Translational Research (TR) provides a set of tools and communication context for scientists and clinicians to optimize the drug discovery and development process. In the proceedings of a Princeton conference on this timely topic, the strengths and needs of this developing field were debated. Outcomes and key points from these discussions are summarized in this article which covers the topics of defining what we mean by translational research , ways in which to engender the TR mindset and embed it in organizations such as the pharmaceutical industry in order to optimize the impact of available technologies (including imaging methods), the scientific basis and under-pinnings of TR including genomics knowledge, information sharing, as well as examples of application to drug discovery and development. Importantly, it should be noted that collaborations and communications between the stakeholders in this field, namely academia, industry and regulatory authorities, must be strengthened in order for the promise of TR to be delivered as better therapies to patients. Successful drug development requires satisfying a matrix of domains from relevance to the disease and the drug-ability of the target through feasibility and convenience of drug delivery, demonstration of favorable benefit-risk profile in order to achieve to a drug label that reflects physician and patient acceptance. Herein lies a key role for TR in helping to navigate this journey.There are many challenges facing pharmaceutical companies in the post-genome era not least of which is declining productivity and innovation. Not surprisingly, there is agreement between Industry, Academia and Regulatory communities that the drug discovery and development process needs to change in order to meet the future needs of patients with effective and desirable drugs. A key part of the strategic solution is to leverage the application of TR principles and practices, which if implemented will go a long way towards addressing the challenge posed by FDA's Critical Path Initiative   that hosted a small group of clinical and basic science researchers and individuals from the pharmaceutical industry. Among the topics discussed were how to define "Translational Research", how to expedite the transfer of pre-clinical findings to influence development plans, how to select biomarkers to ensure support for decisions, how new strategies can be effectively translated to practical tactics, and what team players and collaborations are necessary to conduct successful TR. Success factors identified include: Identification and validation of novel drug targets, development of robust and validated assays to screen drug leads for safety and potential efficacy in humans, and the identification of suitable patients for expedited but informative trials.In order to promote discussion on this topic, the International Quality and Productivity Center (IQPC) organized a in vivo measurements and leverage preclinical models that more accurately predict drug effects in humans, TR itself can be defined in many ways. At its core however, is the thesis that information gathered in animal studies can be translated into clinical relevance and vice versa, thus providing a conceptual basis for developing better drugs. It could in fact be argued that the designation of a special term or definition for TR might be unnecessary or even misleading. Historically the term was assigned to create awareness and advocacy for the general public, clinicians and scientific communities, and especially for the government and other private sponsors [While the goal of TR is to implement sponsors  in this Whatever the precise definition TR, it should serve as a forum to find a "common language" for clinicians and scientist in navigating the complexities of basic scientific approaches, data analysis and information processing. It clearly implies the need for an intensive training for scientists and clinicians in multiple disciplines to acquire expertise and experience to conduct TR. For the purposes of this symposium, the scope of translational research was defined as the application of scientific tools and methods to drug discovery and development. This can be achieved by integrating information concerning a) exposure (pharmacokinetics), b) biological activity (pharmacodynamics including safety profiles) delineating differences between species and leading to the validation of target and mechanism biomarkers, and c) outcomes leading to an understanding of efficacy and safety between species and ultimately to the qualification or linkage of biomarkers to clinical outcome (for a fuller discussion on Biomarkers and Surrogate Endpoints see definitions in ). Thus TTherefore, in taking a pragmatic or operational rather than a definitional approach, a key to a successful translation of non-human research to human clinical trials lies in the choice of biomarkers. While biological pathways tend to be homologous across species and more so than pharmacokinetic parameters such as absorption and clearance, animal models themselves have a poor record of predicting human disease outcome. Nonetheless, biomarkers are the key for prediction of biological activity if not drug efficacy in humans. At least three types of biomarkers can be identified: (1) target biomarkers measuring the interactions between a drug and its target; (2) mechanism biomarkers measuring their downstream biological effects and (3) outcome biomarkers that reflect efficacy and safety. A second dimension can also be ascribed to biomarkers to help drug developers assign risk assessment to such approaches. This sub-classification links desired utility to points on a risk continuum; e.g. low, medium and high, in which 'low' describes a biomarker applied solely to animal models for example for selecting compounds for progression into humans, whereas 'medium' is association with utility for some aspects of early clinical profiling of efficacy and safety including across species correlation, and 'high' is associated with reproducibility and qualification as an outcome or even regulatory tool in humans.Additionally, TR itself undergoes an evolution from pathfinding (hypothesis generating) to discovery research, to development, and finally to application. Each of these operational phases is amenable to being evaluated or supported by biomarkers, either for the definition of objectives, proof of principle or in assessing risk and feasibility. Consequently the right choice of biomarkers can help drive decision-making and lower the costs and cycle-time for progression of a new drug from the bench into the clinic. In summary, whatever the definition or classification ultimately used, in practical terms translational tools should be developed and applied on a "fit for purpose" basis with prior assessment and agreement of attendant risks.Traditional Research and Development  paradigms have accentuated the boundaries between the territories of discovery and development worlds and have not been conducive to bridging key transition points. This is unfortunate since the development world tends to lag behind advances made in discovery, a point recognized by FDA in launching the Critical Path Initiative . In brieWhile advances have been made on streamlining forward progression of R&D through organizational linkages, what has not happened to the same degree is a bi-directional flow of information, namely flow of information from the clinic  back into the hands of the discovery scientist. The consequence of this is that the biological models used to qualify drug candidates may fail to be predictive of subsequent drug responses in the clinical setting. Thus a practical outcome of TR is to improve the overall probability for technical success (POS) in drug development.in silico modeling, but also the need to empower key scientists and clinicians with the task of enhancing the prediction and iteration learning cycle. Since there are different organizational solutions for embedding the TR mindset within an organization, a key element is to provide TR expertise to drug development teams. Furthermore, innovation and productivity values are critically linked through information exchange. Rapid iteration  and transfer of knowledge gained from prototype development experience will enable more rapid compound redesign against the highly desired target and be reflected as enhanced innovation. On the productivity side, the tools outlined in the Critical Path Initiative [Consequently the next paradigm for R&D optimization depends not only on leveraging emerging technologies such as pathway mapping and itiative , once efThe journey however starts at understanding the scientific foundations of physiology and pathophysiology, thus providing a rational linkage between the gene, its expressed product, disease expression and ultimately outcome. The discipline of biomarker identification and development as mentioned previously encompasses these principles and is a core tool in the TR scientist's armamentarium.a priori agreed performance characteristics, such that there is agreement on the utility of the marker.Biomarkers (which are not necessarily Surrogate endpoints and few are in fact) are key tools for escorting the drug candidate from the bench to the bedside and back. That is they can be both animal "diagnostic" as well as human "diagnostic" tools. A key implementation tool is therefore to identify early on which biomarkers may be of value and to study these in the relevant animal models, that is, specifically include them in preclinical screening paradigms, as well as identify their role  in the clinical development plan. Biomarkers, which include imaging techniques as well as protein and genetic markers, may fulfill several roles in R&D from compound screening and selection through dose justification, decision-making and risk mitigation, however the key is to overtly link them to the discovery and development plans with There are many good examples of the value or non-value of preclinical models in predicting subsequent human response and safety. The journey from preclinical experience to the clinic is a well-worn one , albeit without the degree of overall predictiveness we would desire. On the other hand there is a marked paucity of examples in which clinical experience or observation was translated back into a legitimate drug target and discovery effort (e.g. Viagra). Thus, a major opportunity lies in both developing more sensitive and specific animal models of disease (e.g. knock in/out) as well as fully leveraging novel clinical observations. At the same time it is the ultimate validation in the clinic that counts, and rapid feedback of that information will allow the conditional probabilities and learning cycle to be enhanced. By enabling these principles through organizational and cultural change, the impact of TR will be determined by direct impact on high-quality mid-phase transitions as well as reduced cycle-times and resource burdens.The era of genome-scale biology has seen an increase in, and production of, vast amounts of biological data together with an extensive increase in biology-oriented databases. To make the best use of biological databases and the knowledge they contain, different kinds of information from different sources must be integrated in ways that make sense to biologists. A major component of the integration effort is the development and use of annotation standards such as ontologies. Ontologies offer a conceptualization of domains of knowledge and facilitate both communication between researchers and the use of domain knowledge by computers for multiple purposes. Therefore, the Gene Ontology (GO) project was founded in 1998, in an attempt to provide consistent descriptors for gene products, in different databases; and to standardize classifications for sequences and sequence features. Since then, the GO Consortium has grown to include many databases, including several of the world's major repositories for plant, animal and microbial genomes . DespiteIn the past, biological processes and the underlying genes, proteins, other molecules and environmental factors, have been studied separately more than on an integrated basis. The challenge, however, for future research on human disease is to understand not only the mechanistic basis, but also the underlying dynamics of gene product expression. Thus, biological research should emphasize the analysis of pattern of gene expression over individual measurements.Molecular Function describes activities, such as catalytic or binding activities, at the molecular level, e.g. kinase activity. (2) Biological Process describes biological goals accomplished by one or more ordered assemblies of molecular functions, e.g. 'cell death' can have both subtypes, such as 'apoptosis', and subprocesses, such as 'apoptotic chromosome condensation'. (3) Cellular Component describes locations, at the levels of subcellular structures and macromolecular complexes, e.g. 