The delivery of technology-enhanced home care is growing in most industrialized countries. The objective of our study was to document, from the patient's perspective, how the level of user-friendliness of medical technology influences its integration into the private and social lives of patients. Understanding what makes a technology user-friendly should help improve the design of home care services.Four home care interventions that are frequently used and vary in their technical and clinical features were selected: Antibiotic intravenous therapy, parenteral nutrition, peritoneal dialysis and oxygen therapy. Our qualitative study relied on the triangulation of three sources of data: 1) interviews with patients (n = 16); 2) interviews with carers (n = 6); and 3) direct observation of nursing visits of a different set of patients (n = 16). Participants of varying socioeconomic status were recruited through primary care organizations and hospitals that deliver home care within 100 km of Montreal, the largest urban area in the province of Quebec, Canada.both a negative and positive effect on patients' lives. These technologies were rarely perceived as user-friendly, and user-acceptance was closely linked to user-competence. Compared with acute I.V. patients, who tended to be passive, chronic patients seemed keener to master technical aspects. While some of the technical and human barriers were managed well in the home setting, engaging in the social world was more problematic. Most patients found it difficult to maintain a regular job because of the high frequency of treatment, while some carers found their autonomy and social lives restricted. Patients also tended to withdraw from social activities because of social stigmatization and technical barriers.The four interventions have While technology contributes to improving the patients' health, it also imposes significant constraints on their lives. Policies aimed at developing home care must clearly integrate principles and resources supporting the appropriate use of technology. Close monitoring of patients should be part of all technology-enhanced home care programs. The possibility of managing patients outside of the hospital has rarely been so widely considered. Indeed, home care is often seen as less costly and more patient-friendly . HoweverSullivan  showed hdevice they are designing. Users are often expert at the task they are trying to perform with the device" [[The term user-friendly is used to characterize an object – often a computer system – as "easy to operate or understand; not needing special training" . This nodevice" [: p.156].In the field of health care, this specialization often adds to the complexity of the work of designers, who, in addition to not being end users of the device, are simply unfamiliar with the complicated tasks health care providers are achieving through the use of technology. When moving health technologies away from the hospital and into the patient's home, the design characteristics of medical devices become even more salient, since patients have to learn how to operate them safely and with confidence. According to Norman , the useBecause most computer interfaces are used in a somewhat confined environment, the work of scholars who have studied technical aids for the disabled and the elderly brings another dimension to the definition of user-friendliness. Conceptualizing disability as a social phenomenon, the user-friendliness of technical aids has to be gauged with respect to their ability to assist users' move freely in their social environment . From thSocial scientific work on patients learning how to cope with chronic illnesses and life-sustaining technology has offered similar observations, highlighting that each patient tends to go through a personal trajectory, or engage in "biographical work" whose aim is to give meaning to a constellation of unfolding events . Here alTo summarize, the literature underscores the many technical  and human  variables that influence the use of technology and which are affected by the setting  where technology use takes place. The user-friendliness of a technology therefore results from a smooth fit between human and technical features, with the fit varying between and within settings and individuals. Figure We selected four interventions: Antibiotic intravenous therapy (IV), parenteral nutrition (PN), oxygen therapy (O2) and peritoneal dialysis (PD). These were chosen because they are frequently used  and varyOur study relied on the triangulation of three sources of data. We conducted semi-directed, individual interviews with patients (n = 16) and carers (n = 6), and directly observed nursing visits, involving a different set of patients (n = 16). This strategy enabled us to gather data on a broader set of patients. The carers we interviewed were not necessarily related to a patient participating to our study (to reduce pressures on interviewees) and were spouses or family members (often mother or daughter) of a person receiving high-tech home care. Our purposeful sampling strategy was to diversify viewpoints, by including participants of varying socioeconomic status, gender and age Table . These vOur approach was structured according to symbolic interactionism, which focuses on how individuals, through regular interactions, develop shared meanings and conceptualize, perceive and understand the role of technology : p.201].1.20]. InHome care technology transforms the patient's life both inside and outside the homeUser-acceptance was shaped by different types of anxiety . User-acceptance is in fact closely linked to competence. Older patients on IV did not feel comfortable with the electronic components of the programmable pump, which they associated with the "computer age" and about which they felt ignorant. Chronic patients seemed, in general, keener to master the technical aspects. "You can't live without air! So you have to be careful and do it right" (Interview, O2, carer, m1). One PN patient was technically confident and had developed her own technique for preventing air bubbles from forming in the tube (Obs., PN, w1). A similar confidence was shown by the carer of a PD patient: "When you see all that stuff – the reservoir, the wires – you wonder if you'll be able to do it! But, once you know how, it's easy" (Interview, PD, carer, w1). For all four interventions, manual dexterity was required to properly manipulate the different components. "If my eyes were okay, I'd have been able to do it. But I was frightened of not doing it properly, of not seeing the needle, which is so tiny" (Interview, IV, carer, w1). We also observed patients who were not able to read messages on the digital screen due to poor eyesight (due to old age or co-morbidity), limited English linguistic skills, or illiteracy. They relied on their memory or made informed guesses when operating the device.Finally, the technology did not always fit neatly in the home setting: "Well, you wouldn't believe how hot [the room gets] when the door is closed!" (Interview, O2, caregiver, m3). Some O2 patients liked to have an extra set of tubing so they could use a second floor or sit outside on a patio (Obs., O2, w1). One PD patient planned to have an evacuation system installed so he would not have to dispose of the solution exiting the tubing from his peritoneal cavity through the toilet anymore (Obs., DP, m1).While some of the barriers described above can be managed fairly well in the home setting, problems arise in the unpredictable "outside world": "It's great when you're at home where you're all set up. But when you're out, you're always worried about people lighting up a cigarette" (Interview, O2, carer, m3) . One PN patient said it was "impossible to work because of being connected for 12 hours", and receiving lots of fluid at night compromised her sleep (Interview, PN, w2). On the other hand, one PD patient chose a nocturnal exchange regulator, refusing hospital-based hemodialysis, in order to work: "You can't work if you go to the hospital three times a week, and work is very important to me" (Interview, PD, m2). In the latter case, the comparison with an alternative makes PD more acceptable.More crucial was the "black-and-white" definition of ability to work that governs disability pension plans, which is incompatible with what patients experience: "You know, sometimes you feel okay, and sometimes you don't; sometimes you're disabled and sometimes you're not..." (Interview, PN, w3). Overall, due to compromised health and technical barriers, most patients agreed to apply for a disability plan (according to which they cannot accept paid work). A number of patients did volunteer work, such as helping neighbourhood kids with their homework (Obs., PN, w1), doing clerical work for the family business (Obs., O2, w1), or volunteer work for a patient association (Obs., O2, w3).both a negative and a positive effect on the lives of patients and carers. Indeed, our analyses sought to provide more detail on how technology simultaneously improves and constrains patients' lives. This is compatible with Pierret's observation that "Medicine gives the chronically ill reason to hope, even as it produces limitations with which these persons have to live by making adjustments to meet everyday requirements" [[and through effective patient education strategies. Indeed, competence and acceptance are likely to be mutually reinforced, especially if patients are supported and their know-how re-assessed over time. Although this is already part of the nursing staff duties, the experiences shared by our interviewees and the literature [This study, adopting a "technology-in-practice" perspective , shows tements" : p.14]. 4. both aterature  indicateAs observed in a recent U.K. survey on the quality of primary health care provision, crucial factors include educational training, patient education programs and improved communication and teamwork . In the In particular, two issues related to policy-making and clinical practice require prompt consideration. First, technology is often designed as though patients all possess similar abilities, and are neither ageing or incapacitated by other illnesses or physical disabilities . Since tSecond, competence of patients and their carers should be reinforced through adequate education and support from physicians and nurses. Medical specialists who manage hospital-based home care programs for O2, PN, PD patients could emphasize, when enrolling patients, the need for a smooth fit between the technical and human barriers that affect patient compliance. Training programs for nurses could also focus on skills and routines that help increase the user-friendliness of technology ,30. FinaThis study has sought to better understand how the level of user-friendliness of medical technology influences its integration into the private and social lives of patients. In this regard, qualitative methods are particularly well suited for uncovering patients' views. Nevertheless, the limitations of our study should be acknowledged ,31. The both the positive and negative impact of technology on patients' lives, not only brings medicine closer to issues that really matter to patients, but also generates "greater scientific, ethical, and social complexity" [[This paper shows that the barriers facing home care patients can easily be "taken for granted," as though nothing can be done to improve the situation. With a growing elderly population and limited health care resources, this will become a major issue in most industrialized countries. According to McGarry, "The home environment as a location of care provision is largely beyond the public-professional gaze, and therefore, remains potentially hidden from scrutiny" : p.429]..both thelexity" [.The author declares that she has no competing interests.The pre-publication history for this paper can be accessed here:


