes this a hugely worthwhile book. Its breadth of scope and its boldness in creating syntheses have resulted in a stimulating and challenging read.