'nuclear inner membrane' with the synonym 'inner envelope' [GO has been developed to predict behavior of entire biological systems, being assigned to three aspects: (1) nvelope' .The powerful use of comparative gene expression analysis in human disease was exemplified with a recent study on gene expression profiles of gastric cancer patients and their correlation to survival. Leung et al.  have sho provides structured, controlled vocabularies and classifications that cover several domains of molecular and cellular biology and are freely available for community use in the annotation of genes, gene products and sequences.In summary, the application of mathematical models and computer simulations to analyze gene expression profiles and to compare complex data sets of various origins may provide new insight into the pathogenesis of cancer progression and metastasis. The Gene Ontology (GO) project Cancer vaccines are promising therapeutics designed to elicit immune responses against antigens expressed by tumor cells. However, vaccines that have worked well in preclinical models have not translated into consistent responses in the clinic. Since vaccines are comprised of multiple components, multiple immunological endpoints are used to identify the least effective vaccine components in cancer patients. Post-clinical research strategies are subsequently designed with a focus on improving the least effective vaccine components.To improve the performance of cancer vaccines in the clinics, which are traditionally judged by clinical endpoints, novel endpoints and biomarkers are needed to assist in understanding why cancer vaccines are not working. From clinic to bench, a systematic strategy is needed for pre-clinical optimization that addresses vaccine limitations identified in the clinics; and from bench to clinic, performance criteria need to be established for a follow-up clinical study. After gathering the therapeutic options, testing has to be prioritized on the basis of: a) already available data; b) availability of the therapeutic modality; c) models and assays available internally; d) turnaround time; and e) on the patent landscape.Prioritization and rapid evaluation of novel therapeutics will decrease the turnaround time and facilitate decision-making. However, several tools are needed to make this a reality. For example, complex therapeutic strategies require biomarker or even surrogate endpoints from clinical trials to direct development of second-generation therapeutics. The rapid qualification and choice of surrogate endpoints should be based on knowledge gathered by an "early-stage therapeutic opportunities database". This comprehensive database should include data on therapeutic targets, models, assays and published results and indeed the plethora of new therapeutic strategies in preclinical stages can only be managed by accessing informative databases. Moreover, pre-clinical compound optimization can be facilitated by establishing quantitative endpoints of short duration and lastly go / no go decision points must be established for surrogate endpoints and clinical responses in animal models.However, several current issues of scientific basis also have to be addressed, such as the importance of clinical surrogate endpoints, the relevance of animal models, lack of concordances between assays, and the lack of concordance between surrogate endpoints and the clinical response, in order to improve cancer vaccine development strategies., a non-profit organization conducting more than 100 clinical trials and treating 7000 cancer patients yearly.A core principle of TR revolves around validation of targets, biomarkers and treatment modalities in humans. These activities and drug development itself cannot be undertaken without patients or clinical data. How TR can be integrated in a multi-center, multi-cultural organization involving patient accrual from more than 38 different countries worldwide, for the research and treatment of cancer can be exemplified by EORTC Advancement in basic science and immunology and an overwhelming revolution of biotechnology have changed the targets and endpoints in cancer trials from the mere assessment of cytotoxicity to defined mechanisms for potential anti-tumor effect. That is in the era of "targeted therapies" molecular therapeutics are now being designed to target "strategic" checkpoints that underlie the malignant phenotype. The challenges to be met are: 1) dealing with new compounds affecting novel molecular targets, 2) innovation in design and analysis of clinical trials, 3) cooperation between translational researchers and network of clinical investigators and 4) informed patients. The major concerns in conducting clinical trials are rising costs coupled with efficacy rates as low as 5% in cancer patients, making signal to noise detection not only difficult but expensive. and storage of tissue is de-centralized at the institute where it is collected. To assure equal quality of tissue, which in outcome of scientific experiments can be compared, standardization of the collection and storage methods is fundamental. Therefore, protocols for storage, retrieval and tracking of tissues will be standardized and implemented in all participating laboratories.The need for research on tumor tissue requires the set-up of tumor banks and the associated administrative burden often discourages young oncologists. EORTC established a tumor bank comprising real tissue samples but including a "virtual review" by pathologists. This ensures the availability of a well-categorized and prognostically evaluated collection of primary tumors and allows an online-searchable bank for researchers to access. Indeed the tumor bank harbors paraffin-embedded tumors, as well as frozen tumor tissues Access to the tumor bank allows screening of many available tumor samples for the expression of molecular targets and will help to unravel novel biomarkers for diagnosis and treatment. Such access will allow us to overcome the missed opportunities due to lack of tissue collection in clinical trials, which could have allowed better pre-screening of potentially responsive patients based on expression of certain biomarkers e.g., expression of bcl-2, and the treatment of target positive patients may have ensured a better clinical outcome in this target class.The challenge of testing promising new modalities for the cure of disease that had shown efficacy in experimental models lies in a lack of understanding of the underlying mechanism, heterogeneity of human genetic backgrounds and a lack of suitable controls in human studies. Strategies have been developed at the NIH for the global monitoring of patients by studying, with high-throughput technology, the systemic effects of treatments as well as their effect within the target organ. For this bedside to bench effort, a systematic sampling of human tissues of local (site of immunogen application), systemic (circulation) and peripheral (tumor site) origin needs to be standardized to ensure high quality of samples avoiding degradation of protein, RNA and DNA. This TR approach allows experimental studies in human samples during or after therapy through amplification of transcripts for analysis of minimal sample tissue, and the application of monitoring techniques for genetic profiling. Further, proteomic-based approaches allow following the kinetics of the mechanism of actions of therapeutics.Studying the effects of treatment in a bedside to bench approach provides markers for the characterization of disease process and/or testing hypotheses generated by experimental models. Therefore, the nature of research in the clinical setting can realistically be described as 'hypothesis generating", rather than 'hypothesis driven', through a discovery-driven approach. Analysis of the genetic background can reveal polymorphism of genes involved in immune reactions, such as cytokines and their receptors, which might influence the outcome of immunological interventions in different patient populations .Analysis of disease heterogeneity can be approached by transcriptional analysis, through linear amplification of RNA and subsequent analysis by cDNA array and transcriptome array, and/or functional protein analysis, through protein characterization by proteomics ,7. NumerDespite the many obstacles in monitoring therapeutic effect in early phase clinical trials and the lack of hypothesis, the scientific significance of these trials should be reviewed assuming that the new treatment will not be beneficial. Desirable outcomes include learning about the disease process, the primary goal of the therapy and the reasons for its failure. Another concern should be if we have taken advantage of the patient population accrued at least to learn something, although independent of treatment, about the disease process itself. Clinical trials should therefore be designed, within ethical constructs, to look at questions beyond the ones related solely to treatment. This can be achieved through (1) establishment of libraries of relevant clinical samples for immediate or future studies, (2) prospective collection of data into a consistent format, and (3) tight link between clinical and scientific data.Developing better therapies for chronic inflammatory diseases also exemplifies the use of the latest technological advances in TR such as proteomics, transcriptomics and cellomics, for identification or application of biomarkers. Chronic inflammation frequently precedes the development of cancer in adults, such as lung , esophagSeveral factors, such as: 1) the nuclear protein HMGB1, 2) the S100 family of molecules; 3) purine metabolites, ATP, AMP and uric acid, and 4) heat shock proteins have emerged as relevant mediators or "endogenous damage or danger signals" to recruit inflammatory cells, to promote wound healing and associated stromagenesis, angiogenesis; and ultimately to modulate immune functions .Current attempts for cancer therapy focused on vaccination to antigenic targets or application of cytokines have resulted in measurable anti-tumor reactivity in the blood; however, these therapies have mostly failed to show a correlation with tumor outcome or progression. Therefore, to more completely understand and identify factors assessing tumor death could inform and drive the development of more effective biological therapies for cancer patients. Sample acquisition in the blood includes serum/protein collection for Seldi-Tof mass spectrometry; and the collection of cells for microarray, proteomics, and high contents screening via cellomics. Protein chip Seldi-Tof MS has been already successfully used to discriminate serum expression profiles in various cancer types -13. The Beside proper study design, the models chosen to perform data classification and to estimate classification errors are highly critical for the complex data analysis. The identification of diagnostic markers for cancer, or markers to identify responders vs. non-responders to therapy requires systematical analysis of healthy vs. diseased, then of benign inflammatory disease vs. malignant cancer. Thus, methods to perform statistical analysis  are powerful, intuitive and provide an objective position from which to assess results. To handle these complex data analysis problems, the University of Pittsburgh has formed the Pittsburgh Supercomputing Center (PSC) headed by Dr. Arthur W. Wetzel, in a joint effort with Carnegie Mellon University and Westinghouse Electric Company, and is to date the most powerful open-resource computer available.There are many examples of the value of weaving molecular imaging into Investigational New Drug Development. At the same time, the scale of the initial investments required vs. perceived benefits may not gain the necessary support of decision makers for application into development programs. There is a clear need to educate on the power and limitations of nuclear imaging techniques within the context of enhancing new drug development. Within this context, a primary goal for TR is to emphasize the cultural and operational shifts required of various stakeholders including academia, in order to better partner with industry.The term imaging covers a range of available techniques, including discovery autoradiography, small animal imaging (PET and MRI), traditional anatomical imaging , functional imaging  and many new tracers are available as are techniques with increased sensitivity to enable micro-doing studies (AMS) . NuclearFirstly demonstrating drug penetration into the tissue of interest and co-localization or binding with the intended target through receptor occupancy , including describing dose vs. target occupancy curves remains a key objective an done used frequently in early clinical research. A second objective involves the quantification of a compound's pharmacokinetic (PK) profile using radio-labeled compound, an analysis that can be performed on a region of interest basis e.g. to assess time on target as well as potential therapeutic benefits vs. side effects. Additionally, imaging can be used to quantify pharmacodynamic (PD) effects of drug action and their relationship to administered dose. In combination, PK/PD information thus derived can be used to select a dose with which to test the clinical hypothesis or help quantify the therapeutic index. From a TR perspective all these techniques can be applied in the discovery and preclinical phases to facilitate compound selection and optimization as well as in the clinical phases.A key question emerges in applying these technologies: "How best to get it done" and the debate of internal imaging centers vs. external networks and academic relationships quickly emerges. On balance, it is clear that there is not one ideal solution here rather in general a collaborative approach between industry and academia is recommended. As a consumer of medical imaging, industry is a critical player in driving innovation and the paradigm shift towards more frequent yet appropriate utilization. However, a partnership approach ultimately generates better value and cost-effectiveness for the Imaging discipline as a whole.TR is an approach to foster communication between the scientific community and clinical practitioners. To maximize the value this can bring requires that public and governmental education has to be improved in order to leverage understanding and advocacy. There are many benefits to be accrued from this, not least of which being for the patient that is waiting for meaningful therapeutic advances. New drugs have to be developed fast and show effect on the right target at the earliest possible stage of development in order for industry to become more innovative and productive and medicines to be less expensive.Amongst other specific aspects required, are the strengthening of educational opportunities for physician scientists to help prepare them to conduct effective TR. At the same time, discovery science should be conducted by scientists who have been trained in relevant disciplines including cell biology and pharmacology as well as molecular biology. This in turn requires grant support for TR-related projects. Specifically, young scientific investigators should have more access to grants from governmental bodies and foundations in order to conduct research on clinical samples. This funding is largely in the hands of government leadership. Other points for disseminated education include the availability of a plethora of tools available to conduct and advance TR and development opportunities that include high quality clinical sample collection.Dr. Marincola founded the Journal of Translational Medicine, an Open Access, peer-reviewed online journal, so that more therapeutic insights may be derived from new scientific ideas – and vice versa .Lastly, since TR is information intensive, considerable efforts are required to provide accessible databases and share knowledge. To help ameliorate this gap and provide access to information derived from human experimentation and to optimize the communication between clinicians and scientist, In conclusion, TR represents a team effort, since no single constituency can be fluent in all aspects, and thus a concerted effort is needed amongst translational researchers to convince stakeholders and legislators of the need to support TR efforts, and thus maximize its potential.