Laser microdissection allows precise isolation of specific cell types and compartments from complex tissues. To analyse proteins from small cell numbers, we combine laser-microdissection and manipulation (LMM) with mass spectrometry techniques.® Arrays. Initially, to establish the principle, we identified specific protein peaks from 20,000 laser-microdissected cells, combining column chromatography, SDS-PAGE, tryptic digestion, SELDI technology and Tandem MS/MS using a ProteinChip® Tandem MS Interface. Secondly, our aim was to reduce the labour requirements of microdissecting several thousand cells. Therefore, we first defined target proteins in a few microdissected cells, then recovered in whole tissue section homogenates from the same lung and applied to these analytical techniques. Both approaches resulted in a successful identification of the selected peaks.Hemalaun stained mouse lung sections were used to isolate 500–2,000 cells, enough material for complex protein profiles by SELDI-TOF MS (surface enhanced laser desorption and ionization/time of flight mass spectrometry), employing different chromatographic ProteinChipLaser-microdissection may thus be combined with SELDI-TOF MS for generation of protein marker profiles in a cell-type- or compartment-specific manner in complex tissues, linked with mass fingerprinting and peptide sequencing by Tandem MS/MS for definite characterization. Investigation of cell-type specific gene expression and regulation in complex tissues is hampered by the lack of accuracy of cell isolation and sensitivity of post-isolation analysis. Laser-microdissection techniques have proven to be a reliable tool for selectively harvesting cell clusters or single cell profiles from stained tissue sections for mRNA and protein investigation. When combined with qualitative and quantitative PCR, mRNA can be successfully analysed from a few cells -3. The cet al. combined 2D-PAGE with peptide mapping and tandem mass spectrometry to identify a protein significantly higher expressed in tumour tissue and confirmed the identity by immunodepletion assay and immunhistochemistry [Recently, several groups have successfully combined laser-microdissection with surface-enhanced laser desorption/ionization mass spectrometry (SELDI MS) to generate reproducible MS profiles from 200–5,000 cells -14. Chanhemistry .In addition to generating compartment-specific biomarker profiles, we aimed to develop alternative strategies circumventing the laborious 2D-PAGE for definite protein identification using limited cell numbers derived from microdissected material. The strategies were evaluated at septal and vascular compartments of the complex lung tissue.Laser-microdissection and manipulation was used to isolate 500–2,000 alveolar septum cells Figure , which w® Arrays, HEPES/Triton X-100 protein lysate as well as UTC lysate was applied independently to SAX (strong anionic exchanger) and WCX (weak cationic exchanger) ProteinChip® Arrays. Compared to the weaker HEPES lysis, UTC buffer resulted in a remarkably higher yield of peaks  ≥ 3) on WCX arrays. On the other hand, HEPES buffer gave more individual spectra on SAX arrays. 500 cells were sufficient to detect more than 35 peaks on both SAX and WCX arrays. However, immobilization of 2,000 cells resulted in over 50 peaks on WCX arrays  To assess the effect of different lysis buffers and different surface properties of the ProteinChips Figure . In regab) Comparing alveolar septum cells to intrapulmonary vessels, the profiles differed considerably, with only few overlapping peaks. Representative profiles are given in Figure et al. [c) Four representative SELDI TOF MS spectra of alveolar septum cells from four different animals are shown in Figure et al.  who alsoProtein lysate of 10,000 to 20,000 microdissected septum cells was extracted by UTC buffer, the remaining material of the same needles was further extracted by SDS sample buffer. Protein samples from UTC and SDS extracts were separated by SDS-PAGE. Although both extracts showed several colloidal Coomassie Brilliant Blue (CBB) stained bands the SDS extract revealed several proteins in the MW region between 12–16 kD Figure . Some of® spin column (Ciphergen Biosystems, CA). The protein extract equivalent to approximately 50,000 cells was applied to the column and 6 fractions were eluted according to a stepwise pH gradient and concentrated by trichloroacetic acid precipitation. Aliquots of 3 μl  of each fraction were applied to a NP20 ProteinChip Array (hydrophilic chemistry) in order to detect the presence and enrichment of the selected proteins. In the organic fraction (last elution step) all three proteins (13.8, 14.0 and 15.7 kD) were detected on an NP20 ProteinChip. After separation of the complete organic fraction by SDS-PAGE, proteins were stained by colloidal CBB. A protein band with estimated molecular weight between 15–16 kD was excised and subjected to tryptic digestion (not shown).The 15.7 kD protein was enriched by micro-spin column chromatography using a Q HyperD® Arrays with hydrophilic and hydrophobic chromatographic properties, respectively. Peptide mass fingerprinting of the gel bands was done using the PBSIIc instrument. The results are given in Table ® Interface coupled to a Tandem mass spectrometer [Protein bands isolated from the gel were subjected to Trypsin digestion. Gel pieces were extracted twice and the resulting peptide fragments applied to NP20 and H4 ProteinChiptrometer -19. Repr® Array, we were able to recover the target protein markers within the homogenate spectrum  previously known to contain the target proteins from laser-microdissection were collected and proteins were isolated following the procedure as for the laser-microdissected cells. Applying an aliquot of approximately 4,000 cells to a spot of WCX ProteinChipm Figure . Using aThe combination of laser-microdissection and mass spectrometry has been shown to be a reliable tool for compartment and cell type-specific biomarker profiling in complex tissues. Several groups have described the successful generation of mass spectra from as few as 500 to 2,000 cells after microdissection ,15,16 wh® fluorescence staining). While advantageous in gel staining due to higher sensitivity, the problems are shifted towards MS technique: the identification may fail due to minute amounts of protein per gel spot. Therefore, in our study we performed robust and easy CBB staining to be able to detect the required amount of material in MS techniques. Additionally, for low abundant proteins, direct Trypsin digestion of an eluted, gel-resolved protein can be performed directly on the array. Marker protein isolation and enrichment of the protein peak may also be enhanced by application of suitable pH and salt conditions directly on the array.To investigate proteins on 2D-gels, high amounts of cells are required. Therefore, using this approach as a starting point for protein profiling with subsequent MS identification, 50,000 to 250,000 microdissected cells have to be introduced per gel. Our aim was to minimize this laborious procedure, being hardly compatible with microdissection of minor cellular compartments. Thus, we first generated compartment specific profiles from laser-microdissected material by mass spectrometry and subsequently collected high cell numbers to identify the previously selected proteins. Moreover, using an interface to a Tandem MS/MS instrument, analysis of tryptic mass fingerprints can be performed from the same array without need for additional material. To reduce the demand of material on the gel, different staining techniques can be used  were instilled into the airways via a tracheal cannula. Afterwards, lungs were excised and frozen in liquid nitrogen immediately.All animal experiments were approved by local authorities [Regierungspräsidium Giessen, no. II25.3-19c20-15(1) GI20/10-Nr.22/2000]. Lung preparation was performed as described previously [Laser-microdissection and manipulation was performed as described previously -3,5. In ® Arrays and incubated in a humid chamber for 1 h. For profiling, SAX ProteinChip® Arrays were preincubated in SAX Binding Buffer (100 mM Tris, pH 8.5, 0.02% Triton X-100).Needles with adherent cells were transferred to the reaction tubes containing 15 μl of the HEPES/Triton X-100 lysis buffer. After vigorous shaking for 30 min at room temperature, samples were centrifuged at 14,000 g for 10 min. 3 μl of this supernatant were directly applied to the spots of SAX and WCX ProteinChip® Arrays. These were pre-treated with 10 mM HCl for 5 min and equilibrated two times with WCX binding buffer (100 mM NaOAc, pH 4.5, 0.02% Triton X-100) for 5 min. Fifteen μl of UTC supernatant were diluted 1:10 v/v in WCX binding buffer and incubated under vigorous shaking for 45 min. In order to deal with high amounts of volume (150 μl) the Bioprocessor (Ciphergen Biosystems. Inc.) was applied. After removal of the samples every spot was washed twice using binding buffer followed by a final 10 sec water rinse. After air-drying, saturated sinapinic acid (0.6 μl) dissolved in 50% acetonitrile and 0.5% trifluoracetic acid (TFA) was added twice. Subsequently, mass analysis of bound peptides/proteins was performed using the Ciphergen PBS IIc system. ProteinChip® Arrays were analyzed by averaging 100–150 laser shots collected in the positive mode. Optimization range of the time lag focusing was set between 10–30 kD. Deflector settings were used to filter out peaks with <2000 m/z. Calibration was performed externally using purified peptide and protein standards. Obtained spectra were analyzed by the ProteinChip Software version 3.01.After first extraction of the microdissected material using HEPES buffer, 15 μl of UTC buffer (6 M Urea, 2 M Thiourea, 2% CHAPS, 75 mM DTT) were applied to needles/cells. The pellet in UTC buffer was centrifuged for 10 min at 14,000 g and the complete supernatant was used for profiling on WCX ProteinChipSDS-PAGE was performed as described previously  with min® Arrays to reveal the enrichment of selected protein peaks in a certain fraction. The fractions containing the enriched proteins were concentrated by TCA precipitation in order to apply the complete fraction to a single lane of an SDS gel. Staining was performed by colloidal Coomassie brilliant blue (CBB, Roth).For selective enrichment of marker proteins we used small sized anionic exchange spin columns resulting in 6 fractions after elution using a stepwise pH gradient from pH 9–3 and an organic fraction. The protein amount reflecting 50,000 cells was loaded onto a Q HyperD spin column and aliquots of the eluted fractions were analyzed on NP20 ProteinChip® Arrays. A 20% matrix solution of alpha-4 hydroxy-cinnamic acid (CHCA) was applied to the spots. The remaining gel pieces were extracted with a 60% acetonitrile and 0.2% TFA solution for 1 h with a 5 min sonication to extract remaining organic peptides. Supernatants from this second extraction step were also applied to H4 and NP20 ProteinChip® Arrays. All Arrays were measured in the PBS IIc system by averaging 150 laser shots. After subtraction of all peaks also present in the blank gel piece (e.g. Trypsin autolysis peaks), m/z values were submitted to Profound and Mascot for database searching.The CBB stained bands matching the expected molecular weight regions of selected proteins were excised and subjected to Trypsin digestion. Gel pieces were washed three times with 400 μl of 100 mM ammonium bicarbonate and 50% acetonitrile for 15 min followed by 15 min incubation in 100% acetonitrile. After removal, gel pieces were dried shortly in a speed-vac centrifuge. Depending on the gel volume, 10–15 μl of a Trypsin solution (20 ng Trypsin/μl in 25 mM ammonium bicarbonate) was applied and digestion was performed overnight (16 h) at 37°C. Afterwards, reaction tubes were centrifuged and 0.5–1.5 μl aliquots of each supernatant were applied to the spots of H4 (hydrophobic surface coating) and NP20 (hydrophilic surface coating) ProteinChip® Arrays and 2 × 0.6 μl of a saturated CHCA solution was added. For quality control of the peak intensities, the NP20 Arrays were analyzed in a PBS IIc instrument. Afterward, the arrays were transferred to a Tandem MS instrument. Data were acquired on a Micromass QTOF II (Manchester, UK) tandem quadrupole-time of flight (Q-TOF) mass spectrometer equipped with a PCI 1000 ProteinChip® Tandem MS Interface (Ciphergen Biosystems). Ions were created using a pulsed nitrogen laser operating with 30 pulses/sec. Nitrogen gas was used for collisional cooling of formed ions and argon gas was used for all low-energy collision-induced dissociation experiments. The system was externally calibrated in MS/MS mode using the parent ion and selected fragments of adrenocorticotropic hormone (ACTH) human fragment 18–39 .Peptides from tryptic digestion were applied to NP20 ProteinChipGK: laser-microdissection, preparation of samples, PAGE, SELDI-MSMM: SELDI-MS and optimalisation for microdissected materialRB: tandem MS measurementsRMB: instruction to laser-microdissectionWS: design of project, preparation of the manuscriptNW: animal model, preparation of the lungsLF: design of project, preparation of the manuscript


We sought to elicit preference weights from parents for health states corresponding to children with various levels of severity of atopic dermatitis. We also evaluated the hypothesis that parents with children who had been diagnosed with atopic dermatitis would assign different preferences to the health state scenarios compared with parents who did not have a child with atopic dermatitis.Subjects were parents of children aged 3 months to 18 years. The sample was derived from the General Panel, Mommies Sub-Panel, and Chronic Illness Sub-Panel of Harris Interactive. Participants rated health scenarios for atopic dermatitis, asthma, and eyeglasses on a visual analog scale, imagining a child was experiencing the described state.A total of 3539 parents completed the survey. Twenty-nine percent had a child with a history of atopic dermatitis. Mean preference scores for atopic dermatitis were as follows: mild, 91 ; mild/moderate, 84 (95%CI, 83.5 to 84.4); moderate, 73 (95%CI, 72.5 to 73.6); moderate/severe, 61 (95%CI, 60.6 to 61.8); severe, 49 (95% CI, 48.7 to 50.1); asthma, 58 (95%CI, 57.4 to 58.8); and eyeglasses, 87(95%CI, 86.3 to 87.4).Parents perceive that atopic dermatitis has a negative effect on quality of life that increases with disease severity. Estimates of parents' preferences can provide physicians with insight into the value that parents place on their children's treatment and can be used to evaluate new medical therapies for atopic dermatitis. Atopic dermatitis is the most common of childhood skin diseases, with a lifetime prevalence in children of 10% to 20% .Atopic dermatitis can have a negative impact on quality of life by affecting psychosocial adjustment in children. Lapidus and Kerr  report tAs the need to control increasing medical expenditures continues to mount, formal economic evaluations are taking on a prominent role in assessing the value of new medical therapies. To properly evaluate the impact of new therapies for atopic dermatitis, patients' health-related quality of life (HRQOL) must be considered. Although a number of quality-of-life evaluations have been conducted for adults and children affected with atopic dermatitis ,7,9-12, Although the prevalence of atopic dermatitis is highest in children, the existing literature on patient preferences for atopic dermatitis is limited to the adult population ,14. HowePatient preferences can be elicited using standard gamble or time-tradeoff or direct rating methods such as a visual analog scale. Because both the standard gamble and the time-tradeoff exercises involve choices between two alternatives that involve a chance of death or longevity, we believed it was unethical to ask parents to participate in such exercises when children were the subjects. Therefore, our choice for eliciting preferences was the rating scale.We developed case scenarios for 5 levels of atopic dermatitis severity – mild, mild/moderate, moderate, moderate/severe, and severe. These severity levels were created by combining the characteristics of an Investigator Global Assessment (IGA) and the Eczema Area and Severity Index (EASI) score . Each scUsing cognitive interview techniques, we pilot-tested the preference assessment instrument in a convenience sample of 20 parents of children who were being evaluated at a local children's primary care clinic to assess patients' understanding of the instrument and its instructions. The instrument was further revised based on the results of the pilot test.A health state preference assessment asks subjects to make judgments regarding the value of particular health states . PrefereSubjects were invited to participate in the survey from February 12 through 14, 2002, and were asked to register at a specific survey site. After consent was obtained online, subjects completed the survey. Respondents were offered the incentive of a chance to win one of five $100 prizes. Respondents had to be adults with children aged 3 months to 18 years in order to be included in the study. The study was approved by the institutional review board of Duke University Medical Center.Clinical data were based on self-report and included information on diagnosis history and severity level. Specifically, respondents were asked if they had a child between the ages of 3 months and 18 years who had ever been diagnosed by a medical professional with atopic dermatitis. If they responded "yes," they were then asked to describe the child's atopic dermatitis at its worst point by selecting from the following response options: mild, mild/moderate, moderate, moderate/severe, severe.For the preference assessment, each respondent was given the scenarios in the same order – mild through severe atopic dermatitis, asthma, and glasses. Subjects were instructed to indicate on a scale ranging from 100  to 0 (death) how good or bad they believed it would be to be a child experiencing the scenario depicted. Respondents recorded their values using a movable pointer on the scale. Respondents whose Internet browser software did not support the movable pointer entered their numerical responses manually into a required field. All 7 values were summarized at the end of the survey to allow respondents to review and, if desired, revise their ratings.P value of ≤ .05 was used as the criterion for statistical significance.Descriptive statistics were used to describe the sample. Because subjects provided responses across severity levels, a repeated-measures analysis of variance was conducted using polynomial contrasts for the within-subjects (severity) effect. A Of the 28105 subjects contacted, 6131 (22%) responded. Of the 6131 respondents, 3539 (57.7%) met the inclusion criteria, consented to participate in the study, and completed the survey . The linear effect of severity  indicated that preference ratings significantly decreased as the severity of the health states increased . Across all health states, parents of children with atopic dermatitis gave a slightly higher mean preference (72.85 [SD, 13.50]) compared to parents whose children did not have atopic dermatitis (71.34 [SD, 14.19]) , indicating that the differences across health states were the same for both parent groups.There was no significant severity by parent group interaction . Research on adults who rate the health states of other adults has suggested that people who have experienced a particular health state are more likely to assign a higher value to it than others who are asked to imagine the health state -21. The Our study has several limitations. First, by using the Internet, respondents did not have an opportunity to ask questions if they did not understand what they were being asked to do. However, we feel that this limitation was negligible, because the instrument was pilot-tested using in-person interviews, and the very large majority of responses were ranked appropriately (eg, mild ranked higher than severe). Secondly, there may be a sample bias in using the Harris Interactive database. Our sample reflected a predominately white, female cohort from a high socioeconomic class and may present generalizability issues. However, we received responses from 351 (14%) nonwhite respondents and 779 (22%) responses from participants with an annual household income of less than $35000, providing a sufficient number of responses to test for differences by race/ethnicity and income level. Further research will be needed to corroborate our findings using a population-based sample. Third, since these data are self-reported, some parents may have misclassified their children as having or not having atopic dermatitis, potentially biasing our results. Fourth, it is unclear whether physician assessment of severity would correspond with the severity levels that we assigned to the health states. However, physicians could evaluate the descriptions provided to judge whether their assessments are consistent. Finally, people enrolled in the Harris Interactive database are computer users who may be more motivated to participate in a survey than the general population. It is unclear how or in what direction these sample biases might affect the results of our analyses.We had an apparent response rate of 22%. Harris Interactive generally achieves a 15% to 20% response rate when using the Chronic Illness Sub-Panel. While our response rate was higher than Harris' average response rate, possible reasons for why the response was low could be attributed to the nature of the study design. First, because subjects were contacted by e-mail, it is possible that some subjects did not open the e-mail message until after the survey deadline. Second, Harris Interactive panel members agree to be notified about survey opportunities, but do not agree to participate in each survey. Since the characteristics of responders and nonresponders did not differ, we have no reason to believe that nonresponse bias is exerting a substantial influence on our results.The results of this analysis clarify the values that parents of children with atopic dermatitis assign to different atopic dermatitis health states. These assigned values, relative to the comparison states, clearly demonstrate the perceived burden of atopic dermatitis by parents of children suffering from the disease. Understanding the preferences for atopic dermatitis can provide physicians insight into the value that parents place on treatments for their child's disease and in evaluating the cost-effectiveness of therapies for atopic dermatitis.This study was supported by a research agreement between Duke University Medical Center and the Novartis Pharmaceuticals Corporation, East Hanover, NJ, which manufactures a cytokine inhibitor for the treatment of atopic dermatitis.KPW and KAS have received monetary compensation for consultancies, and EBW and KAS have received research grants, from Novartis. KHK is an employee of Novartis.JYF conceived of and designed the study, performed the statistical analysis, interpreted the data, and drafted the manuscript. SDR conceived of and designed the study and assisted in interpretation of the data and drafting of the manuscript. KPW assisted in interpretation of the data and drafting of the manuscript. EBW and KHK conceived of and designed the study and assisted in drafting of the manuscript. KAS conceived of and designed the study, assisted in drafting of the manuscript, and obtained funding. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