This study has evaluated urinary tract injuries and dysfunction after Radical Hysterectomy (RH) performed in patients with cervical cancer and has compared the cystometric parameters and urinary complications occurring in these patients with those occurring in patients who had undergone Simple Hysterectomy (SH).A prospective case-control study was conducted to evaluate urinary tract injuries (intra-operative and post-operative) and dysfunction in 50 patients undergoing RH for cervical cancer and to compare them with the same parameters in 50 patients who underwent SH for benign disease.Mean age in the RH group was 46.3 years and in the SH group was 50.1 (p = 0.63). There were no bladder and urethral injuries in either group of patients. There was one intra-operative ureteral injury in the RH patients but none in those who underwent SH. (p < 0.05). In the two weeks after surgery, 15% of RH patients and 11% of SH patients had experienced a urinary tract infection urinary tract infection (p = 0.61). Two week after surgery 62% of RH patients had no urinary symptoms, compared to 84% in the SH group who did (p < 0.02). Urinary residual volume, first urinary sensation and maximal bladder capacity were higher in the RH group, but this was not statistically significant. The only case of a urinary fistula appeared in a patient who received 5000 cGy radiation therapy pre-operatively, but this spontaneously healed after 3 weeks of catheterization.Intra-operative and post-operative urinary tract complications are comparable in patients undergoing RH and SH and an expert gynaecological oncologist might be able to further decrease complications. However, radiation therapy before surgery may increase the risk of complications. Although the incidence of lower urinary tract complications after RH has been reported with variable rates, up to one half of patients undergoing RH experience at least one lower urinary tract symptom that develops after surgery and at a variable period of time ,2. SeverIn this study, we prospectively evaluated intra-operative urinary tract injuries in addition to post-operative urinary tract dysfunction and infection at 2, 6, and 14 weeks following surgery. We also compared these findings with those at the same times in patients who underwent SH for benign disease.Between October 2000 and December 2002, 50 women who underwent RH and bilateral lymph node dissection (BLND) were considered eligible for inclusion in the study. All patients had squamous cell carcinoma of cervix (SCC Cx.) and were staged as being Stages I and II. The operations were performed by the same gynaecological surgeons, using the same standard technique (class III Piver & Rutledge). Pre-operative management was standardised for all patients. Preoperatively a detailed medical history, physical examination, routine laboratory tests, pelvic CT-scan , urine analysis, and urine culture were carried out.The exclusion criteria were; a history of voiding dysfunction, previous pelvic surgery, brain or spinal cord diseases, diabetes mellitus, and contraindications to urodynamic studies. The latter included a history of vesicoureteral reflux, hydronephrosis, frequent or recent urinary tract infection or urethral stricture. Patients received one pre-operative and three post-operative doses of a second generation cephalosporin (Cephazolin).The duration of surgery, amount of intra-operative haemorrhage and the occurrence of any organ injuries were recorded. A Foley's catheter was inserted at the time of surgery and was left in place for two weeks after surgery. The patient's urinary catheter was removed when their post-voiding residual volume was less than 75 ml.Water cystometry, urinalysis and urine culture were performed at 2, 6, 14 weeks after operation. The test for water cystometry was performed with the subject lying in a supine position. A 12F double-lumen catheter was introduced transurethrally into the bladder to withdraw residual urine. The pressure-volume relationship of the bladder was determined by filling the bladder with isotonic saline at a rate of 30–50 ml/min. The cystometry fill phase ceased when the patient experienced an urge to void urine, the first indication being leakage through the urethra, or a bladder volume of 600 ml (which ever occurred first). The volume at the termination of the fill-phase was designated as the maximum bladder capacity (MBC). We also assessed the bladder volume of each patient at their first desire to void . Post-void residual urine volume (RU) was determined by transurethral catheterization after voiding had ceased. The presence or absence of any urinary symptoms was determined by both questionnaire and direct interview with the patient.Fifty patients with benign disease who had underwent SH, were evaluated at the same time periods in the same way for comparison with the RH group of patients. In the SH group of patients, the Foley's catheter was inserted for 24 hours after operation. Data were analysed by SPSS statistical software using the chi-square and Student's 't' test for data analysis.A and IIB stages, respectively. Patients who had stage IB2 or higher stages of cervical cancer received 4500–5000 cGy of irradiation pre-operatively. None of these patients received adjuvant radiation during the interval between surgery and performance of urodynamic studies.During this study, 50 patients with early stage cervical cancer and who underwent RH for cervical cancer and 50 patients who had undergone SH for benign disease were evaluated. Two patients in the RH group and 3 from the SH group were lost during the study. The mean ages and their BMIs (Body Mass Index) in two groups of patients were not statistically different. However, parity in the RH group was higher (p < 0.05) Table . In the In the SH group, the most common pathological conditions requiring hysterectomy were as follows; dysfunctional uterine bleeding (47.83%), uterine myoma (21.7%), ovarian cyst (10.8%), chronic pelvic pain (4.3%), adenomyosis (4.3%), endometrial cancer (4.35%), CIN (4.35%) and molar pregnancy (2.17%).1 cervical cancer) in the RH group had an intra-operative ureteral injury , and the ureteral anastomosis was carried out at that time. The urinary catheter and ureteral stent were removed four weeks after operation.The average blood loss and mean operative time for both groups are shown in table 2 cervical cancer) had received chemo-irradiation (5000 cGy) pre-operatively. She had a spontaneous urine leakage from the vagina approximately 2 months after surgery. A retrograde cystography revealed a minute vesico-vaginal fistula. After 3 weeks of continuous bladder drainage, the fistula resolved spontaneously and she had no urinary leakage at her follow-up visits.Another patient  are showed in table Modern surgical techniques have resulted in a decrease in the incidence of lower urinary tract complications occurring as a result of radical hysterectomy. In particular, in recent times, various surgical strategies have been developed to avoid damaging the inferior segment of the cardinal ligament as well as the terminal bundle in the uterosacral and pubocervicovesical ligaments. This has made it possible for patients' lower urinary functions to return more rapidly to their pre-operative states ,5. Howevet al, reported a 6.6% rate of intra-operative urinary tract injuries during radical surgery for cervical carcinoma [In a study by Zaino and colleagues intra-operative complications were reported to occur as being 4.5% urinary tract and 8.7% other organs  [arcinoma . In our et al, [Zaino et al,  reportedet al,  and thiset al, . In our et al,  but lesset al, . Also, Cet al, .et al reported that 67% of patients had impairment or absence of bladder sensation after a RH [et al, 84% of patients had an increased first desire to void and maximal capacity in the post-operative period [In our study, urinary tract dysfunctions that followed radical surgery were that 4% had an abnormal post-voiding residual volume at the first post-operative visit. The first voiding sensation at the third visit was 49% and stress urinary incontinence was 17%. However, maximal capacity remained abnormal in 65% of cases by 14 weeks after surgery. Ralph ter a RH . In the e period .Urinary symptoms in our study occurred in 20% 2 weeks after operation and which were higher in patients with pre-operative radiotherapy . Urinary symptoms remained high at the third post-operative visit, although they declined in patients who had undergone surgery alone.et al, (mean was 45 years) [In our study, the patients mean age; BMI, parity, operative time, and blood loss were higher in those undergoing RH. The mean age of our patients was higher than patients in the study by Vervest 5 years) . Also in5 years)  the patiIn recent years, several studies support the role of a gynaecological oncologist who is specifically trained in such aspects of care and who can obtain optimal cytoreductive surgery in patients with ovarian carcinoma ,13. TherThe author(s) declare that they have no competing interests.NB carried out the surgery and participated in drafting the manuscript.FG carried out follow-ups.HA participated in the design of the study and helped to draft the manuscript.HK helped in follow-ups and performed the statistical analyses.PH participated in the design of the study and helped to draft the manuscript.All authors read and approved the final manuscript.


HIV/AIDS is the most dramatic epidemic of the century that has claimed over two decade more than 3 million deaths. Sub Saharan Africa is heavily affected and accounts for nearly 70% of all cases. Despite awareness campaigns, prevention measures and more recently promotion of anti viral regimens, the prevalence of cases and deaths is still rising and the prevalence of systematic condom use remains low, especially in rural areas. This study identifies barriers to condom use based on the Health Belief Model (HBM) in Benin, West Africa.The study was a cross-sectional survey conducted from June to July 2002. Two hundred fifty one (251) individuals were interviewed using a structured questionnaire adapted from a standardized WHO/GAP questionnaire. A logistic regression was used to identify factors associated with condom use.In spite of satisfactory knowledge on HIV/AIDS transmission, participants are still at high risk of contracting the infection. Sixty three (63) percents of the interviewees reported being able to recognize infected people, and condom use during the last occasional intercourse was declared by only 36.8% of males and 47.5% of females. Based on the HBM, failure to use condom was related to its perceived lack of efficacy [OR = 9.76 (3.71–30.0)] and perceived quality [OR = 3.61 (1.31–9.91)].This study identifies perceived efficacy (incomplete protective effect) and perceived utilization-related problem (any reported problem using condoms) as the main barriers to condom use. Hence, preventions strategies based on increasing perceived risk, perceived severity or adequate knowledge about HIV/AIDS may not be sufficient to induce condom use. These data will be useful in designing and improving HIV/AIDS prevention outreach programs in Sub Saharan Africa. One of the current challenging tasks faced by health professionals and scientists worldwide is the prevention and control of HIV/AIDS. This disease claims yearly a huge toll of deaths, productivity and economic losses, especially in sub-Saharan Africa where the population is already weakened by poverty, malaria and tuberculosis ,2. CurtaBenin is a West-African country with a population of 6.2 million people. The male/female sex ratio is 0.96, 48.5% of the population is under age 15, the rate of literacy is nearly 30.0 % and farming as the main occupation [The sample size was calculated based on results from the Benin 2001 Demographic and Health Survey . A stratData were stored in a file using Epi info 2000  and thenThe age distribution of the study population was similar to that of the Benin population . Table 1Table The overall condom use in this population was low 34%). Table 4%. TableHowever, it is interesting to note that whilst females declared ever using condom less often than males, they declared having used the condom during the last occasional intercourse more often. In particular, even though 73% of females declared never using the condom, 47% declared having used it during the last occasional intercourse. None of these differences were significant but they clearly indicated contradictory tendencies and answers to the questionnaire. It may be that among females who ever used condom, they use it more frequently than males. Finally, the median number of sexual partners during the last 12 months varied by gender , by education (2 for some versus 1 for none) and by marital status (2 for single versus 1 for other) but not by age groups.high perceived risk of contracting HIV infection among interviewees: 94% considered themselves as vulnerable to HIV/AIDS. This proportion was higher in females compared to males. Similarly there was a high perceived severity of HIV/AIDS: 99% of females compared to 87% of males perceived HIV/AIDS as a severe and deadly disease. Conversely, there was a relatively low perceived efficacy of condom as a protective measure: only 37% of the interviewees perceived condom as an effective mean in protecting from getting HIV infection. We identified several socio-cultural barriers to behavioural change namely reported problems using condom (88% of the interviewees), the alleged capability to physically recognize an HIV infected person and the denial all together of the disease (only 19% participants believe HIV/AIDS exists). Also, cultural practices such as polygamy (20% of the study population), poverty, the belief that there is a cure for the disease (74%) and religion (9 % of non favorable reaction towards condom are among declared Christians) were all not favorable to HIV infection control.Overall, there was a Table This study was the first ever to use the Health Belief Model (HBM) to assess cultural behaviour in rural Benin towards condom use and HIV/AIDS. The HBM was reported to be one of the most widely used behavioural frameworks for more than five decades but has been criticized for its inability to efficiently predict people's behaviour . There iOur results showed there is a high awareness on AIDS in general and that women knew more about the modes of transmission of HIV/AIDS and its impacts than men. Conversely, women were more likely to feel that they could identify HIV-infected individuals from their symptoms. In addition, females were less likely to declare using condom in general even though a higher proportion declared having used condom during the last occasional sexual intercourse. This finding is disturbing and could be explained by the difference in perception of the question "do you use condoms?" It is difficult to judge what the true answer is but it is likely that rare events are better reported, and thus women may be more prone to recall the use of condom than men during occasional intercourse given that they declared on average fewer sexual partners. It is also possible that among women who do use condom, they will use it more regularly than men.Our measure of perceived vulnerability might not be sensitive enough to capture differences in perceived risks. In fact, all women and most men felt they were at risk of acquiring the infection, yet only a small proportion were using condoms. Another explanation may be that perceived risk is not a driving force in behavioural change in this subset of the population. This is an illustration of the complexity of modeling human behaviour and can thus make a case for further cultural-specific HIV-behavioural research. When only considering the percentage of condom use by gender, females appear to be at a higher risk of acquiring HIV even though they appeared to know more about transmission routes and prevention methods. This might be due to the well established difficulty facing women in negotiating the terms of sexual intercourse. In fact, gender inequality is associated with poverty, condom with distrust and sexual economic exchange is not perceived as prostitution . All theDespite a relatively acceptable knowledge of modes of transmission and prevention methods, only a few of participants declared using condoms, which is an indication that a relatively good knowledge about HIV/AIDS, even though necessary, may not be a key factor in behavioural change in fighting HIV epidemic in the study population. These findings also indicate that programmes which aim only at increasing awareness and knowledge may not succeed.Using the HBM to analyze the determinants of behavioural change in our study population, we can conclude that there is a high-perceived vulnerability and perceived severity, and yet this does not encourage condom use. An important proportion of participants do not believe in the efficacy of condoms and there are barriers to the use of condoms.Our results are comparable to that found in a similar study in the USA  and in aOne limitation of our study was that for ethical reasons, subjects less than 15 years old were excluded even though some may have already been sexually active. Also, there was a potential selection bias by not having equal number of interviewers by gender, which resulted in an over-sampling of males. Our results would be biased if the reason for poor recruitment of women was linked to their behaviours, which is not likely to be the case. There were three males interviewers for one female , and interviewer/participants must be from same gender. For the purposes of the analysis we assumed that reported knowledge and behavioural risk factors are independent. Finally there is no evidence for the validity or reliability for the original WHO questionnaire, however its use allows for comparability of results across settings.Condom use, in our study population, depends on its perceived quality and perceived efficacy. There is an indication that behavioral communication change strategies based on increasing perceived risk or vulnerability of the population or based on fear factor by increasing perceived severity of HIV/AIDS are less likely to be deterrent towards condom use and require more researches. HIV outreach programs must target more barriers of condoms use. Condom outreach programmes should be defined at community level and must be defined in association with the community, using problem-solving techniques and selecting the most relevant targets, based on their importance and changeability . Data frThe author(s) declare that they have no competing interests.SHH conceived of the study, designed the protocol, carried out and supervised the field work and data collection, performed and interpreted the statistical analysis and wrote the manuscript. HC participated in analysis and interpretation of the data, and in the writing of the manuscript. NJH contributed in earlier analysis of the data and reviewed the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Since it has been reported that inhibition of proteasome activities may prevent cancer, the effects of proteasome inhibitors on arachidonic acid release from cells and on prostaglandin I2 production in rat liver cells is inhibited by actinomycin D.The proteasome inhibitors, epoxomicin, lactacystin and carbobenzoxy-leucyl-leucyl-leucinal, stimulate the release of arachidonic acid from rat glial, human colon carcinoma, human breast carcinoma and the rat liver cells. They also stimulate basal and induced prostacycin production in the rat liver cells. The stimulated arachidonic acid release and basal prostaglandin IStimulation of arachidonic acid release and arachidonic acid metabolism may be associated with some of the biologic effects observed after proteasome inhibition, e.g. prevention of tumor growth, induction of apoptosis, stimulation of bone formation. The proteasome degrades many cellular proteins, several with regulatory functions. It is not surprising that proteasome inhibitors affect many biologic processes  includin++-dependent proteases, calpain, papain, chymotrypsin, trypsin and cathepsin are not affected by epoxomicin even at a 50 μM concentration . Some oevention -24. It ievention .3H] AA (91.8 Ci/mmol) was obtained from NEN Life Science Products, Inc. . Epoxomicin, lactacystin and ZLLL were purchased from Biomol . All other reagents were from Sigma Chemical Co. . Rat liver cells incubating with lactacystin (5.4 μM) or ZLLL (1.0 μM) for 6-h have been tested for viability by a tetrazolium-based assay and found not to be toxic  AA (0.2 μCi/ml) were added and the cells incubated for another 24-h. The cells were washed 4 times with MEM and incubated for various periods of time with 1.0 ml of MEM containing 1.0 mg BSA/ml (MEM/BSA) and different concentrations of each compound. The culture fluids were then decanted, centrifuged at 2000 × g for 10 min, and 200 μl of the supernate counted for radioactivity. Radioactivity recovered in the washes before the incubation was compared to input radioactivity to calculate the % radioactivity incorporated into the cells AA, was added after the first 24-h incubation. The cells were incubated for another 24-h, washed three times with MEM, then incubated with the compounds in MEM/BSA for various periods of time. The culture fluids were decanted and analyzed for 6-keto-PGF1α, the stable hydrolytic product of PGI2, by radioimmunoassay  AA release is presented as a percentage of the radioactivity incorporated by the cells. Except for the time-course experiments which used duplicate dishes, three to five culture dishes were used for each experimental point. The data are expressed as mean values ± SEM. The data were evaluated statistically by the unpaired Student's t-test. A P value < 0.05 was considered significant.The [