A number of approaches have been used to grade levels of evidence and the strength of recommendations. The use of many different approaches detracts from one of the main reasons for having explicit approaches: to concisely characterise and communicate this information so that it can easily be understood and thereby help people make well-informed decisions. Our objective was to critically appraise six prominent systems for grading levels of evidence and the strength of recommendations as a basis for agreeing on characteristics of a common, sensible approach to grading levels of evidence and the strength of recommendations.Six prominent systems for grading levels of evidence and strength of recommendations were selected and someone familiar with each system prepared a description of each of these. Twelve assessors independently evaluated each system based on twelve criteria to assess the sensibility of the different approaches. Systems used by 51 organisations were compared with these six approaches.There was poor agreement about the sensibility of the six systems. Only one of the systems was suitable for all four types of questions we considered (effectiveness, harm, diagnosis and prognosis). None of the systems was considered usable for all of the target groups we considered . The raters found low reproducibility of judgements made using all six systems. Systems used by 51 organisations that sponsor clinical practice guidelines included a number of minor variations of the six systems that we critically appraised.All of the currently used approaches to grading levels of evidence and the strength of recommendations have important shortcomings. In 1979 the Canadian task Force on the Periodic Health Examination published one of the first efforts to explicitly characterise the level of evidence underlying healthcare recommendations and the strength of recommendations -28.The original approach used by the Canadian Task Force was based on study design alone, with randomised controlled trials (RCTs) being classified as good (level I) evidence, cohort and case control studies being classified as fair (level II) evidence and expert opinion being classified as poor (level III) evidence. The strength of recommendation was based on the level of evidence with direct correspondence between the two; e.g. a strong recommendation (A) corresponded to there being good evidence. A strength of the original Canadian Task Force approach was that it was simple; the main weakness was that it was too simple. Because of its simplicity, it was easy to understand, apply and present. However, because it was so simple there were many implicit judgements, including judgements about the quality of RCTs, conflicting results of RCTs, and convincing results from non-experimental studies.For example:• Should a small, poorly designed RCT be considered level I evidence?• Should RCTs with conflicting results still be considered level I evidence?• Should observational studies always be considered level II evidence, regardless of how convincing they are?The original approach by the Canadian Task Force also did not include explicit judgements about the strength of recommendations, such as how trade-offs between the expected benefits, harms and costs were weighed and taken account of in going from an assessment of how good the evidence is to determining the implications of the results for practice.The GRADE Working Group is an informal collaboration of people with an interest in addressing shortcomings such as these in systems for grading evidence and recommendations. We describe here a critical appraisal of six prominent systems and the results of the critical appraisal.We selected systems for grading the level of evidence and the strength of recommendations that we considered prominent and that included features not captured by other prominent systems. These were selected based on the experience and knowledge of the authors through informal discussion. A description of the most recent version (as of summer 2000) of each of these systems (Appendix 1 to 6), was prepared by one of the authors familiar with the system, and used in this exercise. The following six systems were appraised: the American College of Chest Physicians (ACCP, see ) 21], A, A21], AThese descriptions of the systems were given to the twelve people who independently appraised the six systems, all of the authors minus GEV appraised the six systems, three of the authors (DH, SH and DO'C) appraised as a group and reported as one (see contributions). The 12 assessors all had experience with at least one system and most had helped to develop one of the six included systems. Twelve criteria described by Feinstein  provided1. To what extent is the approach applicable to different types of questions? -effectiveness, harm, diagnosis and prognosis (No, Not sure, Yes)2. To what extent can the system be used with different audiences? -patients, professionals and policy makers (Little extent, Some extent, Large extent)3. How clear and simple is the system? (Not very clear, Somewhat clear, Very clear)4. How often will information not usually available be necessary? (Often, Sometimes, Seldom)5. To what extent are subjective decisions needed? (Often, Sometimes, Seldom)6. Are dimensions included that are not within the construct (level of evidence or strength of recommendation)? 7. Are there important dimensions that should have been included and are not? 8. Is the way in which the included dimensions are aggregated clear and simple? 9. Is the way in which the included dimensions are aggregated appropriate? 10. Are the categories sufficient to discriminate between different levels of evidence and strengths of recommendations? 11. How likely is the system to be successful in discriminating between high and low levels of evidence or strong and weak recommendations? (Not very likely, Somewhat likely, Highly likely)12. Are assessments reproducible? (Probably not, Not sure, Probably)No training was provided and we did not discuss the 12 criteria prior to applying them to the six systems.Our independent appraisal of the six systems were summarised and discussed. The discussion focused on differences in the interpretation of the criteria, disagreement about the judgements that we made and sources of these disagreements, the strengths and weaknesses of the six systems, and inferences based on the appraisals and subsequent discussion.In order to identify important systems that we might have overlooked following our appraisal of these six systems we also searched the US Agency for Health Care Research and Quality (AHRQ) National Guidelines Clearing House for organisations that have graded two or more guidelines in the Clearing House using an explicit system . These sThere was poor agreement among the 12 assessors who independently assessed the six systems. A summary of the assessments of the sensibility of the six approaches to rating levels of evidence and strength of recommendation is shown in Table The poor agreement among the assessors likely reflects several factors. Some of us had practical experience using one of the systems or used additional background information related to one or more grading systems, and we may have been biased in favour of the system with which we were most familiar. Each criterion was applied to grading both evidence and recommendations. Some systems were better for one of these constructs than the other and we may have handled these discrepancies differently. In addition each criterion may have been assessed relative to different judgements about the evidence, such as an assessment of the overall quality of evidence for an important outcome (across studies) versus the quality of an individual study. Some of the criteria were not clear and were interpreted or applied inconsistently. For example, a system might be clear and not simple or visa versa. We likely differed in how stringently we applied the criteria. Finally, there was true disagreement.There was agreement that the OCEBM system works well for all four types of questions. There was disagreement about the extent to which the other systems work well for questions other than effectiveness. It was noted that some systems are not intended to address other types of questions and it is not clear that it is important that a system should address all four types of questions that we considered (effectiveness, harm, diagnosis, prognosis), although criteria for assessing individual studies must take this into account ,32.Most of us did not find that any of the systems are likely to be suitable for use by patients. Almost all agreed that the ACCP system was suitable for professionals and most considered that the USPSTF system was suitable for professionals. There was not much agreement about the suitability of any of the other systems for professionals or about the suitability of any of the systems for policy makers, although most assessed the USTFCPS system to be suitable for policy makers.There was no agreement that any of the systems are clear and simple, although USPSTF, ACCP and SIGN systems were generally assessed more favourably in this regard. It was generally agreed that the clearer a system was the less simple it was; e.g. the OCEBM system is clear but not simple for categorising the level of evidence. There was some confusion regarding whether we were assessing how clear and simple the system was to guideline developers (as some interpreted this criterion) or how clear and simple the outcome of applying the system was to guideline users (as others interpreted this criterion). Either way, the simpler a system is the less clear it is likely to be.Most of us judged that for most of the systems necessary information would not be available at least sometimes. The OCEBM system came out somewhat better than the other systems and lack of