Relatively few U.S.-based studies in chronic kidney disease have focused on Asian/Pacific Islanders. Clinical reports suggest that Asian/Pacific Islanders are more likely to be affected by IgA nephropathy (IgAN), and that the severity of disease is increased in these populations.To explore whether these observations are borne out in a multi-ethnic, tertiary care renal pathology practice, we examined clinical and pathologic data on 298 patients with primary glomerular lesions  at the University of California San Francisco Medical Center from November 1994 through May 2001. Pathologic assessment of native kidney biopsies with IgAN was conducted using Haas' classification system.Among individuals with IgAN (N = 149), 89 (60%) were male, 57 (38%) white, 53 (36%) Asian/Pacific Islander, 29 (19%) Hispanic, 4 (3%) African American and 6 (4%) were of other or unknown ethnicity. The mean age was 37 ± 14 years and median serum creatinine 1.7 mg/dL. Sixty-six patients (44%) exhibited nephrotic range proteinuria at the time of kidney biopsy. The distributions of age, gender, mean serum creatinine, and presence or absence of nephrotic proteinuria and/or hypertension at the time of kidney biopsy were not significantly different among white, Hispanic, and Asian/Pacific Islander groups. Of the 124 native kidney biopsies with IgAN, 10 (8%) cases were classified into Haas subclass I, 12 (10%) subclass II, 23 (18%) subclass III, 30 (25%) subclass IV, and 49 (40%) subclass V. The distribution of Haas subclass did not differ significantly by race/ethnicity. In comparison, among the random sample of patients with non-IgAN glomerular lesions (N = 149), 77 (52%) patients were male, 51 (34%) white, 42 (28%) Asian/Pacific Islander, 25 (17%) Hispanic, and 30 (20%) were African American.With the caveats of referral and biopsy biases, the race/ethnicity distribution of IgAN differs significantly from that of other major glomerulonephridities. However, among individuals undergoing native kidney biopsy, we see no evidence of a race/ethnicity association with severity of disease in IgAN by clinical and IgAN-specific histopathologic criteria. Further studies are needed to identify populations at higher risk for progressive disease in IgAN. IgA nephropathy (IgAN) is the most common form of glomerulonephritis (GN) worldwide . ApproxiClinical reports suggest that individuals of Asian/Pacific Islander heritage are more likely to be affected by IgAN than whites, African Americans, and persons of Hispanic descent. Reports from U.S. centers have generally compared results of white and African American IgAN patients, with little or no available information on U.S. patients of Asian/Pacific Islander heritage. Studies from Japan and China have reported that more individuals with ESRD in these countries had IgAN, implying that IgAN may have a more severe disease course in certain Asian populations ,7.To explore whether IgAN was more common and severe among Asian/Pacific Islanders in our population, we examined clinical and pathologic data on 149 patients with IgAN and a random sample of 149 patients with other primary glomerular lesions  at the University of California San Francisco (UCSF) Medical Center.The records of 183 percutaneous native and transplant kidney biopsies with a diagnosis of IgAN received between November 1994 and May 2001 at the Renal Pathology Laboratory at UCSF were reviewed. Baseline demographic and clinical data included age, gender, race or ethnicity, history of kidney transplant, date of biopsy, and serum creatinine concentration at the approximate time of biopsy. In addition, the presence or absence of heavy proteinuria  and the presence or absence of hypertension at the time of biopsy were recorded. Major ethnic groups included white, Asian/Pacific Islander, Hispanic, African American, and other/unknown. Ethnicity was determined using information from patient health insurance forms and history provided at the time of biopsy. Any case in which Henoch-Schönlein purpura, systemic lupus erythematosus or chronic liver disease were considered likely diagnoses were excluded, as were cases of IgAN superimposed on a systemic disease involving the kidney . Two examiners unaware of the clinical data independently reviewed the biopsies. Biopsies displaying fewer than six glomeruli by light microscopy or insufficient immunofluorescence staining, as defined below, were also excluded. Fifteen biopsies were additionally excluded due to incomplete recovery of microscopic slides from files. Biopsies from 149 patients, including 25 kidney transplant recipients, satisfied the criteria for inclusion and provided the basis for the IgAN analytic sample.Aside from IgAN, the most commonly diagnosed primary glomerular lesions at our institution over the same time period were focal segmental glomerulosclerosis (N = 314), membranous nephropathy (N = 197) and minimal change disease (N = 147). To establish baseline race/ethnicity prevalences for our region and referral population, we collected demographic data on a computer-generated random sample of individuals with non-IgAN glomerular disease (N = 149), stratified by kidney transplant.Pathologic assessment of the IgAN native kidney biopsies was performed based on Haas' IgA nephropathy classification system . All cas2 test for categorical variables, and analysis of variance  or Kruskal-Wallis test for continuous variables. Two-tailed P-values <0.05 were considered statistically significant. SAS version 8.2 was used for all statistical analyses .Demographic and clinical data are reported as mean ± standard deviation, medians with interquartile ranges, and proportions with 95% confidence limits. Inter-ethnicity comparisons were performed using the Cochrane-Mantel-Haenzsel χPatient clinical characteristics for the IgAN group are summarized in Table P = 0.64). Sixty-six patients (44%) exhibited heavy (≥ 3 g/d) proteinuria, and 74 (50%) had documented hypertension (systolic blood pressure ≥ 140 or diastolic blood pressure ≥ 90 mm Hg) at the time of kidney biopsy. The fractions of patients with heavy proteinuria and hypertension at the time of kidney biopsy were not significantly different among white, Hispanic, and Asian/Pacific Islander groups.The median serum creatinine (SCr) of the IgAN cohort was 1.7 mg/dL (interquartile range 1.1–3.4 mg/dL). The median SCr of the African American group was signficantly higher (5.0 mg/dL) than the other ethnic groups; however, these calculations were based on a small sample size (N = 4) due to the low prevalence (3%) of African Americans with in our IgAN cohort. Median serum creatinine concentrations were not significantly different among white, Hispanic, and Asian/Pacific Islander groups  fell into Haas subclasses IV or V, which are known independent predictors of progressive disease and poor renal outcomes . Only 22P = 0.006) compared to patients with IgAN. In addition, the distribution of race/ethnicity differed significantly between the two groups (P < 0.001). This association of IgAN and distribution of race/ethnicity persisted even when stratified by kidney transplant .Table In a biopsy series of 244 patients with IgAN, Haas found fewer African Americans (in a major urban setting), similar to that noted in other U.S.-based studies of IgAN -11. WhilIn our study, the fraction of biopsies in subclasses I and II (18%) was similar to that observed by Haas (23%). However, we observed a higher proportion of biopsies in subclasses IV and V (64% vs. 31%), and a lower proportion of biopsies in subclass III (19% vs. 45%) compared with Haas, possibly reflecting a temporal trend towards a higher biopsy threshold along with intergrader measurement bias.In a study reviewing the pattern of glomerulonephritis in Singapore over the past two decades, Woo and colleagues reported that IgAN was the most common primary GN occurring in Singapore  . In our In contrast, based on smaller biopsy series, a striking variation in prevalence rates of IgAN has been reported from Europe and South America. In the UK, for example, Ballardie and colleagues noted comparatively low prevalence rates of IgAN in a predominantly white population  in the early 1970's. In the subsequent 15-year period, however, these investigators reported a phenomenal rise in the observed incidence of IgAN , which the investigators felt more likely reflected a higher frequency of detection rather than true rise in disease incidence. Similar prevalence rates have also been documented from isolated white populations in Finland and southern Italy ,17. In cThese differences may be partially attributed to increased screening and disparities in the indication for kidney biopsy. In Japan and South Korea, for example, school-aged children undergo annual screening for urinary abnormalities; kidney biopsy is subsequently recommended for children with evidence of proteinuria or hematuria . More coH. parainfluenzae as a causative agent of IgAN in Japanese children and adults. Such claims are supported by studies showing the glomerular deposition of outer membrane H. parainfluenzae antigens and greater levels of plasma IgA1 antibody against OMHP in Japanese patients with IgAN  [H. parainfluenzae colonization and/or infection has yet to be established.Although the etiology of IgAN remains unknown, there exists a strong suspicion for an environmental antigen trigger combined with a genetic susceptibility factor. Along these lines, several hypotheses have been proposed to account for the reportedly higher prevalence of IgAN in Asian/Pacific Islanders. With respect to potential dietary antigens, Wakai et al. found that high intake of rice and n-6 polyunsaturated fatty acids (PUFA) were associated with an increased risk of IgAN . Recent iseases) ,23. WhetThe presence of either hypertension or proteinuria ≥ 3.0 g/24 hrs at the time of diagnosis significantly correlated with worsened renal survival in IgAN, even when controlling for serum creatinine at the time of kidney biopsy [Despite ongoing investigative efforts, scant data are available regarding genetic markers that may predispose individuals to progressive disease from IgAN. Recent immunogenetic studies have suggested a potential role for the T-cell receptor (TCR) in the development of immune-mediated diseases. Deenitchina and colleagues found that genetic polymorphism of the TCR constant alpha chain was associated with progression of CKD in a cohort of Japanese patients with IgAN. Although promising, such polymorphisms of the TCR gene have yet to be evaluated in large, prospective studies or by genetic analysis of familial IgAN .Our results contest the assertion that IgAN follows a more severe course in individuals of Asian/Pacific Islander descent. One reason for the similar disease severity of IgAN in our study population may stem in part from the large subpopulation of Filipino patients comprising our Asian/Pacific Islander cohort. It is unclear whether certain subpopulations of Asian/Pacific Islanders, including Filipinos, exhibit IgAN prevalence rates similar to those documented by Koyama and Woo. Anecdotal reports from Thailand and India documenting prevalence rates of 4–9% suggest that IgAN may not have the same epidemiology among all southeast Asians . DespiteThere are several important limitations to this report. As with any single-center biopsy series, we may have been underpowered to detect a clinically significant difference due to the limited sample size (type II error). In addition, racial admixture may have also confounded the results, as we were unable to subclassify patients in the Asian/Pacific Islander group or account for the growing population of bi- or multi-ethnic individuals in our population. Furthermore, due to the study's case control design, and breadth of our referral base , we were unable to control for the criteria for kidney biopsy. As a result, a biopsy bias may have confounded our results. In other words, Asian/Pacific Islander patients in our referral base with mild to moderate proteinuria and/or hematuria might have been given a presumptive diagnosis of IgAN without nephrology referral or confirmatory kidney biopsy. With regard to disease prevalence, these potential referral and biopsy biases based on race/ethnicity are largely conservative in nature, and would have biased our results towards the null. Finally, we have included data from a modest-sized IgAN transplant population N = 25), the donor demographics of which were unavailable at the time of the study. However, the association of race/ethnicity and distribution of glomerular lesion persisted, even when stratified by kidney transplant, and thus our overall conclusions remained the same. In addition, a small European study of donor-recipient pairs (average follow-up 7 years) has shown that when a donor kidney with asymptomatic IgA deposits is transplanted into a recipient with ESRD secondary to a disease other than IgAN, the IgA immune deposits in the donor kidney are rapidly removed [, the donIn conclusion, with the caveats of referral bias and biopsy bias, the race/ethnicity distribution of IgAN differs significantly from that of other major glomerulonephridities. However, among individuals undergoing native kidney biopsy, we see no evidence of a race/ethnicity association with severity of disease in IgAN by clinical and IgAN-specific histopathologic criteria. Further studies are needed to identify populations at higher risk for progressive disease in IgA nephropathy.None declared.YH designed the study, collected and analyzed the data, and drafted the manuscript. GC supervised the study design, analyzed the data, and edited the manuscript. EF graded the IgAN histopathologic slides. JO collected, reviewed and graded all histopathologic data, and edited the manuscript. All authors approved the final manuscript.The pre-publication history for this paper can be accessed here:


The study aimed to investigate the role of these nanoparticles on the release of the pro-inflammatory cytokine TNF-α and IL-1α gene expression. We also investigated the role of intracellular calcium signalling events and oxidative stress in control of these cytokines and the effect of the particles on the functioning of the cell cytoskeleton. We showed that there was an increase in intracellular calcium concentration in J774 cells on treatment with PM10 particles which could be significantly reduced with concomitant treatment with the calcium antagonists verapamil, the intracellular calcium chelator BAPTA-AM but not with the antioxidant nacystelyn or the calmodulin inhibitor W-7. In human monocytes, PM10 stimulated an increase in intracellular calcium which was reduced by verapamil, BAPTA-AM and nacystelyn. TNF-α release was increased with particle treatment in human monocytes and reduced by only verapamil and BAPTA-AM. IL-1α gene expression was increased with particle treatment and reduced by all of the inhibitors. There was increased F-actin staining in J774 cells after treatment with PM10 particles, which was significantly reduced to control levels with all the antagonists tested. The present study has shown that PM10 particles may exert their pro-inflammatory effects by modulating intracellular calcium signalling in macrophages leading to expression of pro-inflammatory cytokines. Impaired motility and phagocytic ability as shown by changes in the F-actin cytoskeleton is likely to play a key role in particle clearance from the lung.The effects of PM At 25 μg/ml the [Ca2+]c decreased to a value similar to the 5 μg/ml particle concentration. . The [Ca2+]c following concomitant treatment of cells with particles and calcium antagonists was reduced . In our previous studies c had previously been observed .PMd figure . At timease in Ca+c had pr10 and UfCB particles on TNF-α release by J774 macrophages is shown in figure 10 particles caused significantly more TNF-α release as the same mass of UfCB particles by the mouse macrophage cell line.A comparison of the gram for gram dose effect of PM10 is shown in figure 10 induced TNF-α release with W-7 (W), Trolox (T) or Nacystelyn (N) at any particle dose tested.The release of TNF-α protein by human monocytes after treatment with varying concentrations of PM10 for 4 hours produced a significant increase in IL-1α mRNA content compared with unstimulated cells (p < 0.05) .Treatment of human peripheral blood monocytes with 10 μg/ml PMs p < 0.0 , although the increase in the fluorescence intensity of the PM10-treated cells was modest (a 5% difference).The fluorescence intensity of cells stained for F-actin after treatment with PM10 are considered to drive these effects c changes lead to production of the proinflammatory cytokine TNF-α [10 particles can also stimulate entry of extracellular calcium into both J774 macrophages and human macrophage derived monocytes, and that this process is inhibited by a calcium channel blocker suggesting that the PM10, in a similar fashion to UfCB induces opening of plasma membrane calcium channels leading to a calcium influx. The results obtained using the antioxidant nacystelyn were confusing. In the J774 macrophages nacystelyn was unable to inhibit PM10 induced increases in cytosolic calcium concentration, whereas the same antioxidant was very effective in the human monocyte derived macrophages. This difference could be due to a species difference or a comparison between a cell line and primary cells. A number of cell lines have been demonstrated to exhibit aberrant calcium signalling pathways. Our previous studies using human macrophages suggest that ultrafine particle-induced increases in cytosolic calcium can be mediated by ROS [10 are ultrafine, it is conceivable that much of the calcium increase is ROS mediated, at least in part. However, PM10 also contains other substances, such as metals, that could influence this pathway. Metals would in fact be expected to increase the ROS production by the PM10 particles [We have previously shown that ultrafine or nanoparticles enhanced the calcium influx into cells of a monocytic cell line (MM6) ,34 and tne TNF-α . We demod by ROS  and sincarticles .10 (10 μg/ml) that induces calcium elevation also stimulates significant increases in both TNF-α protein release and IL-1α mRNA production by macrophages. The calcium channel blocker verapamil and the intracellular calcium chelator BAPTA-AM reduced the calcium increase, TNF-α protein release and IL-1 mRNA expression by human monocytes when stimulated with PM10 particles. This is strong evidence to suggest that influx of extracellular calcium plays a key role in upregulating the proinflammatory response induced by PM10 that could lead to disease. However, the calmodulin inhibitor W-7 had little effect on TNF-α release, while it did inhibit IL-1 mRNA expression. The antioxidants also had variable abilities to block cytokine expression, inhibiting IL-1 mRNA production but not TNF-α protein release. These differences could be explained either by divergent pathways controlling expression of the two cytokines, or that TNF-α protein was measured in comparison to IL-1 mRNA. However, clearly both calcium and ROS are important in the regulation of IL-1α mRNA expression while only calcium is important in controlling TNF-α expression in macrophages exposed to PM10.The present study clearly shows that the same dose of PM10 is far more potent that UfCB in terms of its ability to induce TNF-α protein release. This is likely to be due to other components, such as metals and organic compounds other than the carbon core, within PM10 that can promote inflammation. It is also possible that components such as the UF particles and metals could interact to enhance toxicity as has been shown for ROS production in vitro and inflammation in vivro [10 particles, therefore it is unlikely that cytokine release, changes in intracellular calcium, and IL-1α gene expression can be explained solely by endotoxin.These studies indicate that on an equal mass basis PMin vivro . Our pre10 generates ROS [10 induced increase in F-actin staining in this study demonstrates that particle-derived ROS impact on the macrophage cytoskeleton. Our previous studies also demonstrate that Uf particle-induced ROS play a role in elevating the cytosolic calcium concentration of macrophages leading to increased TNF-α production [10 exposure. As has been shown by other workers [10 particles increased the F-actin fluorescence signal in cells stained with FITC-labelled phalloidin, although changes in the distribution of actin filaments was not apparent from microscope analysis there appeared to be more cortical staining. In accord with the role of calcium and ROS in the induction of IL-1 expression, both of these factors appeared to play an important role in modulating the F-actin cytoskeleton.As explained previously, the cytoskeleton is the scaffold of cells, and in the case of motile cells such as macrophages it is responsible for controlling movement. Disruption of the cytoskeleton, particularly via oxidative stress, is thought to disrupt cellular structure and hence function . We haveates ROS . The abioduction . The res workers ,45, in b workers  which ma10 particles may exert their increased pro-inflammatory effects by modulating intracellular calcium signalling in macrophages leading to expression of proinflammatory cytokines. An additional consideration is the effects of particles on the cytoskeleton of the cell. Impaired cellular motility and phagocytic ability is likely to play a key role in particle clearance from the lung, thus perpetuating the effects of PM10. The role of calcium and ROS in other cellular responses are under investigation.The present study has shown that PM