availability of necessary information was considered to be less of a problem for the USTFCPS system. However, the OCEBM and USTFCPS systems were considered by most to be missing dimensions which may, in part, explain why missing information was considered to be less of a problem. This would be the case to the extent the missing dimensions were the ones for which information would often or sometimes not be available. The dimension for which we considered that information would most often be missing was trade-offs; i.e. knowledge of the preferences or utility values of those affected. Additional problems were identified in relationship to complex interventions and counselling, particularly with the USTFCPS and USPSTF systems. It was pointed out that the USTFCPS system addressed this problem by including availability of information about the intervention as part of its assessment of the quality of evidence.Most of the systems were assessed to require subjective decisions at least to some extent. The OCEBM system again stood out as being assessed more favourably, although it may be related to omission of dimensions that require more subjective decisions. Judgement is clearly needed with any system. The aim should be to make judgements transparent and to try to protect against bias in the judgements that are made by being systematic and explicit.Inclusion of dimensions that are not within the constructs being graded was not considered a problem for most of the systems by most of us. Several people considered that it might be a problem for the USTFCPS and USPSTF systems. On the other hand, all of the systems were evaluated to be missing at least one important dimension by at least one person. The challenge of missing dimensions were considered less of a problem for the ACCP and ANHMRC systems. There was not agreement about any of the systems having a clear and simple approach to aggregating the dimensions, although this was considered to be less of a problem for the ACCP, SIGN and USTFCPS systems.There was also not agreement on the appropriateness of how the dimensions were aggregated. This was considered to be more of a problem for the ANHMRC and USTFCPS systems than the other four systems, all of which were considered to have taken an approach to aggregating the dimensions that was at least partially inappropriate by more than half of us.Most of us considered that most of the systems had sufficient categories, with the exception of the ANHMRC system. There was almost agreement that the USPSTF system has sufficient categories. We agreed that it is possible to have too many categories as well as too few, the OCEBM system being an example of having too many categories.There was not agreement that any of the systems are likely to discriminate successfully, although everyone thought that the ACCP, SIGN and USPSTF systems are somewhat to highly likely to discriminate. Lastly, we largely agreed that we were not sure how reproducible assessments are using any of the systems, although half of us considered that assessments using the ANHMRC system are unlikely to be reproducible and about 1/3 considered that assessments using the OCEBM and ACCP systems are likely to be reproducible.We identified 22 additional organisations that have produced 10 or more practice guidelines using an explicit approach to grade the level of evidence or strength of recommendations. Another 29 have produced between two and nine guidelines using an explicit approach. These systems include a number of minor variations of the six systems that we appraised in detail.There was generally poor agreement between the individual assessors about the scoring of the six approaches using the 12 criteria. However, there was general agreement that none of these six prominent approaches to grading the levels of evidence and strength of recommendations adequately addressed all of the important concepts and dimensions that we thought should be considered. Although we limited our appraisal to six systems all of the additional approaches to grading levels of evidence and strength of recommendations that we identified were, in essence, variations of the six approaches that we had critically appraised. Therefore we are confident that we did not miss any important grading systems available at the time when these assessments were undertaken.Based on discussions following the critical appraisal of these six approaches, we agreed on some conclusions:• Separate assessments should be presented for judgements about the quality of the evidence and judgements about the balance of benefits and harms.• Evidence for harms should be assessed in the same way as evidence for benefits, although different evidence may be considered relevant for harms than for benefits; e.g. local evidence of complication rates may be considered more relevant than evidence of complication rates from trials for endarterectomy.• Judgements about the quality of evidence should be based on a systematic review of the relevant research.• Systematic reviews should not be included in a hierarchy of evidence (i.e. as a level or category of evidence). The availability of a well-done systematic review does not correspond to high quality evidence, since a well-done review might include anything from no studies to poor quality studies with inconsistent results to high quality studies with consistent results.• Baseline risk should be taken into consideration in defining the population to whom a recommendation applies. Baseline risk should also be used transparently in making judgements about the balance of benefits and harms. When a recommendation varies in relationship to baseline risk, the evidence for determining baseline risk should be assessed appropriately and explicitly.• Recommendations should not vary in relationship to baseline risk if there is not adequate evidence to guide reliable determinations of baseline risk.Based on discussions of the strengths and limitations of current approaches to grading levels of evidence and the strength of recommendations, we agreed to develop an approach that addresses the major limitations that we identified. The approach that the GRADE Working Group has developed is based on the discussions following the critical appraisal reported here and a pilot study of the GRADE approach . Based o and a discussion list.The GRADE Working Group has continued to grow as an informal collaboration that meets one or two times per year. The group maintains web pages DA has competing interests with the US Preventive Services Task Force (USPSTF), PAB has a competing interest with the US Task Force on Community Preventive Services (USTFCPS), GHG and HS have competing interests with the American College of Chest Physicians (ACCP), DH, SH and DO'C have competing interests with the Australian National Health and Medical Research Council (ANHMRC), BP has competing interests with the Oxford Centre for Evidence-Based Medicine (OCEBM). Most of the other members of the GRADE Working Group have experience with the use of one or more systems of grading evidence and recommendations.DA, PAB, ME, SF, GHG, DH, SH, AL, DO'C, ADO, BP, HS, TTTE, GEV & JWW Jr as members of the GRADE Working Group have contributed to the preparation of this manuscript and the development of the ideas contained herein, participated in the critical assessment, and read and commented on drafts of this article. GHG and ADO have led the process. GEV has had primary responsibility for coordinating the process.The pre-publication history for this paper can be accessed here:American College of Chest Physicians (ACCP), a brief description of the ACCP approach.Click here for fileAustralian National Health and Medical Research Council (ANHMRC), a brief description of the ANHMRC approach.Click here for fileOxford Centre for Evidence-based Medicine (OCEBM), a brief description of the OCEBM approach.Click here for fileScottish Intercollegiate Guidelines (SIGN), a brief description of the SIGN approach.Click here for fileU.S. Preventive Services Task Force (USPSTF), a brief description of the USPSTF approach.Click here for fileU.S. Task Force on Community Preventive Services (USTFCPS), a brief description of the USTFCPS approach.Click here for file


Acidosis is a major cause of death in patients with malaria, although what causes acidosis is still unclear. One possibility is that hypovolemia contributes to the problem, and that rehydration therapy could be of benefit. Now, Sanjeev Krishna and colleagues have shown that in children with severe malaria dehydration is not severe and is not correlated with other measures of disease severity. “The optimum resuscitation approach in severe childhood malaria remains to be defined,” says Nick White (Mahidol University, Thailand), the academic editor of the paper. “The relative advantages of blood, colloids, and crystalloids need to be characterized.”Every year around 200 million people worldwide contract malaria, of whom over a million die. The vast majority of those who die are children under five years, mostly in Africa, since young children have had little chance to acquire any immunity. Fluid resuscitation is generally considered to be a cornerstone of treatment—but how much fluid should be given? Some researchers believe that surrogate signs of fluid depletion—such as tachycardia, reduced capillary refill time, and reduced urine excretion—suggest that there is substantial volume depletion. The reason that the amount of fluid given matters so much is that giving too much, especially of hypotonic solutions, can lead to electrolyte imbalance, especially hyponatremia and hypokalemia.Research efforts have been hampered by not having an easy way to assess in patients the fluid depletion in different compartments of the body, i.e., total body water and extracellular and intracellular water volume. Krishna and colleagues used heavy-water distribution to calculate the total body water and bromide distribution to determine the extracellular volume in 19 children with moderately severe malaria and 16 with severe malaria in Gabon. By subtracting extracellular volume from total body water, they were able to calculate intracellular volume for each child. They also used a less invasive and more rapid method of determining water volumes based on using bioelectrical impedance to calculate the volume.None of the children were severely dehydrated (defined as more than 100 ml/kg depletion), and only three of the children with severe anemia had fluid depletion, which was moderate (60–90 ml/kg depletion). “This challenges the view that dehydration is a major contributor to the pathology of this frequently lethal disease,” says White.So based on these data, obtained from a carefully studied, albeit small group of children, what should people who treat children with malaria do? The authors' first recommendation is that clinicians should think again about how vigorously they rehydrate children, and if they have access to ways of assessing fluid volume more precisely, they should do so . And certainly the methods used by Krishna and colleagues should undergo wider testing in larger groups of children to confirm their usefulness. Until the worldwide efforts to prevent malaria come to fruition, refining the management of infected children will remain a cornerstone of the efforts against this devastating disease.


What defines a sex? Although we tend to think there are only two - males and females - there are many different ways to mix and match the attributes of sexes From a human perspective, sexes seem a relatively simple thing to get one's head around—there are females, and there are males. But our perspective seems biased and narrow when applied to life as a whole, says evolutionary biologist Laurence Hurst of the University of Bath, United Kingdom.“If you were a single-celled alga sitting in a pond, you wouldn't see the world as splitting into males and females.”In fact, different species have evolved a bewildering number of ways to mix and match the attributes of sexes. Some do not have males and females, but have adaptations that mean each individual performs a specific role during sex. There are other species of which every member is sexually equivalent, but individuals nevertheless divide into groups for the purposes of mating. And in some species, individuals make both eggs and sperm . This biPogonomyrmex, queens must mate with two types of males to produce both reproductive individuals and workers . Species with mating types, rather than males and females, aren't limited to two interbreeding groups: the ciliate protozoan Yet most isogamous species have only two mating types. This seems perverse—it excludes half the population as potential mates without gaining the benefits of specialization in sexual biology. With William Hamilton, Hurst came up an explanation for this apparent inefficiency.Two-group mating systems, they proposed, evolved as a way for genes in the nucleus to police the DNA in organelles. Cellular structures with their own genomes, such as mitochondria and chloroplasts, can divide more rapidly than the cells that house them. If the inheritance of organelles was biparental, selfish mutations in their DNA could spread rapidly, Hurst and Hamilton showed. A nuclear gene that enforces uniparental inheritance of organelles, along with a label that allows such cells to recognize each other so that their nuclear genes can share the benefits of cytoplasmic policing, should be favored.C. rheinhardtii, for example, comes in two mating types called plus and minus. When the two fuse, the plastid of the minus cell is detroyed. Most isogamous species that fuse cells have a similar mechanism. Male-killer parasites such as Wolbachia, a parasite of arthropods, show the selection pressure that intracellular passengers can exert . And cellfusion experiments hint that biparental inheritance of organelles does indeed cause problems, says Hurst. “Hybrids are often rubbish, but they can be better if a drug is administered that inhibits the mitochondria of one cell line.”The mating biology of isogamous species offers considerable support for this idea. The aforementioned The species that have lots of mating types, such as ciliate protozoa, exchange nuclear DNA, but not cytoplasm, and hence not intracellular organelles. Since individuals are freed from the need to police their organelles or keep out parasites, selection favors the widest assortment of possible mates, and thus the evolution of a large number of mating types so that one's own type—which one can't mate with—is a small subset of the population. It is possible to imagine species with cytoplasmic policing likewise having many mating types, but such a situation would be much more prone to break down and be invaded by selfish agents than one with two clearly defined types, which is what we usually see in nature. Some have argued that cytoplasmic policing might also be a selective force for different-sized gametes. Sperm could be small so that they do not import mitochondria into the egg.More than a decade after he devised it, Hurst's is still the leading hypothesis explaining the number of mating types in a species. But experimental evidence remains frustratingly elusive. “I wouldn't say I was entirely satisfied,” says Hurst. “We've got all these ideas, and they turn out to be quite hard to test—there's no simple thing one can do on a single species.” There are species where the uniparental inheritance of organelles is not so strictly enforced, says Hoekstra, such as yeasts and plants. “It's not easy to see if selection [on organelles] is strong enough,” he says.S. commune, with its thousands of mating types, each sexual encounter involves only two cells. Nor are we likely to find a species that defies this pattern. The technical difficulties of combining more than two sets of genetic information into one individual, and of parceling out that information during meiosis, must be vast, says Brian Charlesworth of the University of Edinburgh. “We've reached the point of two cells fusing, and stuck with that; two cells are probably just as good as three,” he says.Yet even in a species such as Pogonomyrmex rugosus and P. barbatus. The hybrids have not yet been classed as a new species, but they are well established across the southwestern United States, and there is no evidence of contemporary gene flow between hybrids and their parent species.The ant colonies that Parker suggests have three parents are a hybrid of the species Each ant has one parent if it is male, because male ants are produced from unfertilized eggs, or two if it is female. But each sex also comes in two genetic strains. If a queen mates with a male of her own strain, her offspring will be queens, and if she mates with a male from the other strain, the sperm will give rise to workers. So, for a colony to function fully it—and the queens it produces, because workers raise queens—must have two fathers and one mother. And if any one group were to disappear, the population as a whole would go extinct—unlike fungal mating types, where it's easy to imagine that the species would carry on if a few disappeared. “If you lose any one, the whole thing collapses,” says Parker. “It's really different from any other system.”Pogonomyrmex has four sexes: the males and females of each strain. The idea is particularly potent if one views a social insect colony as a “superorganism,” with the workers equivalent to the cells of a body. It's as if a female mates with one male to produce her offspring's somatic cells, and another to produce its germ cells. The ants form chaotic mating swarms, so most queens have no problem mating multiply and getting sperm from males of both strains, although one would expect that males would strongly favor mating with females of their own strain.So, Parker argues, It's not known how the system originated. Separating the worker and reproductive castes by genetics—other social insects do this by environment, that is, by rearing workers and reproductives differently—may allow selection to operate more efficiently on each lineage, and the workers may benefit from hybrid vigor: field researchers report them as being highly aggressive. In an echo of Hurst's hypothesis, the system also mixes mitochondrial and nuclear genes differently in queens and workers.Pogonomyrmex's mating types sexes, arguing that to define sexes in yet another way only confuses the picture further. “[The ants] are an interesting system, but I wasn't persuaded by Parker's interpretation,” Charlesworth says. “I'm not a fan of the idea that it's useful to use the word ‘sex’ to describe compatibility between mating types—it muddies the waters.” Others are more positive towards Parker's interpretation: “It deserves to be taken seriously,” says evolutionary biologist Eörs Szathmáry of the Collegium Budapest in Hungary. “He's thrown a stone in the water—now we need to see what kinds of ripples it makes. You can't falsify a definition in the way you can a hypothesis; what determines their fate is whether people find them useful or not.”Some evolutionary biologists, such as Charlesworth, do not consider Species in which some individuals give up their reproductive opportunities to form part of a breeding group, such as slime molds, might have a system similar to that of the ants, Parker believes. “There may be hidden mating incompatibilities,” he says. “Now [that] people know to look, we're going to start seeing more of these systems.”One option for dividing up the sexes is “both”—hermaphroditism. This might seem like an ideal solution—everyone becomes a potential partner, and everyone can bear offspring. In practice, however, hermaphroditism is uncommon among multicellular animals. The reasons are similar to those explaining why evolution favors unequal-sized gametes—once sexes have evolved, it's better to commit all one's resources to one role or the other, rather than try and be a jack-of-all-trades. After all, there are many good uses for mating resources other than simply producing eggs or sperm. An animal could defend a territory or provide parental care, for example.Hermaphroditism, however, is useful if one's sexual options are severely limited. In particular, it can be favored when encounters with potential mates are extremely rare. It makes no sense for an animal to invest heavily in the biological equipment of maleness, say, if it will have almost no opportunities to use it: better to hedge your bets. Animals with low or unpredictable population densities and those that are immobile, have poor senses, or lack long-distance signalling are often hermaphroditic. These include sponges, worms—whether flat, nematode, or annelid—and many molluscs (and, of course, plants, the majority of which are hermaphroditic). Most hermaphrodites still need to find at least one mate in their lifetimes: the cost of inbreeding prevents self fertilization from becoming common.Helix aspersa snails shoot calcareous love darts into one another. And when the marine flatworm Pseudobiceros bedfordi mates, each worm has two penises, which they fence with in a battle to smear one another with sperm without being fertilized themselves in the process (Hermaphroditic animals have some weird sexual adaptations.  process  and 2.Such oddities result when the mating opportunities of a hermaphroditic species increase, and specialization starts to become more favorable, says evolutionary biologist Nico Michiels, of the University of Muenster in Germany. In a species with two separate sexes, males and females often have different ideas about whether a mating is a good idea—males tend to be keener, females tend to be choosier. The result can be an evolutionary arms race, with each sex evolving adaptations that help them get their way. By exercising mate choice, each sex has some influence on the types of adaptations that evolve—anything too outlandish is unlikely to be favored. This counterbalance is not present in hermaphrodites, however. Rather than having one half of a species resist a particular mating strategy, the whole species is just as likely to adopt it. “Hermaphrodites run into awkward and bizarre mating conflicts,” says Michiels.Michiels believes that hermaphroditism was the ancestral state for animals, and thinks that we might be able to find the relics of this past in contemporary species with separate sexes. To test these ideas, he is searching for groups containing closely related hermaphroditic and bisexual species. Such taxa are very rare, however.


Mycobacterium ulcerans (MU), as the third most prevalent mycobacterial disease. In Ghana, there have been more than 2000 reported cases in the last ten years; outbreaks have occurred in at least 90 of its 110 administrative districts. In one of the worst affected districts, Amansie West, there are arsenic-enriched surface environments resulting from the oxidation of arsenic-bearing minerals, occurring naturally in mineral deposits.In 1998, the World Health Organization recognized Buruli ulcer (BU), a human skin disease caused by Proximity analysis, carried out to determine spatial relationships between BU-affected areas and arsenic-enriched farmlands and arsenic-enriched drainage channels in the Amansie West District, showed that mean BU prevalence in settlements along arsenic-enriched drainages and within arsenic-enriched farmlands is greater than elsewhere. Furthermore, mean BU prevalence is greater along arsenic-enriched drainages than within arsenic-enriched farmlands.The results suggest that arsenic in the environment may play a contributory role in MU infection. Buruli ulcer (BU) is a skin disease, which usually begins as a painless nodule or papule and may progress to massive skin ulceration. If untreated BU may lead to extensive soft tissue destruction, with inflammation extending to deep fascia. The parts of the body most affected are the extremities. Subsequent complications may include contractural deformities. The main form of treatment is wide excisional surgery, including amputation of limbs, which requires prolonged hospitalization and is thus a significant burden on hospital resources and budgets.In recent years, there has been increased incidence of BU in West Africa (including Benin, Burkina Faso, Cote d'Ivoire, Ghana, Guinea, Liberia and Togo), Mexico, French Guyana, Papua New Guinea and Australia. The disease seems to affect mostly impoverished inhabitants in remote and rural areas; children are the most vulnerable, accounting for about 70% of the cases . The WorMycobacterium ulcerans (MU), which was first described in Bainsdale, Australia, in 1948 [The causative agent of BU is  in 1948 . From th in 1948 ,6. Porta in 1948  have sug in 1948 .Epidemiological data suggest that environmental factors such as climate, soil, geology, geochemistry, etc. may indirectly influence or contribute to MU infection . In addiSince MU is known to be present in nature although its reservoir is not known and since the epidemiology of BU is still unclear, there is a need to have a better understanding of environmental, ecological, and behavioural factors that predispose to infection. Spatial analysis potentially contributes important information leading to the understanding of the epidemiology and etiology of BU. The main objective of this paper is to explore relationships between some spatial environmental factors and the prevalence of BU.P values range