Neisseria species, and phylogenetic trees, to investigate whether genotypic clusters can be resolved among these recombinogenic bacteria and, if so, the extent to which they correspond to named species.It is a matter of ongoing debate whether a universal species concept is possible for bacteria. Indeed, it is not clear whether closely related isolates of bacteria typically form discrete genotypic clusters that can be assigned as species. The most challenging test of whether species can be clearly delineated is provided by analysis of large populations of closely-related, highly recombinogenic, bacteria that colonise the same body site. We have used concatenated sequences of seven house-keeping loci from 770 strains of 11 named N. meningitidis, N. lactamica and N. gonorrhoeae. A few isolates arose from the branch that separated N. meningitidis from N. lactamica leading us to describe these species as 'fuzzy'.Alleles at individual loci were widely distributed among the named species but this distorting effect of recombination was largely buffered by using concatenated sequences, which resolved clusters corresponding to the three species most numerous in the sample, Neisseria where individual loci are inadequate for the task. This approach should be applied by taxonomists to large samples of other groups of closely-related bacteria, and especially to those where species delineation has historically been difficult, to determine whether genotypic clusters can be delineated, and to guide the definition of species.A multilocus approach using large samples of closely related isolates delineates species even in the highly recombinogenic human  The definition of bacterial species, and a concept of species applicable to all bacteria, are problems that have long exercised systematists and microbiologists -4. WhileMolecular approaches to assigning bacteria to species began with the introduction of DNA-DNA hybridization, which allowed an objective assessment of the extent of sequence similarity among a set of genomes, and remains the systematicist's gold standard, defining bacterial species as those isolates whose genomes show at least 70% hybridization under standardized conditions . HoweverIndividual isolates of a named species differ in gene content  and the A multilocus approach has recently been applied to small numbers of isolates of several relatively distantly related named species of enterobacteria , and othNeisseria into genotypic clusters. We chose this example because Neisseria are naturally transformable, are among the most recombinogenic bacteria, and there is good evidence for relatively frequent localised recombination between the named Neisseria species [Neisseria isolates, but the tree based on the concatenated sequences effectively resolves the three major named species within the sample, although the boundaries are fuzzy due to the presence of a small number of intermediate genotypes.In this study we have evaluated the ability of seven individual house-keeping gene sequences, and of the concatenated sequences of these genes, to resolve a large sample of human pathogenic and commensal  species ,16 throuNeisseria MLST database, which includes several thousand sequence types (STs) of N. meningitidis and smaller numbers assigned to several other named human Neisseria species [N. meningitidis were compared with all STs assigned to the other human Neisseria species. The sequences of the seven gene fragments were concatenated in-frame and a tree was constructed (using third codon position sites) using Mr Bayes [N. gonorrhoeae, and all but two of the 171 STs of N. lactamica, descend from single well-supported nodes . The great majority of N. meningitidis also formed a single well-resolved cluster, but a few arise from the branch leading to the N. lactamica isolates. Very similar clustering of these three species was observed using other sets of 500 N. meningitidis STs from the database, and in a neighbour-joining tree constructed using all STs in the Neisseria MLST database (data not shown). The high levels of recombination in the Neisseria make the fine structure of the tree meaningless   for epid species  on the bMr Bayes . Figure N. gonorrhoeae, N. meningitidis and N. lactamica, their boundaries are not perfectly defined and a number of isolates are placed on the branch between N. lactamica and N. meningitidis, representing intermediate genotypes.Analysis of the individual gene trees shows that these fail to resolve the named species and highlights many examples where interspecies recombination has resulted in anomalous clustering Figure . The cleNeisseria species do not cluster clearly. A significant separation is observed between two subtrees  in the MLST database, using Neighbour-Joining, minimum evolution and UPGMA tree-building approaches (data not shown).The small numbers of STs assigned to other human Current molecular definitions of species use rules or cut-off values (e.g. ≥ 70% DNA-DNA hybridization) and rarely take account of the genotypic diversity within and between populations . A more N. meningitidis and N. lactamica  may be a Figure . The conN. meningitidis, N. lactamica and N. gonorrhoeae are reasonably secure; the two N. lactamica that clustered highly anomalously probably represent species mis-identification. The most critical test of the multilocus approach is the ability to resolve N. lactamica from N. meningitidis since these colonise the same body site, the nasopharynx. Resolution of these named species was remarkably good, although the boundaries between N. lactamica and N. meningitidis are somewhat fuzzy, due to the existence of intermediate forms. This is to be expected as recombinogenic bacteria have mosaic genomes, resulting from the occasional replacement of chromosomal segments with those from related populations. Thus, in any large dataset, there may be isolates in which one or more of the loci used in a multilocus approach to species definition will have been recently introduced from a related population. Single unusually divergent replacements, or replacements at more than one of the multiple loci, may place isolates away from the majority of isolates of the species. However, only seven STs in Figure N. meningitidis or N. lactamica), and there was no overlap between these two named species (i.e. a region containing isolates identified as both species interspersed with one another).Network based methods  using the multilocus approach with large datasets, should clarify whether they fall into distinct clusters, or whether the difficulties in defining species by phenotypic methods reflect an underlying genetic reality in which resolved clusters are not evident.Sorting the human commensal es names . We gainIf necessary, further resolution between apparent clusters may be attempted by increasing the numbers of loci sequenced. Provided that the alleles at these loci show a degree of specificity to a given species cluster, then the resolution of that cluster will be enhanced. If this cannot be demonstrated, then it is likely that the isolates under test do not genuinely form separate populations, and should not be considered to be distinct species. This approach lends itself to "electronic taxonomy", in which systematic classification may be evermore finely elucidated through the accumulation of online sequence databases.The work described here obviously begs the question of what forces or mechanisms could generate such separation among recombining bacteria. We offer a simple model for recombining organisms as follows: consider two populations freely recombining within themselves and with each other. New mutations arising in one population will readily spread to the other, and to an observer they appear to form one cluster of related strains. If a barrier to recombination should be erected between them, such that isolates are much more likely to undergo recombination with their own population, then the rate of generation of new genotypes within each population may increase beyond the rate at which such genetic innovation is shared and the two populations begin to diverge. As the populations diverge, decreasing sequence identity will further impede recombination, thus reinforcing the effect of the original genetic barrier and creating a permanent separation ,25.N. gonorrhoeae. Unlike the other human Neisseria, which colonise the nasopharynx, the primary niche of the gonococcus is the genital tract, and it has been proposed that gonococci arose relatively recently due to the successful invasion of the genital tract by a nasopharyngeal Neisseria lineage [It is not difficult to suggest candidate mechanisms. Niche separation is one example, and almost certainly underlies the tight well-defined cluster of  lineage . Similar lineage , limitat lineage  and simiThe point at which such a group is described as a species is a matter more of human interest and attention than any intrinsic evolutionary process. The properties of the species clusters we observe will be determined by the diversification of those strains sharing the speciation loci (i.e those that determine gene flow). Because speciation is gradual, we should be able using estimates of recombination within and between groups derived from multilocus data, to define nascent species which if they continue to diversify in isolation, are expected to form distinct sequence clusters, ie species, in the future.The bacterial domain of life is not uniform. Instead we see clumps of similar strains that share many characteristics, and with an innate human urge to classify, we have defined these as species. This work shows that by applying a simple approach using sequence data from multiple core housekeeping loci, we can resolve those clusters, provided such clusters exist. However, these species clusters are not ideal entities with sharp and unambiguous boundaries; instead they come in multiple forms and their fringes, especially in recombinogenic bacteria, may be fuzzy and indistinct. A multilocus approach using large numbers of isolates will provide data that help us to develop theoretical models of how species emerge, and relate these to the observed population genetic structure of bacteria. This should be enormously helpful to taxonomists, whose foremost duty will remain to provide us with pragmatic species designations which attempt to reflect the underlying genetic reality.Neisseria MLST database [N. meningitidis  were concatenated as described below, and analysed together with the concatenated sequences of N. meningitidis strains with ST numbers from 1 to 500. Species definitions were as recorded at [Neisseria were also used to construct individual gene trees.The contents of the publicly accessible database ,18 were MLST loci were concatenated in-frame to form a 3267 bp sequence, of which only third position sites were used in subsequent analyses. To illustrate clustering in this dataset, a tree was constructed using Mr Bayes 3.0b4 . A startrRNA ribosomal RNAMLST Multi Locus Sequence TypingST Sequence TypeUPGMA Unweighted Pair Group Method with Arithmetic MeanBGS conceived of the study and drafted the manuscript, CF participated in study design and analysis of results, WPH designed the study, carried out the analyses and interpreted the results, and drafted the manuscript.