from 0.09 to 0.46. The buffers with the highest P values (i.e., 0.09) are 100 m for drainage channels and 400 m for farmlands. With these buffers 24 of the 61 settlements (i.e., 39%) fall within 100 m of arsenic-enriched drainage channels  levels of arsenic. Arsenic interacts with and inhibits several enzymes in the body  leading The results of this study reveal spatial dependency of BU prevalence upon proximity to drainage channels and farmlands containing > 15 ppm arsenic. Proximity implies chronic exposure to and/or ingestion of elevated concentrations of arsenic, which influences susceptibility to infection.It has been consistently theorized that BU is acquired when MU enters the body through a skin rupture ,27. HoweSeveral dermatological diseases (e.g., Bowen's disease, hyperkeratosis, hyperpigmentation) are related to arsenic ingestion and exposure . BioaccuIn a case study, Amofah et al.  reportedSpatial analysis of data provides opportunities for epidemiologists to study associations between environmental factors and spatial distribution of diseases . A geogrIn this study, topographic map data, stream sediment geochemical data for arsenic, ASTER satellite imagery and locations of settlements with BU cases were the basic data inputs into the GIS. Spatial data processing was carried out (a) to delineate arsenic-enriched catchment basins based on arsenic concentrations in stream sediment samples, (b) to delineate farmlands from ASTER satellite imagery and determine arsenic-enriched farmlands based on catchment basin data and (c) to extract drainage channels from the topographic map and determine arsenic-enriched drainage channels based on arsenic-enriched catchment basins. Proximity analysis was undertaken to determine spatial relationships between BU-affected areas and the arsenic-enriched areas determined from the data inputs.The study area is in Ghana, where the first case of BU was reported in 1971 and, between 1991 and 1997, more than 2000 cases have been reported . The dis2. The district capital, Manso Nkwanta, is about 40 km south of Kumasi. The district is drained by the Offin and Oda rivers. Vegetation in the district is composed mainly of secondary forests, thicket, forb regrowth (i.e., soft-stemmed leafy herbs, mostly the weeds, which appear on farms and have to be cut regularly) and swamp vegetation. Vegetation thrives in ferric fluvisols, which are the major soil types in the district. These soils have been developed through yearly rainfall ranging from 125 to 200 cm with temperatures of 22 to 30°C. The landscape of the district varies from gentle to broken.The Amansie West District lies between latitudes 6°N and 6°45'N and longitudes 1°30'W and 2°15'W. It covers an area of about 1,136 kmThe district is underlain by Lower Proterozoic Birimian and, to a lesser extent, Tarkwaian rocks. Throughout Ghana, Birimian rocks of West Africa are mainly volcanic greenstones with intervening sedimentary rocks and granitoid intrusions, in places containing deposits composed of pyrite, arsenopyrite, minor chalcopyrite, sphalerite, galena, native gold and secondary hematite .The district has about 310 settlements  with a population in 2000 of 108,726. There are approximately equal percentages of males and females (49% and 51%, respectively), of whom 70% are farmers and 22% are engaged in legal and 'galamsey'  mining.2, comprising 61 settlements and including the Bilpraw goldmine (formerly a treasure mine of the Ashanti Kings). The BU cases per settlement range from 1–29.The study area is the east-central part of the Amansie West District (covered by a single topographic map sheet, 0602C1), with an area of about 623 kmThe following are the sources of spatial data input to the GIS.• Incidence of BU per settlement in 1999, obtained from Korle-BU Teaching Hospital, Accra, Ghana.• Settlement population estimates for 2000, projected by the Ministry of local government and rural development.• Topographic map , a single sheet covering the study area, obtained from the Survey Department, Accra, Ghana.• Location map  of stream sediment samples collected in part of the Amansie West District in 1992 and list of arsenic concentrations determined in these samples, obtained from the Geological Survey Department, Accra, Ghana.• Boundary map  of the district, obtained from the Amansie West District Administration.• ASTER imagery (level 1B) acquired on 15/01/2002, obtained from the US Geological Survey.• Landuse/landcover map of Ghana (traced on Landsat TM data of 1998 and published in the same year), obtained from the Remote Sensing Application Unit (RSAU), University of Ghana, Legon.The GIS operations were carried out in three principal steps: (1) spatial data capture; (2) generation of spatial factor maps; and (c) spatial data analysis. The GIS operations were carried out using ILWIS (Integrated Land and Water Information Systems), a GIS software package developed by the International Institute for Geo-information Science and Earth Observation (ITC) in the Netherlands.The different analog maps were scanned then georeferenced (by defining the x and y coordinates of the corner points of the maps) into a UTM coordinate system. From the scanned map, spatial data were captured by screen digitizing. From the topographic map, rivers, streams and gullies were digitised as line segments as were elevation contours. The boundaries of the district were digitised as line segments and then polygonized. The locations of centres of 61 settlements (identifiable on the topographic map) were digitised as points and the BU incidence in 1999 was recorded as spatial attribute of each settlement.From the stream sediment sample location map, the locations of the samples were digitised as points and the arsenic concentrations (in ppm) were recorded as a spatial attribute of each sample. The ASTER imagery was also georeferenced to the same coordinate system using eight reference points (tie points), which were selected in the image and which could be identified in the topographic map. Using an affine transformation, a root mean square error (RMSE) of 0.58 pixel was obtained in georeferencing the ASTER imagery.For each of the settlements with incidence of BU the percentage prevalence of BU was calculated. Prevalence expresses cases of a disease in terms of the proportion of the population afflicted at a specified time . It is eThe spatial factor maps generated from the stream sediment geochemistry data for use in the spatial analysis were: (a) map of arsenic-enriched catchment basins; (b) map of arsenic-enriched farmlands; (c) map of arsenic-enriched drainage channels.The stream sediment geochemical data for arsenic were initially analysed statistically to determine a threshold value that divides the data into background  classes and anomalous  classes of arsenic concentrations. The data are lognormally distributed and, after removing obvious outliers in the data, a geometric mean of 8.9 ppm As and standard deviation of 2.8 ppm As were obtained. The threshold value was therefore set at 15 ppm As (i.e., approximately the mean plus two standard deviations). The spatial distribution of arsenic was then mapped through the generation of a catchment basin anomaly map in which a sample catchment basin is assigned the geochemical attribute of the corresponding sample ,46. Gene• creation of a raster digital elevation model (DEM) through interpolation of elevation contours;• generation of raster map of drainage lines; and• calculation of sample catchment basin boundaries via an iterative calculation procedure involving the DEM and the raster map of drainage lines.The catchment basin map of arsenic concentrations was then classified into a binary map showing arsenic-normal areas (with ≤ 15 ppm As) and arsenic-enriched areas (with > 15 ppm As) as shown in Figure A supervised classification of ASTER imagery was carried out to distinguish between the major landcover/landuse classes known in the area. These landcover classes are (a) forest areas, (b) residential areas or settlements (bare of vegetation), and (c) farmlands. Using the available landuse/landcover map and topographic map as references, training pixels of known landuse/landcover classes were selected using a colour composite of ASTER bands 2, 3 and 4. These three bands gave the highest optimal index factor (OIF), which indicates the combination of three spectral bands that provide optimum information about landcover . The boxTo determine arsenic-enriched farmlands, a Boolean AND operation was performed by crossing the catchment basin anomaly map and the classified landcover/landuse image. About 21% of the total area of farmlands in the classified image is arsenic-enriched.A Boolean AND operation was performed by crossing the catchment basin anomaly map and the raster map of drainage lines. About 22% of the total length of drainage lines is indicated to be arsenic-enriched.The inhabitants of a settlement earn their livelihoods by exploiting the resources of the surrounding land. This land influences their exposure to infections and to environmental factors that dispose to infections. Proximity analysis was therefore used to determine spatial relationships between BU prevalence per settlement and (i) arsenic-enriched farmlands and (ii) arsenic-enriched portions of the drainage system. The proximity analysis was carried in two principal steps. First, maps of distances from arsenic-enriched farmlands and arsenic-enriched portions of the drainage system were generated. Second, the point map of BU prevalence per settlement was overlaid on (or crossed with) each of these maps.A buffer is a zone of specified distance around a selected map feature. A GIS creates buffer zones around selected map features such as arsenic-enriched farmlands and arsenic-enriched portions of drainage systems. Around each of these, buffers were set at intervals of 100 m up to 1000 m. Each buffer zone map was crossed with BU prevalence data of settlement to determine how many of these fall within and outside of the buffer.At each increasing interval of 100 m, a test of the significance of the difference of the mean BU prevalence within the buffer and outside of the buffer is made using the t-statistic:,  are the sample means,  is the pooled sample variance, i nand j nare the sample sizes from population i and j. Using ij tand degrees of freedom given by i n+ jn-2, a t distribution look-up table provides the probability, P that the means are significantly different.where AAD carried out the research and drafted the manuscript. EJMC guided parts of the research and both EJMC and MH reviewed the manuscript.


Genes use a simple language—written in the molecules of DNA—to build thousands of proteins in a dizzying variety of sizes and shapes. With only four different nucleotide building blocks, DNA codes for the 20 different amino acids (each with their own structures and properties) that provide the foundation for the enormous diversity of protein form and function. This diversity makes the systematic study of all the proteins of a given organism , a challenging enterprise.Interactions between proteins underlie nearly every fundamental process within the cell. They can form higher-order multiprotein complexes like those involved in transcription and replication, help transport proteins to their proper location in the cell, and participate in signaling pathways. Because of their importance, disruption of these interactions can have disastrous consequences. For example, the loss of the ability of a normal cellular protein called Src to bind to certain other proteins can be associated with cancer progression. The determinants of these interactions are poorly understood, but in many cases these interactions are mediated by small pieces of the proteins, which are called peptides. Peptides serve as the starting point for the novel strategy reported in this issue.Gianni Cesareni and colleagues have added to the repertoire of proteomic analysis by devising a global strategy to investigate protein–protein interactions on an organismal level using yeast as a model organism. The authors select a protein of interest from yeast, which can be thought of as the “bait” for which they wish to identify protein-binding partners. They start by looking at a number of different previously identified peptides that bind the bait protein. Commonalities between the sequences of these peptides form the “consensus” binding sequence, a base framework of protein sequence from which many possible variations can be derived. Since the protein sequences of all proteins (the proteome) in yeast can be deduced from the sequenced genome, the authors can scan the proteome for proteins that contain the consensus, or a closely related, sequence. These proteins could potentially bind the bait protein. Peptide sequences from these identified proteins are synthesized chemically and arrayed on a membrane, which is bathed in a solution containing the bait protein. After washing off the excess bait protein, they can figure out where it remains on the membrane and therefore tell which peptides the bait protein has bound. The proteins corresponding to these peptides are candidate binding proteins that are validated by further experimentation.The protein–protein interactions identified by this approach can be used to extend the network of known interactions in the proteome. This will enable researchers to draw functional linkages between proteins, whether they are involved in a basic biological process or in human disease. By examining whole families of proteins, it may also aid in elucidating the underlying determinants of binding specificity, which would provide clues to the biomechanisms underlying cell processes. These insights could lead to methods for manipulating these interactions, perhaps even in cases of human disease, as in the case of Src and cancer. This approach can readily be applied to the proteomes of more complex organisms like humans and adds to the growing number of experimental strategies available to researchers in proteomics.


Cell reports the involvement of an RNA helicase in the pathway by which HIV exports partially spliced and unspliced RNA out of the nucleus. This suggests the ubiquity of RNA helicases in RNA export from the nucleus, and has novel mechanistic implications.Nucleo-cytoplasmic transport of RNA is one of many cellular pathways whose illumination has progressed hand in hand with understanding of retroviral mechanisms. A recent paper in  HIV must export to the cytoplasm not only full length genomic and structural gene-encoding RNA species, but also partially and fully spliced RNAs which code for a variety of overlapping structural and accessory genes. Whereas the fully spliced HIV RNA species readily exit the nucleus and undergo translation, the partially spliced and unspliced species require the interaction of the HIV-1 Rev protein with the cis-acting, viral RRE (Rev responsive element) for expression. Simpler retroviruses, such as the type-D Mason-Pfizer monkey virus (MPMV) solve a similar problem by encoding a cis-acting, constitutively active CTE (constitutive transporter element) in the 3'UTR of the viral genome, which uses a distinct pathway . Of lessThe Rev/RRE pathway involves the Ran-CRM-1, shuttling system: In the nucleus, Ran-GEF converts Ran-GDP to Ran-GTP. Ran-GTP bound CRM-1 binds the NES  domain of Rev, which is in turn bound to the RRE, and enables CRM-1 to transport the resulting RNA/protein complex as cargo into the cytoplasm, presumably through CRM-1 interactions with nucleoporins. In the cytoplasm, Ran-GAP converts Ran-GTP to Ran-GDP, releasing the REV/RNA cargo. The asymmetric distribution of Ran-GEF and Ran-GAP between nucleus and cytoplasm ensures a constant RAN-GTP/GDP gradient to facilitate CRM-1 recycling and continued Rev/RRE export. CTE-mediated RNA export is based on binding to the Tap-NXT complex, and export through an unrelated transport pathway, which, ironically, is responsible for the bulk of mRNA transport .Cell, report the involvement of the RNA helicase, DDX3, in the Rev/RRE pathway [Enter the laboratories of Kuan-Teh Jeang and Larry Kleiman, who, in a comprehensive study in the October 29th issue of  pathway . Interesin vitro (in a manner independent of its NES). Although no data are presented concerning direct in vitro binding of DDX3 to Rev, for such an interaction to exist and to be significant, DDX3 would have to bind one of the three identified functional regions in Rev. These include the NES, the coincident NLS/RBD  and the Rev multimerization domain, which flanks the RBD [A number of findings not only provided evidence consistent with a role for DDX3 in the Rev export pathway, but also provided mechanistic insights. DDX3, though constitutively cytoplasmic, shuttles between nucleus and cytoplasm in a manner dependent on CRM-1, as evidenced by inhibition by leptomycin B (LMB), which inhibits CRM-1 export, but not Tap export. DDX3 also localizes to nuclear outer membranes in a speckle formation and co-immunoprecipitates with nucleoporins, in addition to having a distribution in the cytoplasm. In cell lysates, DDX3 co-immunoprecipitates with either Rev protein or CRM-1. Although the studies on cell lysates fail to distinguish between pairwise binding and ternary complex formation, ternary complex formation is a distinct possibility. DDX3 directly binds CRM-1  the RBD . No regiIn the tradition of good research, the results of Yedavalli et al.  pose as On a highly speculative note, the intimate involvement of a DEAD box helicase in the Rev pathway, and the observation that DDX3 has a cytoplasmic localization, in addition to its nuclear membrane localization, recalls some old data from two independent laboratories, indicating that in some systems Rev may not act on export, but on post export events, to promote Rev/RRE dependent expression ,18. The Finally there is the possibility that specific helicases will be valid therapeutic targets for antiretroviral chemotherapy. Although targeting a cellular gene involved in a viral pathway risks inhibiting a necessary cellular function, it avoids the overwhelming problem posed by rapid viral mutation to resistance.RRE: Rev Responsive ElementMPMV: Mason-Pfizer monkey virusCTE: Constitutive Transport ElementNES: Nuclear Export SignalLMB: Leptomycin BNLS: Nuclear Localization SignalRBD: RNA Binding DomainNone. The opinions expressed are those of the author and do not necessarily express the opinion of the FDA.


Anti-ischemic therapy, in particular beta-blockers, is the most commonly employed drug for the control of myocardial ischemia in patients with stable coronary artery disease. Its widespread use also in patients with suspected coronary artery disease has important practical, clinical diagnostic and prognostic implications because diagnostic tests are heavily influenced by its effects. In the present review, the pathophysiological mechanisms of ischemia protection by antianginal therapy are described. Not all stressors are created equal in front of the different classes of antianginal drugs and on their turn the different classes of drugs exert different levels of protection on inducible ischemia. Several clinical implications can be drawn: From the diagnostic viewpoint antianginal therapy decreases test sensitivity, offsetting the real ischemic burden for a too high percentage of false negative tests. From the prognostic viewpoint test positivity in medical therapy identifies a group of subjects at higher risk of experiencing cardiac death and positivity on medical therapy can be considered a parameter of ischemia severity. Nonetheless in patients with known coronary artery disease the ability of antianginal therapy to modify the ischemic threshold at stress testing represent a powerful means to assess therapy efficacy. From a practical viewpoint, the use of antianginal therapy at time of testing has advantages and disadvantages which are largely dependent on the purpose a test is performed: if the purpose of testing is to diagnose ischemia, it should be performed in the absence of antianginal medications. If the purpose of testing is to assess the protective effects of antianginal therapy, the test should be performed on medications. Anti-ischemic therapy, in particular beta-blockers, is the most commonly employed drug for the control of myocardial ischemia in patients with stable coronary artery disease. Its widespread use also in patients with suspected coronary artery disease has important practical, clinical diagnostic and prognostic implications because diagnostic tests are heavily influenced by its effects. The diagnostic and prognostic impact of anti-ischemic therapy on stress testing is largely ignored but not negligible. The issue raises several questions: How to evaluate patients at time of testing for myocardial ischemia? How to interpret a stress test performed on anti-ischemic therapy? Are the stressors employed for the detection of myocardial ischemia created equal in relation to the different classes of drugs used in clinical practice? Is stress testing able to assess the efficacy of medical therapy in patients with known coronary artery disease? Has the protection of anti-ischemic therapy on inducible myocardial ischemia any impact on long-term survival?The answer to all these issues relies on the mechanism through which myocardial ischemia is induced by the different stressors (exercise or pharmacologic such as dipyridamole and dobutamine) employed during stress testing. Test exploring organic coronary artery stenosis can induce ischemia by two basic mechanisms: 1. an increase in oxygen demand, exceeding the fixed supply and 2. flow maldistribution due to inappropriate coronary arteriolar triggered by a metabolic/pharmacologic stimulus . During In the presence of coronary atherosclerosis, appropriate arteriolar dilation can paradoxically exert detrimental effects on regional myocardial perfusion, causing overperfusion of myocardial layers or regions already well perfused in resting conditions at the expense of regions or layers with a precarious flow balance in resting conditions . Anti-isHowever, experimental studies on the model of the exercising dog have shown that beta-blockers protect myocardium from stress-induced myocardial blood flow-function relation: for a given transmural flow, there is a rise of subendocardial and a fall of subepicardial flow, with an improved regional performance . This saOn the basis of these premises and taking into consideration that the markers of inducible myocardial ischemia  are very different therefore expressing a different sensitivity to the action of anti-ischemic therapy at time of testing, it is clear that medical therapy affects test results  A negative test on medical therapy might represent a false negative result, therefore it is advisable to repeat the test off therapy in order to assess the real ischemic burden through the conventional stress echocardiographic parameters ,37. – i.Patients may be undergoing various forms of antianginal therapy at the time of testing, both an advantage and a disadvantage for stress echocardiography testing. The disadvantage is that antianginal therapy reduces sensitivity, since stress-induced wall motion abnormalities are caused by the development of obligatory myocardial ischemia. The advantage is that the effect of therapy can be assessed using an objective, primary ischemic end-point such as changes in stress-induced wall motion abnormalities. The presence of ischemia can be titrated on the basis of the ischemic-free stress time and the extent and severity of the induced dyssynergy. The various forms of stress are differently affected by various forms of therapy. In patients with known or suspected coronary artery disease, ongoing anti-ischemic therapy at the time of testing heavily modulates the prognostic value of pharmacological stress echo. In presence of concomitant anti-ischemic therapy, a positive test is more prognostically malignant and a negative test less prognostically benign. However, the decision to remove a patient from beta-blocker therapy for stress testing should be made on an individual basis and should be done carefully to avoid a potential hemodynamic rebound effect, which can lead to accelerated angina or hypertension . No majo