We generated transgenic zebrafish in which expression of the firefly luciferase (luc) gene is driven by the zebrafish per3 promoter. Live larvae from these lines are rhythmically bioluminescent, providing the first vertebrate system for high-throughput measurement of circadian gene expression in vivo. Circadian rhythmicity in constant conditions was observed only after 5–6 d of development, and only if the fish were exposed to LD signals after day 4. Regardless of light exposure, a novel developmental profile was observed, with low expression during the first few days and a rapid increase when active swimming begins. Ambient temperature affected the developmental profile and overall levels of per3 and luc mRNA, as well as the critical days in which LD cycles were needed for robust bioluminescence rhythms. In summary, per3-luc zebrafish has revealed complex interactions among developmental events, light, and temperature in the expression of a clock gene.The roles of environmental stimuli in initiation and synchronization of circadian oscillation during development appear to vary among different rhythmic processes. In zebrafish, a variety of rhythms emerge in larvae only after exposure to light-dark (LD) cycles, whereas zebrafish  luciferase gene under a clock gene promoter enables the study of molecular circadian rhythms in vivoGeneration of zebrafish that express the firefly  The circadian clock controls biological processes such as behavior, gene expression, and physiology in diverse organisms, ensuring that these processes to take place at appropriate times of the day. This is crucial for many organisms, such as plants, which must synchronize photosynthesis with day-night cycles. Animals also synchronize to environmental cycles because of more subtle but nevertheless important needs such as predator avoidance, food availability, and optimal temperatures for various processes. Circadian clocks in all species share the following properties: They persist even in constant environmental conditions with periods near 24 h; they can be reset by environmental stimuli such as light and temperature; and their periods are relatively constant at different temperatures.Drosophila, has been extremely valuable in identifying important players of the clockworks and their roles ) were raised as transgenic F1 fish. Three of the five lines emitted bioluminescence above background. The level of bioluminescence varied depending on the line. The strongest-glowing line (#23) was used for this study unless otherwise stated. All the animals used in this study were the progeny of crosses between a transgenic line and the *AB wild-type strain. Therefore, these animals carried the transgene in hemizygous condition.per3-luc transgenic zebrafish is screening for mutations that affect bioluminescence rhythms. Since it is most convenient to screen the youngest possible animals, bioluminescence from transgenic embryos was monitored first. It was also expected that per3-luc-mediated bioluminescence in embryos should cycle from day 1 of development even in constant conditions, because per3 mRNA expression detected by in situ hybridization has been demonstrated to oscillate from day 1 postfertilization in constant conditions  in 2 l of ddH2O) aerated overnight and containing 0.5 mM D-luciferin potassium salt  and 0.013% Amquel Instant Water Detoxifier . Once loaded with animals, four such plates were subjected to automatic monitoring of bioluminescence every 30 min by the Topcount multiplate scintillation counter (Perkin-Elmer) equipped with six detectors and plate stackers. The room temperature was set at 21–22 °C, and the machine at 24 °C. However, due to the heat created by the machine, temperature at the bottom of the stacker was 1–2 °C higher than the room temperature. In order to minimize high background counts under lighted conditions, each plate was dark-adapted for approximately 5 min before being counted for bioluminescence. Each well was counted for 4.8 s every 30 min. The plates were illuminated with two white fluorescent lamps, each facing the left or right side of the stacker. The approximate intensity of the light that reached the plates was 17–35 lux, depending on the position of the plates within the stacker.Embryos or larval fish were placed individually in every other well of a white 96-well Optiplate  with 200 μl of Holtfreter solution . This burst of luminescence is typically followed by a low background level of luminescence (< 50 cps). Furthermore, intermediate levels of spikes were also found in many plots just before the burst of high bioluminescence. Therefore, in order to eliminate data from dead fish, data that exceeded 5,000 cps, or those that went down below 50 cps, at any point of the analyzed portion of the data were first discarded. Then an averaged plot of the remaining data from each clutch of embryos was examined, and the highest count on days 8–9 was determined for experiments presented in Bioluminescence data from the Topcount were imported into Microsoft Excel 2000 by the Import and Analysis macro . In many of the experiments performed on the Topcount, some plates were placed in the machine several days earlier than others in order to monitor fish that experienced different numbers of LD cycles . TherefoPeriod and rhythmicity for each animal were determined by a macro  based on12–18 (Invitrogen) as the primer in a 25–40 μl reaction volume. Real-time PCR was performed in a 25 μl reaction volume containing a probe, forward and reverse primers, and qPCR Mastermix according to the manufacturer's instruction . Each reaction was quadrupled in order to minimize pipetting errors. The primers and TaqMan MGB probes for per3 and luc were designed and synthesized by the Assays-by-Design Gene Expression service : per3 forward, 5′-GCCCTGGCAGCACCA-3′; per3 reverse 5′-GAAAGCTGGAGGACGAGGAA-3′; probe, 5′-6-FAM-CTAAGAGCTCAAAATCC-NFQ-3′; luc forward, 5′-GCAGGTGTCGCAGGTCTT-3′; luc reverse, 5′-GCGACGTAATCCACGATCTCTTTT-3′; probe, 5′-6-FAM-TCACCGGCGTCATCG-NFQ-3′. The ABI Sequence Detection System 7000 (Applied Biosystems) was programmed to perform the following protocol: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.Total RNA was extracted from 9–42 embryos or larval fish raised in petri dishes using TRIzol reagent . The number of animals used for each extraction was recorded. Once extracted, total nucleic acid concentration was determined by a spectrophotometer. In order to prevent genomic DNA contamination, RNA samples were treated with Turbo DNA-free , and the concentration determined again by a spectrophotometer. Total RNA (0.5–1 μg) was subjected to cDNA synthesis by Superscript II Reverse Transcriptase (Invitrogen) using Oligo (dT)per3 or luc cDNA per animal was calculated by the standard-curve method [per3 and luc were compared between two different developmental stages as well as among different times of the day. It was also important to calculate the amount of each mRNA species per animal in order to compare these data to bioluminescence data. The amount of a specific control RNA, as well as the total RNA, may differ among fish of different ages, in which case RNA per animal cannot be calculated by the relative quantification method using a constitutive control. As a concentration standard, a single-stranded DNA oligonucleotide of known concentration was used for each gene. These oligonucleotides span from the 5′ end of the forward primer to the 5′ end of the reverse primer, and including 75 bp for per3 and 110 bp for luc . The standard concentration was varied from 102 to 107 copies per reaction in 10-fold increments. For every qPCR experiment, reactions for standards were performed in four replicates along with reactions for cDNA samples.In this study, relative amount of e method  rather tt-test was performed. The nonparametric Wilcoxon/Kruskal-Wallis test was performed on data that were not normally distributed even after various transformations  were tried. Where multiple tests were performed on a set of data, the experimentwise error rate (α) was adjusted by the Dunn-Sˇidák method [To test whether percentages of rhythmic fish among different experimental groups were equal, the G-test was performed using Microsoft Excel 2000 according to Sokal and Rohlf . If multk method .Protocol S1(27 KB DOC).Click here for additional data file.http://www.ncbi.nlm.nih.gov/) accession numbers of the sequences discussed in this paper are per 3 cDNA (NM_131584) and BAC clone CH211–138E4 (AL929204). The Ensembl (http://www.ensembl.org/Danio_rerio/) ID of the flanking gene of per 3 mentioned in The GenBank (


Microarrays can survey genome-wide expression patterns. Not only can these gene expression profiles be used to identify a few genes of interest, they are now being creatively applied for hypothesis generation and testing Microarrays are used to survey the expression of thousands of genes in a single experiment. Applied creatively, they can be used to test as well as generate new hypotheses. As the technology becomes more accessible, microarray analysis is finding applications in diverse areas of biology. Microarrays are simply a method for visualizing which genes are likely to be used in a particular tissue at a particular time under a particular set of conditions. The output of a microarray experiment is called a “gene expression profile.”Gene expression profiling has moved well beyond the simple goal of identifying a few genes of interest. The notion that this is the major objective of microarray studies has engendered the oft-repeated criticism that the approach only amounts to “fishing expeditions.” The sophistication of microarray analysis very much blurs the distinction between hypothesis testing and data gathering. Hypothesis generation is just as important as testing, and very often expression profiling provides the necessary shift in perspective that will fuel a new round of progress. In many gene expression profiling experiments, the hypotheses being addressed are genome-wide integrative ones rather than single-gene reductionist queries. In general, without a hypothesis only the most obvious features of a complex dataset will be seen, while clear formulation of the scientific question undoubtedly fuels better experimental design. And in some cases, the results of a microarray screen that was initially designed as an effort at cataloguing expression differences are so unexpected that they immediately suggest novel conclusions and areas of enquiry.All microarray experiments rely on the core principle that transcript abundance can be deduced by measuring the amount of hybridization of labeled RNA to a complementary probe. The idea of a microarray is simply to lay down a field of thousands of these probes in perhaps a 5 sq cm area, where each probe represents the complement of at least a part of a transcript that might be expressed in a tissue. Once the microarray is constructed, the target mRNA population is labeled, typically with a fluorescent dye, so that hybridization to the probe spot can be detected when scanned with a laser. The intensity of the signal produced by 1,000 molecules of a particular labeled transcript should be twice as bright as the signal produced by 500 molecules and, similarly, that produced by 10,000 molecules half as bright as one produced by 20,000 molecules. So a microarray is a massively parallel way to survey the expression of thousands of genes from different populations of cells. Trivially, if fluorescence is observed for a gene in one population but not another, the gene can be inferred to be on or off, respectively. With appropriate replication, normalization, and statistics, though, quantitative differences in abundance as small as 1.2-fold can readily be detected. The output of all microarray hybridizations is ultimately a series of numbers, which covers a range of almost four orders of magnitude, from perhaps one transcript per ten cells to a few thousand transcripts per cell .It is the comparison of gene expression profiles that is usually of most interest. This is because the visualization is done at the level of transcript abundance, but just seeing a transcript does not guarantee that the protein is produced or functional. If, however, a difference in transcript abundance is observed between two or more conditions, it is natural to infer that the difference might point to an interesting biological phenomenon.A general approach to performing gene expression profiling experiments is indicated as a flow diagram in The ability to survey transcript abundance across an ever-increasing range of conditions gives geneticists a fresh look at their cellular systems, in many cases providing a more holistic view of the biology, but at the same time feeding back into the classical hypothetico-deductive scientific framework. The technology has rapidly advanced beyond the simple application of fishing for candidate genes and now sees applications as diverse as clinical prediction, ecosystem monitoring, quantitative mapping, and dissection of evolutionary mechanisms.Two of the better-known examples of the interplay between microarray profiling and hypothesis testing are provided by the studies of Much excitement has been generated recently by the potential for clinical applications of gene expression profiling in relation to complex diseases such as cancer, diabetes, aging, and response to toxins. An early foray into this realm was provided by PLoS Biology by DeRisi and colleagues (Plasmodium falciparum might identify a handful of genes that are induced at critical times and hence might be novel drug targets. Employing very careful staging, a platform with low experimental noise, and appropriate statistical procedures, they discovered an extremely tight molecular lifecycle within the organism. Families of functionally related genes are induced as a unit, one after another, in a tightly orchestrated rhythm that testifies to incredible integration of the physiology of the parasite. They show that with microarray analysis it is possible to model the physiology and biochemistry of the pathways instead of just targeting a few genes.A good example of the ability of microarray analyses to simply surprise us is provided by the study reported in this issue of lleagues . They reIn the coming years, expect to see microarrays developed for an extremely diverse range of organisms and applied to an even wider range of questions, from parasitology to nutritional genomics. Consensus on a core set of statistical options will likely emerge, as will agreement on data quality standards. Applications will encompass defining gene function; inferring functional networks and pathways; understanding how variation is distributed among individuals, populations, and species; and developing clinical protocols relating to cancer prognosis and detection of toxin exposure. Similar profiling methods for proteins and metabolites will attract just as much attention as functional genomics, building on the foundations laid by genome sequencing.